Parnas-Conference2011-abstracts - BIO

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Abstracts of the
8th Parnas Conference
Warsaw, Poland
August 27–31, 2011
Warsaw, Poland, August 27–31, 2011
Patronage:
Minister of Foreign Affairs — Mr. Radoslaw Sikorski
Minister of Science and Higher Education — Prof. Barbara Kudrycka
Honorary Committee:
Prof. Jolanta Barańska — Former President of the Polish Biochemical Society
Prof. Hanna Gronkiewicz-Waltz — Mayor of Warsaw
Prof. Michał Kleiber — President of the Polish Academy of Sciences
Dr. Henryk Litwin — Ambassador of Poland in Ukraine
Ms. Agnieszka Magdziak-Miszewska — Ambassador of Poland in Israel
Prof. Markijan Malskyj — Ambassador of Ukraine in Poland
Prof. Maciej J. Nałęcz — Director of the Division of Basic & Engineering Sciences, UNESCO
Prof. Israel Pecht — Secretary General of the Federation of European Biochemical Societies
Mr. Zvi Rav-Ner — Ambassador of Israel to Poland
Prof. Lech Wojtczak — Former President of the Polish Biochemical Society
Special Guests:
Prof. Stefan Angielski
Prof. Lyudmila Drobot
Prof. Janina Kwiatkowska-Korczak
Prof. Piotr Laidler
Dr. Barbara Parnasowa
Prof. Mathias Sprinzl
Scientific Committee:
Prof. Andrzej Dżugaj (Poland) — Chairman
Prof. Michael Eisenbach (Israel)
Prof. Orna Elroy-Stein (Israel)
Prof. Ganna Elska (Ukraine)
Prof. Leszek Kaczmarek (Poland)
Prof. Serhiy Komisarenko (Ukraine)
Prof. Sergey Kosterin (Ukraine)
Prof. Jacek Kuźnicki (Poland)
Prof. Shimon Schuldiner (Israel)
Prof. Maciej Żylicz (Poland)
Local Organizing Committee
Prof. Maria Jolanta Rędowicz — Head
Prof. Jolanta Barańska
Justyna Karolczak
Dr. Joanna Kruszewska
Dr. Katarzyna Kwiatkowska
Dr. Hanna Nieznańska
Iuliia Pavlyk
Dr. Paweł Pomorski
Dr. Elżbieta Rębas
Dr. Oleh Stasyk (Ukraine)
Dr. Joanna Szczepanowska
Ewa Szczepanik
Israela Tishler (Israel)
Dr. Urszula Wojda
VIII Parnas Conference
ORGANIZERS
Polish Biochemical Society
Israel Society for Biochemistry and Molecular Biology
Ukrainian Biochemical Society
CO-ORGANIZERS
Nencki Institute of Experimental Biology PAS, Prof. Adam Szewczyk, Director
International Institute of Molecular and Cell Biology, Prof. Jacek Kuźnicki, Director
Institute of Biochemistry and Biophysics PAS, Prof. Piotr Zielenkiewicz, Director
Faculty of Biology of the University of Warsaw, Prof. Joanna Pijanowska, Dean
University of Wrocław, Prof. Marek Bojarski, Rector
Main Supporting Institution
Supported within 3+ Meeting Programme
The Conference was supported by the EU FP7 Project BIO-IMAGINE:
BIO-IMAGing in research INnovation and Education, GA No. 264173
Dear Participants,
WELCOME TO VIII PARNAS CONFERENCE
We cordially invite you to participate in the 8th Parnas Conference organized by the Polish Biochemical Society,
Ukrainian Biochemical Society and Israel Society for Biochemistry and Molecular Biology.
Parnas Conferences commemorate the world known Polish biochemist Jakub Karol Parnas. Before the Second
World War, J. K. Parnas was a professor of physiological chemistry at University in Lwów (in Polish) or Lviv
(in Ukrainian) or Lemberg (in Yiddish).
The Parnas' laboratory was a unique place where in a friendly atmosphere young Polish, Ukrainian and Jewish
scientists had studied glucose metabolism. The results of their work were highly appreciated as for many years
glycolysis was also termed as Embden-Meyerhof-Parnas pathway.
The first Parnas Conference was organized in 1996 in Lviv by the Ukrainian and Polish Societies, and since
then every two years the Conferences have been organized alternately in Poland and in Ukraine. The coming 8th
Parnas Conference will be organized for the first time by the Polish, Ukrainian and Israel biochemists in Warsaw.
Welcome to Warsaw to meet in a friendly atmosphere, we hope reminding that of the Parnas' laboratory.
Andrzej Dżugaj
President of the Polish Biochemical Society
Serhiy Komisarenko
President of the Ukrainian Biochemical Society
Michael Eisenbach
President of the Israel Society for Biochemistry and Molecular Biology
Warsaw, August 27th 2011.
Contents
Opening lectures:
1
Keynote Lectures
3
Session 1: & 2: Post-transcriptional regulation
5
Session 3: Bioinformatics for gene expression 19
Session 4: Gene expression and brain disease
25
Session 5: Cell guidance
36
Session 6: Transporters in adaptation to hostile environments
48
Session 7: Protein structure and function
52
Session 8 & 9: Calcium in signalling
83
Index of Authors
95
by Andrzej Dzugaj, Rostislav S. Stoika
by Aaron Ciechanover, Ada Yonath, Alexander Wlodawer
Chair: Orna Elroy-Stein, Serhiy Komisarenko
Chair: Ganna Elska
Chair: Leszek Kaczmarek
Chair: Michael Eisenbach
Chair: Shimon Schuldiner
Chair: Maciej Żylicz
Chair: Olga Matyshevska, Jacek Kuźnicki
This issue of Acta Biochimica Polonica contains author-supplied copy of abstracts not
verifed by qualifed reviewers
Sponsors and Exhibitors:
SIGMA-ALDRICH – main sponsoring company
KRZYSZTOF KUCHARCZYK TECHNIKI
ELEKTROFORETYCZNE SP. Z O.O.
LAB-JOT
LABART SP. Z O.O. VWR INTERNATIONAL GROUP
SARSTEDT
ALAB SP. Z O.O.
JOHN WILEY & SONS
EURx SP. Z O.O.
PERKIN ELMER
COMESA POLSKA
Conference supported by the
Polish Ministry of Science and Higher Education
Opening Lectures
O.1
Jakub Karol Parnas Polish biochemist
Andrzej Dżugaj
Department of Genetics and Microbiology, Wrocław University,
Wrocław, Poland
e-mail: Andrzej Dzugaj <[email protected]>
Jakub Karol Parnas was born in 1884 in Mokrzany, a small
village in Galizia, which at that time belonged to AustroHungarian Monarchy. As a result of the WWI, Galizia became part of Poland. Following the Soviet-German agreement this land was turned over to the Soviets. From 1941
to 1944 the land was occupied by Germans and by now it
is a part of Ukraine. Parnas studied chemistry at the universities of Berlin, Strasburg, Zurich and Munich where in
1907, he received the Ph.D. He was associate professor at
Strasburg University in1913 and professor of physiological
chemistry at Lwow University (1920–1941).
Lwów (in Polish) or Lviv (in Ukrainian) or Lemberg (in
Yiddish) was a very special place. It was the Polish city
built on Ukrainian land. In thirties of the last century, the
Lwów population comprised of 52% Poles, 30% Jews,
17% Ukrainians and small percentage of other minorities,
like Germans, Czechs, Armenians. At that time Lwow was
not a peaceful city. Ukrainian Nationalist struggled for free
Ukraine and claimed that Lwów should be the Ukrainian
city. Jews fought against the increasing wave of the antiSemitism. Poles tried to maintain the status quo. Agents
of German Nazi and Soviet Communist were also present
trying to destabilize the situation in the city.
The Parnas’ laboratory was an unique place where in a
friendly atmosphere, young Polish, Ukrainian and Jewish
scientists studied glucose metabolism in Vertebrate skeletal
muscle cells. In order to follow the fate of the phosphate
residue in glucose metabolism, it was necessary to employ
the radioactive phosphor 32P which had been successfully
used in Parnas laboratory. The quintessential discovery of
Parnas and his collaborators was: the breakdown of glucose results in ATP production.
A Time Line of the Discovery of Glycolysis
hexokinase
hexoisomerase
1,6-phosphofructokinase
aldolase
triosephosphate-isomerase
GAPDH dehydrogenase
1,3PDG kinase
3-phosphoglycerate mutase
enolase
pyruvate kinase
Meyerhof 1927
Cori, Cori 1936
Ostern, Guthke, Terszakovec 1936
Meyerhof, Lohmann, Schuster 1936
Meyerhof, Lohmann, Schuster 1936
Warburg, Christian 1939
Bücher 1942
Meyerhof, Kiessling 1935
Meyerhof, Kiessling 1935
Parnas 1934
Two enzymes of glycolysis have been discovered by Parnas
and his collaborators. Ostern, Guthke and Tershakowec
discovered phosphofructokinase and Parnas himself discovered pyruvate kinase. The results of their work were
highly appreciated and for many years glycolysis was also
termed as Embden-Meyerhof-Parnas pathway.
The good time for Parnas and his collaborators has ended
in 1939, when the WWII began. Lwow was occupied by
Soviets and became a part of the Soviet Union. Initially,
Parnas was respected by Soviet authorities and even became the member of the Soviet Academy of Science and
was allowed to continue his research. In 1941, Germans
invaded Soviet Union and Parnas was forced to move to
Ufa in the central part of Soviet Union.
After the WWII, Parnas wanted to go to Polish Republic
but Soviet authorities did not allow him to leave the Soviet
Union. Instead, in 1947, he was arrested and shortly after,
he died in Moscow prison. The circumstances of his death
have never been elucidated.
Parnas’ students and collaborators were pioneers of biochemistry in Poland. Baranowski and Meybaum-Katzenelebogen in Wrocław, Mozolowski in Gdansk, Mochnacka
and Heller in Warsaw. Polish biochemists still remember
Jakub Karol Parnas as the outstanding biochemists and the
great man and consider him as the Founder of the Polish
School of Biochemistry.
Abstracts
2
O.2
Parnas Conferences: 1, 2, 3 …
Rostislav S. Stoika
Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine
e-mail: Rostyslav Stoyka <[email protected]>
Professor Jakub Oskar Parnas was born in Western
Ukraine, and he worked for a long time (1920–1941) at
the University of its big regional center Lviv (Lvov, Lwow,
Lemberg, Leopolis). Parnas is considered to be a father of
Polish biochemistry, and he is also one of the founders of
biochemistry in Ukraine. It should be stressed that being an
outstanding scientist in the field of enzymology of carbohydrate metabolism (Embden-Meyerhof-Parnas pathway)
and energy generation, Prof. Parnas belongs to the whole
world scientific community. Before the Second World War,
Lviv was a very multinational city, and it was reasonable
that in Parnas’s research lab scientists of the dominating
there Polish, Jewish and Ukrainian communities worked.
Prof. Parnas was killed by Stalin’s regime in 1949, and for
a long time there were no official celebrations in his honor. Only in 1995, there was a meeting of the President of
Polish Biochemical Society Prof. Liliana Konarska, its VicePresident Prof. Jolanta Baranska, known Polish biochemist
Prof. Lech Wojtczak, from the Polish side, and Prof. Rostyslav Stoika, the Director of Lviv Division of the Institute
of Biochemistry, Academy of Sciences of Ukraine, from
the Ukrainian side. At that meeting, a decision was taken to
put a commemorative plaque devoted to Prof. Parnas on
the building of the Department of Biochemistry which he
headed at Lviv University. It was proposed by Prof. Stoika
that such action should be done at the bilateral UkrainianPolish scientific conference dedicated to Parnas’s memory,
and this idea was successfully realized in September 9–11,
1996. At that conference, there was only one scientist from
outside Ukraine and Poland — Prof. Simon Shnol who is
Prof. Parnas’s scientific “grandson”. Another good idea
came to my mind at the closing ceremony of the 1st Parnas conference, when I proposed that Parnas conferences
should be organized regularly, once in two years in Ukraine
and Poland. That idea was very welcomed, and it successfully works till now. Together with Prof. Stefan Angielsky,
in 1998, we have co-organized the 2nd Parnas conference
in Gdansk (Poland). It was very successful for two reasons;
1) it coincided with the days of Ukraine in Poland, and
2) around 30 leading biologists from the Western countries (including the USA) participated in that conference.
Since that time, Parnas conferences really got to be international ones, and they gained great scientific respect. The
3rd Parnas conference was co-organized by Prof. Rostyslav Stoika (Ukraine) and Prof. Jolanta Baranska, President
of Polish Biochemical Society. The international scientific traditions were successfully continued at that Parnas
conference. Starting from the 4th Parnas conference, the
President of the Ukrainian Biochemical Society, Prof. Serhiy Komisarenko took the leadership as a Ukrainian coorganizer of Parnas conferences, and the best traditions of
these conferences were not only preserved but also greatly
developed (ex. moving to trilateral organization of Parnas
conferences). I would like to acknowledge all people who
were with us at the start of Parnas conferences which have
really provided an excellent opportunity for initiating collaboration between the biochemists of different countries,
and also for gaining new friends.
Keynote Lectures
K.1
K.2
The ubiquitin proteolytic system —
from basic mechanisms thru human
diseases and onto drug development
Thoughts about the origin of life
and antibiotics resistance
Aaron Ciechanover
Cancer and Vascular Biology Research Center, Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa, Israel
e-mail: Aaron Ciechanover <[email protected]>
Between the 50s and 80s, most studies in biomedicine
focused on the central dogma — the translation of the
information coded by DNA to RNA and proteins. Protein degradation was a neglected area, considered to be a
non-specific, dead-end process. While it was known that
proteins do turn over, the high specificity of the process
— where distinct proteins are degraded only at certain time
points, or when they are not needed any more, or following denaturation/misfolding when their normal and active
counterparts are spared — was not appreciated. The discovery of the lysosome by Christian de Duve did not significantly change this view, as it was clear that this organelle
is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate-specific. The
discovery of the complex cascade of the ubiquitin solved
the enigma. It is clear now that degradation of cellular
proteins is a highly complex, temporally controlled, and
tightly regulated process that plays major roles in a variety
of basic cellular processes such as cell cycle and differentiation, communication of the cell with the extracellular environment and maintenance of the cellular quality control.
With the multitude of substrates targeted and the myriad
processes involved, it is not surprising that aberrations in
the pathway have been implicated in the pathogenesis of
many diseases, certain malignancies and neurodegeneration
among them, and that the system has become a major platform for drug targeting.
Ada Yonath
Department of Structural Biology, Weizmann Institute, Rehovot, Israel
e-mail: Ada Yonath <[email protected]>
Ribosomes, the universal cellular machines, act as polymerases that translate the genetic code into proteins with high
efficiency. They posses spectacular architecture accompanied by inherent mobility that facilitate their efficient performance although they are essentially RNA enzymes, thus
indicating natural mechanisms to turn an inefficient RNA
machine into well performing enzyme.
The peptide bond formation site is located within a universal internal symmetrical region connecting all of the
remote ribosomal features involved in its functions. The
elaborate architecture of this region is capable of positioning both the aminoacylated and peptidyl tRNA substrates
in stereochemistry required for peptide bond formation,
for substrate-mediated catalysis, and for substrate translocation, hence enabling elongation of nascent proteins. The
central location of this region, its connectivity to all of the
functional features of the ribosome this region indicates its
possible key role in intra-ribosome signaling. Its almost full
conservation suggests that it is a remnant of a prebiotic
RNA machine that is still functioning in the contemporary
ribosome.
Adjacent to this site is an elongated tunnel, along which
nascent chains progress until they emerge out of the ribosome. This tunnel is involved in gating and chaperoning functions; provides the binding site of the first cellular
chaperone that encounters the emerging nascent chain, and
hosts a major family of antibiotics that target the ribosome. Analysis of the structure of the pocket hosting these
antibiotics hints at a possible pathway in the evolution of
the species.
4
Abstracts
K.3
Structural biology as a tool for designing
drugs against retroviral diseases
Alexander Wlodawer
Macromolecular Crystallography Laboratory, Center for Cancer
Research, National Cancer Institute, NIH, Frederick, MD, USA
e-mail: Alexander Wlodawer <[email protected]>
It has been 30 years since the first reports of an epidemic
of a new human disease, later called AIDS, have been published. The disease was invariably lethal during the first 15
or so years since its identification, but an unprecedented
effort in basic and applied research has led to a development of more than 20 drugs which, in combination, have
completely changed the clinical outcome, allowing patients
to maintain fairly normal lifespan. This drug development
effort was led, to a very large extent, by the success in determining the properties of HIV, the retrovirus responsible for the disease. A very important role was played by
structural biology, with the structures of almost all virallyencoded proteins, as well as of some human proteins that
play a role in the maintenance of the viral lifecycle, having
been solved and made available to drug developers. In particular, the availability of the structures of the three enzymes encoded by the HIV (protease, reverse transcriptase,
and integrase) played a major role in creation of a variety
of very successful drugs that have changed the outcome
of the epidemic, at least in the developed world. The success of designing anti-AIDS drugs was a major boost for
structure-based drug design in general, and may also be important for the future work on drugs targeting other pathogens, including emerging retroviruses.
Session 1 & 2: Post-transcriptional regulation
Part I
Lectures
L1.1
The role of non coding RNA in
flowering time regulation
Szymon Swiezewski
Institute of Biochemistry and Biophysics PAS, Warsaw, Poland
e-mail: Szymon Swiezewski <[email protected]>
Transcription generates an extensive array of non-proteincoding RNA(ncRNA), the functional significance of which
is mostly unknown. Studies in yeast, humans, drosophila
and bacteria have shown that the majority of nucleotides
in these genomes are transcribed, generating a mixture of
long and short RNAs, most of which are non-coding. This
widespread non-coding transcription changes in a complex
manner during development and in response to environmental signals. This has led to opposing models: some researchers believe that the vast majority of ncRNA constitutes
transcriptional noise, while others suggest that ncRNA is
involved in regulating different aspects of genome function and evolution. Although ncRNA have been intensively
studied in the last years, many questions remain. It is increasingly accepted that whole genome approaches, though
instrumental in discovering the widespread occurrence of
non-coding transcription, are less powerful in exploring
the numerous molecular mechanisms that these transcripts
participate in.
L1.2
Regulation of miRNA repression and
miRNA turnover in mammalian cells
Witold Filipowicz
Friedrich Miescher Institute for Biomedical Research, 4002 Basel,
Switzerland
e-mail: Witold Filipowicz <[email protected]>
MiRNAs regulate gene expression post-transcriptionally by
causing translational repression, and mRNA deadenylation
and degradation. Mechanistic details of how miRNPs repress protein synthesis are still poorly understood. Proteins
of the GW182 family represent effector proteins mediating
miRNA repression in metazoa. Deletion analysis of the human GW182 protein TNRC6C and Drosophila dGW182
revealed that their C-terminal fragments encompassing
PAM2 and RRM domains, but also including other sequences, act as most potent mediators of both translational
repression and mRNA decay. However, other domains of
GW182 proteins also have potential to mediate the repression.
MiRNA-mediated translational repression is a reversible
process in mammalian cells. We previously demonstrated
that target mRNAs containing AU-rich regulatory elements
(AREs) in the 3’UTR, can be relieved from miRNA repression in response to different forms of cellular stress, The
derepression required binding of the ELAV family protein
HuR to the mRNA 3’UTR. Using an in vitro system with
recombinant miRISC and purified HuR and its mutants,
we demonstrate that HuR, by oligomerizing along RNA,
leads to displacement of miRISC from RNA, even when
miRISC is positioned at a distance from the primary HuRbinding site.
We are also investigating function and turnover of miRNAs in retinal and non-retinal rodent neurons. In collaboration with the laboratory of Botond Roska of the
FMI, we found that levels of the sensory neuron-specific
miR-182/183/96 cluster, and miR-204 and miR-211, are
down-regulated in mouse retina during dark adaptation and
up-regulated during light adaptation, with rapid miRNA
decay and increased transcription being responsible for the
respective changes. We found that miRNAs in non-retinal
neurons also turn over much faster than in non-neuronal
cells. miRNA turnover in neuronal cells may be subject to
complex activity-dependent regulation. Blocking action potentials with tetrodotoxin or blocking glutamate receptors
prevented rapid turnover of miRNAs in neurons.
6
Abstracts
L1.3
Oral presentations
MicroRNAs play a dominant
role in cellular processes
O1.1
Noam Shomron
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
e-mail: Noam Shomron <[email protected]>
A central gene regulatory mechanism, present in all animals, is regulation by small non-coding RNAs termed microRNAs. MicroRNAs control gene expression by binding
to mRNA targets and leading them to facilitated mRNA
degradation and translation inhibition. Currently there are
hundreds of reported human microRNAs predicted to
control at least half of the human transcriptome. MicroRNAs were observed to be important for a diverse range
of biological processes such as differentiation and development, and to play a pivotal role during human pathogenesis.
I will present our computational and experimental efforts
in deciphering modes of microRNA biogenesis; global and
specific regulation of target genes by microRNAs; and,
novel functions microRNAs play in the cellular context.
Overall our team effort leads to a deeper understanding
of small RNAs in the development of diseases in order to
generate a significant impact on clinical reality.
Thyroid hormone receptor alpha
(TRα) regulates heterogeneous
nuclear ribonucleoprotein A1
(hnRNPA1)-dependent alternative
splicing of tumor suppressor
CEACAM1 in human renal cancer
Hanna Kędzierska, Agnieszka PiekielkoWitkowska, Anna Wojcicka, Alicja Nauman
The Medical Center of Postgraduate Education, Department of
Biochemistry and Molecular Biology, Warsaw, Poland
e-mail: Hanna Kędzierska <[email protected]>
Objectives: CEACAM1 is a tumor suppressor whose
expression is disturbed in clear cell renal cell carcinoma
(ccRCC). Alternative splicing of CEACAM1 pre-mRNA
results in two splice variants: the long (L) and short (S) one,
due to inclusion or exclusion of exon 7, respectively. This
process is regulated by a splicing factor, hnRNPA1, which
promotes exon 7 exclusion. Previous studies revealed impaired expression of thyroid hormone receptors (TRs) in
ccRCC. Aim of this study was to check whether disturbed
alternative splicing of CEACAM1 is due to aberrant action
of TRs. Methods: ccRCC cell line UOK 171 was transfected with siRNA negative control or siRNA directed against
TRα. Silencing of TRα was confirmed by real-time PCR.
UOK 171 cells and 12 matched-pair ccRCC tumor-control
samples were used for analysis of hnRNP A1 expression
(by real-time PCR) and splicing pattern of CEACAM1 (by
reverse-transcription PCR, gel electrophoresis and densitometric scanning of gel bands). CEACAM1-L expression
was measured by real-time PCR. Results: Silencing of TRα
resulted in significant 31% reduction of hnRNP A1 expression (P<0.0001) and in altering ratio of CEACAM1 splice
isoforms, favoring the expression of 1L. The expression of
hnRNP A1 in ccRCC tumor samples was significantly, 47%
increased in comparison with control samples (P<0.05). In
contrast, the expression of CEACAM1-L isoform was reduced by 28% in tumor samples in comparison with controls (P<0.05). There was strong correlation between the
expression of hnRNPA1 and CEACAM-1L isoform in tumor and control samples (r=0.7649, p<0.05). Conclusion:
TRα affects alternative splicing of CEACAM1 via regulated
expression of splicing factor hnRNP A1. Disturbed alternative splicing of CEACAM1 in ccRCC possibly results
from altered expression of hnRNP A1. This is the first report showing that thyroid hormone receptor can directly
influence the process of alternative splicing.
Acknowledements
The work supported by the Polish National Centre for Science, Grant
NN401532140.
Parnas Conference Warsaw 2011
O1.2
Dynamics of single mRNP nucleocytoplasmic transport through
the nuclear pore in living cells Part II
Lectures
L2.1
Amir Mor, Shimrit Suliman, Rakefet Ben-Yishay,
Sharon Yunger, Yehuda Brody, Yaron Shav-Tal
MicroRNA control in the immune system
and deregulation in immune pathologies
Bar-Ilan University, The Mina & Everard Goodman Faculty of Life
Sciences & Institute of Nanotechnology, Ramat Gan, Israel
Klaus Rajewsky
e-mail: Amir Mor <[email protected]>
The flow of genetic information in eukaryotic cells occurs
via nucleo-cytoplasmic translocation of mRNAs. Knowledge of in vivo mRNA export kinetics through nuclear pore
complexes (NPCs) remains poor in comparison to protein
transport. In this study, we established a mammalian system that allowed the real-time visualization and analysis of
single mRNA-protein complexes (mRNPs) during export.
Using a transcription-inducible system, large mRNPs were
followed during nucleoplasmic travels and NPC translocation. The in vivo rates of mRNP transport and export
were quantified and compared. Bulk mRNP average travel-times from transcription to the NPC occurred within a
long-minute time-frame, with no pileup at the NPC. Export inhibition demonstrated that mRNA-NPC interactions were independent of ongoing export. mRNP export
was rapid (~0.5 sec) and kinetically faster than nucleoplasmic diffusion. The nucleoplasmic transport dynamics of
intron-containing and intronless mRNAs were similar, yet
the presence of an intron did increase export efficiency.
Our results provide visualization on the single mRNP level
of the various steps in gene expression within the nuclear
environment, the interchromatin tracks through which
mRNPs move, and demonstrate the kinetics of mRNPNPC interactions and translocation. 7
Program in Cellular and Molecular Medicine, Children’s Hospital, and
Immune Disease Institute, Harvard Medical School, Boston, USA
e-mail: Klaus Rajewsky <[email protected]>
While miRNAs usually regulate the expression of target
proteins only over a narrow range of concentrations, they
exert critical roles in the control of both hematopoietic
development and the functional activities of the cells of
the immune system. Likewise, small changes in miRNA
concentrations as well as miRNA deficiencies can lead to
dramatic disease phenotypes ranging from fatal autoimmunity to hematopoietic malignancies. This is in line with the
many human disease syndromes caused by genetic haploinsufficiency; in addition, any given miRNA has many target
proteins, which may be coordinately involved in the control
of cellular signaling pathways. I will discuss gene targeting
approaches in the mouse, which have been instrumental in
the elucidation of the functional impact of miRNA control
in the immune system in vivo. These approaches have also
led to the identification of key targets of miRNA control,
but at the same time revealed its enormous complexity,
eluding classical genetic analysis.
Abstracts
8
L2.2
Oral presentations
CTCF and its testis specific paralog BORIS
in shaping of epigenome in cancer
O2.1
Dmitri Loukinov
Laboratory of Immunopathology, NIAID/NIH, Rockville, MD, USA
MicroRNA-directed allelic exclusion
in immunoglobulin gene loci
e-mail: Dmitri Loukinov <[email protected]>
Volodymyr A. Halytskiy
CTCF recently earned the title of “master weaver of the
genome” in appreciation of its versatile and multifunctional interaction with genetic elements. The CTCF protein contains an11 zinc finger DNA recognition domain
flanked with N- and C-termini of about equal size. The
CTCF gene is exceptionally conserved throughout evolution from Drosophila to human; conservation from mice to
men is over 95% identity on the amino acid level. Depending on the genomic context CTCF acts as a transcriptional
repressor or activator, is implicated as a part of hormonedependent composite silencers, plays an important role at
least in some enhancers and is capable of forming intraand inter-chromosomal loops via its intrinsic property to
form di- and multi-mers. BORIS appeared relatively late
in evolution by duplication of the CTCF gene and became
strictly germ cell specific only in mammals. BORIS evolved
rapidly and conservation between mice and men is high in
the zinc-finger DNA recognition domain and significant in
N-terminus, but is insignificant in the C-terminus. In humans, and perhaps other mammals, BORIS is expressed in
multiple (at least two dozen) alternatively spliced isoforms
and the significance of those will be discussed. BORIS is
expressed also in embryonic stem cells and is frequently
detected in various cancers. As a target for immunotherapy,
BORIS has been shown to be safe and successful in elimination of cancer in rodent models. The role of BORIS in
germ cell development and in tumorigenesis will be discussed based on phenotype of knock-out animals and genome-wide ChIP-seq.
Palladin Institute of Biochemistry of the National Academy of Sciences
of Ukraine, Molecular Immunology Department, Ukraine
Acknowledgements
This work was supported by intramural funding from NIAID/NIH
e-mail: Volodymyr Halytskiy <[email protected]>
According to hypothesis of microRNA-directed DNA
methylation, some microRNAs (miRNAs) are involved
in allelic exclusion [1]. The precursors of these miRNAs
should be transcribed from sequences that undergo allelic exclusion. Mature miRNAs can directly hybridize with
complementary DNA untwisted during the transcription,
thereafter involving DNA methyltransferases in de novo
methylation of cytosine within DNA targets. Nevertheless,
RNA polymerase movement along transcribing sequence
must release DNA from miRNAs bound to it. Therefore,
miRNA can initiate DNA methylation in allele, which is
less actively read by RNA polymerase, whereas the concentration of miRNA transcribed from only one allele is
insufficient to overcome the level above which the silencing
of this allele can be initiated. As a result, repression affects
all but one allele that exists in the cell. Recently we have
predicted that immunoglobulin gene loci contain miRNAs
responsible for allelic exclusion [2].
In silico search in miRNA database miRBase shows that
genes of four human miRNAs – miR-4539, miR-4507,
miR-4538, miR-4537 – are clustered within immunoglobulin heavy chain locus (IGH@) of chromosome 14, position
106325653-106323741. This cluster is located in a spacer
between sequences encoding last J-fragment and first exon
of Cμ-fragment on the same strand (negative). Also, miRNA candidate sequence AB019440.1 is transcribed from
positive strand in V-fragments region of IGH@.
The gene of another human miRNA, miR-650, is found
within immunoglobulin lambda chain locus (IGL@) on
positive strand of chromosome 22, position 2316527023165365. This gene overlaps exon 1 of IGLV2-8-001
gene and intron 1 of the gene encoding large intergenic
non-coding RNA (lincRNA) D87024.2. Six miRNA candidate sequences – snoU13.380, D86994.2, D86994.1,
D87015.1, D86998.1 and D87024.1 – are also transcribed
from sequences located in IGL@.
Therefore, localization of miRNAs genes in immunoglobulin gene loci corresponds with the predictions
from hypothesis of miRNA-directed allelic exclusion. Furthermore, each mature sequence of miR-650, miR-4539,
miR-4507, miR-4538 and miR-4537 contains 2-4 C(N)G
sites, which are complementary to DNA methylation sites.
Also, exons of above miRNA candidate sequences contain
several C(N)G sites. These facts confirm that miRNAs can
cause allelic exclusion through methylation of complementary DNA sequences.
References
1. Halytskiy VA (2008) Cell Tissue Biol 2: 97-106.
2. Halytskiy VA, Komisarenko SV (2009) Biopolymers and Cell 25: 12-26.
O2.2
Posters
T-domain of diphtheria toxin may
retard maturation of endosomes
P1_2.1
Andrii Labyntsev, Natalia Korotkevich,
Elizaveta Yurchenko, Andrii Kaberniyk, Denys Kolibo
Palladin Institute of Biochemistry of the NASU, Molecular Immunology
department, Ukraine
e-mail: Andrii Labyntsev <[email protected]>
Diphtheria toxin (DT) is the main pathogenicity factor of
Corynebacterium diphtheriae. Toxin consists of three domains: C-domain (corresponds to the fragment A [SbA]),
T-domain and R-domain (the last two constitute the fragment B [SbB]). R-domain (Rd) mediates binding of DT
to its surface receptor proHB-EGF, which promotes clathrin-mediated endocytosis of the toxin-receptor complex.
Lowering the luminal pH of the endosome vesicles triggers partial unfolding of T-domain and its anchoring into
the endosomal membrane. Thereafter T-domain mediates
translocation of C-domain into the cell cytosol and then
C-domain inactivates translation factor eEF2, which leads
to the inhibition of cellular protein biosynthesis and cell
death.
Today, the mode of DT action is well characterized, but
there remain some unclear aspects of the toxin cell entry,
particularly C-domain translocation mechanism. In order to study the role of T-domain in the process of the
toxin entry into the cell we have constructed two recombinant fluorescent DT-derived proteins (mCherry-SbB
and EGFP-Rd). Both proteins have C-domain replaced by
fluorescent protein (EGFP or mCherry) and one lacks the
T-domain (EGFP-Rd). Flow cytometric analysis showed
strong binding of both proteins to the toxin sensitive cell
line Vero, but their titration curves revealed lower affinity
of EGFP-Rd to the proHB-EGF, which may be associated
with the absence of T-domain. Simultaneous internalization process of these proteins into Vero cells was visualized
via confocal microscopy. After 15 minutes of the observation both proteins were localized in endosomes, but some
differences in their trafficking were observed: mCherrySbB was localized at the near-membrane region of the cell,
whereas EGFP-Rd moved deeply into the cell. EGFP-Rd
was localized in lysosome-like structures after 45 minutes,
whereas mCherry-SbB still remained in endosomes and
only after 60–75 minutes mCherry-SbB was colocalized
with EGFP-Rd in lysosome-like structures. Based on these
data we suggest that difference in the intracellular traffic
of these proteins was due to the presence of the DT Tdomain in mCherry-SbB protein. After anchoring into the
endosomal membrane the T-domain may act as a proton
channel, suchwise it may retard maturation of the endosome. Presumably, retarding maturation of endosomes is
one of the uninvestigated functions of T-domain, which
may promote more efficient translocation of C-domain
into the cellular lumen.
9
Single non-coding transcript
could participate in antisense
regulation of ASCL1 gene family
Alexandra Yu. Filatova1, Andrey V. Marakhonov1,
Ancha Baranova1,2, Mikhail Yu. Skoblov1
1Research Centre for Medical Genetics, Russian Academy of Medical
Science, Russian Federation; 2George Mason University, Molecular and
Microbiology Department, USA
e-mail: Filatova Alexandra <[email protected]>
Examination of the phenomenon of antisense regulation
of gene expression is urgent as recent years it has been
demonstrated that some natural antisense transcripts play
an important role in sense genes expression including those
which are involved in pathogenesis of a number of diseases. However, the functions and the mechanism of antisense
regulation are not enough studied nowadays.
Lately in our laboratory in silico search of overlapping clusters of EST relating to opposite DNA strands has been
conducted. We chose a cluster with a gene ASCL1 (Achaete-Scute Complex-Like homolog 1 [Drosophila]) among
the obtained cases. Protein ASCL1 belongs to a family
of transcription factors, which contain HLH (helix-loophelix)-domain responsible for DNA binding. It was found
that it played a role in the neuronal commitment and differentiation. This cluster is also of particular interest because of its possible role in contributing to progression of
tumors as ASCL1 highly expresses in medullary thyroid
cancer, neuroendocrine tumors of lung and other malignant neoplasms.
Based on the analysis of EST obtained from the dbEST
we suggested a hypothesis about the existence of natural
antisense transcript to ASCL1 gene. This hypothesis was
proved experimentally using quantitative PCR. Then we
identified 3′- and 5′-ends of this transcript using RACEPCR.
The further in silico analysis of a cluster indicated that
there were four ASCL1-homologous genes in human genome, i. e.: ASCL2, ASCL3, ASCL4, ASCL5. It is notable that ASCL2 and ASCL4 are validated, while ASCL3
and ASCL5 are only predicted genes. We identified that
mRNA sequence of the genes ASCL2 and ASCL4 had
significant regions with high homology which were overlapped with the predicted asASCL1 antisense transcript,
whereas ASCL3 and ASCL5 had similar overlapping with
less length and homology. It is worth pointing out that
the overlapping regions of all mRNA contain a sequence
which encodes a conserved and common for all ASCL proteins HLH-domain. On the basis of these facts we made
a hypothesis that a whole gene family could be trans-antisense regulated by one antisense transcript with different
effciency for each of its members depending on homology
level of an antisense transcript and mRNA sequence of
genes. The work aimed to examine and to prove experimentally this phenomenon is in progress.
Abstracts
10
P1_2.2
P1_2.3
Orchestrating myogenesis: microRNAs
processing is influenced by Heme
Oxygenase-1 in myocytes
PPM1A a key player in wound
healing and inflammation
Maciej Cieśla, Magdalena Kozakowska,
Anna Stefańska, Klaudia Skrzypek, Oleksandr Yagensky,
Agnieszka Łoboda, Józef Dulak, Alicja Józkowicz Department of Medical Biotechnology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonain University, Kraków, Poland
e-mail: Maciej Cieśla <[email protected]>
Myoblasts are precursor cells in myogenesis. Pathologies
in their maturation may lead to development of skeletal
muscles derived tumor called rhabdomyosarcoma. Differentiation of myoblasts can be regulated by microRNAs. We
investigated the role of heme oxygenase-1 (HO-1), the cytoprotective enzyme, on microRNAs biogenesis including
class of myocyte specific miRNAs (myo-miRs) in terms of
myoblast maturation and proliferation.
Experiments were performed on murine immortalized
myoblast cell line C2C12, stably transduced with retroviral vectors to express luciferase/GFP reporter genes and
overexpress HO-1 and on human rhabdomyosarcoma
cell lines of embryonal and alveolar origin. High level of
HO-1 activity in C2C12 cells potently inhibited the myoblast differentiation, as indicated by reduced formation of
myotubes, diminished activity of creatine phosphokinase,
and decreased expression of myogenic regulatory factors,
such as MyoD, myogenin and myo-miRs (miR-1, miR-133a,
miR-133b, and miR-206). Accordingly, rhabdomyosarcoma
cell lines of more aggressive phenotype were characterized
by higher amounts of HO-1. Interestingly SDF-1, which
levels are dependent on HO-1 exhibits effect on myo-miRs,
similar to that of HO-1 overexpression. Higher amounts
of HO-1 driven by viral vectors or from endogenous pathways lead to decreased levels of enzymes indispensible for
miRNA processing such as DGCR8, Dicer or lin28. Following this finding we find that levels of total mature and
precursor miRNAs are decreased in HO-1 overexpression.
Finally, transfection with myo-miRs leads to reversal of
HO-1 effect on myocytes differentiation. Thus, HO-1 is
an important factor regulating microRNA biogenesis and
influencing myocytes differentiation in possibly SDF-1 and
myo-miRs dependent pathway. Z. Dvashi1, M. Shohat1, H. Jacobi1, D. Ben-Meir1,
R. Ashery-Padan2, A. Solomon3, M. Rosner3, S. Lavi
1Department of Cell Research and Immunology, George S. Wise Faculty
of Life Sciences, Tel Aviv University, Tel Aviv, Israel; 2Human Genetics
Faculty of medicine,Tel Aviv University, Tel Aviv, Israel; 3Goldschleger
Eye Research Institute, Sheba Medical Center, Tel Hashomer, Israel
e-mail: zeev Dvashi <[email protected]>
Protein phosphatase magnesium dependent 1A (PPM1A),
formerly PP2Ca, belongs to the metal-dependent serine
threonine protein phosphatase family and is highly conserved in evolution. PPM1A plays a significant role in many
cellular processes such as cell cycle, apoptosis, tumorigenesis and differentiation through diverse signaling pathways,
including p38 MAP Kinase, AMP-Kinase, JNK, TGF-beta, WNT etc. Due to the lack of a specific inhibitor the
physiological role of PPM1A is a riddle. We have therefore
generated a conditional PPM1A knockout mouse using the
Cre-LoxP system.
PPM1A was recently reported to be the negative regulator
of Smads in the TGF-beta response that controls wound
healing. In view of the high expression of PPM1A in the
mouse eye we used the cornea to study its role in wound
healing. In vivo, using corneal epithelial debridement, we observed aberrant corneal healing. Interestingly, though PPM1A ablation did not affect the proliferation of the corneal
epithelial cells their migration ability in the cornea of the
KO mice was reduced, causing slower and impaired wound
healing. In vitro, using mouse embryonic fibroblasts (MEFs)
and corneal fibroblast cultures we have demonstrated that
the absence of PPM1A slowed down the migration of the
fibroblasts thus leading to attenuated wound healing. The
cells displayed increased TGF-β response in the PPM1A
KO cells. Unexpectedly, the kinetics of Smad3 phosphorylation, in response to TGF-β was similar in the WT and
KO fibroblasts. Still, the KO cells displayed enhanced p38
activation suggesting that PPM1A downregulates TGF-β
signaling by terminating p38 activation.
Finally, we have shown a novel role for PPM1A in the
regulation of inflammation. Using the alkali burn model
we have found severe inflammation in the KO mice that
prevented healing. The role of PPM1A in wound healing
and inflammation through its control of signaling pathway
engaged in these responses will be discussed.
P1_2.4
P1_2.5
Mitotic modulation of translation
elongation factor 1 leads to hindered
tRNA delivary to ribosomes
Sequence motifs engaged in the
regulation of rosR transcription in
Rhizobium leguminosarum bv. trifolii
Gilad Sivan, Ranen Aviner, Orna Elroy-Stein
Monika Janczarek, Teresa UrbanikSypniewska, Anna Skorupska Tel Aviv Univeristy, Dept of Cell Research & Immunology, Faculty of Life
Sciences, Israel
e-mail: Orna Elroy-Stein <[email protected]>
Translation elongation in eukaryotes is mediated by the
concerted actions of elongation factor 1A (eEF1A) that
delivers aminoacylated-tRNA to the ribosome, elongation
factor 1B (eEF1B) complex that catalyzes the exchange of
GDP to GTP on eEF1A, and eEF2 that facilitates ribosomal translocation. Here we present evidence in support of
a novel mode of translation regulation by hindered tRNA
delivery during mitosis. A conserved consensus phosphorylation site for the mitotic kinase CDK1 on the catalytic
delta subunit of eEF1B (termed eEF1D) is required for its
post-translational modification during mitosis, resulting in
lower affinity to its substrate eEF1A. This modification is
correlated with reduced availability of eEF1A-tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of
regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally-applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at
the elongation level.
References
Sivan G et al. (2007) Mol Cell Biol 27: 6639-6646.
Sivan G, Elroy-Stein O (2008) Cell Cycle 7: 741-744.
Sivan G et al. (2011) J Biol Chem, in Press. 11
Department of Genetics and Microbiology, Institute of Microbiology
and Biotechnology, University of M. Curie-Skłodowska, Lublin, Poland
e-mail: Monika Janczarek <[email protected]>
The rosR gene encodes a transcriptional regulator with a
C2H2 type zinc-finger motif involved in positive regulation
of EPS synthesis in R. leguminosarum bv. trifolii. In silico sequence analysis of the 450-bp long rosR upstream region
revealed the presence of several inverted repeats of different lengths (named IR1 to IR6), among these IR5 was the
longest, with 12-bp inverted repeats. Moreover, motifs with
significant identity to consensus sequences recognized by
PhoB and LysR-type proteins associated with phosphateand flavonoid-dependent gene regulation in R. leguminosarum were identified in this region. We established that rosR
expression was driven from two separate promoters: the
distal strong P1 promoter and the proximal, weaker, P2,
and two rosR transcripts of different lengths were identified. These transcripts contained 273-base and 240-base
long 5’-untranslated regions (5’-UTR), respectively. Such
long upstream regions have often been described as target
sites for the regulation of gene expression. The inverted
repeats IR1, IR2, and IR3 were located upstream of the
two transcriptional start sites TS1 and TS2, suggesting that
they are potential target sites for the action of regulatory
proteins. The IR4 motif was located between TS1 and TS2,
and the IR5 and the IR6 were located below TS2.
Secondary structure analysis of the rosR RNA transcripts
initiated at TS1 and TS2 revealed the presence of several
additional sequences, which stabilized their structures, especially in the upper part of both transcripts. Three stem
structures were generated from the first 120 nucleotides of
transcript 1 with a total energy of –41.4 kcal/mol. These
included inverted repeats IR5, which were located on the
top of this structure. This sequence also played a significant role in stabilizing the upper part of the secondary
structure of the shorter transcript 2. Within the first 90 nt
of the transcript 2, a structure containing two stems was
formed with a total energy of –27.9 kcal/mol. Site-directed
mutagenesis of IR4 indicated that dominant role of this
motif was the repression of rosR transcription.
Acknowledgements
This research was supported by the Ministry of Science and Higher Education, grant no. N N303 092234.
Abstracts
12
P1_2.6
P1_2.7
Interaction of the fragile X
mental retardation protein with
MMP-9 mRNA in neurons
Expression of pleiotropic regulatory
genes in streptomycetes
Aleksandra Janusz1, Jacek Miłek1, Małgorzata Perycz2,
Leszek Kaczmarek1, Magdalena Dziembowska1
1Nencki Institute of Experimental Biology PAS, Warsaw, Poland;
2International Institute of Molecular and Cell Biology, Warsaw, Poland
e-mail: Aleksandra Janusz <[email protected]>
Fragile X Mental Retardation Protein (FMRP) is an RNA
binding protein highly expressed in the brain, implicated
in RNA transport and in the control of local translation.
Fmr1 KO mice have immature filopodia-like dendritic
spines, increased MMP-9 protein level and activity. Treatment with minocycline, that inhibits MMP-9 causes spine
maturation in KO mice, which implies a role for MMP-9 in
Fragile X Syndrome. It has been shown that MMP-9 mRNA
and protein are present at the postsynaptic sites of dendritic spines and that MMP-9 is activated in response to
synaptic stimulation. MMP-9 mRNA contains G quartet,
a sequence pattern found in mRNAs targeted by FMRP.
We have used synaptoneurosomal fraction to precipitate
FMRP-mRNA complexes and we found that MMP-9
mRNA is specifically co-immunoprecipitated with FMRP
protein. By double in situ hybridization and immunofluorescence we have visualized MMP-9 mRNA and FMRP
protein in rat hippocampal neurons and found the colocalization of MMP-9 mRNA with FMRP protein. After stimulation of hippocampal neurons with DHPG (mGluR5 agonist) and bicuculline, we observed mobilization of MMP-9
mRNA and FMRP containing granules from cell soma to
the dendrites. We have also found that the colocalization
of MMP9 mRNA and FMRP protein in granules markedly
decreases after DHPG stimulation. We have confirmed
that FMRP and MMP9 mRNA shift to the polyribosomal
fractions upon glutamate stimulation. Overall, we postulate
that MMP-9 mRNA associates with FMRP in neurons and
is transported to the dendrites.
Dmytro Klymyshin1,2,3, Maria Rabyk1,
Maria Honchar2, Victor Fedorenko1
1Ivan Franko National University of L’viv, Deptartment of Genetics and
Biotechnology, Ukraine; 2Institute of Animal Biology, Deptartment
of Microbiology and Biotechnology, Ukraine; 3Danylo Halytsky Lviv
National Medical University, Deptartment of Biochemistry, Ukraine
e-mail: Dmytro Klymyshin <[email protected]>
Streptomycetes are an abundant source of novel secondary
metabolites with a range of biological activities that may
ultimately find application as anti-cancer agents or other
pharmaceutically useful compounds. The activation of
antibiotic biosynthesis is genetically controlled at several
levels. Pleiotropic regulatory genes exert their control over
two or more antibiotics in the same organism.
Streptomyces ghanaensis is a producer of the phosphoglycolipid antibiotic moenomycin A (MmA) — the only natural
product known to directly target the extracellular peptidoglycan glycosyltransferases involved in bacterial cell wall biosynthesis. Analysis of moe cluster shows that genes moeE5
and moeO5 control the first steps of MmA biosynthesis.
Both aforementioned genes are accompanied by putative
promoters, which should be the primary targets for different transcriptional regulators. The influence of regulators
on these promoters was assessed with the help of titration of promoters. For this purpose we generated S. albus
R1moe38-5+ and S. ghanaensis strains carrying multicopy
plasmid pSOK101 with moeE5p and moeO5p promoters.
LC-MS analysis showed significant changes in moenomycin
production upon introduction of promoters, which point
to the presence of transcriptional activators. A pleiotropic
regulatory gene wblAgh was disrupted in S. ghanaensis. Obtained strain produces not only increased amount of MmA,
but shows severe changes in morphology of S. ghanaensis.
Expression of pleiotropic regulatory genes in heterological hosts is an effective approach in overproduction of
clinically important drugs. Here we focus on the regulation of secondary metabolism in S. peucetius (doxorubicin
producer), S. nogalater (nogalamycin producer) and S. echinatus (aranciamycin producer) strains. Pleiotropic regulatory
genes lndYR, afsS, relA, absA2 were cloned from different
streptomycetes and used to improve production of anthracyclines. Our data shows that amplification of relA gene
conferred overproduction of antibiotics in the host strains.
The production of novel unidentified compound was observed in the case of S. nogalater harboring lndYR gene. Expression of lndYR in S. peucetius also had a dramatic effect
on the sporulation of this strain. This gene is required for
sporulation and also influenced the production of doxorubicin. It was found that expression of absA2 decreased
production of nogalamycin in S. nogalater. Expression of
afsS in S. peucetius, S. nogalater and S. echinatus had no significantly changes on antibiotics production.
P1_2.8
P1_2.9
The possible role of miRNA in OTA-triggered
changes in LLC-PK1 kidney tubular cells
Novel non-protein-coding natural
antisense transcript AFAP1AS participates
in regulation of AFAP1 expression
Anna Stachurska1, Christine Boesch-Saadatmandi2,
Magdalena Kozakowska1, Gerald Rimbach2,
Alicja Jozkowicz1, Jozef Dulak1, Agnieszka Loboda1
1Jagiellonian University, Faculty of Biochemistry, Biophysics and
Biotechnology, Department of Medical Biotechnology, Krakow, Poland;
2Christian-Albrechts-University Kiel, Institute of Human Nutrition and
Food Science, Kiel, Germany
e-mail: Agnieszka Loboda <[email protected]>
Ochratoxin A (OTA) is a nephrotoxin produced mainly by
Aspergillus ochraceus and Penicillum verrucosum with potential
carcinogenic activity. We hypothesized that in kidney tubular epithelial cells (LLC-PK1) 24h stimulation with OTA
may affect expression of various miRNAs.
Among others we found out that expression of miR-132
was upregulated after OTA delivery. One of its possible
targets is nuclear factor (erythroid-derived 2)-like 2 (Nrf2),
which was decreased by OTA in our studies. Nrf2 overexpression by the use of adenoviral vectors diminished OTAenhanced reactive oxygen species (ROS) generation as well
as OTA-inhibited cell proliferation and viability. Moreover,
Nrf2 overexpression reduced OTA-elevated mRNA of
profibrotic transforming growth factor β-2 (TGFβ2) via
reduction of ROS level. Thus, is seems that drop in Nrf2,
which may be caused by induction of miR-132, is crucial in
toxic effect of OTA.
We also demonstrated that expression of miR-122 was increased by OTA. This miRNA was reported to diminish
expression of heme oxygenase-1 (HO-1), an antioxidant
enzyme involved in heme degradation. Additionally, erythropoietin (Epo), which is important for kidney biology, is
one of the possible targets of miR-122. We found that expression of Epo and HO-1 was decreased by OTA and
Nrf2 overexpression attenuated this diminishment. Still, it
is possible that miR-122 may mediate these effects.
Another miRNAs, which may modulate HO-1 expression,
are miR-200b/c. We showed that expression of miR-200b
was reduced whereas of miR-200c was elevated by OTA.
We also detected OTA-evoked induction of p53 expression, which is known to regulate miR-200c. miR-200c may
also affect expression of vascular endothelial growth factor (VEGF). Other miRNAs upregulated by OTA, which
binding sites are present within VEGF sequence, are miR29b/c. We observed diminishment of VEGF expression
by OTA connected with attenuation of expression of SP-1
and hypoxia inducible factor-2 α (HIF-2α). Still, alterations
in miR-29b/c and miR-200c evoked by OTA may also be
responsible in these changes.
Taken together, these results suggest the importance of
alterations in Nrf2 and miRNAs expression in toxicity of
OTA. Still, the role of miRNAs in OTA-triggered effects
needs further examination.
Acknowledgements
Supported by grants No. N N401 297835 and No. N N301 033440 (Ministry of Science and Higher Education). The Faculty of Biochemistry, Biophysics and Biotechnology is a beneficiary of grants No: POIG.02.01.0012 064/08, POIG 01.01.02-00-109/09, POIG.02.02.00-014/08 and
01.01.02-00-069/09.
13
Andrey V. Marakhonov1, Peter E. Prokhorov1,
Ancha Baranova1,2, Mikhail Yu. Skoblov1
e-mail: Andrey Marakhonov <[email protected]>
Sequencing of human genome revealed the paradox: the
complexity in organism’s organization does not correspond
to the number of its genes. Now it is generally accepted
that increasing complexity in organization is concerned
with complication of the regulation of gene expression,
including antisense regulation, rather than with an increase
in number of genes. Antisense regulation is a widespread
but not well-understood mechanism of regulation of gene
expression. Despite its abundance, only few experimentally
validated regulatory models of antisense regulation in human genome have been described. These data pointed out
that different mechanisms exist. Functional study of natural antisense transcripts remains actual.
Recently we have carried out a whole genome in silico search
of cis-antisense clusters of transcripts in human. The created database revealed a significant number of sense–antisense pairs consisting of one EST cluster expressed
predominantly in normal tissues and another cluster with
tumor-specific expression. The role of antisense transcripts
in the carcinogenesis warrants functional research. Here we
describe and characterize an antisense mRNA AFAP1AS
overlapping human AFAP1 gene.
AFAP1 encodes for an actin filament binding protein,
which serves as an adaptor protein interacting with actin
filaments what leads to structure and integrity. It also is
able to relay signals from receptor tyrosine kinases through
PKCα to Src protein kinase. Moreover AFAP1 participates in Src-dependent SRE/AP-1 transcriptional activation. It has been shown that AFAP1 is overexpressed in
prostate cancer and contributes to tumorigenic growth. We
hypothesized that the transcription of antisense mRNA
AFAP1AS may lead to suppression of sense AFAP1 gene
expression and eventually to a compensatory restraint of
the Src mitogenic signaling pathway. The consequences
of AFAP1AS expression are unlikely to be supported by
natural selection in the tumor cell population.
To study the intriguing phenomenon of tumor-specific
AFAP1AS antisense expression we performed detailed
in silico analysis of AFAP1AS sequences, determined potential open reading frames, and experimentally quantified
this transcript and its sense partner in normal and tumor
human tissues. Also we performed comparative genomic
analysis between several mammalian species. Furthermore
we cloned both antisense transcript and sense gene into
expression vectors and carried out functional analysis in
human cell cultures.
Abstracts
14
P1_2.10
P1_2.11
Phosphorylation of ribosomal
protein S6 (rpS6) is a determinant
of pancreatic tumorigenesis
Plasmid-transcribed shRNA in
contrast to siRNA, activate NFκB
signaling pathway and differentially
modulate melanoma cell survival
Avigail Dreazen1, Shlomit Avraham1, Abed Khalaileh2,4,
Areej A. S. Khatib3, Gideon Zamir4,
Yuval Dor2, Oded Meyuhas1
1The
Hebrew University-Hadassah Medical School, Department of
Biochemistry and Molecular Biology, Jerusalem, Israel; 2The Hebrew
University-Hadassah Medical School, Developmental Biology and
Cancer Research, Jerusalem, Israel; 3Bethlehem University, Palestinian
Authority; 4Hadassah Medical Center, Department of Experimental
Surgery, Jerusalem, Israel
e-mail: Oded Meyuhas <[email protected]>
Mice, whose rpS6 cannot be phosphorylated due to Ser>Ala replacement of all phosphorylatable residues (rpS6P-/-), exhibit a small-size phenotype of their β-cells and
impaired glucose tolerance. Contrarily, transgenic mice,
whose β-cells overexpress myr-Akt1 (Akttg), display a
significant increase in both the number and size of their
β-cells accompanied by improved glucose tolerance. A
cross between Akttg and rpS6P-/- mice has established the
dominance of the growth stimulating effect of Akt1 over
the growth defect resulting from rpS6 phosphorylation
deficiency. Interestingly, however, in addition to their large
size, β-cells in Akttg mice tend to undergo transformation
with the development of insulinoma, concomitantly with
a selective rpS6 phosphorylation in this tumor. The causal
relationship between these two variables has become evident by the fact that insulinoma was detected in none of
12 Akttg/rpS6P-/- mice. Intrigued by the possibility that
phospho-rpS6 might exert a pro-oncogenic activity, we examined the involvement of rpS6 phosphorylation in pancreatic ductal adenocarcinoma (PDA). To this end, pellets
of 7,12-dimethylbenzanthracene (DMBA) were implanted
into pancreas head of mice. Like in the case of the endocrine pancreas, rpS6P-/- mice were protected from the
development of PDA and its precursor lesions. Notably,
PDA often involves somatic activation of K-Ras oncogene,
which has also been implicated in various DMBA-induced
tumors. In order to examine the role of rpS6 phosphorylation in the context of K-Ras-induced tumorigenicity,
we crossed rpS6P-/- mice with PDX1-Cre; K-RasLSLG12V
mice. The latter strain expresses an activated K-Ras variant (K-rasG12V) in all cells of pancreatic lineage, causing
the widespread development of precursor lesions for PDA.
Histopathological analysis has demonstrated that K-Rasinduced tumorigenicity is suppressed in rpS6P-/- mice.
In an attempt to study the mechanism underlying the protumorigenic effect of rpS6 phosphorylation, we infected
immortalized WT and rpS6P-/- MEFs with myr-Akt expression vector. Our results indicate that rpS6 phosphorylation
deficiency inhibited colony formation in soft agar, relative
to the number counted in WT cells, concomitantly with
increased cell death. Taken together, these results imply
that phosphorylated rpS6 displays a cell-autonomous prooncogenic activity.
References
1. Tuttle RL et al. (2001) Nat Med 7: 1133-1137.
2. Guerra C et al. (2003) Cancer Cell 4: 111-120.
Kavita Ramji1,2, Dorota Kulesza1,2, Bozena Kaminska1
1Laboratory of Transcription Regulation, Nencki Institute of
Experimental Biology, Warsaw, Poland; 2Postgraduate School of
Molecular Medicine, Warsaw, Poland
e-mail: Kavita Ramji <[email protected]>
Signal Transducer and Activator of Transcription (STAT)
are transcription factors phosphorylated in response to
extracellular ligands that activate cytokine receptor signaling. Compelling evidence demonstrate a critical role of
activated Stat3 in malignant transformation and tumour
progression. One of the aims of this project was to deregulate STAT3 signaling by silencing its expression and
study the interplay between STAT3 and other transcription factors such as the nuclear factor-kappaB (NFκB).
NFκB activation may suppress the apoptotic potential of
chemotherapeutic agents and contribute to drug resistance
in cancer cells. We developed plasmid-transcribed small
hairpin RNAs (shRNAs) which specifically down-regulated
the STAT3 expression in transiently transfected human
melanoma WM239 cells. The STAT3-shRNA and NFκBLuc plasmids were efficiently co-transfected in melanoma
cells using lipofectamine. Western blotting was carried
out to ensure the silencing of STAT3, while the effect on
NFκB activation was inspected by measuring the luciferase
activity and western blotting for phosphorylated inhibitor
IκB. Results show that silencing of STAT3 in melanoma
cells influence the activity of NFκB transcription factor as
determined using both the NFκB reporter luciferase assay
and western blotting for P-IκB, however similar data was
also obtained for the negative control shRNA. Interestingly, such NFκB activation did not occur when cells were
transfected with various siRNA. Furthermore, silencing
with specific shRNA-STAT3, compared to siRNA, affected survival of melanoma cells (as demonstrated using
MTT metabolism test). Lack of reduction in cell viability
in melanoma cells depleted of STAT3 with siRNA suggests that the expression of this transcription factor is not
essential for basal survival. Our data suggest that shRNASTAT3 silencing may induce non-specific or off-target effects related to plasmid transcribed shRNA.
P1_2.12
P1_2.13
Can p53 secure the genomic
fidelity of iPS cells?
miRNA pharmacogenomics:
pharmacogenomics genes
demonstrate varying perceptibility
to microRNA regulation
Noa Rivlin, Ran Brosh ,Tirosh Shapira, Rachel Sarig, Yael Assia, Yoav Shezer,
Alina Molchadsky, Naomi Goldfinger, Varda Rotter
Department of Molecular Cell Biology, Weizmann Institute of Science,
Rehovot, Israel
e-mail: varda rotter <[email protected]>
p53 deficiency enhances the efficiency of somatic cell reprogramming to a pluripotent state. As p53 is usually mutated in human tumors and many mutated forms of p53
gain novel activities, here we studied the influence of mutant p53 on somatic cell reprogramming. Our data indicate
a novel gain-of-function property for mutant-p53, which
markedy enhanced the efficiency of the reprogramming
process compared to p53 deficeincy. Importantly, this novel activity of mutant p53 induced alterations in the characteristics of the reprogrammed cells; while p53-knockout
cells reprogrammed with only Oct4 and Sox2 maintained
their pluripotent capacity in vivo, reprogrammed cells expressing mutant p53 lost this capability, and gave rise to
malignant tumors. This novel gain-of-function of mutant
p53 is not attributed to its effect on proliferation, as both
p53-knockout and mutant p53 cells displayed similar proliferation rates. In addition, we demonstrate an oncogenic
activity of Klf4, as its overexpression in either p53-knockout or mutant p53cells induced aggressive tumors. Overall,
our data show that reprogrammed cells with the capacity
to differentiate into the three germ layers in vitro can form
malignant tumors, suggesting that in genetically unstable
cells such as those in which p53 is mutated, reprogramming
may result in the generation of cells with malignant tumorforming potential. Reference
Sarig S et al. (2010) J Exp Med 207: 2127-2140. 15
Jakob L. Rukov1,2, Roni Wilentzik1, Jeppe Vinther2, Noam Shomron1
1Tel Aviv University, Sackler School of Medicine, Tel Aviv, Israel;
2University of Copenhagen, Department of Biology, Copenhagen,
Denmark
e-mail: Jakob Rukov <[email protected]>
Pharmacogenomics studies how individual differences in
genome and transcriptome composition affects drug efficacy and toxicity. While genomic indicators are intensively
scrutinized, the effects of post-transcriptional regulators
of gene expression remain largely unexplored. MicroRNAs
(miRNAs) are short non-coding RNAs that negatively regulate expression of the majority of animal genes, including
many genes involved in drug efficacy. Consequently, differences in miRNA expression between individuals could
be an important factor contributing to differential drug response. By combining data on pharmacogenomics-related
genes (as annotated by the Pharmacogenomics Knowledge
Base; PharmGKB) and computationally predicted as well
as experimentally verified miRNA targets, we have studied
the regulatory potential of miRNAs in pharmacogenomics.
We describe variations in miRNA perceptibility to miRNA
regulation across functional groups and gene families of
pharmacogenomics-related genes. We have furthermore
collected associations of miRNAs, genes and drugs (what
we term miRNA pharmacogenomic sets) in a database
named Pharmaco-miR and are currently implementing
Pharmaco-miR as a web server, to allow searches on which
specific drugs may be affected by miRNAs.
Pharmacogenomics genes can be divided into drug target
genes termed pharmacodynamic (PD) genes and genes involved in drug metabolism and transport termed pharmacokinetic (PK) genes. To further investigate the regulatory
potential of miRNAs in pharmacogenomics, we have examined the potential regulation by miRNAs of PK and PD
genes. Our analysis identify a striking difference in the level
of miRNA regulation between PK and PD genes, with the
former having less than half predicted conserved miRNA
binding sites compared with the latter. Importantly, this
finding is reflected in a highly significant difference in the
shift in expression levels of PD versus PK genes following
depletion of miRNAs. Our study emphasizes an intrinsic
difference between PK and PD genes and suggests that
miRNA regulation of PD genes will turn out to be important for the efficacy of many drugs.
Abstracts
16
P1_2.14
P1_2.15
Role of HO-1 in alterations evoked
by ochratoxin A in expression of
miRNAs and its target genes Large-scale identification of
microRNAs in transcriptomic data
Anna Stachurska1, Christine Boesch-Saadatmandi2,
Agnieszka Witalisz1, Gerald Rimbach2,
Alicja Jozkowicz1, Jozef Dulak1, Agnieszka Loboda1
1Jagiellonian
University, Faculty of Biochemistry, Biophysics and
Biotechnology, Department of Medical Biotechnology, Krakow,
2
Poland; Christian Albrechts University Kiel, Institute of Human Nutrition
and Food Science, Kiel, Germany
e-mail: Anna Stachurska <[email protected]>
Heme oxygenase-1 (HO-1), an enzyme involved in heme
degradation, is claimed to play an important role in kidney
pathology, mostly due to its anti-apoptotic and anti-oxidant
properties. One of the nephropathic agents, ochratoxin A
(OTA) was previously suggested to affect HO-1.
We injected mice with different level of HO-1 (HO-1+/+,
HO-1+/-) and without HO-1 (HO-1-/-) i.p. with OTA every
other day for 20 days and then analyzed the expression of
specific miRNAs and its target genes. HO-1-/- mice were
more prone to OTA toxicity as we observed ~40% mortality in these animals.
Expression of miRNAs connected with fibrosis was affected by OTA delivery, eg. miR-155, modulating inflammation and TGFβ2 signaling, was diminished by OTA in
HO-1+/+ and HO-1+/- but not HO-1-/- mice. Additionally, expression of miR-200b/c was decreased after OTA
delivery in HO-1+/+ and HO-1+/- but not HO-1-/- mice.
Concomitantly, miR-29a/b/c and miR-192 were reduced
in all groups of animals. We also analyzed expression of
factors connected with fibrosis development and we found
out that some of OTA-evoked alterations were HO-1-dependent.
Additionally, p53 signaling, regulating cell proliferation and
apoptosis, was elevated after OTA delivery. Expression of
p21 and miR-34a, known to be modulated by p53, was upregulated in all OTA-treated animals with the lowest effect
in HO-1-/- mice.
Moreover, we observed OTA-evoked drop of expression
of miR-93 connected with angiogenesis. Concomitantly,
OTA strongly decreased expression of vascular endothelial growth factor (VEGF), known to be regulated by miR93, in all animals tested. We also detected reduced VEGF
level in sera of HO-1+/+ and HO-1+/- but not HO-1-/mice treated with OTA. Additionally, we observed that
HO-1-/- mice had lower basal expression of VEGF. We
isolated and cultured primary renal proximal tubular cells
(PTC) from HO-1+/+ and HO-1-/- mice, as VEGF in kidney is produced mostly by glomeruli and tubuli. We found
out diminished expression of VEGF in PTC from HO-1-/mice with simultaneous elevated level of miRNAs known
to regulate VEGF – miR-29b/c and miR-200b.
Taken together, toxic effect of OTA towards kidney may
be connected with changes in miRNA expression. Still, the
role of miRNAs in elevated susceptibility of HO-1-/- mice
to OTA needs further examination.
Acknowledgements
Supported by grants No. N N401 297835 and No. N N301 033440
(Ministry of Science and Higher Education). The Faculty of Biochemistry, Biophysics and Biotechnology is a beneficiary of grants No:
POIG.02.01.00-12 064/08, POIG 01.01.02-00-109/09, POIG.02.02.00014/08 and 01.01.02-00-069/09.
Michal Szczesniak, Izabela Makałowska
Laboratory of Bioinformatics, Department of Biology, Poland
e-mail: michal Szczesniak <[email protected]>
microRNAs are small RNA molecules, acting as posttranscriptional regulators ofgene expression in Eukaryotes.
The regulation might be through translational repression
ormRNA degradation. The genes under miRNA control
are involved in a number of processes,including development and response to stress. miRNA expression has also
been implicatedin numerous disease states. It is therefore
important to develop methods for effective miRNAsearch,
both through experimental and computational approaches.
As laboratory tests tendto be time-consuming and costly (though necessary to provide experimental evidence
formiRNA), starting with computational approaches is
exceedingly helpful there.We have developed an algorithm
for miRNA search in ESTs on the basis of maturemiRNA
sequence conservation i.e. similarity with known miRNAs.
In contrast to otheralgorithms designed to search for conserved miRNAs, it has several additional features,including
the usage of deep sequencing data (whenever available)
alongside EST sequencesor double-check of secondary
structure of miRNA precursor.The algorithm has been
incorporated into a bioinformatic pipeline and appliedfor
miRNA search in ESTs belonging to 210 plant and 212
animal species. This led toidentification of more than 10
000 miRNA candidates. Most of the candidates have neverbeen predicted before using other tools and also dozens
of species have been consideredin miRNA search for the
first time there. On the other hand, the tool shows high
efficacyin ‘discovering’ already known miRNAs. The predicted miRNAs can be viewed anddownloaded from our
database. It is also possible to run miRNA search in userprovided setof EST sequences.
17
P1_2.16
P1_2.17
Unintended consequences of cucumber
(Cucumis sativus L.) transformation
MYH11 and NDE1: potential sense–
antisense pair in human
Maria Szwacka1, Renata Zawirska-Wojtasiak2,
Michał Gośliński2, Aleksandra Twardowska3 Victoria A. Serzhanova1, Andrey V. Marakhonov1,
Anna V. Baranova1,2, Mikhail Yu. Skoblov1
1Warsaw University of Life Sciences, Department of Plant Genetics,
Breeding and Biotechnology, Warsaw, Poland; 2Poznań University of Life
Sciences, Department of Food Science and Nutrition, Poznań, Poland;
3Jan Wierzchoń and Partners Patent and Trade Mark Attorneys, Poland
e-mail: serzhanova victoria <[email protected]>
e-mail: Maria Szwacka <[email protected]>
Methods used in modern agriculture to improve plant traits
have the potential to generate unintended effects unrelated
to the target traits. These methods include plant genetic
transformation. Transgene DNA integrated into the plant
genome could modify their expression pattern. The transgene could be expressed in an unexpected manner through
interactions with various epigenetic mechanisms with the
result in production of unintended gene products. Unintended processes may result in the formation of new metabolites or altered levels of existing metabolites. The potential occurrence of unintended effects is currently one
of the concerns being raised regarding the application of
recombinant DNA techniques in the production of foods.
The present report aims to point out the scale of unintended effects in transgenic cucumber plants and attempts
to indicate factors that contributed to observed effects. Unintended changes in chemical composition of transgenic
cucumber plants expressing thaumatin II gene from the
West African shrub Thaumatococcus daniellii Benth are
the main focus of this report. Also factors that influence
level of thaumatin II gene expression in transgenic cucumber plants are discussed.
Antisense regulation of gene expression is widespread in
eukaryotes. Mechanisms of this type of regulation are different. Some of them are realized on the transcriptional
level, others work on the posttranscriptional level. But today there is small data about this type of regulation. So biological function and mechanisms of antisense transcripts
interactions still remain unclear.
We performed a computational analysis of human genes,
associated with carcinogenesis. Consequently we chose a
pair of protein-coding human genes MYH11 and NDE1,
which potentially are sense–antisense partners. Both genes
are located on short arm of 16 chromosome and are oppositely oriented. Region of overlap consists of last exon of
NDE1 (9 exon) and 3 exons of MYH11 (29–31). Length
of overlap of sense and antisense transcripts is 479 b. p.
MYH11 encodes a smooth muscle myosin belonging to
the myosin heavy chain family. This protein functions as a
major contractile protein, converting chemical energy into
mechanical forces through the hydrolysis of ATP. It has
been shown that the pericentric inversion of chromosome
16 produces a chimeric transcript with CBFB. This chromosomal rearrangement is associated with acute myeloid
leukemia [1]. NDE1 encodes a member of the nuclear distribution E (NudE) family of proteins. This protein plays
an essential role in microtubule organization, mitosis and
neuronal migration. Chromosomal aberrations, which involve locus of NDE1, produce a chimeric transcript with
PDGFRb and are associated with chronic myelomonocytic
leukemia [2].
Analysis of transcription level of MYH11 and NDE1 was
performed in number of human tissues and eukaryotic cell
lines. As a result we revealed that transcripts of both genes
were presented in most tissues and cell lines in various expression level ratios. On the basis of quantitative estimation of expression level of gene pair we made a conclusion about contribution of NDE1 and MYH11 genes to
antisense regulation of each other and propose a potential
mechanism of such negative antisense regulation.
References
1. Van der Reijden BA et al. (2010) Leukemia 24: 857-859.
2. La Starza R et al. (2007) Leukemia 21: 830–833.
Abstracts
18
P1_2.18
P1_2.19
Implications of microRNA 5p
and 3p strand switching
Revealing functional RNA processing
products from RNA-seq data
Harel Zalts, Noam Shomron
Marek Żywicki, Kamilla Bąkowska-Żywicka, Norbert Polacek
Sackler Faculty of Medicine, Tel Aviv University, Israel
Division of Genomics and RNomics, Innsbruck Biocenter, Innsbruck
Medical University, Innsbruck, Austria
e-mail: Harel Zalts <[email protected]>
MicroRNAs (miRNAs) are a group of ~22 nt non-coding
RNAs, which serve as central factors in post-transcriptional gene expression regulation. In the process of miRNA
biogenesis, the RNAse III enzyme, Dicer, cleaves the hairpin-shaped precursor miRNA to generate an intermediate short double-stranded RNA designated ‘5p’ or ‘3p’. In
most cases, only one of these strands operates as the biologically active miRNA, while its’ cognate opposite strand,
is mostly degraded. Recent evidence, however propose
a functional role in gene regulation for several oppositestrand miRNAs as well, based upon their conservation and
their varying expression. Although 5’ thermodynamics is
known to strongly affect strand bias in several invertebrates
and plants, it does not correlate well in mammals, and the
biological mechanism, which influences the alternating
5p/3p ratio, is still unclear. Here, based on experimental
evidence, such as real-time PCR and deep-sequencing,
combined with computational analysis, we have grouped
miRNAs according to their strand expression and strand
switching between 5p and 3p forms. We present our findings that indicate features of miRNA subgroups, the effect
on their potential targets when strand switching occurs, and
a set of targets selected to be regulated by both strands
of a miRNA. Overall we see that 5p/3p switching might
play an additional regulatory role for controlling genes by
miRNAs.
e-mail: Marek Zywicki <[email protected]>
After the results of the pilot ENCODE project suggesting that up to 90% of the human genome is actively
transcribed [1], the importance of RNA molecules which
does not encode proteins (noncoding RNAs – ncRNAs)
became unquestionable. The main difficulity in identification and characterization of novel ncRNAs is caused by
their complex biogenesis, which often include multiple
processing steps [2]. Thus, current methods are focused on
identification of novel transcription units rather then on
ncRNA species per se. We have addressed this challenge
by developing a computational pipeline named APART
(Automated Pipeline for Analysis of RNA Transcripts),
which in combination with adequate preparation of cDNA
library is able to detect a processing events within the longer RNA precursors. The analysis can be fulfilled in a fully
automated fashion, starting with raw sequence reads and
include genome mapping, contig assembly and annotation.
One of the main advantages of APART is a unique way of
handling of repeat-derived sequence reads. It enables the
identification of expression of distinct loci of the same
gene on one hand and removal of the redundancy caused
by spurious matching of the reads to non-relevant short
repeated sequence fragments dispersed across the genome
on the other.
We have applied the APART analysis on the sequencing
data obtained from a cDNA library of short RNAs associated with yeast ribosomes under different stress conditions.
Within the 174 contigs assembled, we have identified 131
putative processing products of distinct origin, including
intergenic regions, mRNAs, tRNAs rRNAs and snoRNAs.
The most surprising was to observe the snoRNA-derived
small RNAs to be associated with the ribosoms in the cytoplasm. Those small RNAs are believed to be localized
speciffically within the nucleolus and to be involved in ribosomal RNA maturation. In order to verify the results we
have performed a series of experiments, including northern blot analysis and others, which confirmed the cytoplasmic localization of APART-predicted snoRNA fragments
and their association with translating ribosomes. Similar experiments have confirmed the differential stress-dependent
processing of tRNAs and rRNAs which has been predicted
by APART. The result suggest the existance of a new layer
of modulation of gene expression, which is fulfilled by alternative processing of the transcripts.
Acknowledgement
The work was supported with the GenAU grant from Austrian Ministry
of Science and Research no. D-110420-012-012.
References
1. Birney E et al. (2007) Nature 447: 799-816.
2. Peng WT et al. (2003) Cell 113: 919-933.
Session 3: Bioinformatics for gene expression
Lectures
L3.2
L3.1
Specific peptides facilitate
metagenomic analysis
The methods of structural and
computational biology in study
of fidelity of gene expression
M. Tukalo1, O. Kovalenko1, K. Boyarshin1,
A. Raevskyi1, G. Yaremchuk1, I. Kriklivyi1, S. Cusack2
1Institute of Molecular Biology and Genetics, National Academy
of Science of Ukraine, Kyiv, Ukraine; 2EMBL, Grenoble Outstation,
Grenoble, France
e-mail: Mykhaylo Tukalo <[email protected]>
The fidelity of translation is dependent on the specificity
of the aminoacyl-tRNA synthetases (aaRSs). But in some
cases aaRS unable reliably discriminate amino acids with
similar structures. To overcome this problem some aaRSs
have specific editing activities that clear the wrong amino
acid: misactivated noncognate amino acids (pre-transfer editing) or mischarged tRNA (post-transfer-editing). Both reactions are depend on a tRNA cofactor and required translocation to the editing site located in the separate domain.
In this work we have studied molecular mechanisms of
editing by synthetases from two different classes: Thermus
thermophilus leucyl-tRNA synthetase (LeuRSTT) from class
I and Enterococcus fecalis prolyl-tRNA synthetase (ProRSEF)
from class II. Crystal structures of the LeuRSTT revealed
that the same site in CP1 domain can bind analogues of
both pre- and post-transfer editing substrates. To define
the possibility of pre-transfer editing in the editing site
and mechanism of editing we have used biochemical,
X-ray crystallography methods and methods of molecular dynamics. We have shown that editing of norvaline by
LeuRSTT occurs mainly through post-transfer pathway.
We prove the existence of tRNA-independent pre-transfer
editing (hydrolysis of norvalyl-adenylate), which occurs in
the editing domain. For class II ProRSEF we have found
that pre-transfer editing against alanine occurs in the synthetic center of enzyme in the presence of tRNAPro.
Crystal structures of the editing domains of LeuRSTT and
ProRSEF have been used as the platforms for intensive
alanine scanning mutagenesis of the key elements of the
deacylation activity for that type of enzymes. In fact, we
have failed to identify catalytic residues for hydrolysis within the active sites. At the same time the role of the tRNA
A76 hydroxyls in ester hydrolysis has been tested and the
tRNA-assisted mechanisms of post-transfer editing by leucyl- and prolyl-tRNA synthetase are proposed.
David Horn
School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel
e-mail: David Horn <[email protected]>
Specific Peptides (SPs) are deterministic amino-acid motifs, extracted from Swiss-Prot data, that serve as sequence
markers for enzymatic functionality. When found on large
strings of genomic or proteomic origin SPs provide quick
enzymatic annotations. Using the criterion of coverage
length (overall number of amino-acids in consistent SP
hits) ≥ 7 to provide EC annotations at levels 3 or 4, we
specify the biochemical function of an enzyme on the basis
of its sequence. This method has been applied to Sargasso
Sea Data uncovering 220K enzymes among 1 M protein
sequences, and providing an enzymatic spectrum of the
metagenome.
A user-friendly tool that displays occurrences of SPs on
any protein sequence that is presented as a query, together
with the EC assignments due to these SPs, is available at
http://adios.tau.ac.il/DME11.html.
Recently SP usage has been extended to direct search on
short reads. By collecting all short reads where SPs of a
given EC can be located, an estimate is provided for the
abundance of its relevant genes, thus generating an enzymatic spectrum of its genomic or metagenomic source.
Moreover, some of its taxonomic decomposition can be
deciphered using a subset of SPs belonging to aaRS enzymes.
A subset of SPs facilitates the task of taxa counting in metagenomic data. Using a list of 4000 SPs of length ≥ 9,
belonging to a subset of EC:6.1.1. aaRS enzymes that are
single genes in bacterial genomes, we identify their occurrences on given lists of short reads or contigs. Identifying the largest number of reads associated with one SP, we
propose an algorithm that constructs a minimal number
of fused strings that differ from each other, thus serving
as estimates for the different genes that could have led to
the observed reads or contigs. Short reads lead to bounds
on numbers of families, while long reads or contigs lead to
lower-bound estimates of numbers of strains and species.
Abstracts
20
L3.3
Oral presentations
Application of bioinformatics in
pharmacogenomic research O3.1
Michal Korostynski, Marcin Piechota,
Jaroslaw Dzbek, Wiktor Mlynarski, Ryszard Przewlocki
Department of Molecular Neuropharmacology, Institute of
Pharmacology PAS, Krakow, Poland
e-mail: Michal Korostynski <[email protected]>
The therapy of psychotic, depressive and anxiety disorders
is related to cellular and molecular mechanisms in the brain
regions controlling motivation, mood and emotions. It is
believed that psychotropic drugs regulate gene transcription and translation of new proteins that are required for
treatment-induced plastic changes and readaptation of the
target neural systems. The aim of our research is to investigate genetic networks regulated in the brain, in response to
psychoactive drugs from various clinical and pharmacological classes. We applied whole-genome microarray profiling
to evaluate time-course (4 time points) of transcriptome alterations following the drug treatment (18 drugs). The gene
expression data were stored in a data integration system,
based on MySQL in the data layer, Java in the logic layer
and AJAX (GWT) in the presentation layer. Implemented
tools allow for the identification of drug-specific genomic
signatures and drug-regulated transcriptional modules.
Further inspection and visualization of the signatures is
possible using multidimensional data analysis (PCA), coexpression analysis and heatmaps. Transcriptional modules
were identified by hierarchical clustering using the metric
of distance between nodes on the whole transcriptome
correlation tree. The tree was built as minimal spanning
tree using a Pearson correlation between the expression
profiles. Using the bioinformatic tools we reveal drug-regulated gene expression networks in the brain that are formed
by inducible transcriptional networks, as for example group
of CREB/SRF-dependent genes that appears to be related
to drug-induced neuronal activity. The observed alterations are connected to various drug-receptor interactions
and pharmacological effects of the drugs on dopamine,
serotonin and histamine systems. New candidate genes and
novel transcriptional variants with drug and time specific
gene expression profiles were identified.
Acknowledgements
This work was supported by MSHE grants: POIG DeMeTer 3.1, IUVENTUS Plus and NN405274137. Changes in gene expression profile
in primary culture of rat hepatocytes
treated with interferon alpha
Andriy Kuklin1, Bogdan Tokovenko1,
Nataliya Makogon2, Barbara Jarząb3, Maria Obolenskaya1
1Institute of Molecular Biology and Genetics, Ukraine; 2Institute of
Physiology, Ukraine; 3Marie Skłodowska-Curie Cancer Center and
Institute of Oncology, Poland
e-mail: Bogdan Tokovenko <[email protected]>
Interferon alpha (IFN-α) is a cytokine of innate immune
system. IFN-α is widely used in clinical practice for antiviral
and anticancer therapy. Nowadays IFN-α in combination
with ribavirin is the most effective therapy for viral hepatitis and associated hepatocellular carcinoma. The majority
of former and current investigations of IFN-α effects on
liver cells are conducted on transformed cell lines arguably
stressing its importance in treatment of liver cancer but
clearly omitting physiological relevance. In this work, we
aimed to determine the pure response of hepatocytes to
IFN-α. We cultivated primary rat hepatocytes and treated
them during 3 and 6 hours with 250u/ml IFN-α — dose
similar to the IFN-α concentration observed during liver
regeneration (LR). The gene expression profile was assayed
with Affymetrix Rat Genome 230 2.0 microarrays. Inhouse bioinformatics analysis included custom Bioconductor pipeline, computational identification of transcription
factor binding sites, pathways and GO enrichment analysis using 3rd-party tools. 124 genes with the fold change
greater than 2 were defined as differentially expressed. Validation with real-time qPCR confirmed high correspondence with the results of microarray experiment. Differentially expressed genes were attributed, substantially, to GO
categories related to “immune response”, but considerable
enrichment was also observed in GO category “modification dependent protein degradation” pointing to IFN-α activated catabolic processes. We have analyzed whether the
differential expression occurs as a result of activation of
Jak/STAT, Jak/STAT/ISGF3 and p38 signaling pathways
involved in IFN-α response. For this purpose we conducted the search of appropriate transcription factor binding
sites for STATs (1, 3, 4, 5, 6), ISGF3, IRF1, CREB1, CEBP,
NFκB, Max/Myc, MEF2A/C, NFAT, SP1, ELK1 within
promoter regions of differentially expressed genes. Our results support the activation of multiple signaling pathways
and corresponding transcription factors by IFN-α. The signaling pathways Jak/STAT, Jak/STAT/ISGF3 and p38 are
represented in descending order according to the extent of
their involvement in IFN-α response. Majority of differentially expressed genes contained binding sites for more than
one of the transcription factors listed above, which may
be a base for more precise regulation of gene expression,
activated by IFN-α, where each transcription factor makes
certain contribution to the activation of transcription. Current work is the first step towards elucidation of IFN-α role
in the triggering of LR.
O3.2
Posters
Structure-based characterization
of GntR superfamily regulators of
Streptomyces coelicolor A3(2)
P3.1
Olga Tsypik, Nestor Zaburannyy,
Bohdan Ostash, Viktor Fedorenko
Ivan Franko National University of Lviv, Department of Genetics and
Biotechnology, Lviv, Ukraine
e-mail: Olga Tsypik <[email protected]>
Streptomycetes are soil-living bacteria with a complex life
cycle. Members of this genus have large genomes and the
ability of producing a broad spectrum of biologically active substances, in particular, many known antibiotics. The
genome of the main model strain, Streptomyces coelicolor, contains more than 20 secondary metabolite gene clusters and
965 regulatory genes. Given the fact that streptomycetes
are of enormous industrial importance (production of antibiotics and other products), it is essential to further elucidate regulatory mechanisms of metabolic and morphological differentiation in this taxon.
One of the most abundant and widely distributed groups
of transcriptional factors in actinobacteria is the GntR superfamily of regulators. They regulate numerous processes
of primary metabolism and cell differentiation. The S. coelicolor genome encodes 60 putative GntR-like regulatory
proteins (GntRs) but, so far, no exhaustive classification of
this regulators has been undertaken.
We use comparative genomic analysis to describe and characterize the putative GtnRs. Regulators of this superfamily
possess a conserved N-terminal domain involved in the
DNA binding and a quite diverse, heterogenous C-terminal
domain involved in effector binding and/or oligomerization. Based on diversity of C-terminal domain, GntRs are
divided into a few families with conserved secondary structure. The sequence analysis of these regulators showed
distinguishable predicted secondary structure features.
The majority of these proteins represent features of FadR,
HutC, MocR and YtrA subfamilies. Several GntRs show
additional secondary structure elements, suggesting a possible origin of new subfamily within GntR superfamily.
Using the reciprocal BLAST search, we identify that 12
GntRs have the orthologs in all sequenced to date Streptomycetaceae genomes and suggest that they are the most important for morphogenesis and/or secondary metabolism.
Since the expression of many genes for GntRs is autoregulated, we’ve tried to find potential operator sites of GntRs
by analyzing the upstream regions of these genes. Most
of the examined promoters do not possess operators inferred for each GntR subfamily from in silico analysis of
non-actinomycete GntRs. New operators are proposed for
the studied genes. Further identification and verification of
these sites is needed to underpin the functional roles of the
studied genes.
21
Comparison of binding of 44 transcription
factors to putative regulatory regions
identified by histone modifications,
open chromatin and conservation Jakub Lenart, Bozena Kaminska, Michal Dabrowski# The Nencki Institute of Experimental Biology, Laboratory of
Transcription Regulation, Department of Cell Biology, Warsaw, Poland
#presenting author e-mail: Michał Dąbrowski <[email protected]>
We designed and implemented a database, named TRAM
(Transcription Regulatory Areas and Motifs database), unifying representation of the data from Ensembl schemas
relevant for cis-regulation (funcgen, compara and core) to
the analysis layer. Among the key features of TRAM are
pre-computed data on pairwise overlap on the genomic sequence of putative regulatory areas of different types, in
several different cell types.
Here, we report analysis of pairwise overlap among 41
histone modifications, 44 transcription factors (TFs), two
markers of open chromatin, and regions of sequence conservation from two algorithms. We focused on the overlap
between the chip-seq data for the 44 transcription factors
represented in funcgen v.60 and the remaining putative
regulatory regions. We found that the binding of TFs is
highest in regions of histone modifications associated with
active promoters (H3K4me2-3, H3K9ac, Pol II), high in
the regions of modifications associated with active enhancers (H3K27ac) and in cis-RED promoters; and low in the
regions associated with poised enhancers (H3K27me1-3).
We report a notable difference between the high binding of
TFs to the regions of open chromatin identified by DNaseseq and lower binding to the regions identified by FAIRE.
TF binding to the regions identified by sequence conservation was lower than to the regions identified experimentally.
Interestingly, it was also low for the 400 VISTA enhancers
verified experimentally, confirming that enhancer occupancy by TFs is cell and developmental stage specific.
The highest conservation of the same feature localization
in different cell types was found for the markers of active
promoters (H3K4me2-3) and insulators (CTCF); intermediate conservation for regions identified by Dnase1 (but
not FAIRE) and markers of active enhancers (H3K27ac);
whereas localization of histone markers of poised enhancers (H3K27me1-3), as well TFs binding was variable
among the different cell types. To conclude: TF binding
correlates well with regions of regulatory activity computationally predicted from gene expression data. TF binding is
higher, and more conserved across cell types, in promoters
than in enhancers. Regions of open chromatin identified
by Dnase1 are more enriched in TF binding than regions
identified by FAIRE. Abstracts
22
P3.2
P3.3
Genomegems: evaluation of genetic
variability from deep sequencing data Analysis of consequently changed
signaling pathways — a novel method
for analyzing microarray data
Adi Givati1,2*, Sharon Ben-Zvi1,2*, Noam Shomron2 1Tel-Aviv
University, Department of Biomedical Engineering, The Iby
and Aladar Fleischman Faculty of Engineering, Tel Aviv, Israel ; 2Tel-Aviv
University, Department of Cell and Developmental Biology, Sackler
Faculty of Medicine, Tel Aviv, Israel
*equal contribution e-mail: Adi Givati <[email protected]>
Deep Sequencing (also known as Next Generation Sequencing or Massive Parallel Sequencing) is a revolutionary
method that allows myriad amounts of short DNA fragments to be read simultaneously. Deep Sequencing of the
human genome for detection of disease-causing mutations
possesses great challenges. In particular, organizing the
great amount of sequences generated so that mutations,
which might possibly be biologically relevant, are easily
identified is a difficult task. Yet, for this assignment only
limited automatic tools exist. GenomeGems comes to gap
this need by evaluating variability in Deep Sequencing generated genetic data in a simple tabular depiction, graphical representation and visualization for comparing multiple
sequencing datasets. GenomeGems integrates well with
the UCSC Genome Browse in order to create an annotated display of the genetic changes in the acquired data.
As such, via automatic, clear and accessible presentation of
processed Deep Sequencing data, our tool aims to facilitate
ranking of genomic variance calling. GenomeGems runs
on a local PC and is freely available at http://www.tau.
ac.il/~nshomron/GenomeGems.
Jakub Mieczkowski*, Karolina SwiatekMachado*, Bozena Kaminska
Nencki Institute of Experimental Biology Polish Academy of Sciences,
Department of Cell Biology, Warsaw, Poland
*equal contributors
e-mail: Jakub Mieczkowski <[email protected]>
Signaling pathways belong to a complex system of communication that governs cellular processes. They represent
signal transduction from an extracellular stimulus via a receptor to intracellular mediators, as well as intracellular
interactions. Perturbations in signaling cascade often lead
to detrimental changes in cell function and they are a major cause of many diseases, including cancer. Identification
of deregulated pathways may advance understanding of
complex diseases and lead to improvement of therapeutic
strategies. We propose Analysis of Consistent Signal Transduction
(ACST), a novel method for analysis of signaling pathways.
Our method incorporates information regarding pathway
topology, as well as data on the position of every gene in
each pathway. To preserve gene-gene interactions we use a
subject sampling permutation model to assess the significance of pathway perturbations. We applied our approach
to four independent datasets of global gene expression profiling (vulvar epithelial neoplasia and endometriosis versus
control and 2 sets of colorectal cancers-controls). The results of ACST, as well as three other methods used to analyze signaling pathways (SPIA, SEPEA and the maxmean),
are presented in the context of biological significance and
repeatability. We show that our method is characterized by
biologically meaningful results, as well as strong repeatability between similar, yet independent, datasets.
P3.4
P3.5
LEF1 and TCF7L2 are candidates for
terminal selectors of thalamic neurons
Bio-evaluation of novel heterocyclic
4-thiazolidone derivatives designed for
targeting Bcl-XL antiapoptotic protein Andrzej Nagalski1, Michal Dabrowski2,
Jacek Kuznicki1, Marta B. Wisniewska1
1International Institute of Molecular and Cell Biology, Warsaw,
2Nencki Institute of Experimental Biology, Warsaw, Poland
Poland;
e-mail: Andrzej Nagalski <[email protected]>
The concept of ‘terminal selectors’ that regulates terminal differentiation gene batteries (Hobert et al., 2010, Trends
Neurosci), is an attractive way to envisage the role of master
transcription factors in establishing characteristics of specific neuronal populations. We have recently shown that
β-catenin, a cofactor of LEF1/TCF transcription factors,
is constantly nuclear specifically in the thalamus and regulates expression of Cacna1g encoding an ion channel that
is responsible for specific properties of thalamic neurons
(Wisniewska et al., 2010, J Neurosci). We put a hypothesis
that LEF1/TCF factors are terminal selectors of neurons
of the thalamus.
Using Real Time PCR, we quantified expression level of
genes encoding LEF1/TCF factors during postnatal thalamic development and observed that Lef1 and Tcf7l2
fulfill the most fundamental criterion for terminal selector
genes, i.e. the maintained expression throughout the life
of a neuron. Immunohistochemical analysis of β-catenin,
LEF1 and TCF7L2 proteins in the adult mouse brain revealed the presence of these proteins in majority (although
not all) of thalamic nuclei.
In parallel we defined thalamic regions of unique and correlated gene expression, using the Anatomic Gene Expression Atlas (AGEA) from Allen Brain Atlas. AGEA characterizes the multi-scale spatial relationship in the mouse
brain as derived from gene expression data without a prior
knowledge of classical anatomy. Then we collected 200
genes for each defined region using AGEA Gene Finder
and visually inspected for false positives. The final lists
were further used to search for overrepresented cis-regulatory elements in the gene promoters. We analyze LEF1/
TCF binding sites, and other sites for transcription factor
presents in defined gene expression regions. 23
R. R. Panchuk1, V. V. Chumak1,2,
D. Ya. Havrylyuk3, R. B. Lesyk3, R. S. Stoika1,2 1Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine; 2Ivan Franko
Lviv National University, Ukraine, Lviv, Ukraine; 3Danylo Halytsky Lviv
National Medical University, Lviv, Ukraine
e-mail: Rostyslav Panchuk <[email protected]>
Due to poor efficiency of classic cancer chemotherapy,
more efforts are given to design of novel drugs, targeting
tumor-specific vulnerabilities. In silico investigations have
revealed that novel heterocyclic 4-thiazolidone derivatives
can be used as effective inhibitors of Bcl-XL-Bax dimerization, thus, enhancing apoptotis in tumor cells. More
than 5 000 compounds were synthesized at Lviv National
Medical University and pre-screened in vitro at National
Cancer Institute (Bethesda, USA). We have selected three
compounds – Les-3120, Les-3166 and Les-3372 – that
possessed the highest cytotoxicity towards tumor cells.
Docking studies for their interactions with Bcl-XL were
performed using OpenEye software and protein Bcl-XL
structure (code 1YSI) taken from Protein Data Bank.
Western-blot analysis of signaling apoptotic pathways induced by these compounds in Jurkat T-leukemia cells has
shown that different side chemical groups of thiazolidone
molecules can determine specific mechanism of apoptosis
(mitochondrial, receptor-mediated or caspase-independent) that was induced in tumor cells. Taking into account
the results of our in vitro, in vivo and in silico studies, it was
decided to combine in one compound two drug molecules
that belong to Les-3120 and Les-3372. Earlier, we found
that molecular mechanisms of pro-apoptotic action of
these molecules was totally different.
As a result of such approach, two novel isomeric molecules
were synthesized — Les-3661 and Les-3713 differing only
in a place of substitution of phenylpyrazoline side group.
While 2-substituted thiazolidone Les-3713 demonstrated
modest cytotoxic activity (IC50=5 μM), antitumor potential of Les-3661 (4-subsituted thiazolidone) was increased
more than 10 times (IC50=1 μM) reaching a potential of
gold chemotherapy standard – doxorubicin (IC50=0.5 μM).
Les-3661 acted towards target cells not only much quicker (3 h) comparing to Les-3120 (12 h). It also specifically
cleaved initiator procaspases-9 and -10 involved in mitochondrial and receptor-mediated apoptosis, correspondingly. It is hypothesized that such “hybrid” molecule possesses not only tenfold elevated cytotoxicity, but combines
potentials of two distinct apoptotic pathways available in
its predecessors. In vivo studies of Les-3661 action towards
murine NK/Ly lymphoma known by Bcl-XL over-expression, revealed its high therapeutic potential combined with
low toxicity in the experimental mice (LD50=600 mg/kg).
Thus, novel thiazolidones can be proposed for design of
anticancer drugs targeting Bcl-XL protein, and, thus, possessing high selectivity towards tumor cells. Abstracts
24
P3.6
P.3.7
Identification of transcription factor
STAT3 target genes and epigenetic
modifications in glioma cells using whole
genome chromatin immunoprecipitation
Short peptide sequences inhibit
prokaryotic translation
Piotr Przanowski1, Marcin Kruczyk2,3, Karolina SwiatekMachado1, Jakub Mieczkowski1, Michal Dabrowski1,
Claes Wadelius4, Jan Komorowski2,4, Bozena Kaminska1
1Laboratory of Transcription Regulation, Nencki Institute, Warsaw,
Poland; 2ICM-Warsaw University, Warsaw, Poland; 3Postgraduate School
of Molecular Medicine, Warsaw, Poland; 4Uppsala University, Uppsala,
Sweden
e-mail: Piotr Przanowski <[email protected]>
Signal transducer and activator of transcription 3 (STAT3)
is a transcription factor constitutively activated in diverse
human tumors (including gliomas) and contributing to
malignant transformation, tumor progression and resistance to apoptosis. Screening of small molecules inhibitors
(caffeic acid derivatives) targeting Jak2/STAT3 signaling
revealed inhibition of proliferation and induction of cell
death, suggesting a pivotal role of STAT3 in glioma survival. Global gene expression profiling revealed modulation
of numerous genes as a result of treatment and hundreds
of genes were identified as potential STAT3 targets, however a small fraction of those genes was proven to be direct
STAT3 targets. We mapped the genome-wide occupancy
of active, phospho-STAT-3 and epigenetic modifications
(H3K4me3; H3K27me3) in glioma cells by hybridization
of immunoprecipitation-enriched genomic DNA to promoter microarrays (3x720K RefSeq Promoter microarrays,
NimbleGen) or analysis by massively parallel sequencing
(ChIP-Seq). Furthermore, putative STAT binding sites and
motifs were identified computationally in a genome-wide
study. Data from H3K4me3 chromatin immunoprecipitation was intersected with the data on STAT3 ChIP-chip.
This analysis identified a list of 800 genes which promoters
have both STAT3 binding site and H3K4me3 modification
suggesting these are actively transcribed genes. Gene Ontology analysis revealed several groups of genes, including
a few interesting and expected groups such as inflammatory response, cell migration, cell differentiation, positive
regulation of cell proliferation, regulation of blood vessel size. The data from STAT3 and H3K4me3 study were
also intersected with three sets of expression data: a set
identifying genes differentiating C6 glioma cells from nontransformed glial cells and two sets of microarrays data
from C6 glioma cells treated with inhibitors of JAK/STAT
signaling. From this analysis we obtained a list of 250 genes
which are likely to be STAT3 transcriptional targets. The
most interesting hits, representing new identified STAT3
targets, are verified by chromatin immunoprecipitation followed by qPCR.
Acknowledements
Studies supported by Foundation of Polish Science, International PhD
Projects (MPD) program (PP, MK) and a grant N301 239536 from the
Ministry of Science and Higher Education (JK).
Sharon Penias Navon, Tali Schwartzman, Noam Adir
Schulich Faculty of Chemistry Technion — Israel Institute of
Technology, Haifa, Israel
e-mail: <[email protected]>
The ribosome is a highly conserved nucleoprotein machine
whose function is to translate information encoded in an
mRNA into a protein. All nascent polypeptides must pass
through the ribosomal exit tunnel. The tunnel is lined with
chemical groups which can potentially interact with the
growing polypeptide, thus potentially arresting translation.
We hypothesized that the incidence of certain short amino
sequence might be reduced by evolutionary pressure, since
these short amino acid sequences could form strong interactions with the ribosomal exit tunnel leading to translational arrest and loss of cell viability. The existence of
many complete proteomes has afforded us the ability to
search for sequences that exist at frequencies below their
expected frequency (based on each amino acids total usage
in the proteome). We have used “reverse bioinformatics”
and indeed identified such highly under-represented sequences (URSs) in the proteome of E. coli and other organisms. When these URSs were experimentally incorporated
into a protein expressed in E. coli, the rate of translation of
the URS embedded protein, was radically diminished, both
in vivo and in vitro. In addition we found that the presence of
these URSs in one protein also inhibits the translation of
other proteins in vitro, indicating that the rate of ribosome
recycling is also inhibited. Arrest of ribosome translation
by the protein containing URSs also inhibits the translation of other proteins in vivo, to an extent that causes cell
death. We thus propose that knowledge of the chemical
attributes of the URSs may lead to the development of a
completely novel class of antibiotics. We also propose that
the identification of factors lacking from an organisms, using reverse bioinformatics can be a new and useful tool for
understanding the overall physiology of the organism.
Session 4: Gene expression and brain disease
Lectures
L4.2
L4.1
RNA in pathogenesis and experimental
therapy of polyglutamine diseases
Cholinergic-mediated microRNA control of
hnRNPs A/B regulates cortical alternative
splicing and neural functioning
Amit Berson1, Galit Shaltiel1, Shahar Barbash1,
Yael Goll1, Geula Hanin1, Keren Ofek1, Maya Ketzef2,
Yehudit Gnatek2, Alon Friedman2, Hermona Soreq1
1The Edmond and Lily Safra Center of Neuroscience and Institute
of Life Sciences, Department of Biological Chemistry, The Hebrew
University of Jerusalem, 91904 Jerusalem, Israel; 2Zlotowski Center for
Neuroscience, Department of Physiology, Ben-Gurion University of the
Negev, Beer-Sheva 84105, Israel
e-mail: Hermona Soreq <[email protected]>
Genetic studies link neurodegeneration to inherited errors
in RNA metabolism. Here, we report cholinergic-mediated
alternative splicing errors in sporadic Alzheimer’s disease
(AD). AD entorhinal cortices presented impaired exon
exclusions, selectively reduced hnRNP A/B splicing regulators and increased levels of the hnRNP A/B-targeted
miRNA-211. Supporting functional relevance, hnRNP
A/B knockdown in mice induced memory and electrocorticographic impairments, dendrite and synapse loss
and splicing abnormalities. In-vivo neurotoxin-mediated
destruction of cholinergic neurons, but not APP or Tau
mutagenesis caused cortical AD-like decrease in hnRNP
A/Bs and reduced exon exclusions. Inversely, cholinergic excitation increased hnRNP A/B levels, suppressed
miR-211 and elevated the cholinergic enhancer miRNA-132
which was drastically reduced in the AD cortex. Furthermore, suppressing miR-132 elevated miR-211 in primary
neurons whereas miR-211 did not change miR-132. Our
findings uncover hierarchical cholinergic and miRNA-mediated hnRNP A/B loss and consequent RNA metabolism
impairments as novel targets for interference with sporadic
neurodegenerative processes.
A. Mykowska, A. Fiszer, M. Wojciechowska, W. J. Krzyzosiak
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan,
Poland
e-mail: Wlodzimierz Krzyzosiak <[email protected]>
Mutant transcripts containing expanded untranslated CUG
repeats are well documented pathogenic agents in myotonic dystrophy type 1 (DM1). The mutant RNA sequesters
the MBNL1 splicing factor and causes misregulation of
the alternative splicing of multiple genes that are linked to
clinical symptoms of the disease. Here we show that alternative splicing defects are also caused by translated CAG
repeats present in transcripts from mutant genes implicated
in spinocerebellar ataxia type 3 (SCA3) and Huntington’s
disease (HD) as well as by untranslated CAG repeats. We
propose that alternative splicing deregulation by mutant
CAG repeats may contribute to the pathological features
of polyglutamine disorders.
We also demonstrate selective targeting of mutant HD
transcripts, in the presence of their normal alleles and
other human mRNAs containing CAG repeats, by RNA
interference reagents. This selectivity may lead to promising therapeutic modalities for HD and other polyglutamine
disorders.
Abstracts
26
L4.3
Oral presentations
Intersectins and
neurodegenerative diseases
O4.1
Alla Rynditch, Liudmyla Tsyba, Mykola Dergai,
Dmytro Morderer, Inessa Skrypkina, Oleksii Nikolaienko,
Oleksandr Dergai, Sergii Kropyvko
Institute of Molecular Biology and Genetics, National Academy of
Sciences of Ukraine, Department of Functional Genomics, Kyiv, Ukraine
e-mail: Alla Rynditch <[email protected]>
The rich binding capability of the multidomain, adaptor
and scaffolding proteins of intersectin (ITSN) family has
linked them to multiple functions such as clathrin-mediated endocytosis, mitogenic signalling, actin cytoskeleton
rearrangements and apoptosis. One of these family members, ITSN1, is characterized by high level of expression
in neurons. ITSN1 gene is mapped to chromosome 21 in
the Down syndrome (DS) critical region. Abnormalities
of ITSN1 expression were associated with the endocytic
anomalies reported in Down syndrome brains, early stages
of Alzheimer’s disease as well as with neurodegeneration in
Huntington’s disease. DS neurons are characterized by enlarged early endosomes as well as enlarged early endosomes
are the earliest neuropathological alterations identified in
sporadic Alzheimer’s disease.
In order to highlight the role of ITSN1 gene in neurons,
we studied its expression in normal and pathological brains
as well as analyzed the composition of ITSN1-containing
protein complexes in neurons. We have identified neuronspecific alternative splicing of microexon 20 that affects
the structure and binding abilities of the SH3A domain
of ITSN1. Structure modelling and mutational analysis revealed that the insertion encoded by the microexon shifts
negatively charged amino acid residues towards the interaction interface within the n-Src loop of the SH3A domain.
This event leads to dramatic change of the SH3A domain
binding properties, in particular to increasing the binding
affinity to endocytic proteins dynamin and synaptojanin. In
contrary, binding affinity of neuron-specific SH3A domain
for to Ras guanine nucleotide exchange factor Sos1 and E3
ubiquitin-ligase Cbl has decreased.
Analysis of ITSN1-containing protein complexes revealed
novel neuron-specific ITSN1 protein partners: stable tubule-only polypeptide/MAP6 microtubule-associated protein 6 (STOP/MAP6) and synapsin. STOP/MAP6 regulates microtubule stability and is involved in generation of
synaptic plasticity, which is thought to be a molecular background of learning and memory. Moreover, STOP knockout mice were proposed to be used as an animal model of
schizophrenia. Since both, ITSN1 and STOP, are implicated in functioning of synapses and associated with mental
disorders, further investigations of their interactions are of
particular interest.
The neurotrophic factors mRNA expression
in U87 glioma cells and its regulation
by hypoxia and ischemic conditions are
dependent from ERN1 gene function
O. V. Hubenya1, D. O. Minchenko1,2,3,
A. P. Kharkova1, M. Moenner3, O. H. Minchenko1,3
1Department of Molecular Biology, Palladin Institute of Biochemistry
National Academy of Sciences of Ukraine, Kyiv, Ukraine; 2Department
of Pediatrics, Bohomoletz National Medical University, Kyiv, Ukraine;
3INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory,
University Bordeaux 1, Talence, France
e-mail: Olena Hubenya <[email protected]>
Gliomas are characterized by diffuse infiltrative growth
in the surrounding brain parenchyma, which precludes
complete surgical resection and is responsible for local
recurrences. Endoplasmic reticulum to nuclei-1 (IRN1)
is a central mediator of the endoplasmic reticulum stress.
Blockade of ERN1 transduction pathways in experimental
tumor models suggests that its activity is linked to the neovascularization process.
The main goal of this work is to study the role of neurotrophic factors in malignant gliomas progression under
the inhibition of endoplasmic reticulum to nuclei-1 signaling pathways as well as the effect of hypoxia and ischemic
conditions on the expression of several genes encoded the
neurotrophic factors. We have shown that the expression
levels of BDNF (brain-derived neurotrophic factor) and
PRNP (prion protein) are increased but NPDC1 (neural
proliferation, differentiation and control), PSEN1 (presenilin) and SLC1A1 (neuronal epithelial high affinity glutamate transporter) – decreased in glioma cells with suppressed
activity of endoplasmic reticulum to nuclei-1. It was also
shown that hypoxia is induced the expression levels of
BDNF, NPDC1, PNPLA6 (neuropathy target esterase) and
SLC1A1 in control glioma cells, but in glioma cells with
suppressed activity of endoplasmic reticulum to nuclei-1
– NPDC1 and PSEN1 only. At the same time, the expression levels of PRNP and SLC1A1 is decreased in these
genetically modified cells. Glutamine deprivation condition leads to increase the expression levels of PSEN1 and
PRNP in both cell types, however the expression levels of
PNPLA6 is increased in control glioma cells and decreased
in cells with suppressed activity of endoplasmic reticulum
to nuclei-1. We have also shown that glucose deprivation
condition leads to increase the expression levels of PRNP,
PNPLA6 and SLC1A1 in control glioma cells and to decrease the expression levels of NPDC1, BDNF, PNPLA6
and SLC1A1 in genetically modified cells. Thus, our results
showed that blockade of the activity of endoplasmic reticulum to nuclei-1 signaling enzyme changes the expression levels of neurotrophic factors and modulate effect of
hypoxia and ischemia on these genes expression.
O4.2
Posters
Identification of novel targets of stat3
transcription factor in glioma cells
P4.1
Karolina Swiatek-Machado, Michal Dabrowski,
Jakub Mieczkowski, Piotr Przanowski,
Alicja Adach Kilon, Bozena Kaminska
Activation of transcription
factor NFκB in malignant and
nonmalignant glioma cell lines
Nencki Institute of Experimental Biology, Department of Cell Biology,
Warsaw, Poland
Joanna Bem, Magdalena Tyburczy, Bozena Kaminska
e-mail: Karolina Swiatek-Machado <[email protected]>
Signal transducer and activator of transcription 3 (STAT3)
is a transcription factor constitutively activated in diverse
human tumors, including gliomas and contributing to malignant transformation, tumor progression and resistance to
apoptosis. Using microarray techniques, hundreds of genes
have been identified as potential STAT3 targets, however
the small fraction of these genes have been proven to be
direct STAT3 targets. Here we report identification of novel, direct STAT3 target genes using microarray techniques,
computational methods for the genome-wide identification
of transcription factor binding sites and chromatin immunoprecipitation. We determined global changes in gene
expression in C6 glioma cells treated for 24 h with either
DMSO (control) or 25 μM the inhibitor of JAK/STAT3
signaling pathway. The analysis revealed that inhibition of
JAK/STAT3 signaling significantly modulates expression
of numerous genes involved in many different biological
processes such as inflammatory response, apoptosis, signal transduction, proliferation, but also RNA processing or
lipid metabolism. Next, we analyzed promoter regions of
differentially expressed genes. First, we searched for conserved non coding region between human and rat genome
using the global alignment program. Next, we found putative STATs binding sites within conserved non-coding regions using MathInspector, which take advantages of positional weight matrices. Phylogenetic conservation between
rat and human sequences was used to select the binding
sites most likely to be functional. For further validation we
selected 12 genes with at least two putative binding sites
within the promoter regions. All of the tested sites were
bound by STAT3 in C6 glioma cells, as assessed by chromatin immunoprecipitation. Altogether, our results demonstrate identification of a group of novel STAT3 targets,
shed light on complexity of a genetic network regulated
by JAK/STAT3 signaling in glioma cells and its functions.
Acknowledgements
The study was supported by grant N N405 621938 from the Polish Ministry of Science and Higher Education (BK). 27
Warsaw, Poland
e-mail: Joanna Bem <[email protected]>
NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) is knownto be one of the most important
inflammation regulators. It plays a key role inautoimmune
diseases and cancer development. NF-κB regulates transcription of avariety of genes including cytokines, chemokines, adhesion molecules and manyothers. Correlation
between increased Akt phosphorylation and high levels of
NFκB activityin malignant gliomas has been reported. We
recently reported that inhibition of NFκBtranscriptional
activity by inhibitors ofPI-3K/Akt signaling or the NFκB
inhibitor - BAY11-7082, affects MMP-2 expression and
impairs glioma invasion (Kwiatkowska et al., 2011). Elucidating mechanisms of NFκB activation and its role in
different glioma typeswould increase our understanding
of brain tumor invasion. Inactive NF-κB is bound toIκB
(nuclear factor of kappa light polypeptide gene enhancer
in B-cells inhibitor) andretained in cytoplasm. A few pathways of NF-κB activation have been characterized.The
classical pathway involves phosphorylation of IκB by IκB
kinases (IKK-β, IKK-γ) that leads to its degradation by a
proteasome complex. NF-κB is released andtranslocates
to the nucleus, where it can regulate expression of target
genes.Our aim was to elucidate if there are differences in
NF-κB regulation in malignantglioma and nonmalignant
cell lines. First, the relative expression of (IKK-β, IKK-γ)
was determined in various established and primary glioma
cell lines by quantitativePCR. The activity of NF-κB was
further determined in transfected cells using NF-κB binding site -driven reporter luciferase gene using a dual light
reporter luciferaseassay. We found that the expression of
IKK-β and IKK-γ varies between malignantglioma versus
nonmalignant cells Those differences were corroborated
by variouslevels of NF-κB transcriptional activity in malignant gliomas and nonmalignantcells. Our data show that
in malignant gliomas NF-κB activity is higher than innonmalignant tumors (SEGA — subependymal giant cell astrocytoma) and innormal human astrocytes. It corresponds
to the levels of phospho-IκB detected byimmunoblotting.
The classical activation pathway is likely involved in this
process. Inmalignant and nonmalignant cell lines, NF-κB
activity corresponds to the expressionof IKK-β and IKK-γ
kinases.
Abstracts
28
P4.2
P4.3
Lymphocytes from SAD patients
display cell cycle alterations, unlike
lymphocytes from FAD patients
Nanoparticles for delivery of tissue
inhibitor of matrix metalloproteinases
to the brain as means to ameliorate
consequences of ischemic cell death
Emilia Bialopiotrowicz1,2, Bozena Kuzniewska1,
Neli Kachamakova-Trojanowska1,
Jacek Kuznicki1,2, Urszula Wojda1 1International
Institute of Molecular and Cell Biology, Laboratory of
Neurodegeneration, Warsaw, Poland; 2Nencki Institute of Experimental
Biology, Warsaw, Poland
e-mail: Emilia Bialopiotrowicz <[email protected]>
Alzheimer’s disease (AD) is the most common age-related
dementia characterized by progressive neuronal loss. About
95% of AD cases occur sporadically and have unknown
etiology (SAD), whereas the remaining familial AD cases
(FAD) are mostly caused by autosomal dominant mutations in the presenilin 1 (PS1) gene. PS1 is the main enzymatic component of gamma-secretase complex responsible for release of toxic beta-amyloid from its amyloid
precursor protein (APP). Growing evidence suggests that
in AD neurons re-enter the G1 cell cycle phase, which leads
to cell death. Since some alterations in AD are seen also
in peripheral cells, we checked if cell cycle changes occur
in AD lymphocytes. The study involved immortalized lymphocytes derived from a group of Polish patients: 8 FAD
subjects with distinct PS1 mutations, 18 SAD patients, and
34 age-matched individuals. Our PCR arrays experiments
showed that 43% of the 90 investigated cell cycle genes
were down-regulated in SAD, whereas 4% were up-regulated comparing to controls. Interestingly, 19% of the cell
cycle genes were differentially expressed in the SAD versus
FAD. Most significant changes in the expression referred to
the genes engaged in the G1/S control. Therefore we assessed the levels of key proteins involved in the G1/S transition with immunobloting. The most striking difference
we found regarded p21 protein, which was significantly elevated in SAD comparing to control and FAD lymphocytes.
Furthermore, our FACS analysis demonstrated that alterations in the cell cycle genes expression and p21 increase
observed for SAD lymphocytes were accompanied by increased % of cells in G1 phase and decreased % of cells
in S phase. Using FACS cell cycle analysis after nocodazole
treatment and pulse chase BrdU labeling we showed that
observed G1 arrest in SAD cells was a consequence of G1
phase prolongation and S phase shortening. Moreover, we
found that the observed cell cycle changes were independent on gamma-secretase activity.
Our data demonstrate that cell cycle abnormalities are
common for SAD, but not for FAD lymphocytes. Thus,
this study brings to light differences in the mechanisms of
FAD and SAD pathogenesis. Furthermore, these results indicate that human lymphocytes sustain a useful model for
further analyzes of AD pathogenesis and for the development of new diagnostic methodologies targeting cell cycle
proteins, such as p21.
Mayank Chaturvedi1,2, Bojja Sreedhar2, Leszek Kaczmarek1
1Nencki Institute of Experimental Biology, Department of Molecular
and Cellular Neurobiology, Warsaw, Poland; 2Indian Institute of
Chemical Technology, Inorganic & Physical Chemistry Division, India
e-mail: Mayank Chaturvedi <[email protected]>
Introduction: Enhanced expression and activities of matrix metalloprotease (MMPs) have been observed under
numerous pathologic conditions. Therefore inhibition of
MMPs is considered as a potential therapeutic target. After
cerebral ischemia it has been shown that there is a marked
increase of MMP-9 expression which has been implicated
in breakdown of Blood brain barrier (BBB). Tissue Inhibitor of Matrix Metalloprotinase-1 (TIMP-1) is a 28 KDa
endogenous inhibitor of MMP-9.
Objectives: In this study we plan to deliver TIMP-1 using
PLGA Nanoparticles across BBB and investigate whether
this can be an effective strategy to prevent BBB disruption
after ischemia and ameliorate ischemic cell death.
Methods: In order to get TIMP-1, the protein was cloned
in pCMV plasmid with a Histag at C terminal. The plasmid
was expressed in HEK 293T Cell lines and isolated using
Cobalt based affinity column followed by dialysis. TIMP-1
loaded PLGA nanoparticles were synthesized by multiple
emulsion and solvent evaporation method and coated with
Tween 80 for BBB delivery.
Results: TIMP-1 was characterized using western blot, reverse zymography, gelatinase assay and protein sequencing.
PLGA NPs were analysed by using SEM, DLS, PDI, Zeta
potential, protein loading and drug release. The mean size
of NPs as measured using SEM was found to be 90.2 ± 5
nm. The mean hydrodynamic diameter of NPs as measured by DLS was 330 nm. The zeta potential of TIMP-1NPs was 7.78 ± 2.5 mV. The encapsulation efficiency of
TIMP-1 in NPs was 71 ± 5.1% (n=3); i.e., 71% of the added protein was entrapped into NPs. The release of TIMP-1
from NPs was sustained, with 17.1 ± 1.4% of release in
24 h, 65.1 ± 1.1% in 1 wk. At present the formulation is
being tested for BBB penetration, MMP-9 inhibition and
neuroprotection in vivo. Parnas Conference Warsaw 2011
P4.4
P4.5
Cyclosporine A-induced endoplasmic
reticulum stress triggers autophagy
of malignant glioma cells
A novel approach of gene delivery
in cortex and hippocampus of
mouse brain to study mmp-9 gene
regulation in depolarized neurons
Iwona Ciechomska, Bozena Kaminska
Nencki Institute of Experimental Biology, Department of Cell
e-mail: Iwona Ciechomska <[email protected]>
Autophagy is a self-digestion process allowing cell survival
during starvation but functions also as an alternative death
mechanism under certain conditions. Autophagy is accompanied by the progressive formation of vesicle structures
from autophagosomes to autophagolysosomes, and involves both autophagy effectors (Atg proteins) and regulators (i.e. mTOR - mammalian target of rapamycin is a
negative regulator). Malignant gliomas are highly resistant
to available therapies which induce apoptosis, thus induction of the alternative cell death is an attractive strategy. We
demonstrate that cyclosporine A (CsA, an immunophilins/
calcineurin inhibitor) induces cell death with some apoptotic features but also accompanied by the appearance of
numerous cytoplasmic vacuoles, immunostained for endoplasmic reticulum (ER) stress and autophagy markers.
The induction of ER stress in glioma cells by CsA was
evidenced by detection of unfolded protein response activation (phosphorylation of PERK) and accumulation of
ER stress associated proteins (BIP and CHOP). Upon CsA
treatment several autophagy features were observed: formation of the acidic vesicular organelles, increase in punctuate
GFP-LC3 (microtubule-associated protein light-chain 3)
and LC3-II accumulation. Decrease of phosphorylation
4E-BP1, p70-S6K1, and its downstream target molecule S6
ribosomal protein suggests that CsA affects mTOR signaling. Salubrinal, which protect cells from ER stress, partially
blocked CsA-induced decrease of p70-S6K1 and 4E-BP1
phosphorylation, and accumulation of LC3-II. It suggests
that ER stress was primary to CsA-induced autophagy.
Surprisingly, selective silencing of Atg1, Atg5 or Atg7 increased the level of active caspases 3, 7 and PARP degradation in CsA-treated cells. Our results demonstrate that CsA
induces both apoptosis and autophagy in malignant glioma
cells via induction of ER stress and inhibition of mTOR/
p70-S6K1 pathway, however autophagy is cytoprotective in
this context.
Acknowledgements
Studies supported by grant N N301 092036 from The Polish Ministry of
Science and Higher Education.
29
Krishnendu Ganguly, Emilia Rejmak,
Michał Stawarski, Leszek Kaczmarek
Nencki Institute of Experimental Biology, Department of Cellular and
Molecular Neurobiology, Warsaw, Poland
e-mail: Krishnendu Ganguly <[email protected]>
Understanding of the molecular underpinnings of gene
regulation during depolarization-driven synaptic plasticity is a major challenge of modern neuroscience. MMP-9
(matrix metalloproteinase), is a crucial enzyme that helps in
transient alteration of neuronal circuits by affecting extracellular matrix (ECM) components. The molecular mechanism behind mmp-9 gene transcription during depolarization of brain neurons in vivo is poorly understood. Notably,
presently available in vitro reporter assay-based systems for
gene regulation do not simulate adequately the gene regulation that occurs in the brain. Therefore, this study was
directed to develop a strategy to study the transcriptional
regulation of mmp-9 gene in mouse brain neurons in response to pentylenetetrazole (PTZ)-induced seizure. The
transcriptional response of mmp-9 gene was determined by
real time based quantitative PCR and in situ hybridization.
Furthermore, wild type and mutated versions of mmp-9
reporter gene in both plasmid and BAC (Bacterial Artificial Chromosome) vectors were introduced into the brain
by means of electroporation. Both the reporter gene transcripts and proteins levels were increased in PTZ-induced
brain neurons. In conclusion, this is the first effort by
which we studied the in vivo transcriptional regulation of
mmp-9 gene by reporter based assay system, during synaptic plasticity driven by depolarization of mammalian brain
neurons. Abstracts
30
P4.6
P4.7
The effects of antiepileptic agents
on fusion of synaptic vesicles in cellfree model of exocytosis and the
role of cholesterol in this process Changes in homocysteine
thiolactone disposition
and neurotoxicityin bleomycin hydrolase
(Blmh) and paraoxonase 1
(Pon1) knock-out mice
Vitaliy Gumenyuk1, Tamara Kuchmerovska2, Irene Trikash1 Palladin Institute of Biochemistry of National Academy of Sciences
of Ukraine, 1Department of Neurochemistry and 2Department of
Biochemistry of Vitamines and Coenzymes, Ukraine
e-mail: Vitaliy Gumenyuk <[email protected]>
The neurochemical mechanisms underlying seizure-associated dysfunctions of central nervous system are multiple
and poorly understood. The antiepileptic drugs which directly or non-directly attenuate brain dysfunction can use
to restore the balance between the excitation and inhibition
of neurons. To elucidate the effects of ethosuximide, sodium valproate, and gabapentin on the exocytosis process
the calcium-dependent fusion of synaptic vesicles (SVs) in
cell-free model system was investigated. All studies were
carried out on SVs isolated from rat brains. Membrane fusion was assayed with the fluorescence probe R18. To estimate the role of membrane cholesterol in synaptic vesicle
fusion the cholesterol content in membrane was modulated by methyl-β-cyclodextrin used as a cholesterol binding
agent. It has been found that all studied antiepiepileptic
agents increase the Ca2+- and Mg2+/ATP-dependent SVs
fusion. The 20 % reduction of membrane cholesterol leads
to decrease of the rate of calcium-triggered fusion of homotypic membranes of SVs that indicates the disturbances
in function of proteins driving this process. At the same
time, the reduction of cholesterol level in SVs membrane
did not change the ability of antiepileptic drugs to enforce
the fusion process. These findings suggest that antiepileptic
drugs are capable to bind to SVs proteins which are not
located in cholesterol rich lipid microdomains. Thus, it was
shown that SVs proteins can be the functional targets for
the action of ethosuximide, sodium valproate, and gabapentin in regulation of exocytosis in the nerve terminals.
This may be the basis for further studies of new antiepileptic drugs action at the level of SVs proteins and SVs
fusogenic activity.
Kamila Borowczyk1, Joanna Tisonczyk1,
Hieronim Jakubowski1,2
1UMDNJ-New Jersey Medical School, Department of Microbiology and
Molecular Genetics, Newark, NJ, USA; 2Institute of Bioorganic
Chemistry, Polish Academy of Sciences, Poznan, Poland
e-mail: Hieronim Jakubowski <[email protected]>
Genetic or nutritional disorders in methionine metabolism
increase homocysteine (Hcy) editing by methionyl-tRNA
synthetase, which generates Hcy-thiolactone and is linked
to cardiovascular and neurological diseases. Hcy-thiolactone has the ability to form isopeptide bonds with protein
lysine residues, which generates toxic N-Hcy-proteins with
autoimmunogenic, prothrombotic, and amyloidogenic
properties. Bleomycin hydrolase (Blmh) and paraoxonase 1
(Pon1) have the ability to hydrolyze Hcy-thiolactone in vitro.
To determine physiological roles of Blmh and Pon1, we
studied Hcy-thiolactone disposition and neurotoxicity in
mice with inactivated Blmh and Pon1 genes. We found that
endogenous Hcy-thiolactone and N-Hcy-protein levels
were elevated in Blmh-/- and Pon1-/- mice compared with
wild type animals. Exogenous Hcy-thiolactone, injected
intraperitoneally, was hydrolyzed less effectively by Blmh/- than by Pon1-/- and wild type mice. We also found that
Blmh-/- and Pon1-/- mice were more sensitive to Hcythiolactone neurotoxicity than wild type animals. Taken
together, these findings indicate that Blmh and Pon1, by
hydrolyzing Hcy-thiolactone and minimizing protein Nhomocysteinylation, protect against Hcy-thiolactone neurotoxicity in mice.
P4.8
P4.9
Co-regulation of expression of
NFκB-dependent genes by the
HSF1 transcription factor
Identification of signal transduction
pathways re-programming
macrophages into tumor supportive
cells — on the missing link between
inflammation and cancer
Patryk Janus1, Magdalena Kalinowska-Herok1,
Małgorzata Pakuła-Cis2, Wojciech Pigłowski1,
Katarzyna Szołtysek1, Adam Makuchowski3,
Marek Kimmel3, Piotr Widłak1
1Maria Sklodowska-Curie Memorial Cancer Center and Institute of
Oncology, Gliwice, Poland; 2University of Aarhus, Aarhus, Denmark;
3Silesian University of Technology, Gliwice, Poland
e-mail: Patryk Janus <[email protected]>
NFκB- and HSF1-dependent pathways are essential components of cellular responses to stress. They play the major
role in pathogenesis of serious human diseases, including
cancer and response to therapeutic treatments. Both of
these transcription factors regulate several genes involved
in cell proliferation, apoptosis, immune and inflammatory
responses. Here we aimed to identify NFκB-dependent
genes which expression is affected by the active HSF1.
Activation of the NFκB pathway and expression of NFκBdependent genes was analyzed in U2-OS human osteosarcoma cells stimulated with TNFα cytokine. Cells were either
preconditioned with hyperthermia to activate endogenous
HSF1 or engineered to express a constitutively active form
of HSF1 in the absence of heat shock. The expression of
NFκB-dependent genes was analyzed by quantitative RTPCR, using both NFκB-pathway-oriented PCR-Array and
gene-specific reactions. Binding of HSF1 to promoters of
NFκB-dependent genes was analyzed by chromatin immunoprecipitation assay (ChIP) with anti-HSF1 Ab (genes
with hypothetical sites of HSF1 binding were pre-selected
based bioinformatics analysis).
We found that hyperthermia resulted in a general blockade of activation of the NFκB signaling and expression of
NFκB-dependent genes. In marked contrast, the presence
of constitutively active HSF1 did not block TNFα-induced
activation of the NFκB pathway and general expression
of the NFκB-dependent genes in the absence of the heat
shock. However, the presence of HSF1 affected expression
of several specific NFκB-dependent genes activated by
TNFα. Four of these genes, namely TNFA, IL-6, FASLG,
AGT, contained functional binding sites for HSF1 in their
promoter regions.
We concluded that expression of several NFκB-dependent
genes is modulated by HSF1-dependent mechanisms.
Some of these genes could be co-regulated by HSF1 in the
direct mechanism due to the binding of HSF1 transcription factor to their promoter regions.
Acknowledgements
This work was supported by the Ministry of Science and Higher Education, grants N514 411936, PBZ-MNiI-2/1/2005 and by the European
Community from the European Social Fund within the RFSD-2 project.
31
Bozena Kaminska, Konrad R. Gabrusiewicz,
Pawel Wisniewski, Aleksandra Ellert-Miklaszewska,
Maciej Lipko, Michal Dabrowski,
Magdalena Kijewska, Beata Kaza, Malgorzata Porycka
Nencki Institute of Experimental Biology, Laboratory of Transcription
Regulation, Warsaw, Poland
e-mail: Bozena Kaminska <[email protected]>
Malignant tumors benefit from support from the surrounding tumor microenvironment composed of tumorassociated fibroblasts, leukocytes, bone marrow-derived
cells, blood and lymphatic vascular endothelial cells. The
microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells, rare cells
which contribute to tumor propagation and the immunosuppressive tumor milieu. Tumor-infiltrating macrophages
and immunomodulatory mediators present in the tumor
microenvironment polarize host immune response toward
specific phenotypes impacting tumor progression. Aggressive human brain tumors (glioblastomas) contain numerous glioma-infiltrating macrophages, which abundance correlates with malignancy and poor patient prognosis. Using
immunofluorescence and flow cytometry, we demonstrated
an early accumulation of activated microglia followed by
accumulation of blood derived macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the
alternative phenotype, as evidenced by evaluation of the
production of ten pro/anti-inflammatory cytokines and
expression profiling of 28 genes in magnetically-sorted
CD11b+ cells from tumor tissues. In vitro studies revealed
that glioma cells secrete soluble factors which convert
primary microglial cultures into amoeboid cells supporting glioma invasiveness, while attenuating inflammatory
responses. Global gene expression profiling of microglial
cultures, stimulated either by glioma-conditioned medium
or by a classical inflammation inducer — lipopolysaccharide (LPS), revealed different patterns of gene induction,
and activation of distinct signaling amd metabolic pathways. Proteomic analysis of glioma-conditioned medium
using HPLC fractionation followed by a tandem massspectrometry identified two activating proteins which are
small integrin-binding ligand N-linked glycoprotein family
members. Interference with ligand binding to integrins using a blocking RDG peptide or gene silencing in glioma
cells, abolished microglial activation induced by gliomaconditioned medium and their influence on glioma invasion. These data provide strong evidence on the identity of
signals which direct tumor-related inflammation and tumor
progression.
Acknowledgements
Studies supported by the Ministry of Science and Higher Education grant
N N301 7862 (BK).
Abstracts
32
P4.10
P4.11
A role of osteopontin — a small integrinbinding ligand in glioma pathology
M. Kijewska, M. Sielska, A. Ellert-Miklaszewska,
K. Gabrusiewicz, B. Kaminska
Expression of HIF mediates
endogenous neuroprotection of
hippocampal neurons during the
ischemic and anoxic precondition
Laboratory of Transcription Regulation, Nencki Institute of Experimental
Anastasiia M. Maistrenko, Irina V. Lushnikova, Maxim
M. Orlovsky, Victor E. Dosenko, Galina G. Skibo
e-mail: Magdalena Kijewska <[email protected]>
Tumor-associated macrophages represent the major component of the stroma of many tumors, including gliomas,
and their high content correlates with malignancy and poor
patient prognosis. We have demonstrated that glioma cells
release soluble factors which induce accumulation and a
non-inflammatory activation of brain macrophages associated with pro-invasive function of these cells. Proteomic
analysis of glioma-conditioned medium (G-CM) revealed
that one of these factors is Osteopontin (OPN, known as
secreted phosphoprotein 1 — SPP1), a metastasis-associated small integrin-binding ligand and a N-linked glycoprotein family member. OPN was highly overexpressed in rat
and some human glioma cells when compare to non-transformed astrocytes. To evaluate the role of OPN in glioma
biology, we constructed plasmids encoding shRNA against
OPN (shOPN) or negative control shRNA (shNeg). We
obtained C6 glioma cells stable transfected with indicated
plasmids. The silencing of OPN in shOPN clones was
confirmed on RNA and protein level. Silencing of OPN
had no impact on cell proliferation and survival. To evaluate the role of OPN in glioma invasiveness we performed
Matrigel invasiveness assay. We demonstrate that silencing
of OPN diminished invasiveness of glioma cells and abolished the pro-invasive effects of microglia co-cultured with
glioma cells. To follow the role of OPN in glioma pathology in vivo, we injected shOPN and shNeg C6 cell clones into
the striatum of Wistar rats. Tumor volumes and microglia
and lymphocytes infiltration into the tumor were evaluated. Our studies indicate that tumor-derived OPN may
be a crucial factor mediating interactions between glioma
and tumor-associated brain macrophages and is involved in
glioma pathogenesis.
Acknowledgements
The study was supported by grant GP2908 from the Ministry of Science
and Higher Education.
Bogomoletz Institute of Physiology, Department of Cytology, Ukraine
e-mail: Anastasiia Maistrenko <[email protected]>
Brief anoxia or episodic hypoxic preconditioning can enhance a brain resistance to subsequent prolong ischemia.
However, a little is known about events occurring in the
time frame between anoxic preconditioning (APC) and
subsequent formation of ischemic resistance. Existing
reports in this area are predominantly focused on APC
morphological features, in particular, the inhibition of
neuronal apoptosis, neurogenesis or survival of the neurons. Although it has been shown that hypoxia may induce
extensive injury to susceptible brain neurons, it does not
necessarily follow that such injury results in neuronal death,
but rather a marked impairment of brain functioning, such
as behaviours, stress response, learning and memory. Thus
while APC could protect brain from hypoxic/ischemic injury, the mechanisms of this action require further investigations.
Recent investigations indicate that mechanisms of such
stimulated endogenous neuroprotection are related to the
family of hypoxia-inducible factors (HIF), however there
are still little data concerning the role of HIF family members in hippocampus — a brain structure, highly sensitive
to oxygen deficiency. We have used the model of cultured
hippocampal slices and single-cell quantitative RT-PCR to
study HIF-1α and HIF-3α mRNA expression following triple 5-min mild anoxia, 30-min oxygen-glucose deprivation
and their combination. The viability was evaluate with PIstaining. We also tested the effects of HIF prolyl-hydroxylase inhibition with 2,4-pyridinedicarboxylic acid diethyl
ester pre-treatment followed by a 30-min oxygen-glucose
deprivation. It was found that neuronal damage induced
by oxygen-glucose deprivation was accompanied by a significant decrease in both HIF-1α and HIF-3α mRNA levels
in CA1 but not CA3 neurons. Anoxia preconditioning did
not affect cell viability and HIF mRNA levels but applied
before oxygen-glucose deprivation prevented neuronal
damage and suppression of HIF-1α and HIF-3α mRNA
expression. It was also found that effects of the prolyl-hydroxylase inhibitor were similar to anoxia preconditioning.
These results suggest that anoxia preconditioning increases
anti-ischemic neuronal resistance which to a certain extent
correlates with the changes of HIF-1α and HIF-3α expression.
P4.12
P4.13
β-catenin is constitutively present in the
nuclei of postmitotic thalamic neurons
due to WNT-independent mechanism
Loss of snap29 impairs endocytic
recycling and cell motility
Katarzyna Misztal1, Marta B. Wisniewska1,
Mateusz Ambrozkiewicz1, Jacek Kuznicki1,2
2Nencki Institute of Experimental Biology, Warsaw, Poland
Poland;
e-mail: Katarzyna Misztal <[email protected]>
Wnt activation promotes β-catenin accumulation upon
inhibition of β-catenin degradation. Stabilized β-catenin
translocates to the nucleus where it triggers transcription
of the Lef1/Tcf target genes. Wnt/β-catenin signaling is
essential for nervous system development as well as division and maturation of neuronal progenitors in adult brain.
We showed recently that nuclear β-catenin is abundant in
vivo in non-dividing neurons of adult thalamus, where
it is involved in gene transcription of CACNA1G gene
(Wisniewska et al., 2010, J Neurosci). Here we demonstrate
spontaneous accumulation of β-catenin in 40% of cultured
thalamic neurons and lack of such accumulation in cortical
neurons. This phenomenon does not depend on soluble
factors produced by glia or cortical neurons, since neither
conditioned medium of cortical cells nor glial cells coculture affect the number of β-catenin positive cells. This
suggests that nuclear localization of β-catenin in thalamic
neurons is not a consequence of paracrine stimulation.
We also observed that Wnt receptor inhibitor DKK1 and
dnDVL3 had no major effect on the number of β-catenin
positive thalamic neurons. Thus, autocrine WNT stimulation is not responsible for nuclear β-catenin accumulation
in these neurons. We analyzed expression of APC, AXIN1
and GSK3β that are involved in degradation of β-catenin
and detected lower level of APC and GSK3β in thalamus
when compared to cortex and hippocampus. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulsechase experiments confirmed that cytoplasmic β-catenin
turnover was slower in thalamic neurons than in cortical
neurons. Our observations support an idea that β-catenin
accumulation is an intrinsic feature of thalamic neurons,
independent of cellular environment of thalamic neurons
and Wnt stimulation, but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and
subsequent degradation.
Acknowledgements
This work is supported by ”Health-Prot” Grant no 229676 and Polish
MNiSW Grant no 4245/B/P01/2010/38 and 1917/B/P01/2010/39.
33
Debora Rapaport1, Yevgenia Lugassy4,
Eli Sprecher2,3,4, Mia Horowitz1
Tel Aviv University, 1Department of Cell Research and Immunology,
2Sackler Faculty of Medicine, 3Tel Aviv Sourasky Medical Center,
Department of Dermatology, 4Technion, Center for Translational
Genetics, Israel
e-mail: Debora Rapaport <[email protected]>
Intracellular membrane trafficking depends on the ordered
formation and consumption of transport intermediates
and requires that membranes fuse with each other in a
tightly regulated and highly specific manner. Intracellular
trafficking of membrane receptors dictates the kinetics
and magnitude of signal transduction cascades. Membrane
anchored SNAREs assemble into SNARE complexes that
bring membranes together to promote fusion. SNAP29
is a ubiquitous synaptosomal-associated SNARE protein
localized in intracellular membranes. Loss of functional
SNAP29 results in CEDNIK syndrome (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma) causing
abnormalities in epidermal differentiation and in corpuscallosum accompanied by cortical dysplasia. Using fibroblast cell lines derived from CEDNIK patients, we show
that SNAP29 mediates endocytic recycling of transferrin
and β1-integrin. Impaired β1-integrin recycling affected
cell motility, as reflected by changes in cell spreading and
wound healing. Our results emphasize the importance of
SNAP29 mediated membrane fusion in endocytic recycling
and in cell motility.
Abstracts
34
P4.14
P4.15
Identification of a novel CD150 isoform
in tumors of central nervous system
CD150 — a new marker for tumors
of the central nervous system
Olga Romanets1, 2, Mariya Yurchenko1, Tatyana Malysheva3,
Aleksander Taranin4, Branka Horvat2, Svetlana P. Sidorenko1
Svetlana P. Sidorenko1, Olga Romanets1,2, Mariya Yurchenko1,
Yuriy A. Zozulya3, Tatyana A. Malysheva3,
Alexander M. Najakshin4,
Alexander V. Taranin4, Branka Horvat2
1R. E. Kavetsky Institute of Experimental Pathology Oncology and
Radiobiology of NAS of Ukraine, Department of Cell regulation, Kyiv,
Ukraine; 2INSERM U758-ENS de Lyon, Lyon, France; 3Romodanov
Institute of Neurosurgery of AMS of Ukraine, Kyiv, Ukraine; 4 Institute of
Molecular and Cellular Biology, Novosibirsk, Russia e-mail: Olga Romanets <[email protected]>
CD150 receptor (IPO-3/SLAM) is a member of CD2 family within the immunoglobulin superfamily. It is expressed
on thymocytes, activated T and B lymphocytes, dendritic
cells and monocytes. However, it is still little known about
CD150 expression in non-lymphoid cells, especially in tumors. The aim of our work was to study the expression
of CD150 in human glioma cell lines and in primary tumors of central nervous system (CNS). Using reverse
transcriptase reaction, followed by polymerase chain reaction (RT-PCR) with several pairs of specific primers to
the CD150 extracellular, transmembrane and cytoplasmic
domains, we showed differential expression of the gene
among human glioma cell lines. The A172 cell line highly
expressed mRNA for full transmembrane form of CD150,
but the protein production was not detected in these cells.
We found a low level of CD150 mRNA expression in
the U87, U343, NCH89 and NCH92 cell lines. Expression of CD150 at the protein level was observed in fixed
U87, U343, NCH89, NCH92 and TE671 cell lines using
immunofluorescent staining. However, FACS analysis did
not reveal CD150 on the surface of live cells. All studied
glioma cell lines were not sensitive to the infection with
wild type measles virus (which uses CD150 as entry receptor) in contrast to laboratory strain of measles virus (which
uses CD46 as receptor as well). These results showed that
CD150 is absent on the surface of the human glioma cells.
Sequencing of the RT-PCR fragments from the U87 cell
line revealed an unusual CD150 transcript containing an
83 bp insert between exons for the transmembrane and
cytoplasmic regions of the full-length mRNA. Bioinformatics analysis showed that the insert was derived from a
previously unrecognized exon located 511 bp downstream
of the exon for the transmembrane region. The exon designated Cyt-new is flanked with canonical AG/GT splice
sites. The use of the Cyt-new exon results in the reading
frame shift in the exons Cyt1-Cyt3 and production of an
isoform with cytoplasmic tail lacking any known signaling
motifs. Specific primers for this novel CD150 isoform were
designed and used for study its expression in cell lines of
different origin, primary human CNS tumors and human
normal brain. Taken together, we found a novel CD150
isoform that is expressed in malignant cells of central nervous system tumors.
1Kavetsky Institute of Experimental Pathology Oncology and
Radiobiology of NAS of Ukraine, Department of cell regulation, Kyiv,
Ukraine; 2INSERM U758-ENS de Lyon, Lyon, France; 3Romodanov
Institute of Neurosurgery of AMS of Ukraine, Kyiv, Ukraine; 4Institute of
Molecular and Cellular Biology, Novosibirsk, Russia
e-mail: Svetlana Sidorenko <[email protected]>
The big progress was made in characterization of tumorassociated markers in central nervous system (CNS), which
helps to identify cell of origin and the level of tumor cell
differentiation. The efforts are focused now on new marker
disclosure for routine immunohistochemical diagnostics,
evaluation of invasiveness and tumor cell proliferation rate,
prediction of treatment outcome and possible targets for
therapy. CD150, encoded by the SLAMF1 gene, is a dualfunction receptor, expressed in hematopoietic system and
involved in immune regulation. Four isoforms of CD150
were identified: trans-membrane (mCD150), secreted that
lacks transmembrane region, cytoplasmic, lacking the leader sequence and a variant membrane CD150 with truncated
cytoplasmic tail. CD150 is one of two identified receptors
for measles virus (MV) and binds hemagglutinin of both
wild-type (wt) and vaccine MV strains. MV holds promise
as a novel oncolytic agent in the treatment of tumors of
CNS, but expression of wild type measles virus receptor
CD150 was not reported on cells of the nervous system
yet. Our immunohistochemical studies of primary human
CNS tumors (108 cases) revealed CD150 expression in
diffuse astrocytoma (66.7% of analyzed cases), anaplastic
astrocytoma (81.3%), glioblastoma (88.9%), ependymoma
(66.7%) and anaplastic ependymoma (75.0%). Expression
of CD150 in different regions of human normal brain was
not observed. Immunostaining of all samples with the antibodies to GFAP, nestin and CD68 validated the CD150
expression in the malignant cells. The majority of tested
glioblastoma cell lines also expressed CD150, however we
did not detect its expression on the cell surface and cells
were resistant to MVwt infecton. To find which splice
isoforms of CD150 were expressed by glioblastoma cells
we performed RT-PCR and 3’RACE of CD150 mRNA,
from the cell line U87. A variety of aberrant and normal
mCD150 transcripts were found of which one contained
a previously unrecognized 83 bp exon downstream of
the exon for the transmembrane region. The exon insertion caused the reading frame shift and translation of a
novel isoform with alternative cytoplasmic tail. Thus, we
show that CD150 is frequently expressed in CNS tumors.
Further studies will clarify the biological significance of
CD150 isoforms expression in CNS tumors and validate
the potential use of this marker in differential diagnostics
and targeted therapy.
35
P4.16
P4.17
Crosstalk between NFκB- and p53dependent signaling pathways in
HCT116 colon carcinoma cells
Expression of pro-epileptic protease Matrix
Metalloproteinases 9 is regulated by DNA
methylation during epileptogenesis
Katarzyna Szołtysek1, Patryk Janus1,
Adam Makuchowski2, Marek Kimmel2, Piotr Widłak1
Katarzyna Zybura-Broda1, Renata Amborska1,
Leszek Kaczmarek1, Marcin Rylski2
1Maria Sklodowska-Curie Memorial Cancer Center and Institute of
Oncology, Gliwice, Poland; 2Silesian University of Technology; Gliwice,
Poland
1Nencki Institute of Experimental Biology, Department of Molecular
and Cellular Neurobiology, Warsaw, Poland; 2The Medical Centre of
Postgraduate Education, Department of Clinical Cytology, Warsaw,
Poland
e-mail: Katarzyna Szołtysek <[email protected]>
e-mail: Katarzyna Żybura-Broda <[email protected]>
Signaling pathways that depend on NFκB and p53 transcription factors are essential elements of cellular responses to stress. Both factors participate in regulation of a network of genes involved in control of the cell cycle, DNA
repair, apoptosis, immune response and inflammation.
Here we aimed to analyze the interference between these
signaling pathways at the level of expression of selected
NFκB- and p53-dependent genes.
Experiments were performed using human colon carcinoma HCT116 cells in two congenic lines either containing or lacking transcriptionally competent p53. Cells were
incubated with TNFα cytokine to activate NFκB, and/or
exposed to ultraviolet/ionizing radiation to activate p53
pathway; both factors were used in two different time combinations: stimulation with TNFα was placed either 3 hours
before or 6 hours after irradiation. Activation of the NFκB
and p53 pathways was monitored by Western-blot detection of selected proteins (IκBα, p53, p21, MDM2, PTEN,
PARP, AKT). Expression of selected p53-dependent
genes (MDM2, p21/WAF1, PTEN, NOXA) and NFκBdependent genes (BCL3, NFKBIA, NFKB1, REL, IL1A,
IL8, TNFA, TNFAIP3, JUN, LTA) was assessed by quantitative QRT-PCR; p53-dependeant genes were analyzed 6,
12 and 24 hours after irradiation while NFκB-dependent
genes were analyzed 1 and 6 hours after stimulation with
TNFα.
We observed that radiation-induced activation of p53dependent genes was affected in cells with activated
NFκB: UV-induced expression of p53-dependent genes
was further up-regulated by either mode of stimulation
with TNFα. TNFα-stimulated activation of expression of
NFκB dependent genes was different in cells with different status of p53. Irradiation of p53-competent cells before stimulation with TNFα resulted in down-regulation of
analyzed NFκB-dependent genes. Our data revealed interference between regulation of p53-dependent genes and
activation of NFκB signaling and between regulation of
NFκB-dependent genes and activation of p53 signaling.
Acknowledgements
This work was supported by the Ministry of Science and Higher Education, Grant NN 301 264536.
Matrix Metalloproteinase 9 (MMP-9) acts pro-epileptically,
stimulating aberrant synaptic plasticity occurring during
epileptogenesis. Here, we have studied epigenetic changes
occurring on Mmp-9 proximal promoter during pentylenetetrazole-induced epileptogenesis in the rat hippocampus.
Rats received multiple intraperitioneal injections of pentylenetetrazole in non-seizure inducing doses, what led to a
precipitation of seizures and to a gradual progression in
their strength and duration. We have shown that MMP-9
activity and expression of MMP-9 mRNA are gradually
upregulated during PTZ-dependent epileptogenesis in the
hippocampus. Moreover, using Methylated DNA Immunoprecipitation (MeDIP) and Methylation Specific PCR
(MSP) we have demonstrated that the induction of MMP-9
gene expression is associated with a progressive demethylation of its proximal promoter. It’s probably performed by
DNA demethylase Gadd45β, which increasingly binds to
the proximal Mmp-9 promoter in vivo during PTZ-induced
kindling. Accordingly, our results imply that important epigenetic mechanism — active DNA demethylation, play an
essential role in a regulation of MMP-9 expression during
development of epilepsy.
Session 5: Cell guidance
Lectures
L5.2
L5.1
Mechanisms of axonal guidance and
degeneration during development
Robust information processing
in bacterial chemotaxis
Victor Sourjik
ZMBH, University of Heidelberg, Heidelberg, Germany
e-mail: Victor Sourjik <[email protected]>
Bacterial chemotaxis has been extensively used as a relatively simple model system of environmental signal processing,
to gain general insights into evolutionary optimization of
biological networks. One of the central questions in quantitative analyses of cell signalling is how optimal function of
the network is achieved under conditions of constant perturbations that are experienced by any biological system.
In our analysis of chemotaxis as a model system, we could
show that most of the pathway features can be indeed explained by the evolutionary selection for high response sensitivity and robust signal processing under conditions of
such intra- and extracellular perturbations as stochastic ligand binding, stochastic variation in the levels of signalling
proteins and variations in temperature. Even in this simple
system, perturbations are almost perfectly compensated by
the robust pathway topology and by the specific regulation
of chemotaxis genes, to ensure extremely efficient and robust sensing of chemoeffector gradients over large span of
concentrations.
Erez Romi, Zohar Schoenmann, Adi Minis,
Liat Haklai-Topper, Irena Gokhman, Avraham Yaron
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot, Israel
e-mail: Avraham Yaron <[email protected]>
During development, the basic wiring of the nervous system is established by connecting trillions of neurons to
their target cells. To reach their correct targets, neurons
extend axons that are guided by cues in the extracellular
environment.
The talk I will describe our studies on two aspects of the
somatosensory system development: the mechanisms of
axonal guidance and axonal elimination.
In the first part I will focus on developmental control of
the Semaphorins receptor complex through ecto domain
cleavage by a protease of the ADAM family.
In the second part on I will concentrate on the intra cellular
pathways the mediate axonal destruction.
L5.3
Oral presentations
A cellular module for singlemolecule sensitivity in sperm
O5.1
U. Benjamin Kaupp
Center of Advanced European Studies and Research (caesar),
Molecular Sensory Systems, Germany
e-mail: U. Benjamin Kaupp <[email protected]>
Sperm of the sea urchin Arbacia punctulata respond to a
single molecule of chemoattractant released by eggs. The
mechanism underlying this extreme sensitivity is unknown.
Crucial signaling events include the synthesis of the intracellular messenger guanosine 3′,5′-monophosphate (cGMP),
the ensuing membrane hyperpolarization due to the opening of K+-selective cyclic nucleotide-gated (CNGK) channels, and Ca2+ entry through unknown Cav channels.
Calibrated photolysis of caged cGMP shows that ≤45
cGMP molecules are required for a single-molecule response. This corresponds to a change in the total cGMP
concentration of 45 nM in the flagellum. The changes in
free cGMP concentration might be much smaller due to
buffering by cGMP-binding sites on phosphodiesterase.
The CNGK channel can respond to such small cGMP
changes because it is exquisitely sensitive to cGMP (K1/2=
25 nM) and activated in a non-cooperative fashion.
The structure of the CNGK channel differs from that of
classical CNG channels of photoreceptors and olfactory
neurons. These channels form heterotetramers composed
of different subunits. In contrast, like voltage-activated
Cav and Nav channels, the CNGK polypeptide consists of
four homologous repeat sequences. Each repeat harbors
all the functional motifs of a classical CNG channel subunit. Disabling each of the four cyclic nucleotide-binding
sites through mutagenesis revealed that binding of a single
cGMP molecule to repeat 3 was sufficient to activate the
CNGK channel. Thus, CNGK has developed an activation
mechanism that is different from that of classical CNG
channels, which requires the cooperative binding of several
ligands and operates in the micromolar rather than the nanomolar range. At nanomolar concentrations and in small
compartments such as cilia, when only few molecules are
available, the simultaneous binding of several ligand molecules to the same channel is a rare event; thus cooperative
activation would impair rather than enhance channel sensitivity. This study provides a framework that might be also
relevant for pheromone signaling in vertebrates and insects.
37
Are energy complexes associated with the
switch-motor complex of bacterial flagella?
Gabriel Zarbiv1, Hui Li2, Amnon Wolf1, Gary Cecchini3,
S. Roy Caplan1, Victor Sourjik2, Michael Eisenbach1
1The Weizmann Institute of Science, Department of Biological
Chemistry, Israel; 2Universität Heidelberg, Zentrum für Molekulare
Biologie, Germany; 3University of California, Molecular Biology
Division, Veterans Administration Medical Center, and Department of
Biochemistry and Biophysics, California, USA
e-mail: Gabriel Zarbiv <[email protected]>
The switch-motor complex at the base of bacterial flagella
powers flagellar rotation and acts as a gearbox that switches the direction of the rotation. Recently we found that
the electron-transport protein, fumarate reductase (FRD),
forms a 1:1 complex with the switch protein FliG and that
this interaction is essential for flagellar assembly and function [1]. The functional association of FRD with the switch
raised the question of whether additional energy-linked enzymes are associated with the switch-motor complex. We
addressed this question by first comparing the ATP synthase activity and respiratory activity between two membrane preparations isolated from Escherichia coli: a preparation of vesicles enriched with membrane areas adjacent
to the flagellar motor and a preparation deficient in such
areas. We found that ADP phosphorylation, ATP hydrolysis, and respiration from NADH to oxygen were all higher
near the motor. We, therefore, investigated whether the
enzyme FoF1 ATP synthase (responsible for the first two
activities) and NADH-ubiquinone oxidoreductase (NDHI,
involved in the third activity) interact with the switch-motor complex. The findings were positive for both enzymes.
For FoF1 ATP synthase, the major evidence involved in vivo
demonstration that the level of the β subunit of FoF1 ATP
synthase radically affects flagellar behavior, and that this
subunit binds to FliG at the switch both in vivo and in vitro.
For NADH-ubiquinone oxidoreductase, the evidence involved in vivo and in vitro demonstration of association of
the NuoCD subunit of NADH-ubiquinone oxidoreductase to each of the three switch proteins. The functional
interaction of so many proteins with FliG suggests the existence of energy complexes in bacteria, associated with the
flagella and required for their function. Further studies are
required to reveal the structure and function of these putative energy complexes and to address intriguing questions
that emerge from these findings.
Reference
1.Cohen-Ben-Lulu GN et al (2008) EMBO J 27: 1134-1144. Abstracts
38
O5.2
Posters
The molecular mechanism of lectininduced signal transduction in
leukocytes under diabetes mellitus P5.1
M. Zdioruk, I. Brodyak, N. Sybirna Hyperactivation is a component
of human sperm chemotaxis
Ivan Franko Lviv National University, Lviv, Ukraine
Leah Armon, Michael Eisenbach
e-mail: Mykola Zdioruk <[email protected]>
Changes in the morphofunctional state of leukocytes causing disturbances to their interaction with vassal endothelia
are regarded as an etiological precondition for the development of diabetic complications and chronic diseases,
deteriorating physical health of diabetics. Our previous research has shown that lectin-induced leukocyte aggregation
in healthy donors and type 1 diabetics decreases considerably after being treated with wortmannin, selective uncompetitive inhibitor of phosphatidylinositol-3’-kinase, which
participates in key signal ways in the cell.
It was also noted that wortmannin slows down chemotaxis,
which testifies to participation in chemokine-induced cell
migration. Furthermore, it was proved that PI-3’-kinase
and its lipidic products play an mportant role in the reorganization of the actin cytoskeleton and cell mobility.
The overall aim of our research was to reveal the dynamics
of translocation between the cytosolic leukocyte fractions
of p85α/PI-3’-kinase regulatory subunit when the cell aggregation was induced by means of lectins with different
carbohydrate-binding specificity affected by wortmannin.
Diabetes-associated hyperglycaemia leads to the transition
of leukocytes to the preactivated state, which, to a great
extent, is caused by PI-3’-kinase and translocation into
cytoskeletal sites, mediating integrin-dependent cell focal
adhesion.
The following lectins were lymphocyte aggregation modulators: MAA — maackia amurensis lectin, and WGA —
wheat germ lectin. It was found out that signal transduction caused by the linkage between sialospecific lectins and
carbohydrate determinants of leukocyte membrane glycoconjugates was mediated through phosphatidylinositol-3’kinase way.
The research revealed that the cell response under diabetes
mellitus, which was registered as the translocation of PI-3’kinase enzyme regulatory subunit caused by lectin induction on neutrophils and mononuclear of MAA, SNA and
WGA lectins, differed in intensity and duration which may
be connected with changes in the number or structure of
plasma glycoprotein receptors. The Weizmann Institute of Science, Department of Biological
Chemistry, Israel
e-mail: Leah Armon <[email protected]>
When mammalian spermatozoa become capacitated they
acquire, among other activities, chemotactic responsiveness and the ability to exhibit occasional events of hyperactivated motility — a vigorous motility type with large
amplitudes of head displacement. Although a number of
roles have been proposed for this type of motility, its function is still obscure. Here we provide evidence suggesting
that hyperactivation is part of the chemotactic response.
By analyzing tracks of spermatozoa swimming in a spatial
chemoattractant gradient we found that, in such a gradient, the level of hyperactivation is significantly lower in
a chemotactically responsive sperm population than in a
non-responsive population or in the no-gradient control.
In a temporal chemoattractant gradient produced by photorelease of progesterone, roughly one third of the cells
responded, the response being a delayed turn. Most of the
turns involved hyperactivation events or an even more intense response in the form of flagellar arrest. These results
suggest that the function of hyperactivation is to cause a
rather sharp turn during the chemotactic response of capacitated cells.
P5.2
P5.3
Auxin’s production ability of rhizobia
and phythopathogenic bacteria in vitro
The influence of Angiotensin II and
Relaxin 2 on the cell proliferation
in prostate cancer cell lines
Liudmyla Dankevich1, Natalia Leonova2, Igor Dragovoz3
1Zabolotny Institute of Microbiology and Virology of the NASU,
Department of Phytopatogenic Bacteria, Ukraine; 2Zabolotny Institute
of Microbiology and Virology of the NASU, Department of General and
Soil Microbiology, Ukraine; 3Zabolotny Institute of Microbiology and
Virology of the of the NASU, Department of Antibiotics, Ukraine
e-mail: Liudmyla Dankevych <[email protected]>
Hormones and hormone-like substances play not only role
of intracellular signals in plants, but also mediate interactions between macro- and microorganisms. Therefore, microorganisms associated with the plants are able to synthesize plant hormones themselves. Plant’s growth substances,
produced by phytopathogenic bacteria, determine the development of the initial stages of pathogenesis, leading to
plant’s infection. At the same time hormones play a regulative role in initiation of symbiotic relationships between
plants and rhizobia. In microbe-plant’s integration auxins
play an important role, as it’s mainly responsible for the cell
elongation, division and tissues differentiation. That is why,
the subject of our study was a qualitative and quantitative
analysis of the ability of some rhizobial and pathogenic
bacterial strains to produce auxins in vitro.
The objects of research were effective B. japonicum UKM
B-6018 B. japonicum UKM B-6035 and low effective B. japonicum 21110 strains of rhizobia. We also used typical representatives of pathovars: P. syringae pv. pisi 9177, P. savastanoi
pv. phaseolicola B-1123, P. savastanoi pv. glycinea 8571, “P. lupini” 8531, P. savastanoi pv. savastanoi 9174, P. syringae pv. syringae B-1027 of phythopathogenic bacteria. Detection of
exogenous auxins was performed by high resolution TLC.
All strains of rhizobia contain next exogenous auxins: indole-3-acetic acid and indole-3-carboxylic acid. It should
be noted that the synthesis of these auxins was higher in
effective strains to comparison with the low effective rhizobial strain. It was shown the ability of rhizobial strains to
produce abscisic acid (ABA). Level of ABA synthesis in
culture medium of effective rhizobia strains was higher to
comparison with low nodules forming strain. These results
corresponded with published, according to which the ABA
is a signaling molecule that controls the metabolic processes in the nodules of legumes. In comparison to rizobia strains all pathogenic bacteria strains produce a lot of
auxins: the indole-3-acetic acid and indole-3-carboxylic acid
complex, indole-3-carboxylaldehyde, hydrazine indole-3acetic acid. Perhaps, the synthesis of a so wide spectrum
of auxins by phytopathogenic bacteria associated with it’s
influence on plant cell growing by elongation and more
effective pathogen penetration in plant’s tissues. We also
detected the ability to synthesize ABA by phytopathogenic
bacteria strains. The highest level of ABA was founded in
classical polyphages — P. syringae pv. syringae B-1027, which
could induce disease more than 50 plant’s species.
39
Kamila Domińska, Agnieszka Wanda Piastowska,
Anna Gajos-Michniewicz, Tomasz Ochędalski
Department of Comparative Endocrinology, Medical University of Lodz,
Lodz, Poland
e-mail: Kamila Domińska <[email protected]>
It is common knowledge that sex steroid hormones regulate a wide range of prostatic metabolic functions. An increasing number of in vitro and in vivo studies have shown
that also local hormones, such as angiotensin and relaxin
may influence the proliferation, differentiation, migration
and metabolic activity of prostate cells. The both of peptides are present in human seminal plasma at concentration
higher than found in blood. The results of many study reported that same processes, for instance inflammation or
carcinogenesis involve changes in particular elements of
the renin-angiotensin system and relaxin system. Nevertheless, the role of Ang II and Rel-2 peptides in prostate
physiology and pathology is still not well known. We examined the relationship between angiotensin, relaxin and
prostate cancer induction and progression. The weakly
tumorigenic prostate cancer cells LNCaP and aggressively
tumorigenic cells PC3 we used as models of early stage
androgen-dependent prostate cancer and late androgenindependent stage disease, respectively. The cell viability
was measured using the easy and safe colorimetric method
based on the reduction of tetrazolium salts by viable cells
(WST-1). As an indicator of cell proliferation we using
BrdU Assay, based on the detection of bromodeoxyuridine
incorporation into DNA during the S-phase of cell cycle.
Finally, we compared the changes in expression levels of
AT1, AT2 and LGR7, LGR8 receptors, before and after
peptides treatment (Western Blot). Our dates clearly indicate that Ang II and Rel-2 can regulate cell viability and
proliferation in prostate cancer lines. However, the biological properties of this peptides are associated with the
hormonal status and invasion potential of examined cells.
Our study demonstrated as well that the influence of Ang
II and Rel-2 on prostate cells can proceed also by ability
to modify the levels of receptors: AT1, AT2 and LGR7,
LGR8. In conclusion, the clinical potential of manipulating
the angiotensin and relaxin system, seems promising in the
treatment prostate cancer.
Acknowledgments
The study was supported by the Grant no. NN 403 208 139 from the
Ministry of Science and Higher Education, Poland
Abstracts
40
P5.4
P5.5
The influence of μ-opioid receptor agonist
and antagonist peptides on peripheral
blood mononuclear cells (PBMCs)
Kinetic characteristic of amino acid
derivatives of 1,4-naphthoquinone
action on Na+, K+-activated Mg2+-ATP
hydrolysis depending of coldblooded
Ewa Fiedorowicz#, Małgorzata Iwan#, Bartłomiej Kocbach
Chair of Biochemistry, Faculty of Biology, University of Warmia and
Mazury, Poland #equal contribution
e-mail: Ewa Fiedorowicz <[email protected]>
Milk is one of the main source of biologically-active peptides that may function as regulatory substances called
food hormones. After passing the gut-blood barrier, the
μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the
human organism. The aim of the conducted research was
to determine the influence of μ-opioid receptor agonist
peptides: human and bovine β-casomorphin-7 (h/bBCM7) and antagonistic peptides: casoxin-6 and –D (CXN6/D) on proliferation and cytokine secretion of human
peripheral blood mononuclear cells (PBMCs). The PBMCs
proliferation was measured by the use of the BrdU test,
which assesses the DNA synthesis activity and the WST1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated
peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was
determined by the use of the ELISA tests. Incubating the
cells with the peptides has not caused any changes to their
enzymatic activity, which has been proved by a WST-1 test.
When using a BrdU test, however, it has been observed
that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated
into the cellular DNA. Moreover, changes to secretion of
IL-4 and IL-13 by the cells under the influence of agonists
were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The
obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may
be of clinical significance especially in the case of allergic
diseases in newborns. Anastasiya Heneha1, Marta Bura1, Svitlana Mandzynets1,
Dmytro Sanagurski1, Volodymyr Novikov2
1Lviv National University of Ivan Franko, Biophysics and Bioinformatics,
Ukraine; 2National University “Lviv Politechnic”, Biotechnology of
Biologically Active Substances, Ukraine
e-mail: Anastasiya Heneha <[email protected]>
The substances with structure quinone ring and amino acid
residues are interested today. New synthesized substance
amino acid derivatives of 1,4-naphthoquinone have low
toxicity, showing antyhypoxic, anticonvulsant effects. Its
can be used in the pharmaceutical industry as drug component with cerebroprotective action. It doesn’t clear cellular mechanisms of influence of substances on biological
systems. We used cold-blooded animal embryos (Misgurnus
fossilis L.) in synchronous stages of blastomere division as
a suitable model for these studies. Previous results demonstrate ultrastructural changes in blastomeres with alanine
and aspartic derivatives of 1,4-naphthoquinone in the first
hours of embryo development revealed: destructive changes hyaloplasm density and inner membrane of mitochondria, partial destruction smooth and rough endoplasmic
reticulum, increasing the number of lysosomes. We shown
that the aminoacid derivatives of 1,4-naphthoquinone
(alanine, asparagine, glutamine) can act as inhibitors. These
substances can cause dose-related changes in activity Na+,
K+-ATPase embryos during early embryogenesis. The aim
of this study was to establish a possible molecular mechanism action of new synthesized substances. We analyzed
the kinetic parameters of ATP hydrolysis investigated enzyme system of active transport of Na+ and K+. We used
the system of Dixon coordinate for kinetic analysis of
ATPase activity. We shown that the investigated substances inhibit ATP hydrolysis by competitive inhibition type.
This suggests direct interaction of molecules of amino
acid derivatives with catalytic center of ATPase. Linearization of the concentration dependence (potassium salt of
2-α-alanin derivatives of 1,4-naphtoquinone) rate of Na+,
K+-depended ATP hydrolysis in embryonic Hill coordinate
was found a negative cooperativity (Hill coefficient of <1),
that indicate inhibition of transport cycle enzyme molecule
by interaction with the studied substance.
41
P5.6
P5.7
Effective application of novel polymeric
nanocomposites for delivery of anticancer
drugs at in vivo treatment of murine
LPS activates motility of macrophages
governed by actin cytoskeleton reassembly
Rostyslav Horbay1, Natalia Boiko2,
Yelyzaveta Shlyakhtina2, Natalia Mitina3,
Anna Ryabtseva3, Olexander Zaichenko3, Rostyslav Stoika2
1Ivan Franko National University of Lviv, Lviv, Ukraine; 2Institute of Cell
Biology, NAS of Ukraine, Lviv, Ukraine; 3Lviv National State University,
Lviv, Ukraine
e-mail: Rostyslav Horbay <[email protected]>
We found that a single dose (50 µg) of vinblastine (Vbl) injected to C57-black mice with implanted NK/Ly lymphoma increased to 43% a ratio of tumor cells of bigger size
(giant (average diameter equaled 15.4 µm)equaled 15.4 µm),
while the parental tumor cell diameter equaled 12.7 µm.
The third Vbl injection led to an increase in the ratio of giant lymphoma cells to 83% with average diameter 19.5 µm.
When Vbl was conjugated with novel polymeric nanocomposites, a single dose of this drug increased the number
of giant lymphoma cells to 85% with average diameter 28
µm. In the last case, the diameter of individual tumor cells
in Vbl treated mice achieved 85 µm. It was shown that 10
times lower dose of Vbl (5 µg) delivered by the nanocarriers induced an appearance of tumor cells of average size
of 17 µm, and their ratio achieved 53%.
Giant cell cycle arrested tumor cells are known to be
more resistant to cytostatic and cytotoxic treatments, and
can spend weeks in such state (Wheatley, 2008). After the
chemotherapeutic action stops, these cells can reenter cell
cycle and give rise to daughter cells which possess an inherited resistance to anticancer drug. Vbl was shown to arrest cells in G2/M phase, and these cells could later enter
apoptosis, mitotic catastrophe, or return to cell cycling. We
found that conjugation of Vbl with the nanocarriers considerably increased cytostatic effect of this drug. We also
demonstrated that pre-treatment with Vbl (either free or
with nanocarrier) significantly increased tumor cell sensitivity to cytotoxic action of doxorubicin (Dx) conjugated
with nanocomposites and injected in 0.1 mg/kg dose. The
combined action of two drugs (Vbl and Dx) caused an increase in cell size (27.5 µm), as well as in ratio of giant
cells (86%), and significantly decreased the amount of tumor cells. Lymphoma bearing mice treated with Vbl with
the nanocarrier or treated with free Vbl and then with Dx
immobilized on the nanocarrier, survived while the mice
treated with only free Vbl died.
We hypothesize that Vbl delivered by the nanocarrier might
affect drug resistant NK/Ly cells, probably, due to better
action of the drug towards these cells. As a result, tumor
cells became more sensitive to the action of Dx. Study of
other potential mechanisms of more efficient anticancer
action of Vbl delivered to tumor cells by the developed
nanocarriers is in progress.
Galyna Kleveta1,2, Natalia Sybirna1,
Andrzej Sobota2, Katarzyna Kwiatkowska2
1Ivan Franko Lviv National University, Department of Biochemistry, Lviv,
Ukraine; 2Nencki Institute of Experimental Biology, Department of Cell
Biology, Waesaw, Poland e-mail: Galyna Kleveta <[email protected]>
Lipopolysacharide (LPS) is a bacterial component which
upon infection induces accumulation of macrophages in
tissues and production of pro-inflammatory cytokines,
leading to sepsis. The aim of these studies was to examine
molecular mechanisms governing LPS-stimulated motility
of macrophages.
We found that macrophage-like J774 cells exposed to 100
ng/ml LPS underwent polarization concomitant with
translocation of CD14 and TLR4, LPS receptors, toward
the leading edge of the cells. To estimate colocalization
of the two receptors, Pearson’s correlation coefficient was
calculated on enlarged confocal sections through the leading lamella; the coefficient was in the range of 0.5 with
1 as the maximal value, indicating significant but transient
character of the interaction between CD14 and TLR4. This
interaction triggered an accumulation of actin filaments
and PI(4,5)P2 at the leading edge of cells, with PI(4,5)P2
being visualized by means of GFP-fused PH domain of
phospholipase C. Fractionation of Triton X-100 cell lysates
revealed that after 20 min, 100 ng/ml LPS induced polymerization of cytoskeletal actin filaments by 50%. This
microfilament population appeared at the expense of actin monomers and short filaments composing the plasma
membrane skeleton of unstimulated cells. The peak of
actin polymerization coincided with maximal elongation
of LPS-stimulated cells and formation of ruffles, features
typical of motile cells. During 20 min of LPS action the
length of cells increased 1.5-fold and the amount of cells
with ruffles doubled. Simultaneously, in LPS-stimulated
cells an increase of phosphorylation of two actin-regulatory proteins, paxillin at tyrosine 118 by 80% and N-WASP
at serine 484/485 by 20%, was detected. Protein phosphorylation and reorganization of the actin cytoskeleton were
diminished by PP2, an inhibitor of tyrosine kinases of the
Src family.
Our data indicate that LPS-stimulated motility of macrophages is driven by reorganization of the actin cytoskeleton
controlled by Src family kinases and PI(4,5)P2. An inhibition of leukocyte motility can provide a means to limit the
inflammatory reactions toward LPS.
Abstracts
42
P5.8
P5.9
A new open reading frame of
Saccharomyces cerevisiae encoding
a protein likely involved in the
regulation of Pol II transcription
Tyrosine kinases of the Src
family participate in activation
of macrophages by LPS
Piotr Kowalec, Anna Chełstowska, Anna Kurlandzka
Kinga Borzęcka, Andrzej Sobota,
Kazimiera Mrozińska, Katarzyna Kwiatkowska
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Warsaw, Poland
Nencki Institute of Experimental Biology, Laboratory of Plasma
Membrane Receptors, Warsaw, Poland
e-mail: Piotr Kowalec <[email protected]>
e-mail: Katarzyna Kwiatkowska <[email protected]>
Sister chromatid cohesion complex is responsible for accurate segregation of chromosomes in mitosis and meiosis. In Saccharomyces cerevisiae the complex is composed of
three core proteins Scc1/Mcd1, Smc2 and Smc3, and the
Irr1/Scc3 protein whose role is poorly understood. All
these proteins are evolutionarily conserved. It is becoming evident that Irr1p and its mammalian homologues
participate not only in chromosome segregation but also
in the regulation of transcription serving as transcription
co-activators. In the heterozygous diploid strain irr1‑1/
IRR1, bearing one wild-type and one non-functional allele,
we found a significant decrease of transcription concerning ca. 30 genes. These genes were identified by microarray
and RT-PCR analyses. To identify proteins which may be
involved in transcription regulation by Irr1p we performed
a two-hybrid assay with C-terminal part of Irr1p as bait.
We identified a prey clone carrying a fragment of chromosome IV. According to data bases, this region contains
two open reading frames which encode vaguely characterized hypothetical proteins. By de novo sequencing we identified, however, a single 1183 amino acids-long ORF. In a
genome-scale genetic analysis (Constanzo et al., 2010) deletion of a fragment of this ORF interacted genetically with
numerous proteins involved in transcription and chromatin
remodeling. In an independent two-hybrid assay we found
another Irr1p interactor, involved in chromatin remodeling.
We identified new interactors of this protein involved in
the regulation of transcription — subunits of the mediator complex. Mediator is recruited to enhancer regions by
activator proteins and associates with RNA polymerase II.
Since deletion of any of the new Irr1p interactors-encoding genes causes chromosome segregation errors we believe that we found links between transcription and chromosome segregation.
Upon infection with Gram-negative bacteria, lipopolysaccharide (LPS) activates macrophages and neutrophils leading to life-threatening sepsis. Double knock-out mice of
hck and fgr genes encoding two protein tyrosine kinases
of the Src family gained resistance to lethal doses of LPS,
which suggests that these kinase can be crucial for stimulation of cells by LPS.
The aim of these studies was to establish an engagement
of Src tyrosine kinases in stimulation of macrophages by
LPS.
We found that an exposure of J774 and RAW264 macrophage-like cells to 100 ng/ml LPS induced tyrosine phosphorylation of several proteins including those in the range
of 70–50 kDa. Among these proteins we identified paxillin/Hic-5 which underwent phosphorylation at Tyr118 residue. Phosphorylation of paxillin/Hic-5 increased two-fold
at 20 min of cell stimulation with LPS and returned to its
basal level within 1 h of LPS action. Paxillin/Hic-5 interact,
among others, with kinases of the Src family. Inhibition of
the activity of these kinases with PP2 led to an inhibition
of phosphorylation of paxillin/Hic-5 and concomitant inhibition of LPS-stimulated motility of cells.
To analyze an engagement of Lyn, one of the major kinases of the Src family in macrophages, in LPS-induced
protein phosphorylation we analyzed activity of the kinase,
reflected by phosphorylation of its Tyr396 residue. To our
surprise we found that activity of Lyn was inhibited shortly
after exposure of cells to 100 ng/ml LPS. At 20 min of
LPS action, phosphorylation of Tyr396 was reduced by
40% and remained at this level during 1 h of LPS stimulation. This Lyn inactivation was also seen in cells serumstarved prior to LPS stimulation. After overexpression of
Lyn WT and stimulation of cells with LPS, an inhibition of
the kinase activity indicated by dephoshorylation of Tyr396
was detected, similarly as found in mock transfected cells
Our data suggest that activity of some of the kinases of
the Src family is required for LPS-induced phosphorylation
of proteins, including that of paxillin/Hic-5. However, Lyn
kinase of the family does not seem to be engaged in these
events as its activity is inhibited in LPS-treated cells.
References
Costanzo M et al. (2010) Science 327: 425-431.
P5.10
P5.11
EGF and Herceptin influence on
proliferation, adhesion and glucose
consumption of tumor cells MCF-7
Beta-catenin functions in embryonic
and postnatal heart formation
K. Nepyyvoda, K. Lavrova, L. Garmanchuk, L. Ostapchenko
Educational and Scientific Centre “Institute of Biology” of
Taras Shevchenko National University of Kyiv, Department of
Biochemistry, Kyiv, Ukraine
e-mail: Khrystyna Nepyyvoda <[email protected]>
Aim: to investigate proliferation, adhesion and glucose
consumption of tumor cells MCF-7 under the action of
EGF and Herceptin.
Methods: MCF-7 (breast adenocarcinoma) cells were cultured in DMEM medium (Sigma, USA) supplemented with
10% FBS (Sigma, USA) at the standard conditions. Tumor
cells were incubated with EGF (obtained from submandibular mouse glands) and Herceptin (Roshe, France) during
72 h. Proliferation was determined using MTT-test and
analyzed on flow cytofluorimeter. Adhesion was evaluated
after staining of cells with crystal violet. The level of glucose in the incubation medium were analyzed using standard kits (Philisit Diagnostics, Ukraine).
Results: It was shown that proliferation of tumor cells
MCF-7 amplified for 49% (p<0.05) by incubation with
EGF, while Herceptin caused decrease of this index on
16% compared with control. Adhesion of MCF-7 cells
under the action of EGF increased on 47% (p<0.05)
compared with control, whereas Herceptin did not cause
significant changes of studied parameter. There also were
identified changes in the consumption of glucose by tumor
cells as a result of incubation with test agents. Thus, under
the influence of EGF the level of glucose consumption by
MCF-7 decreased 36% (p<0.05), while under the action of
Herceptin this setting increased 32% (p<0.05) compared
with control.
Thus, we showed opposing effects of EGF and Herceptin, which is an anti-idiotype toward EGF, on tumor cells
MCF-7, in particular on the proliferative activity, adhesion,
as well as the level of glucose consumption.
43
Оksana О. Piven1, Іgor E. Коsteskii1,
Larysa L. Маcewicz1, Glenn Radice2, Lubov L. Lukash1
1Іnstitute of Molecular Biology and Genetic, Humans Genetics
Department, Ukraine; 2Jefferson Medical College, USA
e-mail: Oksana Piven <[email protected]>
The adherens junction (Ajs) provides strong cell-cell adhesion, which is mediated by the cadherin/catenin complex
via linkage to the actin cytoskeleton. In addition, members
of the cadherin/catenin complex play important roles in
cell signalling. One of the member of Ajs, β-catenin is necessary for zygotic, blastocyst development throws its adhesive and signaling function. Loss of zygotic β-catenin allows normal blastocyst development results ethality. These
phenomena are not caused by the loss of cell–cell adhesion, which is maintained by plakoglobin, but because of
a deficiency in canonical Wnt signalling. Inhibition of Wnt
signalling is required for early cardiogenesis. Conversely,
β-catenin signalling is required for the septation of cardiac
chambers. Aims: Requirement of β-catenin for embryo
and postnatal heart development. Result: In our investigations, we have demonstrated that ablation of β-catenin
after embryonic day 5.5 (after chamber specification) does
not lead to lethality, probably due to functional redundancy
of β-catenin by plakoglobin, similar to that found in the
adult heart. For studying how the ablation of β-catenin reflected on postnatal heart formation and function we crossed
β-cateninflox/+, αMHC-Cre mice with β-cateninflox/flox. Genotyping of β-catenin newborn mice revealed that homozygous
mutant mice are underrepresented, suggesting that some
mice may be dying in utero, probably at late gestation. Thus,
only 10% of mice born had a genotype of Cre-αMHC+,
β-cateninflox/β-cateninflox, and 33.3% of these mice were
dead. In these experiments we obtained 20% heterozygous
animals (genotype Cre+, β-catflox/-) and 16.6% of these heterozygotes were born dead.
Abstracts
44
P5.12
P5.13
Impact of izatison on nucleic
metabolism of wheat sprouts Stem cell-like properties of CD133+ cell
subpopulation of mouse and human
colon cancer cell lines — a potential
target for novel anti-cancer therapies
Olena Martynenko1, Tamara Kyrylenko1, Andriy Stepanyugin1, Nadezhda AdamchukChala2, Dmytro Plodnik3, Anatoliy Potopalsky1
1Institute
Ukraine; 2Institute
of Molecular Biology and Genetics, NAS of
of Botanics, NAS of Ukraine; 3National University of Food Technologies,
Ukraine e-mail: Dmytro Plodnik <[email protected]>
Developed technologies of controlled plant growing using
effects of various moderating external factors, e.g., Izatison (Iz) — antivirus, antitumor and antibacterial preparation — explain the necessity to study mechanisms through
which these factors affect plant organisms. The goal of this
research was to study how Iz affects the state of genetic apparatus of plant cells using RNA/DNA analysis and electronic microscopy of apikal meristem cells nuclei.
We used wheat sprouts in the experiments: the wheat seeds
were treated with Iz for 1 hour prior to planting in the
sand. We were studying the effect of this chemical factor
on changes in the indicators of nucleic gomeostasis (RNA
& DNA) in plants every day for 20 days. Based on the obtained data we calculated the RNA/DNA ratio, which is a
multifunctional characteristic of cells that also reflects the
intensity of nucleic metabolism process (NMP).
As a result of the experiment we established that one-time
treatment of seeds with Iz led to changes in NMP of experimental sprouts in almost all time periods of the study.
These changes were in opposite direction for experimental versus control sprouts. The minimum value of RNA/
DNA ratio was 2.8–3.9 for all studied samples except for
control samples of 5-days plants with the ratio of 11.5. The
maximum levels of intensity observed in NMP of control
sprouts attained 16.0 (by day 4), 14.0 (by day 6) and 12.3
(by day 10), whereas in the experimental samples these
numbers ranged from 14.7 (by day 5) to 11.1 (by day 17).
Specifically, we found that NMP switches on later in treated
plants as compared to control ones, however, its level was
merely identical to the level of control plants. Further, we
show that under the effect of Iz in the process of wheat
sprouts development, increases and decreases in the intensity of NMP were accompagnied by corresponding changes in ultrastructural organization of wheat meristem cells
and their nuclei.
In sum, the obtained data show the sensitivity of wheat
cells genetic apparatus to the effect of Iz. The study of
biological effects of Iz continues.
References are available from authors. Małgorzata Przybyszewska1, Agnieszka Kotlarz1,
Ewa Bielczyk2, Joanna Miłoszewska1, Halina Trembacz1,
Paweł Swoboda1, Agnieszka Lipiec1, Beata Grygalewicz1,
Marzena Biernacka1, Andrzej Kutner3, Sergiusz Markowicz1
1Maria Skłodowska-Curie Memorial Cancer Center - Institute of
Oncology, Warsaw, Poland; 2University of Warsaw, Department of
Cytology, Poland; 3Pharmaceutical Research Institute, Warsaw, Poland;
e-mail: Małgorzata Przybyszewska <[email protected]>
CD133/Prominin-1 is proposed as a stem cell surface
marker that can be used to identify and isolate cells of cancer stem cell (CSC) phenotype in a number of human and
mouse tumors and tumor-derived cell lines. We have previously identified a minor CD133+ cell subpopulation in
CT-26 mouse colon cancer cell line by flow cytometry. A
proportion of CD133+ cells among CT-26 cells in vitro decreased after exposure to cytoreductive drug doxorubicin
followed by exposure to tyrosine kinase inhibitor imatinib.
We developed a two-step therapy to target in vivo CT-26
cells resistant to doxorubicin by a subsequent treatment of
mice with imatinib. Such therapy inhibited growth of CT26
tumors in mice more efficiently than treatment with doxorubicine or with imatinib alone. In this study we evaluated
properties of mouse CD133+ CT-26 colon cancer cells and
human CD133+ HT-29 adenocarcinoma cells with a high
expression of glycosylated AC133 epitope of CD133 protein. Such subpopulations are potential targets for therapy
aimed to eliminate cancer stem-like cells. CD133+ cells
were isolated using MACS Microbead system to test their
growth potential, clonogenicity, migration, and resistance
to cytostatic drugs. A clonogenic and growth potential of
CT-26 cell fraction enriched in CD133+ cells was at least
two times higher than that of non-separated CT-26 cells,
but decreased to the control level after two/three weeks of
culture. CT-26 cells overexpress receptors associated with
stem-like phenotype, i.e. PDGFRA and B; the molecular
targets of specific tyrosine kinase inhibitor, and therefore
are sensitive to imatinib treatment. Imatinib decreased viability of CD133+ CT-26 cells measured in the MTT assay and reduced their capability to migrate from spheroids
in vitro. Percentage of CD133+ cells decreased following
treatment of HT-29 cells with 5’FU in vitro. HT-29 cells
pre-treated with 5’FU were separated to CD133+ and
CD133- cell fractions. Cell growth and clonogenicity of
CD133+ cell fraction were significantly higher as compared
to CD133- cell fraction. It suggests that cells responsible
for cell repopulation and cancer recurrence after exposure
to conventional anti-cancer drugs are found preferentially
among HT-29 and CT-26 cells expressing AC133 epitope
of CD133. Therefore such pre-selected subpopulation of
CD133+ cells will be used in further studies to search for
drugs targeting CSC directly or inducing CSC differentiation to reduce their drug resistance. Acknowledgements
Supported by Grant MNISW NN402139738.
P5.14
P5.15
Low-level laser irradiation effect
on membrane-related processes
in loach embryo cells
Effect of low intensity diod laser
irradiation towards normal and
transformed fibroblast cell lines
Myroslava Romaniuk, Oksana Neofita,
Natalia Holovchak, Maria Dyka, Dmytro Sanahursky
L. Rudavska1,2, I. Novitskyi1, O. Kluchivska3, R. Stoika3 Ivan Franko National University of Lviv, Department of Biophysics and
Bioinformatics, Lviv, Ukraine
e-mail: Myroslava Romaniuk <[email protected]>
Recently, in clinical practice is commonly used laser irradiation. However, its mechanism of action remains at the
hypothesis. Biological membranes play an important role
not only in maintaining ionic homeostasis of the cell but
also participate in regulation of cell division, cell proliferation and development of the organism, and is the center of
morphogenetic processes of early development. The cell
changes depends of the state of the membrane and process
lipid oxidation of membrane components. We researched
the impact of laser irradiation on lipid peroxidation and
membrane transport properties, include the functioning of
Na+,K+-ATPase in loach embryo cells. The useful model
for our issue is a loach embryo cell during early embryogenesis. We noted that lipid peroxidation (LPO) to get maximum level on stage 2 blastomers with laser irradiation and
it is 130%. We found that laser irradiation provides low
energy stimulation to cells, which results in increased activity of the superoxide dismutase (SOD) on stage 64 blastomers at 170%, following 5 min irradiation. When on stage
2 blastomers the laser irradiation following 1 min leads to
decrease activity of the this enzyme on 12.3% compared
with control. Laser irradiation can cause Na+,K+-ATPase
dysfunction through the intensification of LPO in membrane. We noted laser irradiation exposure at 1 min and 5
min on stage 2 blastomers of embryos leads to decrease
membrane enzyme activity on 63.70% and 58.94% compared with control respectively. Our results extend the
notion of probable mechanisms of action at laser irradiation membrane-related processes, including the functioning of Na+,K+-ATPase and membrane lipid peroxidation
and antioxidant protection of organism. The aim next our
study was to establish whether laser irradiation is able to
cause changes in compounds membrane proteins. For control on the dynamics of fractions membranes proteins of
the embryo using method PAAG electroforesis with SDS
and β-mercaptoethanol.
45
1Lviv National Medical University; 2Lviv Regional Military Hospital;
3Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine
e-mail: Lidiya Rudavska <[email protected]>
Application of laser irradiation in industry and medicine
is rapidly growing. Effect of low intensity laser light interaction with human skin fibroblast has been studied by
using nano-probes with emission of He–Ne laser [1]. An
enhancement of cell proliferation dependent on the energy
dose of laser irradiation was found.
The aim of this study was to evaluate the effect of Diod
810 nm laser emission towards proliferation of the fibroblast lines differing in the level of malignant transformation.
Methods: The effect of Iridex IQ 810 multifunctional laser system on the population of normal (ВALB 3T3) and
transformed (L929) murine fibroblast lines was studied.
The cells were irradiated under different exposure schemes:
1) irradiating cells with micropulse mode (22 impulses were
applied with 200 milliseconds each); 2) continuous laser
emission was applied with 2000 and 1000 mW power).
During the next seven days after laser irradiation, cytomorphometric study (counting the number of alive and dead
cells, and evaluating metabolic potential of cells by using
MTT-test) was performed.
Results: During the first 2 days after laser irradiation, there
were no significant differences in the proliferation activity
of two fibroblast cell lines subjected to either micropulse
or continuous laser emission. On the third day, cell cultures
treated with continuous laser emission mode demonstrated
higher amount of alive cells in comparison to cell cultures
exposed to the micropulse laser irradiation, although, in
general, micropulse laser treatment was less damaging for
the fibroblastic cells. There was no significant difference
in proliferative activity between cell cultures treated with
either 1000 mW or 2000 mW modes, but in both cases
such activity on the fifth day was higher than in control
(untreated) cell cultures. In general, the laser-enhanced proliferative activity of normal (ВALB 3T3) was higher than
such activity of the malignant transformed (L929) murine
fibroblast line. The results of MTT-testing of the treated
cells confirmed this dynamics.
Conclusion: Diod 810 nm laser irradiation enhances proliferation of both normal (ВALB 3T3) and transformed
(L929) murine fibroblast lines. This effect is more pronounced in normal cell cultures. Micropulse laser emission
is less damaging for cells than continuous laser irradiation.
Acknowledgements
1. Gopalendu Pal et al. (2007) J Photochem Photobiol B: Biol 86: 252–261.
Abstracts
46
P5.16
P5.17
Influence of n-3 polyunsaturated fatty acids
on GJP-43 gene and connexin-43 protein
expression in experimental models of
ischemia-reperfusion and diabetes mellitus MGST1 downregulation increases PC12
cells susceptibility to oxidative stress
A. Shysh1, A. Zhukovska1, J. Radosinska2, B. Bacova2,
N. Tribulova2, V. Dosenko1, A. Moibenko1
1Bogomoletz
2Institute
Institute of Physiology, Kyiv, Ukraine;
for Heart
Research, Slovak Academy of Sciences, Bratislava, Slovakia
e-mail: Anzhela Shysh <[email protected]>
Background: Many studies indicate that dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have cardioprotective
properties. It is not clear still whether n-3 PUFAs have beneficial effects on connexin-43 (Cx43) expression, the major
gap junctions cardiac protein, crucial for cell-to-cell communication and functioning of the heart. The aim of the
work was to investigate the effect of n-3 PUFAs on Cx43
protein and GJP-43 gene expression in the condition of
experimental myocardial ischemia-reperfusion and diabetes
mellitus.
Methods: Male Wistar rats were divided into 2 groups: the
control group including intact rats, and the group receiving
n-3 PUFAs in diet (0.1 mg/kg for 4 weeks). The isolated
hearts of animals from both groups were exposed to global 20 min lasting ischemia followed by 40 min reperfusion
(I/R). Model of streptozotocin-induced diabetes mellitus
in rats was used and n-3 PUFAs diet was applied on the 3rd
day after STZ injection for 4 weeks. Results: In hearts undergoing I/R n-3 PUFAs treatment
resulted in increased expression of total myocardial Cx43
protein by 14.8 % compared with control. Moreover, n-3
PUFAs elevated GJP-43 (to a code connexin 43) mRNA
expression in 2.27 folds in rats hearts. In addition, n-3
PUFAs elevated mRNA expression of fatty acid transport protein (FATP) and interleukin-1 receptor antagonist
(IL-1ra) genes in 2.25 and 8.4 folds respectively in adult rats
tissues. In diabetic hearts the total level of Cx43 protein
and its phosphorylated forms were increased, while application of n-3 PUFA normalized Cx43 expression as well as
its phosphorylated forms. Conclusions: Results indicate that omega-3 PUFAs stimulate GJP-43 mRNA expression most likely via stimulation
of PPAR target genes expression likewise to FATP and
IL-1ra. Application of n-3 PUFAs modulation of Cx43
protein expression and suppression of diabetes-induced
hyper-phosphorylatation of Cx43 may be beneficial for
protection of cell-to-cell communication and heart function.
Acknowledgements
Supported by SRDA SK-UA-0022-09 grants. Monika Sobczak, Tomasz Boczek, Ludmila Zylinska
Department of Molecular Neurochemistry, Medical University, Lodz,
Poland
e-mail: Monika Sobczak <[email protected]>
Microsomal glutathione transferase 1 (MGST1) belongs
to the superfamily of Membrane Associated Proteins in
Eicosanoid and Glutathione Metabolism (MAPEG) enzymes. It constitutes about 3% of reticulum endoplasmic
membrane proteins and approximately 5% in the outer mitochondrial and plasma membranes. MGST1 possesses a
glutathione transferase as well as peroxidase activity, and it
plays an important role in the cellular protection. The aim
of our study was to analyse of MGST1 role on cell function using a stable-transfected PC12 lines with suppressed
MGST1 (PC12_M). Real-time PCR and Western blot assays confirmed decline of MGST1 expression. Also an immunocytochemical staining showed its lowered amount in
PC12_M line. The first morphological changes in PC12_M
cells we observed after 48 hours, including larger tendency
to grow in clusters, irregular shape of bodies and short
neurites outgrowth. Moreover, flow cytometry analyses revealed a higher percentage of necrotic cells. To elucidate
the role of MGST1 in protection against oxidative stress,
we studied the cytotoxic action of hydrogen peroxide on
viability of PC12 cell lines using WST assay. After treatment with 1mM of hydrogen peroxide for 24, 48 and 72
h the viability of PC12_M decreased more significantly
than in control cells.These results indicate on unique role
of MGST1 in the cell resistance to the stress conditions. In
addition, it suggests that MGST1 activity cannot be compensated by other glutathione transferases.
Acknowledgements
Supported by the grant 502-03/6-086/502-64-003 from the Medical University of Lodz, Poland.
P5.18
P5.19
Effects of silver nanoparticles on
some parameter of rabbit’s granulose
cells and its ability to promote the
maturation of rabbit’s oocytes in vitro
Changes of the levels of reactive
oxygen species in cells exposed
to ionizing radiation
Syrvatka1,
Hevkan1, Y.
Slyvchuk1,
V. J.
I. I.
I.
G. O. Milovanova2, O. J. Yrkiv1, O. V. Shtapenko1,
S. V. Fyodorova1, I. O. Matuha1, I. I. Rozgoni1
1Institute of Animal Biology NAAS, Department
Ukraine; 2Taras Shevchenko National University
Biochemistry, Ukraine
of Biotechnology,
of Kyiv, Department of
e-mail: Vasyl Syrvatka <[email protected]>
Among antibacterial agents, silver nanoparticles (AgNPs)
have the greatest toxic effect against multi-resistant gramnegative and gram-positive bacteria, viruses, fungi and
some protozoa. It is used in different fields of medicine
since years, e.g. as prophylaxis of infection diseases, in
wound healing or in biomaterials. However, it’s little known
about influence of AgNPs on oocyte-granulosa cell interactions at oocytes maturation to metaphase II. The aim of
this study was to investigate the effect of different concentrations (0, 0.01, 0.1, 1 and 10 µg/ml) AgNPs (size 5 nm)
in cultural medium on ability of rabbits granulosa cells to
promote the maturation of oocytes, some biological functions (proliferation, synthesis of progesterone) and same
biochemical parameters of cultural medium (lactate dehydrogenase activity, concentration of total protein, albumin,
Ca, P, glucose and cholesterol) in oocyte-granulosa culture
system after 24 hours of in vitro cultivation.
Oocytes and granulosa cells were collected from ovaries of
slaughtered rabbits. Maturation media consisted of TCM
199 supplemented with sodium bicarbonate, FCS, FSH,
LH, estradiol, gentamicin and different concentration of
AgNPs. 20 oocytes of high quality and 106 granulosa cells/
ml were seeded in cultural dish and kept in maturation medium in CO2 incubator at 38oC. After 24 hours of cultivation culture medium was sampled to determinate some
biochemical parameters using commercial kits. Number
of viability granulose cells was counted after Trypan blue
staining. Oocytes maturation was evaluated for the presence of first polar body using Giemsa stain.
The results showed the concentration-dependent effect
AgNPs on granulosa cell proliferation and some biochemical parameters. There were no significant difference in all
measured findings between 0, 0.01, 0.1, 1 µg/ml AgNPs in
medium. However, concentration of 10 µg/ml AgNPs impacted changes in some biochemical parameters (increasing of lactate dehydrogenase activity and concentration of
total protein, albumin and Ca) compare without AgNPs
in culture medium. There were no significant differences
among all investigated concentrations of AgNPs on rate of
rabbit’s oocytes maturation.
The data obtained from given study showed that silver nanoparticles in concentration 0.01-1 µg/ml had no toxic effect on granulosa cells and oocyte maturation in vitro. Concentration, higher that 1 µg/ml influences changes in some
biochemical parameters. 47
Artur Cieślar-Pobuda1, Yuriy Saenko2,
Magdalena Skonieczna1, Sebastian Student1,
Joanna Rzeszowska-Wolny1
1Biosystems Group, Institute of Automatic Control, Silesian University
of Technology, Gliwice, Poland; 2Center of Nanotechnology, Ulyanovsk
State University, Ulyanovsk, Russian Federation
e-mail: Artur Cieślar-Pobuda <[email protected]>
The production of reactive oxygen species (ROS) is one
of the key events in the response of cells to exposure to
ionizing radiation, but it may occur also in response to
other stressing factors. ROS induced directly by radiation
are short-lived, but ROS levels can increase further in the
later stages of the cellular response to radiation, even hours
or days after exposure. Significant differences are observed
between cell types in the levels and time of appearance of
these second ROS waves. Increased levels of ROS in irradiated cells are important because they can influence many
cellular processes and cause different types of damage to
cellular macromolecules.
Here we present results of experiments in which ROS levels, DNA damage, mitochondrial potential, and mitochondrial mass were measured at different time after exposure
of human lymphoblastoid (K562), melanoma (Me45) and
fibroblast (NHDF) cell lines to 4 or 12 Gy of ionizing radiation. Intracellular ROS were measured by flow cytometry
using the probe 2,7-dichlorofluorescein (DCF), and mitochondrial membrane potential and mass were assayed by
methods using tetramethylrhodamine ethyl ester (TMRE)
and 10-nonyl bromide acridine orange (NAO), respectively. The poly (ADP-ribose) polymerase (PARP-1) activity was inhibited with NU-1025 inhibitor. In experiments
aiming to study the influence of mitochondrial status on
ROS levels, rotenone was used to inhibit the mitochondrial
respiratory chain. In all cell lines, exposure of cells to Xrays caused an accumulation of ROS, increased levels of
DNA strand breaks and, except for Me45 cells, a cell cycle
block in the G2 phase. A 30–40% increase in ROS level was
observed in melanoma cells immediately after irradiation,
which persisted for the next 48 hours. A rise in ROS level
was observed in K562 cells only during the first minutes
after irradiation, and then the level decreased and a second ROS wave occurred 12–48 hours later. This second increase of ROS level was accompanied by an increase of the
mitochondrial membrane potential, and could be diminished by inhibition of the mitochondrial respiratory chain,
and surprisingly increased by the inhibition of PARP-1 activity in first minutes after irradiation. The changes of ROS
levels observed in irradiated cells seem to be a common
phenomenon with cell type specificity, and may play a role
in the global response of cell populations to radiation. The
first steps of DNA damage repair which engage PARP-1
activation may influence further ROS induction steps however the mechanism remains unclear.
Session 6: Transporters in adaptation to hostile environments
Lectures
L6.2
L6.1
Mechanism and selectivity in the
bacterial Na+/H+ antiporter
On the molecular mechanism of
the AcrB RND-efflux pump
Klaas Martinus Pos
Institute of Biochemistry, Goethe-University Frankfurt am Main,
Germany
e-mail: Klaas Pos <[email protected]>
AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents
from the cell. The functional unit of inner membrane component AcrB is a homotrimer and the monomers adopt
three different conformations designated loose (L), tight
(T) and open (O). It is postulated that the AcrB monomers
cycle consecutively through these three conformational
stages in a concerted fashion, from L to T to O and back
to L, much in analogy to the functional rotation observed
for the synthesis of ATP by the F1FO-ATPase. For the
asymmetric AcrB homotrimer, binding of drugs has been
shown in the T monomer at an internal hydrophobic binding pocket. The initial drug binding step and the transport
towards the internal periplasmic drug binding site in the T
conformation presumably mediated by an intrinsic tunnel
system has been elusive thus far due to the lack of high
resolution AcrB/drug co-crystal structures. Here we report
high resolution structures (2.0–2.25 Å) of AcrB/DARPin
complexes with bound minocyclin or doxorubicin. The
binding and release of drugs has its structural basis in large
conformational changes of the periplasmic porter domain
implied to be energized by protonation and deprotonation
events in the transmembrane domain. The four functionally essential transmembrane residues D407, D408, K940
and R971 constitute a proton relay network in the AcrB
membrane domain. The presence of ordered waters exhibited by the high resolution structures allows us to deduce a
pathway for proton-transfer.
Isaiah T. Arkin
The Alexander Silberman Institute of Life Sciences, Department of
Biological Chemistry, The Hebrew University of Jerusalem, Edmund J.
Safra Campus, Jerusalem, Israel
e-mail: Isaiah Arkin <Israel. [email protected]>
Na+/H+ antiporters are found in all kingdoms of life and
are essential for maintaining salt and pH homeostasis.
Therefore the solved structure of NhaA from E. coli provides a unique opportunity to investigate the mechanism
of this central protein family. Using experimental and computational analyses we provide a model for the mechanism
and cation selectivity of the protein. The mechanism of
the protein is based on two adjacent Asp residues. One
serves as the Na+ binding site, while the other controls the
alternating accessibility of the protein. Together we show
how the two residues act in concert to pump the cation at
the expense of two protons. Secondly, we show that the
mechanism of selectivity amongst cations in the protein
is unexpected. Specifically, all alkali cations are capable of
binding the protein despite the fact that only Li+ and Na+
are transported, thereby distinguishing between futile and
productive binding.
L6.3
Oral presentations
Molecular mechanism of betaine
transport and stress regulation by
the Na+-coupled symporter BetP
O6.1
Christine Ziegler
Department Structural Biology, Max-Planck Institute of Biophysics,
Frankfurt, Germany
e-mail: Christine Ziegler <[email protected]>
Na+-coupled
The
symporter BetP, a member of the
Betaine-Choline-Carnitine-Transporter (BCCT) family
catalyzes uptake of the osmolyte betaine in Corynebacterium glutamicum. BetP also senses hyperosmotic stress and
regulates its own activity according to the stress level. Four
X-ray structures of BetP in different conformations reveal
the translocation pathway of sodium and betaine towards
the cytoplasm. While the coordination of the conserved
high affinity sodium binding site was shown by structural
and functional studies, the location of a second Na+ binding site that varies in transporters sharing the LeuT-fold
was predicted based on symmetry consideration between
the inverted repeats and confirmed by mutagenesis studies. Moreover, the binding order of betaine and the two
sodium ions during secondary transport was determined.
Furthermore structural and functional data on single point
mutants of BetP that exhibit changed substrate and cosubstrate specificity elucidate the coupling mechanism in
BetP.
BetP adjusts its transport rate according to the external
osmotic conditions. The stress stimulus sensed by BetP is
the cytoplasmic K+ concentration, which increases immediately with increasing external osmolality. BetP regulates
activity only when it is trimeric as shown by mutagenesis
and functional analysis. X-ray structures and electron microscopy data revealed that intra-trimeric interactions
between protomers lock individual protomers in distinct
conformations of the catalytic and regulatory cycle as observed by a pronounced conformational asymmetry within
the BetP trimer. Specific lipid-protein interactions play an
additional role in transport activation and regulation. Negatively charged POPG lipids are bound to the hydrophobic
central cavity of the trimer and to functional important
scaffolds of one protomer contributing to the observed
conformational asymmetry. X-ray structures of BetP in
K+-activated conformations reveal a sophisticated regulatory mechanism in which lipids participate in the formation
of K+ binding sites.
49
Heterogeneous expression of
the human sodium-dependent
phosphate co-transporter 2b in
ovarian cancer and normal ovaries M. A. Shyian1, 2, V. S. Gryshkova1, O. I. Kostianets1,2,
V. V. Filonenko1, R. G. Kiyamova1 1Institute of Molecular Biology and Genetics, NAS of Ukraine,
Department of Cell Signaling, Ukraine; 2Taras Shevchenko National
University of Kyiv, Institute of Biology, Kyiv, Ukraine
e-mail: Maksym Shyian <[email protected]>
Recently, sodium dependent phosphate transporter
NaPi2b was revealed as potential marker for breast, thyroid, lung and ovarian cancer. In vivo, NaPi2b is involved
in maintenance of phosphate homeostasis and mutations
or aberrant expressions of its gene (SLC34A2) are associated with several diseases including cancer. However, data
about NaPi2b mRNA expression in different types of tumors and correspondent normal tissues are controversial
and restricted.
The main purpose of this study was to estimate the
SLC34A2 gene expression in normal ovary and different
types of ovarian tumors.
We investigated SLC34A2 gene expression level in normal
ovarian tissues and different histomorphological types of
ovarian tumors (papillary serous, endometrioid, unspecified adenocarcinoma tumors, benign tumors) using realtime PCR analysis. It was found that SLC34A2 gene was
highly expressed in well-differentiated endometrioid and
papillary serous tumors, but was not expressed in lowdifferentiated tumors, benign tumors and in most normal
tissues. mRNA expression of SLC34A2 in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples
by Western blot analysis and immunohistochemistry in our
previous investigation. Immunohistochemical analysis of
ovarian cancer samples confirmed that NaPi2b expresses
predominantly in well-differentiated ovarian carcinomas at
the cytoplasmic membrane of more differentiated cancer
cells, but in some cases NaPi2b in ovarian cancer cells was
also observed in cytoplasm and nucleus, where its role is
completely unclear and needs to be further investigated.
Analysis of SLC34A2 gene expression according to tumor
differentiation level (poor- and well-differentiated) showed
that SLC34A2 tends to increased level of gene expression
in well differentiated tumors and decreased level in poor
differentiated tumors.
Better differentiated tumors usually have lower neoplastic
grade and therefore have better prognosis. It is possible
that the molecular pathways leading to low grade (welldifferentiated) tumors require the expression of SLC34A2.
Thus, upregulation of SLC34A2 gene expression in welldifferentiated cells of ovarian tumors may reflect cell differentiation processes during ovarian cancerogenesis and
could serve as a potential marker for ovarian cancer diagnosis and prognosis. Abstracts
50
O6.2
Posters
Regulation of amino acid/carnitine
transporter B0,+ (ATB0,+) by protein kinase C
P6.1
Łukasz Samluk1, Magdalena Czeredys1, Krzysztof
Skowronek1,2, Katarzyna A. Nałęcz1
1Nencki Institute of Experimental Biology, Warsaw, Poland;
Poland
e-mail: Łukasz Samluk <[email protected]>
Plasma membrane transporter B0,+ (ATB0,+) transports
neutral and basic amino acids, as well as carnitine (with low
affinity) in a Na/Cl-dependent way, being a member of a
superfamily coded by SLC6 genes, containing neurotransmitters, amino acids and osmolytes transporters, whose activity can be modulated post-translationally. There are 2 potential protein kinase C (PKC) phosphorylation sites within
ATB0,+ amino acid sequence, therefore the possibility of
post-translational regulation of ATB0,+ by this kinase were
investigated with PKC activator (phorbol-12-myristate13-acetate, PMA) and inhibitor (bisindolylmaleimide, BIS
II). The experiments were performed both, in the primary
culture of rat astrocytes and HEK 293 cells overexpressing rat ATB0,+ after transfection with p3xFLAG-CMV/
B0,+ vector containing cloned SLC6a14 gene tagged with
3xFLAG coding sequence at a C-terminus of the fusion
protein. Immunoprecipitation experiments with anti-B0,+
antibodies (astrocytes) and anti-FLAG antibodies (HEK
293 cells overexpressing B0,+) demonstrated in B0,+ presence of phosphoserine, the amount of which augmented
after PMA, a process reversed after BIS II pretreatment.
An increase of phosphoserine amount was correlated with
higher rate of [3H]leucine transport, both in astrocytes and
HEK 293 cells and the sensitivity of leucine accumulation
to arginine as well as to Na+ and Cl- removal confirmed
functioning of ATB0,+. Biotinylation of plasma membrane
proteins, followed by purification with Avidin Agarose Resin demonstrated an increased amount of ATB0,+ in plasma
membrane after PKC activation, without any change of
the total amount of the transporter. This observation, together with an increased Vmax of leucine transport, point to
activation of ATB0,+ due to its phosphorylation by PKC
by a process of trafficking to plasma membrane from a
pre-existing intracellular pool. All the effects observed after
PMA treatment indicate an involvement of classical and/
or novel isoform(s) of PKC, the identity of which has been
under investigation.
Acknowledgements
Work supported by grant 4427/B/P01/38 from the Polish Ministry of
Science and Higher Education and by the statutory funds of the Nencki
Institute.
[1,2-3H(N)]cholesterol redistribution
and sodium-dependent glutamate
uptake in cholesterol-enriched
rat brain nerve terminals
Roman Sivko, Natalia Krisanova, Tatiana Borisova
Palladin Institute of Biochemistry, NAS of Ukraine, Kiev, Ukraine
e-mail: T Borisova <[email protected]>
The effects of the complex of cholesterol acceptor methylbeta-cyclodextrin (MCD) with cholesterol, which enriched
the plasma membrane by cholesterol, and MCD alone on
the activity of sodium-dependent glutamate transporters
were assessed in rat brain synaptosomes. MCD did not alter
membrane integrity, the addition of 10 mM MCD from the
both side of planar lipid bilayer did not change the transmembrane current in 100 мМ NaCl. It was shown that
the application of the complex 15 mM MCD-cholesterol
(1:0.2) increased the level of cholesterol in rat brain nerve
terminals from 0.7 ± 0.04 mmol/mg of protein to 1.4 ±
0.08 mmol/mg of protein (P≤0.05, n=4). The application
of 15 mM MCD alone decreased the level of cholesterol in
synaptosomes by ~25%.
Using [1,2-3H(N)]cholesterol, we demonstrated that the
treatment of nerve terminals with complex MCD-[1,23H(N)]cholesterol (1:0.2) (following by depletion of the
complex from the incubation media by washing of the synaptosomal preparation) accompanied with the transfer of
[1,2-3H(N)]cholesterol from the plasma membrane to synaptic vesicles. The level of [1,2-3H(N)]cholesterol in synaptic vesicle fraction consisted of ~20% from total amount
of synaptosomal radioactivity. Thus, extra cholesterol was
redistributed between the plasma membrane and intracellular membranes of nerve terminals.
It was shown that the initial velocity of L-[14C]glutamate
uptake was increased from 2.5 ± 0.3 nmol × min–1 × mg
of protein–1 to 3.5 ± 0.3 nmol × min-1 × mg of protein –1
during application of the complex MCD-cholesterol (1:0.2)
(P≤0,05, n=8), whereas decreased two times after the treatment with MCD alone. Thus, cholesterol is a potent endogenous modulator, which can modulate glutamate uptake processes.
51
P6.2
P6.3
Permeability coefficient determination of
bovine erythrocytes for glycerol and DMSO
Research of dogs’ bone marrow cells safety
after cryopreservation in liquid nitrogen
Olga Denysova,1 Larisa Vodopyanova,1 Gennadiy
Zhegunov1, Johannes M. Nitsche2
Larisa Vodopyanova1, Olga Denysova2
1Department
of Biochemistry, Kharkov State Veterinary Academy, Mala
Danilovka, Dergachy district, Kharkov region, Ukraine; 2Department
of Chemical and Biological Engineering, University at Buffalo, State
University of New York, Buffalo, NY, USA
1Department of Physiology, Kharkov State Zooveterinary Academy,
Malaya Danilovka, Kharkov region, Ukraine; 2Department of Chemistry
and Biochemistry, Kharkov State Zooveterinary Academy, Malaya
Danilovka, Kharkov region, Ukraine
e-mail: Larisa Vodopyanova <[email protected]>
e-mail: Olga Denysova <[email protected]>
Insofar as cryoprotectant molecules must enter erythrocytes to be effective, quantitative understanding of the
permeability of the cell membrane to these molecules has
long been key to the mechanistic understanding of their
function. This talk describes a novel revisitation of permeability determinations by osmotic shock, in which: (i) crenation occurs essentially instantaneously upon immersion
of erythrocytes in a concentrated cryoprotector solution,
followed by slow membrane penetration of the cryoprotector over a specific period Dt, and (ii) hemolysis occurs
upon subsequent removal of the erythrocytes from the
concentrated cryoprotector solution and immersion in saline if Dt was long enough for so much cryoprotector to
have entered that the final osmotically equilibrated cell volume exceeds the mechanical threshold of the membrane.
Classical approaches rely on a separate experiment to determine the “critical” threshold volume at which 50 percent
of erythrocytes undergo hemolysis. We instead introduce
a probability distribution for cumulative percentage hemolysis as a function of cell volume with an adjustable width
parameter. Both the cryoprotector permeability coefficient and the width of the distribution are determined in
a self-contained manner from best fits to osmotic shock
data alone expressed in terms of percentage hemolysis as
a function of permeation time Dt. We find that the initial
crenation apparently renders erythrocytes more fragile and
makes the critical volume applicable to the actual osmotic
shock experiment smaller than usually determined separately. The new, more self-consistent data analysis yields
permeablities smaller than classical determinations by up
to an order of magnitude. We demonstrate our procedure
with reference to glycerol and DMSO membrane permeability for bovine and equine erythrocytes at various temperatures between 0 and 37oC. Mechanistic conclusions
are suggested by comparing our results with a detailed new
critical assessment of the intrinsic permeability properties
of bilayer membranes.
Long-term storage of bone marrow cells (BMC) has remained an actual problem in contemporary cryobiology
and transplantology. The methods for BMC cryopreservation of any animals have not been developed yet. However,
the low temperature conservation of BMC of domestic
animals are the necessary stage of biomaterial storage before transplantation to treat diseases connected with haemopoesis disorder.
The aim of the investigation was to study the properties
and to work out the cryopreservation methods for dogs’
BMC. The task was to study the efficacy of cryoprotectants (glycerol, Me2SO, PEG m.m.400) during dogs’ BMC
freezing.
Incubation of dogs’ BMC at low temperature (4oC) with
cryoprotective solutions during 30 minutes reduces the viability not so much. Glycerol in all used concentrations was
non effective for freezing. Cells cryopreserved with PEO400 had better viability rate and Me2SO was the most effective among cryoprotectors used. 7% Me2SO solution had a
better effect on almost all types of dogs’ BMCs.
On the basis of the results obtained we can make the following conclusions: dogs’ BMC cryopreservation with
PEO-400 and Me2SO saves cells in the cryopreservation
process. Me2SO is a more effective cryoprotertant, as the
cell content remains on proper level in cells influenced by
the influence of incubation and freezing-thawing.
The 7% Me2SO occurred to be the most efficient among
the used cryoprotectants solutions. Indeed, the long-term
storage of dogs’ bone marrow cells is one of the biggest
challenges faced by researchers working in the field of cell
transplantation in veterinary and the driving force for continuing research that aims to extend cryopreservation technology.
Session 7: Protein structure and function
Lecture
L7.2
L7.1
Hsp100 proteins — disaggregating
chaperone machines
CG methylation and depletion
Marek Wojciechowski,
Honorata Czapinska, Matthias Bochtler
International Institute of Molecular and Cell Biology (IIMCB), Warsaw,
Poland
e-mail: Matthias Bochtler <[email protected]>
DNA methylation occurs in prokaryotes and eukaryotes,
but in different forms and with different functions. In
prokaryotes, methylation is very diverse. Mechanistically,
the modification can affect the N4 or C5 of cytosine or N6
of adenine, in a wide variety of sequence contexts. Functionally, DNA methylation plays a role in restriction-modification systems for genome defense, in DNA repair for
the distinction of parental and daughter strand, and also
in the control of bacterial lifestyle. Some features of DNA
methylation are conserved in primitive eukaryotes, but in
higher eukaryotes, particularly vertebrates, methylation is
predominantly reduced to the C5 methylation of cytosine
in a single sequence context (CG, or more traditionally
CpG). Its main role is the epigenetic control of DNA state,
in crosstalk with appropriate histone modifications. For eukaryotic organisms that feature pronounced CG methylation, it is known that this sequence is not only important,
but also rare: in fact, the actual number of CGs is about
fourfold lower than statistically expected. In my talk, I will
discuss the mechanistic connection between methylation
and the depletion of target sequences and I will address the
generality of the link across the kingdoms of life.
Krzysztof Liberek1, Szymon Ziętkiewicz1, Natalia Litwińczuk1,
Alicja Kochanek1, Ewa Morawiec1, Wojciech Potocki1,
Magdalena Ślusarz2, Sylwia Rodziewicz-Motowidło2
1Department of Molecular and Cellular Biology, Intercollegiate Faculty
of Biotechnology, and 2Faculty of Chemistry, University of Gdańsk,
Gdańsk, Poland
e-mail: Krzysztof Liberek <[email protected]>
The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in
the cell. Many factors leading to unfolding and misfolding
of proteins eventually result in protein aggregation. Stress
imposed by high temperature was one of the first aggregation-inducing factors studied, and remains one of the main
models in this field. With protein aggregation occurring in
response to heat exposure, the cell needs chaperones to
control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides
or their proteolysis. Here we focus on the molecular mechanisms by which Hsp100 chaperones in cooperation with
Hsp70, Hsp40 and small Hsps chaperones liberate and refold polypeptides trapped in protein aggregates.
Analysis of homology models obtained by us for Hsp100
chaperones (Hsp104 from S. cerevisiae and ClpB from
E. coli) suggests the presence of characteristic network
of ionic interactions between the first ATPase domain
(NBD1) and the coiled-coil M subdomain that is characteristic for disaggregating chaperones. To test the importance of this structural aspect we have designed a series of
point mutations. Mutants intended to disrupt ionic interactions resulted either in inactive proteins or in proteins that
display unexpected biochemical properties. These include
increase of ATPase activity, significant increase of in vitro
substrate renaturation rate and partial independence on
Hsp70 chaperone in refolding process. The increased activities paradoxically result in serious growth impediment and
rapid loss of mutant Hsp104-encoding plasmids in yeast
cells. We were able to suppress those effects by introducing
complementary changes that restore particular salt bridges
within suggested network. Therefore we propose the presence of novel structural aspect of disaggregating Hsp100’s
that is responsible for regulation of the disaggregating/
refolding activity.
L7.3
Oral presentations
Structural aspects of PR10 protein
— ligand interactions
O7.1
Mariusz Jaskolski
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz
University and Center for Biocrystallographic Research, Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
53
The effect of macromolecular crowding on
DNA replication: A structure-function study
Barak Akabayov, Charles C. Richardson
e-mail: Mariusz Jaskolski <[email protected]>
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts, USA
Pathogenesis-related proteins of class 10 (PR10) are
uniquely found in plants where they are associated with
stress-related processes. Despite their abundance and existence in many variants, their precise biochemical role remains unknown. However, their structure is well studied
and defines a canonical PR10 fold consisting of a sevenstranded antiparallel β-sheet gripped over a long C-terminal α-helix, with a huge cavity (up to 4500 Å3) enclosed
between these main structural elements. On the other
hand, we have shown that plant proteins identified as specific binders of plant hormones from the cytokinin group
(CSBP) belong to the same structural class despite very low
sequence similarity. Cytokinins regulate plant development
and senescence processes and are also involved in plant response to stress. We were able to show that trans-zeatin, a
classic cytokinin hormone, is bound in the internal cavity
of mung bean CSBP not in one but in at least three different, highly specific ways. Following this discovery, we have
been able to show that also classic PR10 proteins (from yellow lupine) can bind both natural and synthetic cytokinin
hormones. Others have shown that the proteins are capable
of binding a wide range of different ligands, including the
non-physiological (in plants) deoxycholate, which however
shows resemblance to plant hormones from the brassinosteroid group. We also found out that a PR10 protein from
St John’s wort is most likely and contrary to expectations
not a hypericin-synthesizing enzyme but that it can bind
in its internal cavity such exotic ligands as PEG molecules.
Recently we have made the most puzzling discovery, showing that CSBP from lucerne can bind with very high specificity a completely unrelated hormone, gibberellic acid, from
a diterpenoid group. Gibberellins are extremely important
plant hormones regulating processes of cell growth and
division. We have thus a very perplexing situation, where
proteins that are regarded to be specific binders of specific
ligands (CSBP) bind also other physiological ligands with
what can be called structural specificity, and that structurally related proteins with unknown function bind a broad
range of diversified ligands. These observations force us
to rethink the notion of molecular recognition between a
ligand and its binding protein.
e-mail: Barak Akabayov <[email protected]>
Enzymatic activities in vivo occur in a highly crowded and
confined environment mainly composed of protein and
nucleic acid macromolecules. A crowded environment:
1) enhances the apparent concentration of the measured
macromolecule, 2) leads to desolvation of water molecules
around the proteins, 3) decrease degrees of freedom for
diffusion and molecular hopping of proteins on their DNA
substrates, and 4) enhances binding equilibria and catalytic
activities of those macromolecules. However, the effect of
macromolecular crowding on the structure of the proteins
involved is poorly understood. We have characterized the
effect of molecular crowding on DNA replication using
the replication system of bacteriophage T7 as a model system. We probe the structural effects that are involved in a
crowded environment using small angle X-ray scattering.
Our results show the consequences of macromolecular
crowding on DNA replication mediated by the T7 replisome.
Abstracts
54
O7.2
Posters
Changes in human ednothelial cell proteom
caused by N-homocysteinylated proteins
P7.1
Joanna Suszynska-Zajczyk1, Magdalena Łuczak1,
Łukasz Marczak1, Edyta Gendaszewska-Darmach2, Tomasz Twardowski1, Hieronim Jakubowski1,3
1Institute
of Bioorganic Chemistry, Polish Academy of Sciences,
Poznan, Poland; 2Institute of Technical Biochemistry, Faculty of
Biotechnology and Food Sciences, Technical University of Lodz,
Lodz, Poland; 3University of Medicine and Dentristy, Department of
Microbiology and Molecular Genetics, International Center for Public
Health, Newark, NJ, USA
e-mail: Joanna Suszyńska-Zajczyk <[email protected]>
Homocysteine (Hcy) is a focus of scientific inquiry in the
context of human diseases. Elevated plasma Hcy, termed
hyperhomocysteinemia, caused by genetic defects in Hcy
metabolism, leads to pathological changes in many organs
and premature death due to cardiovascular complications
[1]. Hcy is formed from dietary protein methionine as a
by-product of biological methylation reactions. If not
removed by transsulfurattion and transmethylation reactions, Hcy becomes toxic to human cells. A mechanism
involving the conversion to Hcy-thiolactone (HTL) in the
error editing pathway catalyzed by methionyl-tRNA synthetase [2] and indirect incorporation into protein by a
post-translational modification of protein lysine residues
(N-homocysteinylation) [3] contributes to Hcy toxicity.
Protein N-homocysteinylation affects protein structure
and function [3], causes cellular toxicity, elicits autoimmune
response leading to the generation of antibodies against
N-Hcy-proteins[4], and prothrombotic N-Hcy-fibrinogen
[5]. To determine how HTL affects human endothelium,
we studied cultured human umbilical vein endothelial cells
(HUVECs), which are frequently used as a model of vascular cells and are known to efficiently metabolize Hcy to
HTL and generate N-homocysteinylated proteins (N-Hcyprotein). Protein extracts were analyzed using 2-dimensional IEF/SDS/PAGE electrophoresis, followed by MALDITOF analysis. In HUVECs treated with HTL (0.5-5mM)
for 24h, we found 24 differential proteins. Expression of
most of them decreased and only expression of vimentin, UCHL1 and TXNDC5 protein increased. In HUVECs
incubated with N-Hcy-FBS we recognised 34 differential
proteins. We observed changes in the levels of protein expression dependent on the extent of FBS modification.
After incubation with N-Hcy-FBS modified in the highest
extent, the protein expression level was similar to the control. Chlorine intracellular channel protein I and dnaK-type
molecular chaperone demonstrated other pattern. Many of
the proteins whose expression levels were affected by HTL
or N-Hcy-FBS are known to play role in an apoptosis. This
result suggest high toxicity of the protein N-homocysteinylation to the endothelium.
References
1. Mudd SH et al. (2001) The metabolic and molecular bases of inherited disease,
8th ed. New York: McGraw Hill, 2007-2056.
2. Jakubowski H (1997) J Biol Chem 272: 1935-1942.
3. Jakubowski H (1999) FASEB J 13: 2277-2283.
4. Undas A et al. (2004) Stroke 35: 299-304.
5. Jakubowski H (2008) J Physiol Pharmacol 59 (Suppl 9): 155-167.
Characterization of the interaction
between Hsp70’s SBD and a ligand
Itzhaq Azoulay, Esther Nachliel,
Yossi Tsfadia, Abdussalam Azem
Tel Aviv University, Department of Biochemistry, Tel Aviv, Israel
e-mail: Itzhaq Azoulay <[email protected]>
Molecular chaperones of the 70 kDa Heat Shock Protein
family (HSP70) are present in all organisms, and have an
essential roles in a wide range of inter cellular functions
such as folding of nascent proteins targeting into different
compartments of the cell and preventing their aggregation,
these actions are coupled with ATP binding and hydrolysis. Its two functional domains are the 44 kDa Nucleotide
Binding Domain (NBD), which responsible for the binding
of the ATP and its hydrolysis, and the 26 kDa Substrate
Binding Domain (SBD) is responsible for the binding and
the releasing of polypeptide chains. In the ATP state the
peptide substrate exhibits low affinity to the HSP70, while
in the ADP state the substrate has high affinity to the SBD.
The chosen model in this study was the Escherichia coli
Hsp70, the DnaK, focusing on the SBD domain (PDB entry 1dkx). The structure consists of two sub domains: a
β sandwich domain containing the hydrophobic cavity to
which the NRLLLTG heptapeptide is bound, and a α helical segments followed after, that sit on top of it, and also
known as lid.
By using Molecular dynamics we have characterized the
properties of the interaction of the peptides with the protein. Several simulations with different initial conditions of
the SBD complex were carried out, in order to investigate
the effect of electrical charge upon binding, and determine
the lid’s role of the binding.
Our interesting findings would be shown in the poster.
P7.2
P7.3
Protein and carbohydrate composition of
the exopolymeric matrix as the main factor
in the thionic bacteria biofilm formation
Life and death of proteins destined to the
intermembrane space of mitochondria
Mariia Boretska, Andryi Ostapchuk, Irina Kozlova
Zabolotny Institute of the NAS of Ukraine, Ukraine
e-mail: Maria Boretska <[email protected]>
Microbial populations on the solid surfaces are capable to
form highly organized structures — biofilms. Major role in
these biofilms has the exopolymer matrix — the polymer
structure in which cells are immersed. Studying the biofilms formed by corrosion bacteria is needed to develop
ways to protect large-scale metal structures as the primary
site of microbial influence corrosion. The objective of
our study was to investigate the composition of exopolymer complex and structural features of biofilm formatted
by acidophobic bacteria Thiobacillus thioparus on the mild
steel surface. We used an Albersheim method for allocating monosaccharide composition (analysis was performed
on GC-MS system Agilent 6890N/5973 inert), Bradford
method, the method of confocal laser scanning microscopy (LSM Pascal 5, Carl Zeiss, Germany). The greatest
variety of monosaccharide composition of the samples is
presented in association matrix biofilms (rhamnose, xylose,
ribose and galactose were present only in samples of biofilms associated culture) were showed that. To visualize the
carbohydrate part of the matrix was chosen lectin ConA +
FITC (Sigma) is specific to glucose (the predominant monosaccharide), for cells visualization — DAPI (Sigma). The
biofilm formed by the associative culture in an average of
40 mkm thick, the surface is more uniform in comparison
with the biofilm’s monocultures it was shown. Localization
of cells for both mono- and for the associative cultures
was above matrix, the localization of carbohydrates - from
the bottom. Specific production of exopolymers associated
culture was 2.5 times more than for the monoculture. The
study of the matrix protein composition of mono- and associated cultures thionic bacteria showed that the method
of Bradford supported by microscopic observations (Thiazine Red was used to visualize the protein components of
the matrix). It is shown the number of protein components
of associative biofilms more than in monoculture (almost
than 20 times), the location of protein structures has been
mainly in the center of the matrix as one of main factor of
creating of biofilm’s structure. Obviously, associated culture is able to produce more exopolymers and its characterization such as quantity and localization of the proteins
and carbohydrates provides a powerful, dangerous and corrosive biofilms, formed by acidophobic thiobacteria.
55
Piotr Bragoszewski, Agnieszka Gornicka,
Agnieszka Chacinska
International Institute of Molecular and Cell Biology in
Warsaw, Laboratory of Mitochondrial Biogenesis, Warsaw, Poland
e-mail: Piotr Bragoszewski <[email protected]>
Nearly all mitochondrial proteins are synthesized in the cytosol and translocated into the organelle. Many of them
have signal presequences that are proteolytically removed
by specific peptidases upon their selective import into one
of mitochondrial compartments: the matrix, the inner
membrane or the intermembrane space (IMS). Also various types of non-cleavable targeting and sorting signals exist. This variety of signals is matched by the specific import
pathways and translocases that recognize the signals and
direct the precursor proteins into a final destination within
mitochondria. Many IMS proteins share a coiled coil-helixcoiled coil-helix (CHCH) domain, which is stabilized by the
formation of disulfide bonds within characteristic cysteine
motifs. Upon synthesis and release to the cytoplasm these
proteins stay reduced. Once the IMS precursor proteins
enter the IMS via the protein channel formed by the translocase of the outer mitochondrial membrane TOM, they
are recognized and oxidized by the mitochondrial IMS import and assembly machinery MIA. The majority of the
cysteine-containing proteins are relatively small, typically
smaller than 20 kDa. It is likely that such small proteins can
traverse freely through the TOM complex (or other pores)
in the outer membrane of mitochondria. Subsequently, the
oxidative folding catalyzed by MIA is required to trap them
in the IMS. In our project, we aim to understand the fate
of IMS proteins under the conditions of oxidative folding restrictions. The conditional mutants of two essential
components, Mia40 and Erv1, do not accumulate the precursor proteins in mitochondria that results in their death
under restrictive conditions. We undertook a systematic
analysis of the MIA substrate proteins to categorize them
into two major classes: the proteins found in the cytosol or
destroyed under the conditions of oxidative folding failure.
This study forms a basis for the identification of mechanisms involved in the biogenesis of mitochondrial proteins
and their influence on cellular protein homeostasis.
Abstracts
56
P7.4
P7.5
Antistreptokinase antibodies prevent
inhibition of platelet reactivity
Adaptor protein Ruk/cin85 regulates
NGF-dependent neuronal differentiation
Nataliia Burlova-Vasilieva,
Oleksiy Savchuk, Ludmila Ostapchenko
Nadiya Byts, Lyudmyla Drobot
Educational and Scientific Centre “Institute of Biology” of National
Taras Shevchenko University of Kyiv, Department of Biochemistry, Kyiv,
Ukraine
e-mail: Nataliia Burlova <[email protected]>
Objective: to investigate platelet reactivity under thrombolytic theraphy.
Methods: ADP-inducible platelet aggregation, anti-Sk
(streptokinase) antibodies and plasminogen activators inhibitor type 1 (PAI-1) were determined by light transmission aggregometry Elisa and Western Blot respectively.
Results: Sk and heparin inhibited АDP-inducible platelet aggregation by 30% in AMI patients. Next day after
heparin canceling the platelet aggregation returned to the
initial high level. PAI-1 activity in blood plasma of AMI
patients exceeded the rate of relatively healthy donors by
200%. Heparin canceling didn’t prevent normalization of
inhibitor activity.
Anti-Sk antibodies were not detected in rabbit study population before and 7 days after Sk administration. Sk infusion increased PAI-1 level in rabbit platelet fraction from
41±5 ng/ml (baseline) to 95.8±7.4 ng/ml. ADP-inducible
platelet aggregation in rabbit platelet rich plasma did not
increase the baseline level and was inhibited by Sk in dosedependent manner. Sk in a concentration of 16 mkg/ml
was associated with significantly reduced aggregation rate.
ADP-inducible platelet aggregation peaked at 7 weeks after
intravenous administration of Sk and approached 81±5%,
considerably exceeding the baseline value (46±7%). The
titers of anti-Sk antibody were 1:100. Inhibition effect of
Sk on ADP-dependent platelet aggregation was not observed. Sk in concentration of 16 mkg/ml and 32 mkg/ml
caused insignificant increment of aggregation rate. Platelet
fraction treated with Sk released free and complex forms
of PAI-1.
Conclusion: We suggest that Sk influence platelet reactivity not only through plasmin and Sk-antibody complex
formation, but also interacting with Anti-Sk antibodies attached to the platelet surface.
Palladin Institute of Biochemistry, Laboratory of Cell Signalling, Kyiv,
Ukraine
e-mail: Nadiya Byts <[email protected]>
Despite extensive investigations of Cbl-interacting protein of 85 kDa (Ruk/CIN85) in receptor trafficking and
cytoskeletal dynamics, little is known about its functions in
central nervous system. Previous studies showed that the
specific isoforms of adaptor protein Ruk/CIN85 are expressed in central nervous system and reported their function in dopamine receptor endocytosis in striatal neurons.
Here we investigated the role of Ruk/CIN85 in NGF-induced differentiation and neurite outgrowth. PC12 rat pheochromocytoma cells were used as a model. We found that
upon chronic NGF treatment the pattern of Ruk/CIN85
isoforms expression is changed in wild type PC12 cells as
well as in PC12 cells stably overexpressing GFP-tagged
Ruk/CIN85. While the levels of Ruk/CIN85 150 kDa
isoform were increased, the middle 56 kDa isoform was
downregulated and no changes in full-length 85 kDa isoform levels were observed. It is known that the activation
of NGF/TrkA pathway leads to the transient expression
of inhibitor of differentiation (ID1) gene, which is necessary for axonal elongation of immature neuron. In PC12
cells that overexpress Ruk/CIN85 the elevation of ID1
upon NGF treatment was almost undetectable. Moreover,
the overexpression of GFP-tagged Ruk/CIN85 in PC12
cells inhibited NGF-induced neurite outgrowth. In conclusion, our data indicate that adaptor protein Ruk/CIN85 is
involved in neuronal differentiation and NGF-dependent
signalling.
57
P7.6
P7.7
Differential ubiquitination and
degradation of intersectin 1 isoforms
Shb scaffold mediates interaction between
adaptor ITSN1 and viral protein LMP2A
Mykola Dergai, Oleksandr Dergai, Olga Novokhatska,
Liudmila Tsyba, Inessa Skrypkina, Alla Rynditch
Oleksandr Dergai1 , Mykola Dergai1 ,
Inessa Skrypkina1, Liudmyla Matskova2,
Liudmyla Tsyba1, Gosta Winberg2, Alla Rynditch1
Institute of Molecular Biology and Genetics, NASU, Kiev, Ukraine
e-mail: Mykola Dergai <[email protected]>
Intersectin 1 is an evolutionarily conserved adaptor protein implicated in endocytosis, exocytosis, propagation
and attenuation of mitogenic signal and cell survival. Its
role also was shown in nervous system functioning and
development. Possible role of ITSN1 in Alzheimer and
Huntington`s diseases and Down syndrome was demonstrated. Despite intense study of ITSN1 nothing is known
about posttranslational regulation of this adaptor protein.
Here we present investigation of ubiquitination of ITSN1
isoforms. We studied major intersectin isoform in nonneuronl cells — ITSN1-s and recently identified shortest
known isoform ITSN1-22a. It was shown that ITSN1-s
undergoes monoubiquitination whereas ITSN1-22a is multiply ubiquitinated. Ubiquitination of ITSN1 isoforms is
dependent on clathrin-mediated endocytosis and is not
affected by mitogen starvation/stimulation. AIP4 ubiquitin ligase provides posttranslational regulation of ITSN1
isoforms. Moreover, another ubiquitin ligase Cbl modifies
ITSN1-22a additionally. It is noteworthy that ubiquitin
ligase activity of Cbl impact on association of ITSN1-s and
ITSN1-22a isoforms in vivo. ITSN1-22a isoform undergoes
combined mode of ubiquitination. It is polyubiquitinated
on its specific C-terminal domain (CTD) and is monoubiquitinated on the N-terminal part common for ITSN1-s
and ITSN1-22a isoforms. Polyubiquitination results in instability of ITSN1-22a in vivo in comparison to ITSN1-s
and causes proteosomal degradation of ITSN1-22a. Furthermore, overexpression of ITSN1-22a causes enhanced
degradation of ITSN1-s in HEK293 cells. Thus, ITSN1
isoforms are differentially ubiquitinated and degraded.
1Institute of Molecular Biology and Genetics NAS of Ukraine, Kiev,
Ukraine; 2Karolinska Institute, Stockholm, Sweden
e-mail: Oleksandr Dergai <[email protected]>
Epstein-Barr virus (EBV) is a member of the herpesvirus
familyp. EBV is associated with a number of human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma
and the epithelial cell malignancy nasopharyngeal carcinoma (NPC). Only restricted set of viral genes is expressed
within latent phase: LMP1, LMP2A, LMP2B, EBNAs and
EBERs. Latent membrane proteins are key players in infected cells transformation. But little is known about mechanism governing internalization of LMP2A from plasma
membrane and trafficking through cells compartment. The
aim of current work is to identify protein-protein interaction that allows latent membrane proteins to get access to
the host endocytic machinery. Here we report interaction
between viral protein LMP2A and endocytic adaptor intersectin 1. Our immunoprecipitation data evidenced about
complex formation between LMP2A and ITSN1 in vivo
in different cell types. SH3-domains of ITSN1 were sufficient to precipitate LMP2A in vitro. Mutational analysis
of LMP2A was undertaken to map sites of interaction
with ITSN1. Interaction between LMP2A and ITSN1
was found to be a superposition of interaction of SH3domains of ITSN1 with -PXXP- motives of LMP2A and
tyrosin-phosphorylated ITAM-motives of LMP2A with
SH2-domain of adaptor protein Shb that binds simultaneously ITSN1 and LMP2A. Shb was shown to be phosphorylated and activated by Syk kinase in this complex. Moreover it was found negative cooperativity of Syk engagement
to complex of ITSN1 and LMP2A. Current findings provide new data about LMP2A-driving signalosome assembly
and functioning.
Abstracts
58
P7.8
P7.9
Expression, purification and evaluation
ATPase activity of four essential
tellurite resistance proteins
SCA14 mutations affect the intramolecular clamp between the c1 domain
and carboxyl-terminus of pkc-gamma
Vinh Phu Hoang, Silvia Vávrová,
Lenka Valkovičová, Mahesh Madyagol, Jan Turňa
Justyna Jezierska1, Joachim Goedhart2,
Jan T. Westerink3, Eric A. Reits3, Dineke S. Verbeek1
Department of Molecular Biology, Faculty of Natural Sciences,
Comenius University in Bratislava, Mlynská dolina, 842 15 Bratislava,
Slovak Republic
1Department of Genetics, University Medical Center Groningen,
Groningen, Netherlands; 2Swammerdam Institute for Life Sciences,
University of Amsterdam, Amsterdam, Netherlands; 3Department of
Cell Biology and Histology, Academic Medical Center, University of
Amsterdam, Amsterdam, Netherlands
e-mail: Vinh Phu Hoang <[email protected]>
This work is focused on many years of tellurite operon
study in clinical strain Escherichia coli KL53. The main objective of our work is to study tellurite operon at the the
protein level. In our previous work we studied this at the
DNA level. We created a precise bioinformatics analysis
of all essential Ter proteins (TerBCDE) to find their cell
localization. We focused mainly on protein TerC as only
putative trans membrane protein with regard to analysis of
its membrane segments. The TerC protein has been predicted as innermembrane protein with 9 transmembrane
domains. The BLAST search of this protein sequence
showed that 80–85% of homology was assessed with the
H(+)-transporting ATPase Lactobacillus johnsonii FI9785,
cation-transporting ATPase Lactobacillus johnsonii ATCC
33200 and sodium/proton antiporter, CPA1 family Sulfurimonas autotrophica DSM 16294. We prepared recombinant
plasmids harboring cloned ter genes to express and isolate
proteins. We found out that C terminus of these proteins
is crucial to maintain their original function. We also determined ATPase activity of Ter proteins. Activity of purified
TerC protein was 30 times higher than ATPase activity in
cell lysate of E. coli K12 and 65 times higher than activity in
B. subtilis and 51 times higher than in Pseudomonas FH2. We
also revealed ATPase activity of TerB protein which was
2.4 times lower than ATPase activity of TerC. To compare
with ATPase activity in E. coli K12 it is 12 times higher and
27 times higher than B. subtilis and 21 times higher than in
Pseudomonas FH2. Proteins TerDE have no ATPase activity.
e-mail: Justyna Jezierska <[email protected]>
Spinocerebellar ataxia type 14 (SCA14) is an autosomal
dominant neurodegenerative disease, caused by mutations
in PRKCG encoding for protein kinase C gamma (PKCγ).
Most of the known mutations are missense mutations and
are concentrated in the diacylglycerol (DAG)-binding C1B
subdomain, which is important for PKCγ protein translocation and proper function. PKCγ is abundantly expressed
in Purkinje cells (PCs) and has been shown to regulate the
morphological development of PCs and motor learning
functions and participates in various cell functions, including cell growth, differentiation and maintaining oxidative
homeostasis. PKCγ undergoes a complicated activation
cycle to be able to perform its kinase function. In order
to effectively transduce extracellular signals, PKCγ proteins must be properly primed and positioned for optimal
downstream signalling. Perturbations in their phosphorylation state, conformation or localization can disrupt kinase
signalling and lead to altered physiological states, such as
ataxia. We previously showed that SCA14 mutations in the
C1B subdomain of PKCγ enhance translocation to the cell
membrane upon phorbol ester stimulation due to an open
protein conformation. However, the increased translocation kinetics did not result in an increased kinase activity,
but the mutant PKCγ proteins showed decreased kinase
function causing aberrant MAPK signalling. Our recent
findings reveal that SCA14 mutant PKCγ has an open Cterminus, which causes the enhanced accessibility of C1B
for DAG. As a consequence, the C-terminus of mutant
PKCγ was shown to bind more efficiently to the kinase
PDK1. The mutant PKCγ proteins are not efficiently degraded, and therefore subsequently accumulate in the insoluble fraction of cells. In conclusion, we show that SCA14
mutations affect the intra-molecular clamp between the C1
domain and the C-terminus of PKCγ leading to altered
protein conformation affecting its degradation.
P7.10
P7.11
Calcium binding protein S100A6
(calcyclin) in Wharton’s jelly –
localization and interacting partners
Does the lack of GRHL1 activity increase
the chance of skin cancer development?
Ewelina Jurewicz1, Irena Kasacka2,
Edward Bańkowski2, Anna Filipek1
1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Medical
University of Bialystok, Białystok, Poland
e-mail: Ewelina Jurewicz <[email protected]>
Wharton’s jelly, a connective tissue in the umbilical cord, is
composed of high amount of extracellular matrix and of
a very low number of cells (myofibroblasts mainly) (Ferguson & Dodson, 2009). The mechanical properties of
Wharton’s jelly, mainly elasticity, protect the umbilical vessels from compression and absence of this tissue around
the umbilical arteries is associated with fetal death (Kulkarni et al., 2007).
The S100A6 protein binds calcium ions and belongs to
the S100 protein family (Leśniak et al., 2009). Originally
S100A6 was isolated from Ehrlich ascites tumor cells in our
laboratory (Kuźnicki & Filipek, 1987). Under normal conditions high expression of S100A6 is found in fibroblasts
and epithelial cells (Kuźnicki et al., 1992) and interestingly
its expression is characteristic for tissues which are specific
for pregnancy such as deciduas (Thordarson et al., 1991).
In the present work we show that S100A6 is present in
human umbilical cord. The use of Phenyl-Sepharose chromatography served to obtain a fraction enriched in S100A6
in relation to the bulk of proteins contained in this tissue.
Further investigation revealed that S100A6 was present
both in the walls of the umbilical cord vessels (artery and
vein) and in the surrounding Wharton’s jelly. Immunohistochemical studies confirmed the presence of S100A6 in the
umbilical cord. An intensive immunoreaction for S100A6
was observed in Wharton’s jelly, both in the cytoplasm
of myofibroblasts and in the extracellular matrix. Affinity
chromatography performed on S100A6 resin indicated that
Wharton’s jelly contains some proteins which could bind to
S100A6. Thus, our results might suggest the involvement
of the S100A6 protein in intra- and extracellular signaling
pathways in this tissue.
Acknowledgements
This work was supported by grant N N301 293637 from the Ministry of
Science and Higher Education of Poland to A.F. and by statutory funds
from the Nencki Institute of Experimental Biology.
References
Ferguson VL, Dodson RB (2009) Eur J Obstet Gynecol Reprod Biol 144 (Suppl 1): S108-S113.
Kulkarni ML, Matadh SP, Ashok C, Pradeep N, Avinash T, Kulkarni MA
(2007) Ind J Ped 74: 787-789.
Kuźnicki J, Filipek A (1987) Biochem J 247: 663-667.
Kuźnicki J, Kordowska J, Puzianowska M, Woźniewicz BM (1992) Exp
Cell Res 200: 425-430.
Leśniak W, Słomnicki PŁ, Filipek A (2009) Biochem Biophys Res Commun
390: 1087-1092.
Thordarson G, Southard JN, Talamantes F (1991) Endocrinology 129: 12571265. 59
Agnieszka Kikulska, Michał Mlącki, Tomasz Wilanowski
Nencki Institute of Experimental Biology PAS, Laboratory of Signal
Transduction, Warsaw, Poland
e-mail: Agnieszka Kikulska <[email protected]>
Grainyhead-like (GRHL) family consists of highly conserved homologs of Drosophila Grainyhead (GRH) transcription factor. In mice, GRHL3 maintains the integrity
of skin barrier formation through the regulation of transglutaminase 1. In Drosophila melanogaster, GRH controls the
expression of DOPA decarboxylase which serves analogous function preserving the integrity of fly cuticle. Given the conserved role of GRHL in maintaining epithelial
integrity, it is noteworthy that the evolutionary origin of
GRHL family appears coincident with evolutionary origin
of the epithelium. GRHL1 is normally active in the developing epidermis and hair follicles. Mice lacking this factor
display an abnormal hair coat (defective hair anchoring)
and reduced expression of desmoglein 1, a member of the
desmosomal cadherin family and a direct target of GRHL1
regulation.
It is still uncertain whether GRHL1 is involved in skin
cancer development induced by UV radiation and what is
the role of GRHL1 in skin barrier formation. The main
goal of my research is to investigate skin cancer formation
in Grhl1-deficient mice upon exposure to UV radiation.
Experiments are performed on mice with three different
Grhl1 genotypes: wild type, heterozygous and null. Grhl1
knock-out mice were generated in the Royal Melbourne
Hospital (Australia) using genetic engineering methods.
These animals were provided to us under a Material Transfer Agreement.
Consensus DNA binding sequence of GRHL1 transcription factor is already known, which makes it possible to
predict its putative targets genes that are linked to skin cancer development. My research is likely to discover novel
signal transduction pathways that are relevant to skin cancer formation. I will utilize various molecular biology, histological and bioinformatics methods.
References
Wilanowski T et al. (2008) EMBO J 27: 886-897.
Auden A. et al. (2006) Gene Expr Patterns 6: 964-970.
Ting SB. et al. (2005) Science 308: 411-413.
Boglev Y et al. (2011) Dev Biol 349: 512-522.
Wilanowski T et al. (2002) Mech Dev 114: 37-50. Abstracts
60
P7.12
P7.13
ERK1/2 is dephosphorylated by a
novel phosphatase — CacyBP/SIP
Soluble form of heparin-binding EGFlike growth factor promotes EGF
receptor intracellular traffic distinct
from the lysosomal degradation
and receptor recycling
Ewa Kilanczyk1, Slawomir Filipek2, Anna Filipek1
1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Faculty of
Chemistry, Warsaw University, Warsaw, Poland
e-mail: Ewa Kilanczyk <[email protected]>
ERK1/2 kinase-dependent signaling pathways are involved
in diverse cellular functions including gene expression,
proliferation, differentiation etc. The activity of ERK1/2
kinase is regulated by phosphatases, among them there is
MKP-3 phosphatase the association of which with ERK2
is highly specific and results in down-regulation of ERK2
kinase activity. Recently we have found that ERK1/2 binds
to the CacyBP/SIP protein (Kilanczyk et al., 2009) and in
this work we examined whether CacyBP/SIP might demonstrate phosphatase activity toward ERK1/2 kinase. We
have found that CacyBP/SIP indeed dephosphorylates
ERK1/2. The Km and Vmax values established for a standard phosphatase substrate, p-NPP, are 16.9±3.6 mM and
4.3±0.4 μmol/min, respectively. The CacyBP/SIP phosphatase activity was decreased by okadaic acid (IC50 =
45 nM). These experimental results were supported by a
theoretical analysis which revealed important sequence
similarities between CacyBP/SIP and the phosphatase-like
proteins as well as certain MAP kinase phosphatases. We
also found that decreased ERK1/2 activity in the nuclear
fraction of NB2a cells after CacyBP/SIP overexpression
results in a lower level of β-catenin which suggests that
CacyBP/SIP, through dephosphorylation of ERK1/2, may
affect β-catenin degradation and cell differentiation. Altogether our results show that CacyBP/SIP, as a phosphatase,
might play a role as a new negative modulator of ERK1/2
kinase and that, in consequence, this protein might be implicated in differentiation of NB2a cells.
Acknowledgements
This work was supported by grants: N N401 057837 to E.K. from the
Ministry of Science and Higher Education of Poland and by statutory
funds from the Nencki Institute of Experimental Biology.
Natalia Korotkevich, Andrii Labyntsev, Alla
Honcharenko, Denis Kolibo, Serhiy Komisarenko
Palladin Institute of Biochemistry of the NASU, Molecular Immunology
Department, Ukraine
e-mail: Natalia Korotkevich <[email protected]>
Heparin-binding EGF-like growth factor (HB-EGF) is a
member of the EGF family growth factors. It has high affinity for heparin and heparan sulfate. HB-EGF precursor
is synthesized as type I transmembrane protein (pro-HBEGF), which is cleaved by proteases resulting in shedding of soluble HB-EGF (sHB-EGF). sHB-EGF acts as
mitogenic signal through the EGF receptor (EGFR). The
ligand binding to EGFR results in EGFR activation and recycling or further lysosomal degradation of ligand-receptor
complex. On the other hand, EGF binding to EGFR could
promote EGFR nuclear translocation. In nucleus EGFR
acts as transcription factor able to activate genes required
for highly proliferating activities. But, the probable role of
HB-EGF as a ligand of EGFR in this process is still unclear. HB-EGF has some peculiar properties comparing to
other EGF family members. HB-EGF belongs to a heparin
binding ligands, which implies that proteoglycans may also
be involved in translocation complex. In addition, some
other important questions remain to be answered, e.g. does
the nuclear translocation complex consist of both ligand
and receptor, or EGFR is active on its own, and whether
EGFR kinase activity is required? In this work we focused
on the investigation of the ability of sHB-EGF to induce
EGFR nuclear importation.
We have demonstrated that sHB-EGF induced EGFR internalization and its further intracellular importation into
the Golgi apparatus and endoplasmic reticulum via retrograde movement. Increasing amount of EGFR in the
nuclear fraction of sHB-EGF treated cells was observed,
which implies that sHB-EGF could induce EGFR nuclear
importation. In addition we demonstrated that the translocation complex consisted of both ligand and receptor.
It has been also shown that addition of EGFR kinase
inhibitor — AG1478 dramatically increased EGFR internalization. These data indicate that EGFR kinase plays an
important role in induction of intracellular internalization
process. We found here the new sHB-EGF activity, its ability to induce EGFR nuclear importation. We suggest that
sHB-EGF is an essential element of translocation complex
and is required for maintaining EGFR in active conformation. Parnas Conference Warsaw 2011
P7.14
P7.15
PPAR-gamma modulation
influences EPC functions
Association of phosphoglycerate mutase
with glycolytic complex regulates
energy production in cancer cells
Jerzy Kotlinowski1, Anna Grochot-Przęczek1,
Magdalena Kozakowska1, Ewa Zuba-Surma1,
Rafał Derlacz2,3, Józef Dulak1, Alicja Józkowicz1
61
Wojciech Kowalski1, Dominika Nocoń1,
Andrzej Gamian2, Dariusz Rakus1
1Jagiellonian University, Faculty of Biochemistry, Biophysics and
Biotechnology, Department of Medical Biotechnology, Krakow,
Poland; 2Adamed Ltd, R&D Department, Pieńków, Poland; 3University
of Warsaw, Faculty of Biology, Department of Metabolic Regulation,
Warsaw, Poland
1Department of Animal Molecular Physiology, Wroclaw University,
Wroclaw, Poland; 2Department of Medical Biochemistry, Wroclaw
Medical University, Wroclaw, Poland
e-mail: Jerzy Kotlinowski <[email protected]>
In contrast to normal, differentiated cells, cancer cells rely
on glycolysis, even in the presence of oxygen. It has been
hypothesized, based on in vitro experiments, that the association of the glycolytic enzymes, especially enzymes of
triose phosphate metabolism, in a metabolic complex leads
to efficient degradation of glucose to lactate.
Here we provide a strong evidence that in cancer cells, all
the enzymes of triose phosphate metabolism, from aldolase to pyruvate kinase consecutively, function as a macromolecular complex and that disruption of this complex
inhibits lactate release and ATP synthesis in glycolytic pathway.
Results of our experiments indicate that composition of
the complex and modulation of glycolytic flux is affected
by elevated lactate concentration and by glucose deprivation which regulates association of the phosphoglycerate
mutase (PGAM) C-terminal region with the complex.
High concentration of exogenous lactate not only disrupts
the glycolytic complex and inhibits ATP synthesis, but also
affects nuclear localization of PGAM and ceases cell proliferation.
The studies unraveling the nuclear function of PGAM and
the components of signaling pathway linking energetic metabolism with proliferative machinery are in progress.
Our results open new insight into the role of metabolic
complexes in the cancer cell biology and might provide
new prospects for the cancer disease treatment using lowmolecular weight competitors which destabilize the glycolytic complex.
PPARγ nuclear receptor is a target for thiazolidinediones
(TZD), the commonly used insulin sensitisers. Besides
improving insulin action and normalising glycemia in diabetic patients TZD can also improve some functions of
endothelial progenitor cells (EPC). Although activation of
PPARγ was shown to be beneficial for EPC both in vitro
and in vivo more experiments must be performed to demonstrate pathways involved in this actions. Our aim was to
find expression changes in diabetic and healthy EPC and
check how modulation of PPARγ activity affects EPC biology.
Experiments were performed on the 12-weeks old wild type
(wt) and diabetic (db/db) mice. For in vivo tests EPC were
characterised as the CD45-/KDR+/Sca-1+ cells, whereas
in vitro studies bone marrow population enriched in EPC
(approx. 600 times) was used. Importantly, percentage of
CD45-/KDR+/Sca-1+ cells was significantly reduced by
40–80% in the bone marrow of diabetic mice in comparison to wt (in db/db only 0.0003% of all cells were EPC).
Reduction related to blood glucose levels suggested that
diabetes influences the number of EPC according to its severity. These defect was partially or fully reversed by an oral
application of rosiglitazone 10 mg/kg, for 14 or 28 days,
respectively. We observed a similar trend for CXCR4+ cells
in bone marrow: a reduction in db/db animals and stimulation upon rosiglitazone treatment. Although EPC number
was reduced by 40% in blood of db/db mice the oral rosiglitazone administration did not change it. Furthermore,
in in vitro assays, EPC isolated from db/db mice displayed
impaired migratory and angiogenic potential. Migration of
db/db cells was decreased by 50%, whereas ability to form
tubes on matrigel by 40%. Importantly, in both tests functions of diabetic EPC were restored by rosiglitazone (10
μmol/L, 24 h) in a PPARγ-dependent manner.
Next, based on the EPC transcriptome analysis we found
that proteoglycan 4 (PRG4) was one of the strongest
downregulated gene in db/db cells (7-fold decrease). Since
PRG4 stimulates proliferation and survival of hemangioblasts we tried to induce its expression in vitro by rosiglitazone (10 μmol/L, 24 h) stimulation. We observed 2.5 fold
PPARγ dependent induction in PRG4 expression both in
wt and diabetic EPC. Thus, PPARγ is an important regulator of EPC biology and its induction may be used to improve the EPC functions.
e-mail: Wojciech Kowalski <[email protected]>
Abstracts
62
P7.16
P7.17
Actin polymerization by lysenin
Phosphorylation of p66Shc under oxidative
stress in fibroblasts of NARP patients
Edward A. Czuryło, Natalia Kulikova, Andrzej Sobota
Nencki Institute of Experimental Biology, Warsaw, Poland
e-mail: Natalia Kulikova <[email protected]>
Lysenin is a 33.5kDa protein isolated from coelomic fluid
of an earthworm Eisenia foetida. It interacts with lipids,
specifically recognizing sphingomyelin. It also causes longlasting (more than 30 min) contraction of isolated rat aorta
strips. The mechanism underlying induction of contraction
as well as the biological action of lysenin in animals has not
been clarified.
Our studies of the effect of lysenin on the ATPase activity
of regulated actomyosin system has shown that addition of
lysenin resulted in the abolishment of caldesmon inhibition
and further activation of the ATPase activity up to the level
of its activation by tropomyosin. This effect was observed
at very low molar ratios of actin to lysenin.
Pelleting of reconstituted smooth muscle actomyosin system with or without lysenin revealed that lysenin did not
induce dissociation of the filament constituents. Moreover,
previous electron microscopic observations have shown
that addition of lysenin to F-actin resulted in filament
cross-linking. Thus it may be assumed that the abolishment
of caldesmon inhibitory properties is related to the suppression of caldesmon effect on the actin filament structure.
It has been suggested that lysenin invokes structural changes in the F-actin helix favoring the ATP hydrolysis by myosin and suppressing the effect of caldesmon on the actin
filament structure. To understand the process of G-actin
polymerization at addition of lysenin we have performed
studies of stoichiometric ratio of actin–lisenin interaction.
Pelleting of actin with lysenin followed by the subsequent
Western blotting analysis has shown that actin binds lysenin
with the 1000:1 ratio. Therefore it may be suggested that
lysenin changes actin conformation at the same time preventing the binding of next lysenin molecules. Magdalena Lebiedzinska1, Agnieszka KarkucinskaWieckowska2, Jan M. Suski1,3, Gyorgy Szabadkai4, Grzegorz
Wilczyński1, Jakub Wlodarczyk1, Maciej Pronicki2, Jerzy
Duszynski1, Paolo Pinton3, Mariusz R. Wieckowski1
1Department of Biochemistry, Nencki Institute of Experimental Biology,
Warsaw, Poland; 2Department of Pathology, The Children’s Memorial
Health Institute, Warsaw, Poland; 3Department of Experimental and
Diagnostic Medicine, Section of General Pathology, Interdisciplinary
Center for the Study of Inflammation (ICSI) and LTTA Center, University
of Ferrara, Ferrara; Italy; 4University College London, Department of Cell
and Developmental Biology, Consortium for Mitochondrial Research,
London, United Kingdom
e-mail: Magdalena Lebiedzińska <[email protected]>
In our previous studies we have shown that p66Shc, the
adaptor protein regulating mammalian lifespan is involved
in pathology associated with oxidative stress observed in
cases of mitochondrial disorders. Affected mitochondrial
respiratory chain generates reactive oxygen species (ROS)
which activates phosphorylation of p66Shc at serine 36,
what in turn triggers further ROS production in mitochondria and may lead to serious cell damage or apoptosis.
Activation of p66Shc phosphorylation also contributes to
decrease of antioxidants enzymes levels. However, inhibition of p66Shc phosphorylation decreased ROS production and improved antioxidant defense [1]. Thus we examined whether p66Shc phosphorylation depends on the type
of defect and if the decrease of ROS level also attenuates
p66Shc phosphorylation. In fibroblasts of NARP patients,
respiratory chain is working properly, and the oxidative
stress results from hyperpolarization of inner mitochondrial membrane. In these fibroblasts abnormalities in bioenergetic parameters and antioxidant defense, together with
high level of carbonylated proteins, lipid accumulation and
affected mitochondrial structure and motility are observed.
Interestingly, in cells of NARP patients we observe strong
induction of p66Shc serine 36 phosphorylation pathway.
Also in this cellular model, inhibition of p66Shc phosphorylation, similarly to antioxidants treatment, led to a decrease in superoxide anion production and to an increase in
antioxidant enzymes levels. Our data suggest that serine 36
p66Shc phosphorylation is an important factor contributing to cell damage in cases of mitochondrial dysfunction
(i.e. associated with ATP synthase defect). Both, inhibition
of kinase phophorylating p66Shc (PKCβ) and antioxidants
treatment result in decreased level of ROS, restrain phosphorylation of p66Shc and its pro-oxidative function.
Reference
1. Lebiedzinska M et al. (2010) Biochim Biophys Acta – Bioenergetics 1797:
952-960.
63
P7.18
P7.19
Dissecting the function of the ArfGAP2/3
proteins in the COPI trafficking machinery
Evaluation of horizontal gene transfer from
genetically modified plants to bacteria
Lena Lifshitz1, Irit Pevzner1,
Joelle Bigay2, Anna Parnis1, Dan Cassel1
Mahesh Madyagol, Vinh Phu Hoang,
Stanislav Stuchlík, Jan Turňa
1Technion-Israel Institute of Technology, Department of Biology, Israel; 2Université de Nice Sophia-Antipolis et CNRS, Institut de
Pharmacologie Moléculaire et Cellulaire, France
Department of Molecular Biology, Faculty of Natural Sciences,
Comenius University in Bratislava, Bratislava, Slovak Republic
e-mail: Lena Lifshitz <[email protected]>
From yeast to mammals, two types of GTPase activating
proteins, ArfGAP1 and ArfGAP2/3, control GTP hydrolysis on the small G protein Arf1 at the Golgi apparatus.
Although functionally interchangeable, they display little
similarity outside the catalytic GAP domain. ArfGAP1
is controlled by membrane curvature through its ALPS
(amphipathic lipid packing sensor) motifs, whereas Golgi
targeting of ArfGAP2 depends on heptameric protein
complex named coatomer, the building block of the COPI
coat. Using a reporter fusion approach and in vitro assays,
we identified several functional elements in ArfGAP2/3.
We show that the Golgi localization of ArfGAP3 depends
on a central basic stretch. This region interacts directly
with coatomer, which we found essential for the catalytic
activity of ArfGAP3 on Arf1-GTP. In order to further understand the role of ArfGAPs in the COPI system we are
performing detailed analysis of the interactions between
the GAP2/3 and COPI constituents at the molecular and
structural level.
e-mail: Mahesh Madyagol <[email protected]>
Horizontal gene transfer (HGT) is today recognized as being the most important factor for the rapid global dissemination of antimicrobial resistance. One of the main concerns regarding the use of GM food in human and animal
nutrition is the effect that newly introduced sequences may
have on the organism. Public and scientific concerns about
the environmental and food safety of genetically modified
(GM) crops overshadow the potential benefits offered by
crop biotechnology to improve food quality. The main objective of this work is to quantify the risk of horizontal
gene transfer from from the GM plants to (GMOs), and
food derived thereof, to the bacterial microflora. For studying potential gene transfer we have chosen CP4 EPSPS
(5-enolpyruvylshikimate 3-phosphate synthase) gene from
Round Ready maize (Monsanto) chromosome. The CP4
EPSPS enzyme is a key enzyme in the biosynthesis of aromatic amino acids in microorganisms and plants. It is the
target of the broad-spectrum herbicide glyphosate. During
our earlier studies of HGT of the CP4 EPSPS gene from
GM maize through gastrointestinal tract to bacteria living in animal gut we have observed a functional truncated
CP4 EPSPS gene form present in bacteria. This truncated
CP4 EPSPS gene was cloned into the T7 expression vector pET-28b(+) along with native full-length CP4 EPSPS.
We have tested the truncated form of CP4 EPSPS indetail
and carried a comparative study with native full-length CP4
EPSPS. We have expressed, purifed and characterized both
forms of CP4 EPSPS synthases. We have studied enzyme
activity of both forms of purified CP4 EPSPS proteins.
We have found that both enzymes shown positive for both
forms of CP4 EPSPS. This will help to decide that some
truncated forms of CP4 EPSPS could confer full function
of native full-length CP4 EPSPS and these findings also
should be taken into account in risk assessment of possible
HGT from GM plants.
Abstracts
64
P7.20
P7.21
Energy metabolism in cells with glycogen
branching enzyme deficiency
Dominika Malinska, Malgorzata Bejtka,
Joanna Szczepanowska, Jerzy Duszynski
Cannabidiol inhibits expression and
constitutive signaling of Kaposi’s sarcomaassociated G protein-coupled receptor
and induces programmed cell death
Nencki Institute of Experimental Biology, Department of Biochemistry,
Laboratory of Bioenergetics and Biomembranes, Warsaw, Poland
Yehoshua Maor1,3, Jinlong Yu1, Paula M. Kuzontkoski1,
Bruce J. Dezube2, Xuefeng Zhang1, Jerome E. Groopman1
e-mail: Dominika Malinska <[email protected]>
Glycogen storage disease type IV (GSD IV) results from
mutation in the GBE1 gene encoding a glycogen branching enzyme. The disease manifests itself by dysfunctions
of liver, skeletal muscle and nervous system. The hallmark
of GSD IV are intracellular aggregates of linear glycogen
molecules, which are characterized by poor water solubility
and high damage potential towards intracellular structures.
Additionally, the efficiency of glucose release from such
abnormal glycogen is strongly impaired, which may lead
to energetic imbalance in the cell. It is not clear to which
extent the disruption of glucose metabolism contributes
to the cell injuries occurring in GSD IV and, on the other
hand, how much it results from a mechanical damage due
to presence of intracellular glycogen aggregates. In our
study we compare energy metabolism of fibroblasts with
functional GBE and with GBE deficiency (cell lines derived from GSD IV patients as well as cells with suppressed
expression of GBE). The preliminary data will be presented regarding mitochondrial morphology, respiratory
parameters as well as growth and survival rate of the cells
under different accessibility of substrates for glycolysis and
for the oxidative phosphorylation.
1Division of Experimental Medicine and 2Department of Hematology
and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, USA; 3 Institute of Drug Research, Medical Faculty of
the Hebrew University of Jerusalem, Jerusalem, Israel
e-mail: Yehoshua Maor <[email protected]>
Kaposi’s sarcoma (KS) is the most common neoplasm
caused by Kaposi’s sarcoma associated herpesvirus
(KSHV), also termed human herpesvirus 8 (HHV-8). It is
prevalent among the elderly in the Mediterranean, inhabitants of sub-Saharan Africa, and among immunocompromised individuals like organ transplant recipients and AIDS
patients. The virus encodes for several gene products that
function like cytokines and chemokines. Of special interest
is the KSHV G protein-coupled receptor (vGPCR), which
by its constitutive activity can alter key signaling pathways
and cell functions, implicating these pathways in the initiation of the neoplasm.
Cannabidiol (CBD), a plant-derived cannabinoid, exhibits
promising anti-tumor effects without inducing psychoactive side effects. In this study we investigated the effects of
CBD both on the infection of endothelial cells by KSHV,
and on the growth of KSHV-infected endothelium, an in
vitro model for the transformation of normal endothelium to Kaposi’s sarcoma. We found that while CBD did
not affect the efficiency of KSHV infection of endothelium, this cannabinoid reduced proliferation and induced
apoptosis in KSHV-infected endothelial cells. CBD appeared to have these effects by inhibiting the expression
of KSHV viral G protein-coupled receptor (vGPCR), the
chemokine growth-regulated protein alpha (GROα), vascular endothelial growth factor receptor 3 (VEGFR-3), and
the VEGFR-3 ligand vascular endothelial growth factor C
(VEGFC). Our study suggests that CBD may be a novel
agent for the treatment of Kaposi’s sarcoma.
65
P7.22
P7.23
Recombinant perfringolysin O, a useful
probe for cholesterol detection
The influence of elevated level of
homocysteine on human placental function
Ewelina Marszałek, Piotr Koprowski, Andrzej Sobota
Olga P. Martsenyuk1, Berthold Huppertz2 ,
Kateryna L. Romanets1, Maria Y. Obolenskaya1
Warsaw, Poland
e-mail: Ewelina Marszałek <[email protected]>
Cholesterol is one of major lipids in mammalian cells and
is essential for plasma membrane structure and function.
Cholesterol content and distribution in cells are strongly
regulated by de novo synthesis as well as by endocytic uptake from media. Disturbances in these processes affect
cholesterol deposition in lysosomes, evoking neurodegenerative diseases, such as Niemann-Pick type C disease. To
detect cholesterol deposits in lysosomes we prepared a recombinant toxin, perfringolysin O, which interacted specifically with the lipid. Perfringolysin O (PFO) from Clostridium perfringens is a member of cholesterol-dependent
cytolysins. These toxins are responsible for life-threatening
pathogenicity of a number of bacteria. The toxins bind to
plasma membrane cholesterol and induce cell lysis. Due to
selective interaction with cholesterol, PFO can be used as
a probe to visualize or even quantitate cholesterol in cell
membranes. We decided to optimize production of a fusion protein of PFO and glutathione transferase (GST).
To avoid potential secretion of the toxin, the open reading
frame of PFO was modified in such a way that it lacked
sequence for first 29 amino acids that serve as a leader peptide. We mutated the only cystein, Cys459 to alanine, since
this modification was shown to be responsible for toxin
inactivation. On the other hand, we introduced new cystein
in position 397 to facilitate fluorophore modification of
PFO. The codon bias of PFO gene was optimized for Escherichia coli expression and the gene was synthesized by a
commercial vendor. The gene was subcloned from pUC57
to pGEX4T vector for expression of N-terminal GST fusion protein. The protein was expressed in E. coli BL21
cells and purified by GSH-agarose.
The produced PFO, when incubated with erythrocytes, induced hemolysis at 50 pg/ml. Incubated with liposomes
composed of different lipids the recombinant protein
bound to vesicles containing cholesterol. In the result of
interaction with cholesterol-containing membranes oligomers of PFO were formed, as demonstrated by SDS/
PAGE analysis. Altogether, the presented data indicate that
the prepared recombinant PFO can be used as a probe to
detect and visualize cholesterol in cells.
Reference
Flanagan JJ et al. (2009) Biochemistry 48: 3977-3987.
1Institute of Molecular Biology and Genetics, National Academy
of Science of Ukraine, Kyiv, Ukraine; 2Department of Cell Biology,
Histology and Embryology, Medical University of Graz, Graz, Austria
e-mail: Olga Matsenyuk <[email protected]>
Background: Elevated level of homocysteine (Hcy) increases the risk of placental insufficiency, spontaneous
abortion, preeclampsia and developmental defects of
the fetus. Hcy is an aminothiol which is remethylated to
methionine by methionine synthase or transsulfurated to
cysteine by cystathionine β-synthase (CBS). The lack of
CBS makes tissues more sensitive to Hcy and causes its accumulation leading to hyperhomocysteinemia (hHcy). The
goal of our study was to examine the influence of hHcy on
the placental functions — proliferation and apoptosis and
on the ability of transsulfuration pathway to protect the
tissue from the adverse effect of Hcy.
Objectives: Placental samples were obtained from the I
(8–10 weeks) and the III (38–40 weeks) trimesters of gestation, three samples from each term. Explants were cultivated in DMEM/F12 in the presence of 20, 40, 80 µM Hcy
with or without 20 nM folic acid for 48h at 37°C and 5%
CO2, 5% oxygen. Ki-67 positive nuclei of cytotrophoblast
cells and the square of M30 positive regions were counted
and related to the villous circumference and the square,
correspondingly. The expression, localization and enzymatic activity of CBS were examined by RT-PCR, WB, IHC
and incorporation of [14C]serine residue into cystathionine.
Results: The proliferation index is higher (4.2 vs 0.7) and
the apoptotic index is lower (0.8 vs 1.5) in the explants from
the I trimester in comparison with the III one. Hcy induces
concentration-dependent down-regulation of proliferation
index and up-regulation of the apoptotic one. The apoptotic changes are more pronounced in the explants from
the III trimester. The highest concentration of Hcy (80
mkM) induces the separation of syncytiotrophoblast from
cytotrophoblast.
The expression of CBS gene at RNA and protein levels
and the catalytic acidity of the protein (~50 mU/mg protein) were ascertained for the first time. The CBS protein
was localized in trophoblast and stroma cells of villous
chorion. The elevated concentrations of Hcy induce upregulation of CBS content.
Conclusion: The elevated level of homocysteine provokes
the disbalance between the proliferation and apoptotic
processes in human placenta. We speculate that the presence of CBS and its activation under mild hHcy provides
the evidence that placenta has metabolic resources to withstand the elevation of Hcy and maintain redox homeostasis
via transsulfuration pathway. Abstracts
66
P7.24
P7.25
The activity of adenosine deaminase in
erythrocytes and leukocytes is normal
even in cases of neoplastic pathology
Grainyhead-like 1 (GRHL1) transcription
factor in development of skin cancers
Elena M. Bakurova, Kseniya O. Mironova,
Tatiyana V. Moroz, Berta G. Borzenko
National Medical University of M. Gorkiy, Department of Biochemistry,
Ukraine
e-mail: Ksenia Mironova <[email protected]>
Biochemist Otto Varburg concluded that tumours are more
frequent in the tissues poorly supplied with oxygen, and
actually, normal cells are transformed into malignant ones
due to the oxygen lack. It has been proved at present that the duration of the relapse-free period depends on the oxygen starvation of the tumor while cancers characterized
with pO less than 10 mm Hg are expected to relapse earlier,
it also depends, to great extent, on the severity of anemia.
Dysmetabolic processes inside the blood cells lead to the
cells dysfunction, they also impact cancer capacity to
progress. Our task was to determine the activity of adenosine deaminase (ADA), it being the enzyme of adenosine catabolism in erythrocytes and leukocytes in patients
with stomach cancer, lung cancer. ADA deficiency in lymphocytes is known to lead to the development of severe
combined immunodeficiency. Under simultaneous estimation there was also a sorption capacity of the erythrocytes
membranes as well as the reaction of the blast transformation (stimulation index) of lymphocytes. The examination included 15 patients with lung cancer T3-4N1-nM0,
30 patients with stomach cancer T3-4N1-nM0, 40 healthy
volunteers. Spectrophotometric method has been applied
in the process of the study. Decrease of ADA activity in
lymphocytes to 19.02±2.33 nmol/min·mg in stomach cancer and 26.93+5.89 nmol/min·mg in lung cancer to be
compared to 51.79±8.09 nmol/min·mg in control (p<0.01)
while the index of lymphocytes stimulation decreased from
65.44±2.4 nmol/min·mg in control to 16.38±2.33 nmol/
min·mg in cancer (p<0.001). The activity of the enzyme in
erythrocytes was also reliably lower than in the control, respectively 5.65±1.29 nmol/min·mg in stomach cancer and
5.84±2.78 nmol/min·mg in lung cancer to be compared to
20.04±2.78 nmol/min·mg in the control (p<0.01). It has
also been specified a decrease of sorption activity of erythrocytes membrane in the patients. Thus the rate of correlation between sorting mean molecules on the erythrocytes
membrane and free-moving in the plasma molecules is
4.23±1.13 in stomach cancer and 5.41±0.55 in lung cancer to be compared to 9.68±1.22 (p<0.05). To sum it up,
the decrease of ADA activity was accompanied with the
decrease of lymphocytes stimulation index as well as the
drop of sorption activity, so it had a direct correlation to
cells dysfunction.
Michał Mlącki, Agnieszka Kikulska, Tomasz Wilanowski
Laboratory of Signal Transduction, Nencki Institute of Experimental
Biology PAS, Warsaw, Poland
e-mail: Michał Mlącki <[email protected]>
GRH family of proteins is an ancient family of transcription
factors which has appeared in evolution in early unicellular
ancestor of animals, choanoflagellates, and fungi. In extant
animals, as evolutionary distinct as insects and man, they
are involved in regulation of development, functioning,
regeneration of epidermal barrier and in other processes.
Grainyhead (GRH) is a Drosophila melanogaster’s protein
that has been identified as a key regulator of exoskeleton
development, as it has been shown to induce expression of
main cuticular cross-linking enzyme DOPA-decarboxylase
(Ddc). In addition, by regulating Fasciclin 3 and Coracle,
this transcription factor is also involved in forming septate
junctions between embryonic epidermal cells. Mutant flies
are not able to hatch because of flimsy cuticle.
In mammals there are three homologs of GRH — Grainyhead-like 1/2/3 (GRHL1/2/3). All of them are highly expressed in mice epidermis and appear to be crucial for the
maintenance of this organ. GRHL3 was shown to regulate
the expression of transglutaminase 1 (TGase 1) which in
mammalian skin performs a cross-linking function analogous to that of Ddc in the fly. GRHL1 regulates expression
of keratinocytes’ cell junction component — desmosomal
cadherin desmoglein 1 (Dsg 1). Knock-out mutations of
GRHL2 and GRHL3 ale lethal in mice. Mutant embryos
do not develop proper epidermal barrier and die shortly
before birth because of extensive water loss. Only GRHL1
knock-out mice are viable but they also have some minor
epidermal defects — delay in coat growth, hair loss in older
mice and palmoplantar keratoderma.
There are some premises that suggest not yet investigated
roles of GRHL proteins in carcinogenesis — GRHL2 regulates overexpression of human telomerase reverse transcriptase (hTERT) during immortalization of human oral
squamous cell carcinoma cells and GRHL3 is a responsive
gene in the TNFα signaling pathway and stimulates migration of endothelial cells. Mechanisms of involvement of
the factors in carcinogenesis have not yet been studied. I
would like to present results of my investigation on role of
GRHL1 transcription factor in development and progression of epidermal carcinomas.
References
Auden A (2006) Gene Expr Patterns 6: 964-970.
Boglev Y, Wilanowski T et al. (2011) Dev Biol 349: 512-522.
Ting SB (2005) Science 308: 411-413.
Wilanowski T (2008) EMBO J 27: 886-897.
Wilanowski T (2002) Mech Dev 114: 37-50.
67
P7.26
P7.27
Secondary structure of SsoII-like
(Cytosine-5)-DNA methyltransferases
N-terminal region determined by
circular dichroism spectroscopy
Interaction of endocytic adaptor intersectin
1 with microtubule stabilizer STOP, which
is involved in synaptic transmission
Molochkov1,
Ryazanovaa2,
Nikolay V.
Alexandra Yu.
Lyudmila A. Abrosimovaa3, Elena A. Kubarevaa4
1Institute
of Theoretical and Experimental Biophysics, Russian Academy
of Sciences, Laboratory of NMR Research Biosystems, Russia; 2Faculty
of Bioengineering and Bioinformatics, Chemistry Department, and
A.N. Belozersky Institute of Physico-Chemical-Biology, M.V. Lomonosov
Moscow State University, Moscow, Russia
e-mail: Nikolay Molochkov <[email protected]>
Abstract: (Cytosine-5)-DNA methyltransferase SsoII
(M.SsoII) has a long N-terminal region (1–71 residues)
preceding the sequence with conservative motifs, which
are characteristic for all DNA methyltransferases of such
kind. The presence of this region provides M.SsoII capability to act as a transcription regulatorin SsoII restrictionmodification system. To perform its regulatory function,
M.SsoII binds specifically to a 15-mer inverted repeat in
the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein
Δ(72–379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII like DNA methyltransferase M.Ecl18kI
and is homologous to M.SsoII N-terminal region. Δ(72–
379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of
high protein excess relative to DNA, which couldindicate
an altered structure in the deletion mutant in comparison
with the full length M.SsoII. Circular dichroismspectroscopy demonstrated that Δ(72–379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32%
α-helices and 20% β-strands. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is
made how α-helices and β-strands are arranged in M.SsoII
N-terminal region.
Dmytro Morderer1, Oleksii Nikolaienko1, Inessa Skrypkina1,
Liudmyla Tsyba1, Volodymyr Cherkas2, Alla Rynditch1
1Institute of Molecular Biology and Genetics, Department of Functional
Genomics, Ukraine; 2Bogomoletz Institute of Physiology, Department
of General Physiology of Nervous System, Ukraine
e-mail: Dmytro Morderer <[email protected]>
ITSN1 is one of the candidate genes to be involved in
development of Down syndrome phenotype as well as in
early stages of Alzheimer’s disease. This gene encodes multifunctional evolutionally conserved adaptor protein intersectin 1, which is implicated in clathrin-mediated endocytosis, as well as in actin nucleation and mitogenic signaling.
ITSN1 is characterized by high level of expression in neurons. Moreover, several ITSN1 alternative splicing events
occurring predominantly in neuronal cells were reported.
These data suggest that intersectin 1 could play an important role in neuronal functioning.
In order to determine the composition of neuronal intersectin 1-related protein complexes we performed search
for novel intersectin 1 neuron-specific interacting partners.
For this purpose, we performed GST pull-down assay with
mouse brain lysate using intersectin 1 SH3 domains as a
bait. Bound proteins were subjected to MALDI-TOF mass
spectrometry analysis. For the SH3A domain of intersectin 1 a band of 125 kDa was observed. It was identified
as STOP (stable tubule-only polypeptide). This protein is
a main factor that determines Ca2+/calmodulin-regulated
microtubule cold and drug stability and also takes part in
generation of synaptic plasticity. Since glial cells contain
STOP isoforms with smaller molecular weight; we concluded that STOP is neuron-specific binding partner of intersectin 1. To confirm these results we performed several
independent experiments. GST pull-down assay revealed
that STOP can bind the SH3A and with less affinity the
SH3C and SH3E domains. Intersectin 1–STOP complexes
were coimmunoprecipitated from mouse brain lysate using anti-STOP and anti-ITSN1-EH2 antibodies. Finally,
we determined subcellular localization of these proteins in
rat primary hippocampal neurons using both fluorescent
antibodies and recombinant FP-tagged proteins. We found
that intersectin 1 and STOP partially co-localize both in cell
body and in neurites. Additional experiments will be performed to elucidate functional meaning of this interaction.
Acknowledgements
This work was supported by The State Fund for Fundamental Researches
and Joint Project Programme of European Scientific Unions “Early Stages
of Human Oncological, Autoimmune and Neurodegenerative Diseases”.
Abstracts
68
P7.28
P7.29
In vitro effect of pH and cadmium on malic
enzyme from herring sperm from the Gulf
of Gdansk — similarities in structure and
activity profiles under adverse conditions
Thermodynamics of molecular
recognition of mRNA 5’ cap by yeast
eukaryotic initiation factor 4E
Natalia Niedźwiecka, Edward F. Skorkowski
Gdansk University Biological Station, Laboratory of Comparative
Biochemistry and Ecotoxicology, Gdansk, Poland
e-mail: Natalia Niedźwiecka <[email protected]>
Introduction: Our knowledge about the enzymes expression and theirs properties in fish spermatozoa is very inconspicuous. The overall tertiary structures of these malic
enzymes are similar, but there are still some differences
that may be significant for catalysis and regulation of the
enzyme. ME usually exists as tetramer. It catalyzes the reaction of malate decarboxylation to form pyruvate in the
presence of divalent cations like Mn2+ or Mg2+ and NAD+
or NADP+ coenzyme. NADH or NADPH is generated in
this reaction.
The kinetic properties of malic enzyme from Baltic herring
sperm have been not previously described.
Baltic herring is a common species which is exposed on
high dose of cadmium and other hazardous substances.
The mechanism of cadmium toxicity is still not yet established.
Material and Methods: Milt was collected from mature
male herring (Clupea harengus membras) specimens from the
Gulf of Gdansk. The malic enzyme (EC 1.1.1.39) from
spermatozoa was purified by chromatography on DEAESepharose, Red Agarose and Sepharose 6B. Malic enzyme
activity was calculated spectrophotometrically by observing
the appearance of NADPH at 340 nm at 30°C (E mM 340 =
6.22 for NADPH in 1 cm light path quartz cell) and by native polyacrylamide gel electrophoresis. The malic enzyme
was dissociated in buffers with different pH values and also
after incubation with cadmium. Samples were analyzed using native electrophoresis and, after cross-linking, SDS/
PAGE electrophoresis.
Results: 1. Effect of the range of pH 4.5–8 on malic enzyme activity was studied spectrophotometrically. The results shows that the optimum pH for NAD(P)-dependent
malic enzyme was 7 and 7.5 (for NADP and NAD respectively).
2. The native page electrophoresis results confirm that the
native malic enzyme exists as active tetramer. The intensity
of tetramer band decreases with the decrease of pH.
3. In low pH ME tetramers partially dissociate to dimers
(observed in pH 5) and monomers (pH <5).
4. Similar gel profiles were observed in the case of malic
enzyme incubated with 2 mM cadmium. The activity of
malic enzyme decreases in a time-dependent manner and
tetramer dissociation can be observed. Katarzyna Kiraga-Motoszko1, Anna Niedzwiecka1,2,
Anna Modrak-Wojcik1, Janusz Stepinski1,
Edward Darzynkiewicz1, Ryszard Stolarski1
1University of Warsaw, Faculty of Physics, Institute of Experimental
Physics, Division of Biophysics, Warsaw, Poland; 2Institute of Physics,
Polish Academy of Sciences, Laboratory of Biological Physics, Warsaw,
Poland
e-mail: Anna Niedźwiecka <[email protected]>
Molecular mechanisms underlying the recognition of
the mRNA 5’ terminal structure called “cap” by the eukaryotic initiation factor 4E (eIF4E) are crucial for capdependent translation. To gain a deeper insight into how
the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van’t Hoff analysis based
on intrinsic protein fluorescence quenching upon titration
with a series of chemical cap analogs were performed,
providing a consistent thermodynamic description of the
binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed
similarities and differences between yeast and mammalian
eIF4Es. The yeast eIF4E complex formation was enthalpydriven and entropy-opposed for each cap analog at 293 K.
A non-trivial isothermal enthalpy-entropy compensation
was found, described by a compensation temperature, Tc
= 411±18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected,
ΔCp°= +5.2±1.3 kJ ∙ mol-1∙K-1. The charge-related interactions involving the 5’-5’ triphosphate bridge of the cap and
basic amino acid side chains at the yeast eIF4E cap-binding
site were significantly weaker (by ΔΔH°vH of about +10
kJ ∙ mol-1) than those for the mammalian homologs, suggesting their optimization during the evolution.
Acknowledgements
The work was supported by N N301 267137; N N301 035936, N N301
096339 and the HHMI 55005604.
P7.30
P7.31
Estrogen receptors expression and
clinical peculiarities of breast cancer
Functional diversity of intersectin
family adaptor proteins
Olga Shatova, Igor Sedakov, Igor Zinkovych
Olga Novokhatska1, Mykola Dergai1, Oleksandr
Dergai1, Liudmyla Tsyba1, Inessa Skrypkina1,
Jacques Moreau2, Alla Rynditch1 Donetsk National Medical University named after M. Gorkiy,
Biochemistry Department, Ukraine
e-mail: Shatova Olga <[email protected]>
Rate of distant metastases (M1) and positive nodes status
(N1-N2) was analyzed in the moment of hospitalization
of 1916 patients with breast cancer, including of 1309
with estrogen receptor-positive tumors. In 25 patients the
level in the blood serum of some enzymes of exchange
of carbohydrates and nucleosides was determined. It was
shown that probability of distant metastases was significant higher at estrogen-negative tumors of women at the
small sizes (T1-2) of tumor or at the stored menstrual
function. Opposite, the metastases in regional lymphatic
nodes were more frequently registered among women in
a premenstrual period and with the expression of estrogen receptors. High levels of serum adenosine deaminase
13.7±2.9 nmol/min ∙ mg (as compared with 9.5±3.2 nmol/
min ∙ mg, р=0.045) and lactate dehydrogenase 3.2±0.15
nmol/min ∙ mg (as compared with 3.0±0.2 nmol/min ∙ mg,
р=0.023), combining with the low serum activity of thymidine phosphorylase 18.5±5.7 nmol/min ∙ mg (as compared
with estrogen receptor-negative – 10.6±1.4 nmol/min ∙ mg)
can be indirect index of absence of estrogen receptors. 69
1Institute of Molecular Biology and Genetics NASU, Department of
Functional Genomics, Ukraine; 2Jacques Monod Institute, Department
of Molecular Mechanisms of Development, France
e-mail: Novokhatska Olga <[email protected]>
Mitogenic signalling from receptor tyrosine kinase (RTK)
is tightly linked to endocytosis that regulates the fidelity of
signal transduction. Intersectins (ITSN1 and ITSN2) are
evolutionarily conserved adaptors implicated in the earliest stages of clathrin-mediated endocytosis. However, their
increased levels lead to distinct effects on signal transduction. Overexpression of ITSN1 has mitogenic effect
whereas high expression level of ITSN2 is associated with
prolonged disease-free survival during breast cancer. A role
for ITSN1 was also shown in Alzheimer’s and Huntington’s neurodegenerative diseases and Down syndrome. The
aim of this study was to compare these two highly related
adaptor molecules functionally. For this, we investigated
subcellular distribution of ITSNs, their binding properties
towards interaction partners and regulation via posttranslational modifications.
Immunofluorescent analysis together with coimmunoprecipitation showed that endogenous ITSN1 and ITSN2 significantly colocalized in HEK293 cells and formed a complex in vivo. In vitro binding assay revealed no interaction
between ITSN1 and ITSN2 coiled-coil regions capable of
dimerization. This data suggest that association of ITSNs
is not mediated by their direct interaction but rather depend on the N-terminal EH domains responsible for the
intracellular localization.
To investigate whether differences of ITSNs lie in their
binding specificity we compared protein interactions of
these adaptors. Seven well-established protein partners of
ITSN1 were tested for binding to ITSN2. In addition, three
novel binding partners for ITSNs were identified. Interaction data showed that all protein partners tested are common for ITSNs. However, binding of ITSN2 to signalling
proteins Cbl, CIN85 and Sema6A was mediated by partially
distinct interaction domains.
Investigation of posttranslational modifications of ITSNs
revealed that both proteins are monoubiquitilated. ITSN2
in contrast to ITSN1 specifically underwent tyrosine phosphorylation. The level of phosphorylation did not depend
on inhibition of endocytosis but was reduced in serumstarved cells.
Obtained data showed that ITSN1 and ITSN2 differ in
their binding properties and posttranslational regulation.
Further investigation is needed to distinguish the precise
roles of ITSN1 and ITSN2 in cell processes.
Abstracts
70
P7.32
P7.33
Novel DNA-binding protein from
Nanoarchaeum equitans Kin4-M
binds all kinds of nucleic acids
Expression and purification of
recombinant Paramecium Rab7 proteins
Marcin Olszewski, Jan Balsewicz, Marta Nowak, Józef Kur
Gdańsk University of Technology, Department of Microbiology, Gdańsk,
Poland
e-mail: Marcin Olszewski <[email protected]>
Nanoarchaeum equitans is the only known representative of
Archaea phylum Nanoarchaeota and stands out as one
of the tiniest known living organism. What is more it has
smallest genome, which is only 490.885 base pairs long. It is
also one of the most compact genomes. According to predictions about 95% of the DNA encodes proteins or stable
RNA. Nanoarchaeum equitans lacks genes for most vital
metabolic pathways including lipid, cofactor, amino acid, or
nucleotide biosynthesis. However contrary to most known
organisms with reduced genome has a full set of enzymes
involved in DNA replication, repair and recombination,
one of which is a single-stranded DNA binding protein.
To date four nanoarchaeal proteins have been identified:
tRNA splicing endonuclease, reverse gyrase, neelaredoxin
and family B DNA polymerase. Interestingly all of them
possess unusual features. Sequence analysis of Nanoarchaeum equitans SSB protein indicates that it may also fit the
trend. We report the identification and characterization of
SSB from Nanoarchaeum equitans Kin4-M. NeqSSB is one of
the largest known SSB proteins consisting 243 amino acid
residues with a calculated molecular mass of 27.78 kDa.
It is functional as a monomer with encoding one singlestranded DNA binding domain (OB-fold), which is the
smallest and consists of 75 amino acids residues only. Nanoarchaeum SSB is the completely new kind structure of
SSB protein. SSBs proteins identified to date are homotetramers or homodimers and theirs OB-fold have about
100 amino acids residues. In fluorescence titrations with
poly(dT), NeqSSB protein binds single-stranded DNA
with a binding site size about 42 nt per monomer. In addition, a unique feature is the high protein binding capacity
of RNA and DNA, both linear and circular. The half-lives
of NeqSSB was 5 min at 100°C. These results showed that
Nanoarchaeum SSB as the first characterized SSB with
ability to binding all kinds of nucleic acids is thermostable
SSB protein with unique structure, offering an attractive
alternative for other SSB proteins in their applications for
molecular biology techniques.
Magdalena Osińska, Kamil Kobyłecki,
Krzysztof Skowronek, Elżbieta Wyroba
e-mail: Magdalena Osińska <[email protected]>
Recent data indicate that changes in post-translational
modification represent rapid routes to the sub- and neofunctionalization of duplicated genes (Amoutzias et al.
2010). Recombinant proteins may be useful tool in studies
of such modifications and we expressed in Escherichia coli
BL21(DE3) Rab7 proteins encoded by Paramecium octaurelia
rab7a and rab7 paralogous genes.
Although the Rab7a and Rab7b proteins are exhibiting
97.6% amino acid sequence identity, they differ in their
expression, properties and function. Up to now liquid
chromatography-tandem mass spectrometry (LC-MS/MS)
revealed eight identical phosphorylated residues in both
these proteins: S17, S23, S71, T34, T40, T63, Y77 and Y89.
In 2D gel electrophoresis two immunoreactive spots were
identified for Rab7b at pI~6.34 and ~6.18 and only one
spot for Rab7a of pI~6.34 suggesting post-translational
modification of Rab7b.
In order to elucidate this problem the constructs of the
native Rab7a and Rab7b proteins as well as of their two
variants undergoing putative unique modifications were
created by the site directed mutagenesis and were subcloned into expression vectors pPR-IBA2 for Streptag
and pET28 Novagene for (His)6 tag. Prior to this step full
coding sequences of both genes were synthesized to replace TAG encoding glutamine in Paramecium in order to
prevent termination of the protein translation in E.coli.
All the constructs were sequenced to prove their correctness. By changing the conditions of both the cultivation of
Escherichia coli BL21(DE3) bearing the expression vectors
and the induction of lac operon (time, temperature, IPTG
concentration) the efficient overproduction and purification of Rab7a-Strep and Rab7b (His)6-tagged recombinant
proteins was achieved. The effectiveness of these procedures was confirmed by double immunodetection (Western
Blot) using two sets of specific antipeptide antibodies recognizing Strep or His tag and the Rab7a or Rab7b proteins,
respectively. This was followed by mass spectrometry that
brought the evidence of required amino acid sequence of
the recombinant proteins.
Acknowledgements
This work was supported by grant N N303 615038 of the Ministry of
Science and Higher Education.
P7.34
P7.35
The impact of adaptor protein
Ruk/CIN85 in the maintenance of
cancer stem cell phenotype
The effect of Sgt1 phosphorylation
on its nuclear translocation
N. Byts, A. Fedoseenko, G. Pasichnyk,
A. Basaliy, A. Samoylenko, L. Drobot
Palladin Institute of Biochemistry, Laboratory of Cell Signalling, Kyiv,
Ukraine
e-mail: Ganna Pasichnyk <[email protected]>
Introduction: Cancer stem cells (CSCs) are tumor cells
with a self-renewal activity and ability to generate new heterogeneous tumors. Several mechanisms are involved in the
development and maintenance of CSCs phenotype such as
increased NF-κB signalling, overexpression of membrane
transporters (ABCG2 etc.) as well as the expression of
several membrane markers. Nevertheless, the impact of
adaptor proteins in the regulation of CSCs biological responses remains mostly unknown. It was shown that adaptor protein Ruk/CIN85 is involved not only in the control
of the normal cell homeostasis but is implicated in the
mechanisms of malignant transformation of mammalian
cells and as a result might serve as a molecular marker of
carcinogenesis. Here we investigated the possible role of
adaptor protein Ruk/CIN85 in the development of phenotypic and functional features of CSCs.
Methods: All experiments were carried on human breast
adenocarcinoma MCF-7 cell line. Either wild type MCF-7
cells or MCF-7 cells with stable overexpression of Ruk/
CIN85 were used. Toluidine blue staining that can discriminate between stem/early breast progenitor cells (“pale”
stained cells) and more differentiated epithelium cells
(“darker” stained cells) was used for analysis of MCF-7 adherent and suspension cells according to Cioce et al. (2010)
Cell Cycle 9: 2878-2887. The activation of transcription factor NF-κB was analyzed using appropriate luciferase gene
reporter construct. MCF-7-derived mammosheres were
cultured on low-adhesive plastic in growth factor-supplemented medium. The expression of CD44 surface marker
was analyzed in CSC-like subpopulations by Western blotting.
Results: We demonstrated that relatively high percentage of suspension cells as compared to adherent cells
were stained pale with Toluidine blue. Interestingly, the
number of “pale” stained cells positively correlated with
the level of Ruk/CIN85 expression. When subjected to
a mammoshere forming conditions floating MCF-7 cells
with Ruk/CIN85 overexpression quickly developed mammosheres. Overexpression of Ruk/CIN85 also leads to the
activation of the transcription factor NF-κB and elevated
expression of CD44 surface marker. These features are
known to be typical for CSCs.
Conclusion: Briefly, the data obtained indicate the potential regulatory role of adaptor protein Ruk/CIN85 in the
development of CSC-like phenotype in breast adenocarcinoma MCF-7 cells.
71
Wiktor Prus 1, Magdalena Zabka1,
Paweł Bieganowski2, Anna Filipek1
1Nencki Institute of Experimental Biology, Warsaw, Poland;
2Mossakowski Medical Research Center PAS, Warsaw, Poland
e-mail: Wiktor Prus <[email protected]>
Sgt1 (suppressor of G2 allele of Skp1) is a conserved protein present in yeast, plant and mammalian cells. Sgt1 interacts with several ligands such as Skp1 [1], members of
the S100 family [2] and some heat shock proteins such as
Hsp90 or Hsp70 [3]. Interestingly, the binding of S100A6
with Sgt1 inhibits the Sgt1-Hsp90 complex formation [4].
Since it was shown that the Sgt1 protein has co-chaperone
properties [5] and that it translocates to the nucleus after
heat shock [6], in this work we examined the influence
of phosphorylation on Sgt1 translocation. The theoretical analysis and in vitro kinase assay revealed that Sgt1 is
phosphorylated at the C-terminus by casein kinase II [2].
To check the influence of Sgt1 phosphorylation we prepared non-phosphorylable (S249A, S299A, S249/299A) or
phosphorylation-mimic (S299D) mutants of human Sgt1.
Human Sgt1 containing serine 299 is a homolog of yeast
Sgt1 containing serine at position 361 [7]. Non-phosphorylable Sgt1 mutants were then overexpressed in HEp-2 cells
and afterwards cells were heat shocked. We have found that
after heat shock the non-phosphorylable S299A, but not
the phosphorylation-mimic S299D, mutant of Sgt1 translocates to the nucleus more efficiently than wild type Sgt1.
Additionally, we revealed that S100A6 is required for translocation of the non-phosphorylable Sgt1 mutant and that
S100A6 also translocates to the nucleus after heat shock.
To elucidate the role of S100A6 and Hsp90 in Sgt1 translocation we checked the binding of Sgt1 with these two
ligands. Our results indicate that non-phosphorylated Sgt1
binds S100A6 stronger and Hsp90 weaker than wild type
Sgt1. This suggests that S100A6-Ca2+-mediated Sgt1 dephosphorylation promotes its nuclear translocation most
likely due to the disruption of the Sgt1-Hsp90 complex.
Acknowledgements
This work was supported by grants N N301 293637 from the Ministry of
Science and Higher Education of Poland to A.F. and by statutory funds
from the Nencki Institute of Experimental Biology.
Reference
1. Kitagawa et al. (1999) Mol Cell 4: 21-33.
2. Nowotny et al. (2003) J Biol Chem 278: 26923-26928.
3. Lee et al. (2004) J Biol Chem 279: 16511-16517.
4. Spiechowicz et al. (2007) Biochem Biophys Res Commun 357: 1148–1153.
5. Zabka et al. (2008) Biochem Biophys Res Commun 370: 179-183.
6. Prus, Filipek (2009) Amino Acids 2010 Mar 6. [Epub ahead of print].
7. Bansal et al. (2009) J Biol Chem 284: 18692-18698.
Abstracts
72
P7.36
P7.37
Two different regions of the fibrin
coiled-coil domain important for
fibrin protofibril lateral association
Adaptor protein Ruk/CIN85 forms complex
with prolyl hydroxylase-2 influencing
hypoxia-inducible factor 1 alpha stability
Nikolay A. Pydiura, Eduard V. Lugovskoy,
Evgeniy M. Makogonenko, Serhiy V. Komisarenko Anatoly Samoylenko1, Nina Kozlova1, Nadiya Byts1,
Thomas Kietzmann2, Lyudmyla Drobot1
O. V. Palladin Institute of Biochemistry, Department of Protein Structure
and Function, Ukraine
1Palladin Institute of Biochemisry, Laboratory of Cell Signalling, Kyiv,
Ukraine; 2University of Oulu, Department of Biochemistry, Oulu, Finland e-mail: Nikolay Pydiura <[email protected]>
e-mail: Anatoliy Samoylenko <[email protected]>
Previously we obtained fibrin-specific monoclonal antibody
(mAb) FnI-3C and synthetic peptide imitating fibrin(ogen)
a. a. sequence Bbeta L121-V138 that inhibit the lateral stage
of fibrin polymerization. The exposition of the epitope
(Bbeta M118-V134) for fibrin-specific mAb FnI-3C is a result of FpA cleavage from fibrin(ogen) molecule [1]. This
testifies that desAA fibrinogen molecule after FpA cleavage undergoes structural changes which permit protofibril
lateral association. We performed a bioinformatical analysis
and structural modelling of the human fibrin molecule to
investigate this process.
Dynamical properties of the 3D models of human fibrinogen and fibrin X fragments were analyzed with the help of
the method of correlation molecular dynamics in package
CONCOORD 2.1. Analysis of the alpha, beta and gamma
interchain interactions shows the differences in the spatial
organization of coiled-coil domain between fibrinogen and
fibrin X fragments. Coiled-coil region of fibrin fragment
X molecule proved to have 40% greater RMSF compared
to fibrinogen, reflecting an increase in molecular flexibility. At the same time coiled-coil region containing Bbeta
M118-V138 fragment showed less critical change in RMSF
– 10% increase. The part of this domain including Bbeta
M118-V138 is less stiff then the rest of coiled-coil domain.
It contains the unstructured gamma chain region T68-M78
and plasmin sensitive sites. We more accurately identified
the “hinge” regionin the Bbeta with the center at amino
acid residues Q129-V133 according to chain beta.
Our results testify that coiled-coil domain plays a crucial
role in the process of fibrin protofibril lateral association.
Analysis of the structural localization of point mutations
gives us the reason to distinguish 2 different regions of the
coiled-coil domain of fibrin molecule important for protofibril lateral association. First is the structurally important “hinge” fragment in the middle of coiled-coil region,
containing Bbeta M118-V138. Such factors as mutations in
conserved position or interaction with antibody or peptide
changing the structure and thus flexible properties of this
region lead to impairment of protofibril lateral association.
Second – the “functional” region surface of which is important for fibrin protofibril lateral association. This region
is located in the D-part of the coiled-coil domain.
Tumor hypoxia is a well-known microenvironmental factor
that influences cancer progression and resistance to cancer
treatment. This involves multiple mechanisms of which the
best-understood ones are mediated through transcriptional
gene activation by the hypoxia inducible factors (HIFs).
Among a plethora of biological effects, HIFs may contribute to invasion and metastasis by inducing the expression
of components of the plasminogen activation system including plasminogen activator inhibitor-1 (PAI-1). A recent
study has shown that the adaptor/scaffold protein Ruk/
CIN85, which plays an important role in apoptosis, adhesion, invasion and downregulation of receptor tyrosine kinases, is involved in the hypoxic response (Samoylenko et
al., 2010). Thereby, Ruk/CIN85 induced PAI-1 expression
via enhancing the protein levels of the HIF-1 α-subunit.
The HIFα-subunit abundance is primarily regulated on the
levels of protein stability and activity by iron- and 2-oxoglutarate-dependent dioxygenases, the HIF prolyl hydroxylases (PHDs) and asparaginyl hydroxylases. To investigate
whether Ruk/CIN85 may interfere with HIF-1α stabilization or transactivation, the cells were co-transfected with the
luciferase reporter construct pG5-E1B-Luc that contains 5
copies of a Gal4 response element and vectors allowing
expression of fusion proteins consisting of the Gal4-DNA
binding domain (Gal4) and either HIF-1α N-terminal
(NTAD) or C-terminal (TADC) transactivation domains
along with the Ruk/CIN85 expression vector. It was found
that Ruk/CIN85 interfered with the prolyl hydroxylationdependent HIF-1α protein destabilisation but not with asparaginyl hydroxylation-dependent HIF-1α transactivation.
By continuing to unravel the mechanisms by which Ruk/
CIN85 increases HIF-1α levels we next investigated whether Ruk/CIN85 may interact directly with PHDs. By using
co-immunoprecipitation and GST “pull down” assays it
was demonstrated that Ruk/CIN85 forms complex with
PHD2. When different fragments of Ruk/CIN85 protein
fused to GST were investigated by “pull down” assay it was
found that SH3 domains of Ruk/CIN85 are responsible
for Ruk/CIN85-PHD2 interaction. On the other hand, it
was shown by in vitro hydroxylation assay that Ruk/CIN85
is not hydroxylated directly by PHD-2.
In conclusion, the Ruk/CIN85-PHD interplay may provide the unique opportunity to regulate PHD enzymatic
activity, resulting in distinct modulation of HIF activity under hypoxia and normoxia. References
1. Lugovskoy EV et al. (2009) Thromb Res 123: 765-770.
P7.38
P7.39
The role of PKD1 and PKD2 protein kinases
in regulation of gastric tumor cell biology
Novel application of the advanced mass
spectrometry approaches to characterize
acetylation of CheY, a response regulator
in chemotaxis of Escherichia coli
Mariana Yu. Shabelnik1, Larisa M. Kovalevska1,
Mariya Yu. Yurchenko1, Larisa M. Shlapatska1,
Yuriy Rzepetsky2, Svetlana Sidorenko1
1R.E.Kavetsky
Institute of Experimental Pathology, Oncology and
Radiobiology NASU, Kiev, Ukraine; 2 Institute of cell biology NASU, Lviv,
Ukraine
e-mail: Mariana Shabelnik <[email protected]>
Protein kinase D (PKD) genes are ubiquitously expressed
in different human tissues. Recently the differential expression of PKD genes was found in tumors of different
histogenesis. These protein kinases could be activated by
growth factors, antigen stimulation and oxidative stress, the
processes that usually are observed during tumor progression. PKDs regulate cell-cell contacts by affecting cell adhesion. These kinases are involved in the regulation of cell
proliferation and apoptosis, and also participate in epigenetic regulation of gene expression. Nevertheless, the role
of different PKD isoforms in these processes in normal
and malignant cells is not completely clarified. That is why
studies of differential expression and activity of PKD1 and
PKD2 in the context of tumor invasiveness and prognosis
could be of interest for translational research in oncology.
The aim of our research was to study the role of PKD1
and PKD2 protein kinases in regulation of gastric tumor
cell biology. For this we developed a model system based
on gastric adenocarcinoma сell line AGS. AGS cells were
transfected with human PKD1 or PKD2 in pcDNA3.1
plasmid or empty vector. After selection with G418 we received a stable sublines, which were used for comparative
analysis in MTT and proliferation assays, and also tests for
cell migration and survival. Q-PCR analysis with specific
primers showed that in AGS cell line the level of PKD1 expression was lower than PKD2. Moreover, PKD1 was not
detectable in these cells on protein level. The AGS transfection with PKD1 decreased about two times the level of
PKD2 expression on mRNA level. PKD1 transfection led
to inhibition of cell proliferation, migration and survival;
however, adhesive properties of cells were increased. The
PKD2 overexpression enhanced cell proliferation, migration and survival, but reduced cell adhesion. Our data will
help to evaluate the significance of PKD1 and PKD2 differential expression in primary gastric adenocarcinomas.
This will contribute to the development of new approaches
for differential diagnostics of tumors and identification of
a novel targets for therapy, and also help to reveal the prognostic factors for the prediction of clinical outcome.
73
Tevie Mehlman1, Bassem Ziadeh1, Gabriel Simon2,
Atim Atte Enyenihi3, Milana Fraiberg4,
Michael Eisenbach4, Roman Zubarev3, Alla Shainskaya1 1Weizmann Institute of Science, Biol MS Facility, Israel; 2Washington
University in St. Louis, USA; 3Karolinska Institutet, Molecular Biometry
Lab, Sweden; 4Weizmann Institute of Science, BiolChem Department,
Israel e-mail: Alla Shainskaya <[email protected]>
CheY, the response regulator of bacterial chemotaxis,
transduces the chemotactic signal from receptor supramolecular complex to the switch element in flagellar-motor
to generate clockwise rotation of bacteria. The rotation is
regulated by two covalent modifications of CheY – phosphorylation and acetylation. Recently we showed that CheY
undergoes in-vitro acetylation by a) acetyl-CoA synthetasecatalyzed transfer of acetyl groups from acetate to CheY
and b) autocatalyzed transfer from AcCoA [1–3]. Our goal
now was separation and quantitation of the heterogeneous
pools of chemically acetylated CheY using mass spectrometry (MS) approaches. To separate acetylated species, CheY
was resolved by native PAGE and by ion-exchange chromatography. Gel bands excised from Native Blue Gels and
fractions were subjected to proteolytic digestion, followed
by LC-ESI-MS/MS on Orbitrap-XL for identification of
acetylated residues and Protomap quantitation. Top-down
MS analyses were performed using a Thermo Scientific
LTQ Orbitrap Velos instrument and Q-TRAP-4000. Previously we showed that the acetylation sites of non-treated
CheY and CheY after in-vitro, in vivo and auto acetylation
are mostly clustered at the C-terminus of the protein, with
lysine residues 91, 92, 109, 119, 122 and 126 being the main
acetylation sites. Chemical acetylation extends acetylation
to the N-terminal lysine residues 4, 7 and 26 being additional sites for acetylation. We assume that the result of
the chemical acetylation is an incremental increase in the
extent of acetylation of the same lysine residues. To prove
this assumption we applied recently described Protomap
Approach [4], modified so to enable relative quantitation
of the acetylation sites, distributed among 11 Lysine residues in the CheY sequence. Implementing Top-Down approach we achieved HPLC separation of heavily acetylated
CheY species, up to 11 AcLys, and their MS analysis. To
our knowledge it is the first aattempt to apply complex MS
studies for the analysis of CheY acetylation. These findings
may have far reaching implications for the structure–function relationship of CheY a key protein, involved in a signal
transduction to generate clock-wise rotation of bacteria.
References
1. Barak R, Eisenbach M (2004) J Mol Biol 342: 383-401.
2. Barak R, Eisenbach M (2006) J Mol Biol 359: 251-265.
3. Liarzi O, Eisenbach M (2010) Mol Microbiol 76: 932-943.
4. Dix M, Simon GM, Cravatt BF (2008) Cell 134: 679-691. Abstracts
74
P7.40
P7.41
Autophagy inhibition promotes cell death
in the human ovarian carcinoma SKOV3
Study of a functional role of
KCTD-family proteins
Galyna Y. Shuvayeva1, Oxana B. Groholska1,
Nataliya I. Igumentseva1, Ciro Isidoro2, Oleh V. Stasyk1
Мikhail Yu. Skoblov1,
Andrey V. Marakhonov1, Anna V. Baranova1,2
1Institute of Cell Biology, National Academy of Sciences of Ukraine,
Department of Cell Signaling, Lviv, Ukraine; 2Amedeo Avogadro
University, Department of Medical Science, Laboratory of Molecular
Pathology and Nanobioimaging, Novara, Italy
Sciences, Russia; 2Molecular and Microbiology Department, George
Mason University, USA e-mail: Mikhail Skoblov <[email protected]>
e-mail: Galyna Shuvayeva <[email protected]>
Autophagy is a major mechanism of cellular protein and
organelle recycling, induced in response to nutrient starvation or metabolic stress. The role of autophagy in cancer
progression and in response to various therapies is a topic
of debate in biomedical science.
Recently we showed that single amino acid arginine (ARG)
deprivation strongly induces autophagic response in the
human ovarian carcinoma SKOV3 cells, highly resistant
to this metabolic stress. ARG deprivation achieved with
recombinant ARG-degrading enzymes is currently considered as anticancer therapy. On the model of SKOV3 cells,
we addressed the question, whether autophagy modulation
may enhance potential of this therapy.
We examined cell viability upon autophagy inhibition or
overinduction in ARG-free medium, and ability to restore
proliferation upon ARG resupplementation. Antimalarial
and potential anticancer drug chloroquine (CQ) was utilized as autophagy inhibitor, whereas resveratrol (RV) was
applied as autophagy inducer.
We demonstrated that RV did not affect SKOV3 cells viability upon ARG deprivation. However, inhibition of autophagy with CQ decreased cell viability under ARG deprivation and dramatically affected cell proliferation upon
ARG resupplementation. Transcriptional silencing of autophagic protein Beclin 1, required for the initiation of autophagy, led to a similar phenotype. These results suggest
an important role of autophagic process for maintaining
SKOV3 cell viability upon ARG deprivation. Thus, autophagy may serve as a prosurvival mechanism upon single amino acid restriction providing the recycling of ARG
from preexisting proteins.
Although ovarian carcinomas were not considered as potentially sensitive to the therapy based on ARG deprivation,
it was recently reported that tumor relapses after treatment
with platinum compounds concomitantly become sensitive
to ARG deprivation due to the loss of the expression of
ARG biosynthetic enzyme, argininosuccinate synthetase
(ASS). Our results point at potential efficacy of the combinational approach based on ARG deprivation and autophagy inhibition as a second line therapy for such difficult to cure ovarian carcinomas. Studying the causes of B-cell chronic lymphocytic leukemia, we found several protein-coding genes. One of the
interesting candidates for the role of tumor suppressor
gene was KCNRG. Bioinformatic analysis showed that its
amino-acid sequence contains the T1 domain responsible
for protein-protein interactions through tetramerization.
Genome search revealed 27 other human genes with T1domain, which groups them into a family of KCTD-proteins.
We have hypothesized that the KCNRG disrupts the assembly of tetramers of potassium channels, also possessing the T1 domain. We have shown that the KCNRG protein inhibits voltage-dependent potassium currents in cells
of prostate carcinoma LnCaP, and stable over-expression
of the gene KCNRG leads to suppression of proliferation
and increased apoptosis in model cell lines of prostate
carcinoma LnCaP, multiple myeloma RPMI-8226 and in
promyelocytic leukemia HL-60 cells. This proves that the
KCNRG gene has tumor suppressor properties.
Despite the high degree of similarity with other KCNRGlike proteins, KCTD5 protein in solution does not form
tetramers, but pentamers, whereas the tetramers are very
unstable. Interestingly, the KCTD5 protein actively interacts with other proteins (non-potassium channels) through
its C-terminal module, as well as through other epitopes.
For the rest of the members of this family information
about their interaction is scarce.
We carried out the analysis of literature on KCNRG-like
proteins and analyzed the participation of each unique
members of the KCTD-family proteins in various signaling cascades and cell systems. In functional terms, family of
KCTD proteins is quite diverse.
In order to understand the function of KCNRG-like proteins and their interaction with potassium channels, we conducted a multiple alignment of amino-acid sequences of
N-termini (with the T1 domain) and C-termini of KCTD
proteins family. Also, we performed expression analysis
to reveal groups of KCNRG-like proteins and potassium
channels which co-expressed in the same type of tissues.
P7.42
P7.43
Hpy188I–DNA pre- and post-cleavage
complexes — snapshots of the
GIY-YIG nuclease mediated catalysis
Does CacyBP/SIP dimerization
modulate ERK2 activity?
Monika Sokolowska1,2,
Honorata Czapinska1,2, Matthias Bochtler1,2,3
1International Institute of Molecular and Cell Biology, Warsaw, Poland;
2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; 3Schools of Chemistry and Biosciences, Cardiff University,
Cardiff, UK
e-mail: Matthias Bochtler <[email protected]>
The GIY-YIG nuclease domain is present in all kingdoms
of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1Slx4, the prokaryotic nucleotide excision repair proteins
UvrC and Cho, and in proteins of selfish genetic elements.
Here we present the structures of the ternary pre- and
post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or
catalytic metal ion, respectively. Our structures suggest that
GIY-YIG nucleases catalyze DNA hydrolysis by a single
substitution reaction. They are consistent with a previous
proposal that a tyrosine residue (which we expect to occur
in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our
data identify the general base with the GIY and not the
YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation
in the active site. This metal ion contacts the phosphate
proS oxygen atom and the leaving group 3’-oxygen atom,
presumably to facilitate its departure. Taken together, our
data reveal striking analogy in the absence of homology
between GIY-YIG and ββα-Me nucleases.
Acknowledgements
This work was supported by grants No. N N 518497639 from the Polish
Ministry of Education and Science and BK 214/Rau1/2011 from the Silesian University of Technology, Supported by the European Community
from the European Social Fund within RFSD 2 project.
Reference
Sokolowska M, Czapinska H, Bochtler M (2011) Nucleic Acids Res 39:
1554-1564. Epub 2010 Oct 8.
75
Agnieszka M. Topolska1, Walter J. Chazin2, Anna Filipek1
1Nencki Institute of Experimental Biology, Warsaw, Poland; 2 Vanderbilt
University, Center for Structural Biology, Nashville, TN, USA
e-mail: Agnieszka Topolska <[email protected]>
CacyBP/SIP was originally discovered as a S100A6 (calcyclin) binding partner [1] and then as a Siah-1 interacting protein [2]. Up to now the role of CacyBP/SIP is not clear but
it has been suggested that it might be involved in cell cytoskeleton rearrangement (via interaction with tubulin and
actin) or signaling pathways leading to cell differentiation
(via binding to ERK1/2 kinases) [3, 4]. It has been shown
that CacyBP/SIP dephosphorylates ERK2 and that the
C-terminus of CacyBP/SIP (residues 178-229) is responsible for interaction with ERK2 [5].
Theoretical analysis of the C-terminus of CacyBP/SIP
showed the presence of two α-helices: H-A (residues 189203) and H-B (residues 203-219). Since in helix B there
is a universal sequence responsible for interaction with
kinases, called KIM (Kinase Interaction Motif), it seems
that residues 203-219 of CacyBP/SIP are involved in
CacyBP/SIP–ERK2 interaction. Theoretical analysis of
the N-terminal CacyBP/SIP fragment revealed that it has
very similar structure to the C-terminus (two α-helices) and
also contains KIM sequence.
In this work we studied CacyBP/SIP dimerization and its
effect on the interaction with ERK2. By cross-linking experiment using NB2a cell extract we found that CacyBP/
SIP indeed forms dimer which is present both in cytoplasmic and nuclear fractions. However, a higher amount of
the dimeric form is present in the nuclear fraction. Also, it
seems that formation of CacyBP/SIP dimer is associated
with stress induced by hydrogen peroxide. Presence of the
CacyBP/SIP dimer was confirmed by sedimentation velocity and sedimentation equilibrium experiments. Theoretical
analysis of CacyBP/SIP model indicates that monomers
interact through their N-terminal domains. Our hypothesis
is that CacyBP/SIP dimer formation might occlude the
N-terminal KIM sequence and in consequence might inhibit the interaction with ERK2 and phosphatase activity
of CacyBP/SIP.
Acknowledgements
This work was supported by the European Union through the European
Regional Developmental Fund within the scope of the International PhD
Studies in Neurobiology (MPD4-502) and by statutory funds from the
Nencki Institute of Experimental Biology.
References
1. Filipek A, Wojda U (1996) Biochem J 320: 585–587.
2. Matsuzawa S, Reed JC (2001) Mol Cell 7: 915-926.
3. Schneider G et al. (2010) Biochim Biophys Acta 1803: 1308-1317.
4. Kilanczyk E et al. (2009) Biochem Biophys Res Comm 380: 54-59.
5. Kilanczyk E et al. (2011) Biochem Biophys Res Comm 404: 179-183.
Abstracts
76
P7.44
P7.45
Cloning, expression and antimicrobial
activity of the human cathelicidin LL-37
Palmitoylcarnitine affects localization
of growth associated protein
GAP-43 in plasma membrane
subdomains of NB-2a cells
Csaba Tóth, Roland Pálffy, Stanislav Stuchlík, Ján Turňa
Comenius University Bratislava, Faculty of Natural Sciences,
Department of Molecular Biology, Bratislava, Slovakia
e-mail: Csaba Toth <[email protected]>
Antimicrobial peptides are evolutionarily conserved components of the innate immune response and they can be
found among all classes of life. These peptides are able
to kill Gram-negative and Gram-positive bacteria, mycobacteria, enveloped viruses, fungi and even transformed
or cancerous cells. They possess a variety of antimicrobial
activities ranging from membrane permeabilization to action on a range of cytoplasmic targets. Therefore they are
excellent candidates for the development of novel therapeutic agents and complements to conventional antibiotic
therapy since fighting dangerous microbes has become
increasingly difficult because many bacterial strains have
become resistant to antibiotics. Among the AMPs, LL-37
is a very promising candidate for its possible clinical application as agent for the treatment of numerous diseases.
LL-37 is a cationic, amphipathic α-helical peptide and it is
the only AMP from the cathelicidin family found in human.
It is mainly expressed in myeloid cells, where it is located in
specific granules, but it was also described in inflamed skin,
testis, wound fluid, lung epithelia, sweat and saliva. Apart
from its’ wide spectrum of bactericidal activity, LL-37 also
plays an important role in the regulation of the inflammatory response, neutralization of LPS and the promotion of
wound healing.
As the therapeutical and commercial importance of these
peptides is rising, simple expression and purification systems will be needed for their large-scale production. We
have designed and prepared a cost-effective and efficient
expression system for the production of LL-37 in fusion
with ketosteroid isomerase in E. coli cells. After the purification and activation of the peptide, we have tested its’
biological activity on various pathogens.
Keywords: antimicrobial peptides, cathelicidins, LL-37, biological activity
Karolina Tułodziecka, Katarzyna A. Nałęcz
e-mail: Karolina Tułodziecka <[email protected]>
Palmitoylcarnitine, synthesized in the cell from palmitoylCoA and L-carnitine, is known to be an intermediate of
fatty acid metabolism, anyhow, some other physiological
functions have been ascribed as well to this long-chain
acylcarnitine. In neuroblastoma NB-2a cells, characterized
by negligible β-oxidation, palmitoylcarnitine was observed
to promote differentiation and to affect signal transduction
pathways, in particular activation and localization of protein kinase C (PKC). Palmitoylcarnitine was also observed
to increase palmitoylation of several proteins, including
growth associated protein GAP-43, whose expression
augments during differentiation of neural cells. GAP-43
undergoes two reversible post-translational modifications,
phosphorylation by PKC (a process decreased in the presence of palmitoylcarnitine) and palmitoylation (stimulated
by palmitoylcarnitine), which should promote anchoring
in plasma membrane microdomains. Since the phosphorylated form of GAP-43 is capable of binding phosphatidylinositol 4,5-bisphosphate (PIP2), the present study has
been focused on a possible effect of palmitoylcarnitine
on localization in plasma membrane, when comparing its
reversed influence on GAP-43 modifications. Solubilization of membranes in the presence of Triton X-100, followed by sucrose gradient centrifugation and Western blot
analysis allowed the detection of GAP-43 presence in the
floating fractions (rafts). Long-term (96h) palmitoylcarnitine treatment of NB-2a cells was shown to promote appearance of GAP-43 in rafts, a process completely reversed
by β-methylcyclodextrin, a cholesterol binding agent, while
there was no change upon sequestration of PIP2 with
neomycin. A direct interaction with raft proteins (caveolin-1, flotillin-1) was excluded by immunoprecipitation,
while immunocytochemistry analysis showed a possibility
of a small fraction of GAP-43 interacting with PIP2 in
microdomains not containing cholesterol, a phenomenon
not affected, however, by palmitoylcarnitine. Treatment
with bromopalmitate (inhibitor of protein acyltransferases)
did not reverse the effect of palmitoylcarnitine and did not
remove GAP-43 from the raft fractions. These observations point to palmitoylation of GAP-43 as a non-reversible modification, leading to localization of this protein in
microdomains of plasma membrane rich in cholesterol,
different, however, from microdomains in which PIP2 is
present.
77
P7.46
P7.47
Cloning, expression, purification and
characterization of recombinant ADH
from Saccharomyces cerevisiae
Uncoordinated expression of
the elongation factor 1 subunits
in human lung carcinomas
Pavol Utekal1, Stanislav Stuchlik1, Pavol Kois2, Jan Turna1
M. Veremieva1, V. Zakcharychev2, B. Negrutskii1, A. El’skaya1
1Comenius University Bratislava, Faculty of Natural Sciences, Molecular
Biology, Bratislava, Slovak Republic; 2Comenius University Bratislava,
Faculty of Natural Sciences, Organic Chemistry, Bratislava, Slovak
Republic
1Institute of Molecular Biology and Genetics NASU, Laboratory of
Protein Biosynthesis, Kiev, Ukraine; 2P. Schupik Kiev National Medical
Academy of Postgraduate Education UMH, Kiev, Ukraine
e-mail: Maryna Veremieva <[email protected]>
e-mail: Pavol Utekal <[email protected]>
C-6 aldehydes and alcohols contribute to the fresh green
odor in plants and are widely used in perfumes and in food
technology. Important member of this family is trans-2hexenol. It can be produced by transformation of trans2-hexenal which is extracted from plant tissue. Carbonyl
compounds such as aldehydes and alcohols are reduced
by chemical methods in industry but it is not appropriate for production of compounds used in food industry.
Therefore, in recent decades biocatalysis is used for these
purposes. The enzymes suitable for reduction of aldehydes
are oxidoreductases, which catalyze the reduction of carbonyl groups of aldehydes and alcohols. The most suitable
enzyme from this class is alcoholdehydrogenase (ADH),
which is the last enzyme involved in lipoxygenase pathway
in several species of higher plants, which converts natural
trans-2-hexenal to trans-2-hexenol. In this work we have
focused on the cloning, expression, purification and characterization of the enzyme activity of recombinant Saccharomyces cerevisiae alcoholdehydrogenase I produced in
Escherichia coli as a host.
Keywords: trans-2-hexenol, biocatalysis, alcoholdehydrogenase
Translation elongation complex eEF1H comprises the
subunits responsible for GTP-dependent delivery of
aminoacyl-tRNA to ribosome (eEF1A) and for providing
GDP/GTP exchange in the eEF1A molecule (eEF1Bαβγ
complex). Cancer-induced increase of mRNAs coding for
some subunits of eEF1 in different types of cancer was reported previously, however, the related changes in the level
of corresponding proteins were rarely investigated. We
have compared the expression of mRNAs and corresponding subunits of eEF1 in 25 clinical lung adenocarcinoma
specimens by Northern blot and Western blot analyses.
Elevated expression of mRNAs was observed in 44% cancer samples while increase in protein amount was detected
in 52% cases. Unexpectedly, the alterations in the mRNAs
and corresponding proteins levels were found to be uncoordinated when analyzed in the same tumor samples.
To determine whether the eEF1 components change their
cell localization in cancer tissues the immunohistochemical
analysis was performed. Perinuclear localization of eEF1A
and eEF1Bα was found in the normal and cancerous lung
tissues. eEF1Bβ was present in the perinuclear region of
normal lung tissue, however, it was detected in the nucleus
of cancerous lung tissue. Nuclear staining of eEF1Bγ was
found in both normal and cancerous specimens. Also, the
intracellular distribution of the eEF1 subunits in the lung
carcinoma A549 cell line was investigated by Western blot
analysis of nuclear and cytoplasmic fractions. Surprisingly,
in cultivated cells all components of the eEF1 complex
demonstrated both nuclear and cytoplasmic localization.
The non-coordinated expression and nuclear localization
of some eEF1 components in cancer cells indicate that
the release of individual subunits from the complex might
be of specific cancer-related importance beyond its established role in protein biosynthesis.
Abstracts
78
P7.48
P7.49
Posttranslational modifications
of CacyBP/SIP under normal and
pathological conditions Molecular dynamics and substrate
docking studies on paraoxonase 1
Urszula Wasik1, Beata Jastrzebska1, Serge Weis2,
Grazyna Niewiadomska1, Anna Filipek1 1Nencki
Institute of Experimental Biology, Warsaw, Poland; 2WagnerJauregg Hospital, Linz, Austria
e-mail: Urszula Wasik <[email protected]>
The CacyBP/SIP protein was discovered as a S100A6 binding partner (Filipek & Wojda, 1996) and later as the Siah1 interacting protein (Matsuzawa & Reed, 2001). The role
of CacyBP/SIP is not clear but it has been suggested that
CacyBP/SIP is involved in cytoskeleton rearrangement
through its interaction with actin and tubulin (Schneider
et al., 2007 and 2010), and in cell signaling pathways since
it has been shown recently that CacyBP/SIP has phosphatase activity toward ERK1/2 kinase (Kilanczyk et al.,
2009 and 2011). To elucidate the cellular role of CacyBP/
SIP in this work we studied its posttranslational modifications (PTMs). We also checked the effect of S100A6 on
the level of various CacyBP/SIP forms. By applying 2D
electrophoresis we found that in neuroblastoma NB2a cells
the CacyBP/SIP protein might undergo phosphorylation
by protein kinase C. Treatment of NB2a cell extract with
alkaline phosphatase resulted in disappearance of acidic
forms of CacyBP/SIP and appearance of its basic forms.
The same result was obtained when the extract from differentiated NB2a cells, in which the level of S100A6 increases,
was used. Moreover, the same alterations in the isoelectric point of CacyBP/SIP forms were caused by S100A6
overexpression. The influence of S100A6 on CacyBP/SIP
phosphorylation by casein kinase II was checked in an in
vitro experiment and again S100A6 was found to inhibit
this phosphorylation. Since CacyBP/SIP is highly expressed in brain and its translocation from dendrites to the
neuronal cell body during aging was observed, we checked
CacyBP/SIP forms in neurodegeneration. We performed
2D electrophoresis on brain extract from thauopatic mice
and from AD patients. In both cases the ratio between the
two major forms of CacyBP/SIP was similar but it differed
from control groups. Altogether, our results indicate that
CacyBP/SIP undergoes PTMs that affects its isoelectric
point and that the level of different CacyBP/SIP forms
might change due to pathological conditions. At present,
we focus on the identification of the phosphorylation site
in the CacyBP/SIP molecule and on identification of other
PTMs by mass spectrometry.
References
This work was supported by grants N N303 548439 from the Ministry
of Science and Higher Education of Poland to AF, Wista Laboratories
Ltm and by statutory funds from the Nencki Institute of Experimental
Biology.
Grzegorz Wieczorek1, Moshe Ben-David1, Clifford Felder1,
Yacov Ashani1, Dan Tawfik2, Israel Silman3, Joel L. Sussman1
1Weizmann Institute of Science, Department of Structural Biology,
Israel; 2Weizmann Institute of Science, Department of Biological
Chemistry, Israel; 3Weizmann Institute of Science, Department of
Neurobiology, Israel
e-mail: Grzegorz Wieczorek <[email protected]>
Paraoxonase (PON1) is a Ca+2-dependent enzyme that is
found in mammalian sera in which it is associated with
high-density lipoprotein (HDL) particles. Although its biological function(s) remain(s) to be established, it is believed
to fulfill anti-oxidative and anti-atherosclerotic roles. It displays promiscuous specificity, acting on organophosphates
(OPs), esters and lactones, and there is substantial evidence
that the latter are its physiological substrates. Its ability to
hydrolyze OPs raised the possibility that it could serve as
a bioscavenger that could provide protection against OP
nerve agent intoxication, and be used for decontamination
of both nerve agent and insecticide OPs, which attack human and insect acetylcholinesterases (AChEs), respectively.
The activity of wild-type (wt) PON1 towards OP nerve
agents is too low to provide protection. Consequently, directed evolution has been used, together with mutagenesis
of specific amino acid residues based on structural data
obtained by X-ray crystallography, to enhance the catalytic
activity of PON1 towards nerve agents, as well as its stereospecific preference for the chiral forms of the OPs that
are preferentially active on AChE. Despite the fact that
substantial enhancement of the bioscavenger activity of
wt PON1 has thus been achieved, open questions remain
concerning the mechanism of action of PON1 both on
OPs and on its other substrates, and the mode of binding
of substrates and inhibitors at the active site. We are complementing the experimental studies by molecular docking
protocols and by molecular dynamics. The preliminary results of these studies will be presented.
P7.50
P7.51
Chemical phosphorylation of thymidylate
synthase on histidine residue(s)
Crystal structure of human muscle
fructose-1,6-bisphosphatase
mutant E69Q in T conformation
Piotr Wilk1, Zbigniew Zieliński1,
Tomasz Ruman2, Wojciech Rode1
1Nencki Institute of Experimental Biology, Polish Academy of Sciences,
Warszawa, Poland; 2Rzeszów University of Technology, Faculty of
Chemistry, Rzeszów, Poland
e-mail: Piotr Wilk <[email protected]>
Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the C(5)
methylation of 2’-deoxyuridylate (dUMP) in a concerted
transfer and reduction of the one-carbon group of N5,10methylenetetrahydrofolate, and with concomitant production of dihydrofolate and thymidylate. As the sole de novo
source of thymidylate synthesis in cells, it is a target in
anticancer, antiviral, antifungal and antiprotozoan chemotherapy.
Potential phosphorylation of TS protein was first shown
in 1997 after incubation of rat hepatoma cells with 32P labeled ATP (Samsonoff et al., 1997, J Biol Chem 272: 1328113285). Recently, phosphorylated residues were determined on both endogenous and recombinant eukaryotic
TS. Phosphorylated fractions (separated by MOAC; constituted always ~1% of total protein) of purified preparations of recombinant enzyme from various sources were
studied by 31P-NMR, allowing to determine involvement
of phosphate groups in acid-labile phosphoramidate bond
with histidine residue. Comparison of histidine-phosphorylated TSs with the corresponding fractions of non-phosphorylated protein showed a significant influence of this
posttranslational modification on enzymes catalytic (decreasing Vmax) and non-catalytic (enabling mRNA translation repressing) properties (Frączyk et al., 2009, Pteridines
20: 137-142).
Our current studies are aimed at obtaining of histidinephosphorylated TS quantities sufficient for crystallographic trials. With no histidine kinase capable of TS
phosphorylation known, chemical reaction with potassium
phosphoramidate (KPA) was employed. After optimization
of reaction conditions, recombinant mouse TS protein was
confirmed by 31P-NMR analysis to undergo phosphorylation on histidine residue(s). The apparent phosphorylation
level was similar to that found in recombinant protein expressed in bacterial cells, and comparative studies showed
the chemical modification to influence the enzyme’s properties in a way similar to that observed for recombinant TS
phosphorylated in bacteria. Conditions are being sought
enabling localization of the modification(s) by MS analyses
and crystallization of phosphorylated protein. 79
Marek Zarzycki1, Robert Kolodziejczyk2,3, Ewa Maciaszczyk4,
Robert Wysocki4, Mariusz Jaskolski2,5, Andrzej Dzugaj1
1Institute of Genetics and Microbiology, University of Wroclaw,
Poland; 2Department of Crystallography, A. Mickiewicz University,
Poznan, Poland; 3Macromolecular X-ray Crystallography Group,
Institute of Biotechnology, University of Helsinki, Helsinki; 4Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland;
5Institute of Plant Biology, University of Wroclaw, Poland
e-mail: Marek Zarzycki <[email protected]>
Human fructose-1,6-bisphosphatase is an allosteric enzyme
that is regulated by different ligands. There are two known
isozymes in human tissues – liver isozyme (the key enzyme
of gluconeogenesis), which is regulated by fructose-2,6bisphosphate, and its muscle counterpart (participating in
glycogen synthesis) regulated by calcium ions. AMP being an allosteric inhibitor of both inhibits muscle isozyme
with I0.5 35 to 100 times lower than liver one – the reason
of which still remains obscure. In our studies leading to the
explanation of the main differences in regulation of those
two isozymes we have shown that only one residue in position 69 regulates sensitivity towards calcium ions Zarzycki
et al (2007) FEBS Lett. 581: 1347-1350. As a consequence
of this finding we have crystallized and solved the structure
of E69Q muscle isozyme mutant, that became insensitive
to calcium ions, but preserved all other kinetic properties resembling liver isozyme. We present here two crystal structures with and without fructose-6-phosphate (the
product of the catalytic reaction) both being in complex
with AMP. The binding pattern of muscle isozyme toward
AMP is quite similar to the corresponding pattern of the
liver isozyme and the T conformations of both isozymes
are nearly the same. The product and the AMP binding
sites are compared with the corresponding sites in human
and porcine liver FBPases.
Abstracts
80
P7.52
P7.53
Three-dimensional structure
and function of human
immunoglobulin G subclasses
Structural analysis of the second catalytic
cysteine half-domain (SCCH) from
mouse ubiquitin-activating enzyme
E1 based on NMR data in solution
Vladimir P. Zaviyalov1,
Sergey N. Ryazantsev2, Vladimir M. Tischenko3
1University
2University
of Turku, Department of Chemistry, Turku, Finland;
of California, Department of Biological Chemistry, Los
Angeles, USA; 3Institute for Biological Instrumentation, Russian
Academy of Sciences, Pushchino, Russia
e-mail: Vladimir Zaviyalov <[email protected]>
Pro-inflammatory activity of IgG is initiated by the binding of two or more molecules of IgG to the surface of
the target cell, followed by multivalent interaction between
the IgG and complemen (Clq) or cell FcγR. C1q and FcγR
show marked differences in their reactivity towards human IgG subclasses. The hIgG1 and hIgG3 are essentially
equivalent in their abilities to activate the classical complement cascade (CCC). However, neither hIgG2 nor hIgG4
have been convincingly shown to activate the CCC. 3D
structure of the hIgG2 Mat has been studied by us using
single-particle 3D reconstruction and negative staining approach. Model of the 3D structure of hIgG2 was created
at resolution of 1.78 nm. Molecule is asymmetrical so that
one Fab is in close interaction with CH2 domains and hinge
region; another Fab is located at some distance from Fc,
there is some weak electron density connecting Fab with
Fc; Fc shape is deformed possible due close interaction
with Fab. The side view of the molecule shows that long
axes of Fabs are nearly perpendicular to the long axis of Fc
(“tripod-like” shape). Model is in great agreement with the
data of difference scanning calorimetry (DSC) and fluorescent measurements obtained by us for a few myeloma
hIgG2 as well as with other data previously obtained for
hIgG2. It may explain why C1q- and FcγR-binding sites are
less accessible in the hIgG2 and therefore it less effective
or even lack of main pro-inflammatory activities. The EM
studies of the hIgG3 revealed that the extremely long hinge
region of this subclass exists under physiological conditions in a compact globule-like form. The DSC shows that
the melting of a globule-like form of the hIgG3 hinge is
a cooperative process. The ‘two-state’ model accepted for
small globular proteins well describes the transition. Thus,
in the hIgG3 molecule the core hinge folds into the 13th
globular domain. The globular form of the hinge affects
the conformation of CH2 domains that correlates with less
effective inhibition of the CCC activation by the hIgG3
compact form. Thus, the 13th globular domain modulates
the hIgG3 effector functions. The new insights gained by
us into the molecular anatomy of hIgG subclasses are important not only for the understanding of molecular physiology of immune response but also for creation a new generation of therapeutic human antibodies. Emilia A. Lubecka1, Krzysztof Kazimierczuk2,
Anna Zawadzka-Kazimierczuk2, Jan Stanek2,
Krystian Stodus3, Jerzy Ciarkowski1,
Wiktor Kozminski2, Igor Zhukov3,4,5
1Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland; 2Faculty
of Chemistry, University of Warsaw, Warsaw, Poland; 3Intitute of
Biochemistry and Biophysics PAS, Warsaw, Poland; 4Slovenian NMR
Centre, National Institute of Chemistry, Ljublljana, Slovenia; 5EN-FIST
Center of Excellence, Ljubljana, Slovenia
e-mail: Igor Zhukov <[email protected]>
Proteins degradation by ubiquitin-proteosome pathway
require jointed action of three enzymes responsible for
ubiquitin-activation (E1), conjugation (E2) and ligation
(E3). Second catalytic cysteine half-domain (SCCH) is a
276 amino acid residues long fragment of mouse ubiquitinactivating E1 enzyme contained catalytic cysteine. It was
shown that the catalytic domain consist two autonomously
folded half-domains – first catalytic cysteine half-domain
(FCCH) and second catalytic cysteine half-domain (SCCH)
[1]. Moreover, both half-domains formed stable 3D structures available in pdb databank as 2V31 and 1Z7L, respectively. Unfortunately, in spite of existence structural data
the initial stages of ubiquitin-activation process are still not
understood in details [2].
NMR spectroscopy is a good method for detail characterization of enzyme functioning from both structural and
dynamical point of view [3]. In presented work we demonstrated initial structural analysis of SCCH half-domain
from the mouse E1 enzyme in solution based on backbone
and side chains chemical shifts. The backbone sequencespecific assignments were achieved on 2H,13C,15N-triple
labeled recombinant SCCH using standard heteronuclear
3D NMR experiments [4]. Side chains assignments were
obtained from analysis 4D 13C,15N-edited NOESY-HSQC
and 4D HCCH-TOCSY data sets acquired with arbitrary
sampling in evolution time space [5]. That data sets were
processed with additionally suppression mathematical artifacts by on-grid algorithm [6]. All NMR experiments were
recorded at 298 K on Varian VNMRS 800 NMR spectrometer equipped with cryogenic probeheads with inverse
detection. Structural analysis is performed by TALOS+
and CS23D programs and will be presented in detail on
a poster.
Acknowledgments
This work is financially supported by the grant from Polish Ministry of
Higher Education N N301 318539 as well as European FP7 projects EastNMR (contract no. 228461) and BioNMR (contract no. 261863) inside of
transnational access programs. EL has financial support from European
Erasmus exchange program.
References
Szczepanowski RH et al. (2005) J Biol Chem 280: 22006-22011.
Lee I, Schindelin H (2008) Cell 134: 268-278.
Bhabha G et al. (2011) Science 332: 234-238.
Lubecka E et al. (2011) Euromar 2011 (Frankfurt am Main, Germany, August 21–25), Book of Abstracts.
Kazimierczuk K et al. (2007) J Magn Reson 188: 344-356.
Stanek J, Kozminski W (2010) J Biomol NMR 47: 65-77.
P7.54
P7.55
Mitochondrial protein import: a structurefunction analysis of the TIM23 complex
Nicotinic acetylcholine receptor
subtypes in b lymphocytes
Keren Zohary, Lada Gevorkyan Airapetov, Abdussalam Azem
L. M. Koval, O. Yu. Lykhmus, S. V. Komisarenko, M. V. Skok
Tel Aviv University, Department of Biochemistry, Tel Aviv, Israel
e-mail: L. M. Koval <[email protected]>
e-mail: Keren Zohary <[email protected]>
The mitochondrion is under the control of two genetic
systems – its own genome, and that of the cell nucleus.
However, only 8 proteins are encoded by the mitochondrial genome while approximately 1000 mitochondrial proteins are nucleon encoded, synthesized in the cytosol and
then imported into the mitochondria. The recognition and
sorting of nucleon encoded mitochondrial preproteins are
mediated by four protein translocases: TOM (Translocase
of the Outer Membrane), SAM (Sorting and Assembly Machinery), TIM22 (Translocase of the Inner Membrane) and
TIM23. The latter mediates mainly the import of proteins
that are translocated into the matrix.
The TIM23 complex is a hetero-oligomeric complex that
is composed of two sub complexes — the import motor
(PAM complex) and a core complex that consists of the
integral membrane proteins: Tim23, Tim17, Tim50 and
Tim21. It is suggested that, during import, the inter-membrane space (IMS) domains of Tim23 and Tim50 interact
and form one complex. The goal of this study is a structure-function analysis the Tim23-Tim50 interaction. For
this purpose, we have purified the IMS domains of Tim23
and Tim50 and studied the interaction between them using
Surface Plasmon Resonance and cross-linking. Using site
directed mutagenesis, we have been able to detect amino
acids that most likely participate in the formation of the
Tim23-Tim50 complex. Furthermore, we have found that
introducing the mutations into yeast caused a growth defective phenotype when compared with the wild type.
81
Palladin Institute of Biochemistry, Kyiv, Ukraine
B lymphocytes express nicotinic acetylcholine receptors
(nAChRs) involved in regulating their development and
activation (Koval et al., 2008, Int J Biochem Cell Biol 40: 980990). However, the molecular mechanisms of nAChR immunoregulating activity remain a question. In addition,
the role of some nicotinic acetylcholine receptor subtypes
is not completely understood. We investigated the exposure of a9-containing nAChR subtype in B lymphocytes
of the spleen and peritoneal cavity of C57Bl/6 mice and
the involvement of a4b2, a7 and a9(a10) nAChRs in B
lymphocyte activation. By means of flow cytometry, a7
nAChRs were found in about 60%, while a4b2 and a9(a10)
nAChRs in about 10% and 20% of spleen B lymphocytes,
respectively. The increased level of both a7 and a9(a10)
nAChR subtypes was found in marginal zone B lymphocytes in the spleen and in B1 cells from peritoneal cavity. Then we established that a4b2 and a7, but not a9(a10)
nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies
demonstrated that a7 and a9(a10) nAChRs are coupled to
CD40, whereas a4b2 nAChR is coupled to IgM. By using
knockout mice and selective ligands of nAChR subtypes
we showed, that B lymphocytes of both a7-/- and b2-/- mice
responded to anti-CD40 stronger than those of the wildtype mice, whereas the cells of b2-/- mice responded to anti-IgM worse than those of the wild-type or a7-/- mice. Inhibition of a7 and a9(a10) nAChRs with methyllicaconitine
resulted in considerable augmentation of CD40-mediated
B lymphocyte proliferation in cells of all genotypes; stimulation of a4b2 nAChRs with epibatidine increased the IgMmediated proliferation of the wild-type and a7-/-, but not
b2-/- cells. Inhibition of a9(a10) nAChRs with a-conotoxin
PeAI exerted weak stimulating effect on CD40-mediated
proliferation. Neither of nAChR ligands used influenced
IgM to IgG class switch of B lymphocytes stimulated with
anti-CD40 plus IL-4.
Conclusions: (1) Mouse B lymphocytes express a4b2, a7
and a9(a10) nAChR subtypes, which regulate mitogenic
pathways of B lymphocyte activation. (2) a4b2 nAChR is
the minor subtype in mature B lymphocytes; it is co-localized with the BCR (IgM) and produces a stimulatory IgMrelated effect; (3) a7 nAChR is the major subtype of mature
B lymphocytes; it is co-localized with CD40 and fulfills inhibitory CD40-related function; (4) a9(a10) nAChR is a “reserve” receptor, which partly compensates the absence of
a7 nAChR in a7-/- cells; (5) Both a7 and a9(a10) nAChRs
represent the ancient regulatory system of natural memory
immune response of B1 and marginal zone B cells.
Abstracts
82
P7.56
P7.57
Nicotinic acetylcholine receptors in
monocytic, astrocytic and lymphoid
cells studied by flow cytometry
and confocal microscopy
2’-5’-Oligoadenylates and their analogs as
modulators of activity of protein kinases
Olena M. Kalashnyk, Olena Yu. Lykhmus, Maryna V. Skok
Laboratory of Molecular Pharmacology, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine
Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv, Ukraine
Nicotinic acetylcholine receptors (nAChRs) are pentameric
ligand-gated ion channels that mediate fast synaptic transmission in muscles and autonomic ganglia and regulate
dopamine release in the brain. They are also expressed in
many non-excitable cells where their functions are less understood. The aim of our work was to compare the subunit
composition of nAChRs expressed in the cells of astrocytic
(U373), monocytic (U937) and lymphoid (Daudi) origin,
found either intracellularly or on the plasma membrane,
and to reveal whether these receptors can be internalized
with specific antibodies. By using nAChR subunit-specific
antibodies in flow cytometry and confocal microscopy, we
have found that glioblastoma U373 cells contained a lot
of intracellular α3 and α7 nAChRs, which were also found
on the plasma membrane. In contrast, U937 cells did not
express α3 subunits on the plasma membrane but have a lot
of intracellular α3 nAChRs; also they contained significant
level of intracellular α4 nAChRs, which appeared on the
membrane after certain period of cell cultivation. Daudi
cells expressed α3, α4 and α7 nAChRs found both on the
membrane and intracellularly. The binding of fluorescently
labeled antibody raised against the whole extracellular domain of α7 subunit (1-208) resulted in receptor internalization in all cell lines examined. As expected, the fastest and
most effective internalization was observed in monocytes
U937: the antibodies were found under the membrane in
5 min after their binding and a widespread network of intracellular vesicles was formed during an hour. Astrocytes
(U373) internalized the bound antibody in 15 min after its
binding; the vesicles didn’t merge to form large intracellular
structures. In contrast, lymphocytes (Daudi) demonstrated very weak and slow internalization: the first antibody
clusters tending to move under the membrane were observed in one hour after the antibody binding. The results
obtained demonstrate that non-excitable cells of monocytic, astrocytic and lymphoid origin express different sets
of nAChR subtypes; the export of certain subtypes to the
plasma membrane varies upon cultivation. The ability of
studied cells to internalize the antibody-bound receptors is
also different that may account for their different resistance
to nAChR-specific autoimmune attacks.
Zenovii Yu. Tkachuk, Olexander V. Kozlov,
Igor Ya. Dubey, Larysa V. Tkachuk
e-mail: Zenovii Yu. Tkachuk <[email protected]>
It is known that protein kinases are key mediators of
various cellular reaction. We have previously shown that
“core” 2’-5’A3 form complexes with some proteins and
change their conformation. The mechanism of action
of “core” oligoadenylates is not completely explored. In
this study we tried to find direct evidence of the ability
of “core” 2’-5’-oligoadenylates to modulate the activity of
kinases (PKs): FGFR, TIE2, ROCK1, Aurora, JNK1, CK2,
ASK. PKs activity was determined using peptide substrate
RRREEETEEE and labeled ATP. 2’-5’А3 and its analogs
were found to have diverse, inhibiting as well as stimulating, effects on the activity of protein kinases. In most cases
the 3’-epoxy analog 2’-5’epoА3 showed more pronounced
stimulating activity as compared to the natural 2’-5’А3.
The highest inhibiting activity demonstrated 2’-5’aminoА3
analog containing the residue of 8-aminoadenosine. The
curves of titration of Aurora protein kinase with 2’-5’А3
and 2’-5’epoА3 show no linear dependence on oligoadenylate concentration and are V- or W-like shaped. 2’-5’epoА3
analog demonstrated more pronounced inhibition effect on Aurora characterized by high activity in broader
range of concentrations. The inhibitory effect of 2’-5’А3
and 2’-5’epoА3 depends on ATP concentration. The IC50
values for oligoadenylates were determined from the titration curves. Since the curves are V- or W-shaped, there are
IC50 values obtained at lower and higher concentrations.
The IC50 at low concentration area for natural 2’-5’A3 was
19.3 µМ, while for the epoxy-derivative it was 11.6 µМ in
the presence of 100 µМ ATP. At higher concentrations, the
IC50 for 2’-5’A3 was found to be 23.8 and for 2’-5’epoA3
85 µМ, respectively. Possible mechanism of biological action of oligoadenylates is suggested where the binding of
2'-5'А3 and its analogs to protein kinases results in the conformational changes which lead to the modification of enzymatic activity. This suggestion is supported by the character of action of tested compounds on protein kinases
since not only inhibition but also stimulation of enzymes
occurs. So in this study we have managed to find direct
evidence of the ability of “core” 2’-5’-oligoadenylates to
modulate the activity of protein kinases and thus to affect
various cellular processes.
Sessions 8 & 9: Calcium in signalling
Part I
Lectures
L8.1
L8.2
Ca2+ signalling in macro- and microvessels
L. Borisova
AMPA receptor trafficking in spinal dorsal
horn neurons under inflammatory pain
Department of Cellular and Molecular Physiology, Institute of
Translational Medicine, University of Liverpool, Liverpool, UK
Nana Voitenko
Although it has long been recognized that macro-and microvessels have different mechanisms controlling their mechanical behaviour, underlying mechanisms are still poorly
understood. The aim of the present work was to investigate Ca2+ signalling in the smooth muscle cells (SMC) of
macrovessels and all branches of microvessels in situ using
Nipkow disc based confocal imaging system. The effects of
central (phenylephrine, PE) and local (endothelin-1, ET-1)
factors on Ca2+ signalling in SMC of rat tail artery and
all branches of terminal arterioles (TA) have been investigated. SMC of artery responded to PE with generation of
the complex Ca2+ transient which consisted of the initial
spike followed by the sustained plateau component superimposed by asynchronous Ca2+ oscillations. This complex
Ca2+ transient was associated with the generation of force
which consisted of the initial phasic followed by the sustained tonic components. In SMC of TA, PE induced asynchronous Ca2+ waves causing regional vasomotion. ET-1
(1–10 nM) had no effect on Ca2+ signalling in SMC of tail
artery but induced asynchronous Ca2+ oscillations in SMC
of TA with the frequency being the highest in the SMC of
the terminal branchings. Removal of external Ca2+ for 60
min abolished Ca2+ transients in SMC of tail artery but had
no effect on agonist induced Ca2+ oscillations in SMC of
TA. Inhibition of RyR channels by ryanodine (50 µM) had
little effect on the sustained component but fully abolished
the initial spike component and asynchronous Ca2+ oscillations of the complex Ca2+ transient in SMC of tail artery,
however in SMC of TA it had no effect on Ca2+ oscillations
induced by either PE or ET-1. Inhibition of IP3R channels
by 2-APB blocked Ca2+ oscillations in SMC of macro-and
microvessels but had little effect on sustained component
of the complex Ca2+ transient in SMC of tail artery. Inhibition of SERCA pump by CPA (20 µM) blocked Ca2+
oscillations in SMC of macro- and microvessels causing
sustained elevation of baseline Ca2+ in SMC of macro- but
not microvessels. The data obtained suggest that there is a
significant difference in the mechanisms controlling Ca2+
signalling between SMC of macro- and microvessels. In
macrovessels Ca2+ signalling involves Ca2+ release from
the SR mediated by both RyR and IP3R channels and Ca2+
entry via receptor- and store-operated Ca2+ channels while
in SMC of the microvessels it is controlled by Ca2+ release
from the SR mediated exclusively by IP3R channels.
Bogomoletz Institute of Physiology, Kiev, Ukraine
e-mail: Nana Voitenko <[email protected]>
AMPA receptors (AMPARs) are responsible for the majority of excitatory synaptic transmission and play a critical
role in synaptic plasticity in the mammalian CNS. In adult
mammalian CNS, most AMPARs have low Ca2+ permeability because GluR2 subunit is fully edited throughout development and widely expressed. However, AMPAR Ca2+
permeability in adult CNS is not static. It can be modified
dynamically by neuronal activity, sensory experience, and
neuronal insults via changes in trafficking, expression, and
RNA editing of the GluR2 subunit.
We reported recently that synaptic AMPAR subunit GluR2
is internalized in dorsal horn neurons of the spinal cord
during the maintenance period of Complete Freund’s Adjuvant (CFA)-induced persistent inflammatory pain. This
internalization depends on spinal cord dorsal horn (DH)
protein kinase C α (PKCα) activation and is causally linked
to pain hypersensitivity during the maintenance period of
persistent inflammatory pain. We have also shown that
CFA-induced inflammation causes an increase in functional expression of extrasynaptic AMPARs in rat substantia gelatinisa neurons during the maintenance rather than
development of persistent pain. This increase, revealed as
a significant enhancement of AMPA-induced membrane
currents and [Ca2+]i transients, was observed only in neurons characterized by an intrinsic tonic firing properties;
whereas no changes were observed in neurons exhibiting
a strong adaptation. The increase was also accompanied
by an enhancement of surface GluR1 expression and of
the total amount of cobalt-positive neurons indicating an
increase in a pool of GluR2-lacking AMPARs in extrasynaptic plasma membrane.
Our recent results show that PKCα inhibition by both AS
ODN and Chelerythrine (intrathecal administration) attenuate CFA-induced increases in the calcium permeability of synaptic AMPA receptors in the superficial dorsal
horn neurons. Concomitantly, such inhibition resulted in
major anti-hyperalgesic effects, suggesting that PKCα plays
a major pronociceptive role in chronic pain states. Taken
together, the results provide direct evidence linking dorsal
horn PKCα to pain perception and suggest that it may offer a specific molecular target for the treatment of pain.
Acknowledgements
This work was supported by NASU Biotechnology grant (N.V).
e-mail: Ludmila Borisova <[email protected]>
Abstracts
84
L8.3
Oral presentations
Calcium signaling in normal and
transformed T-cells, possible
ways of modification
O8.1
Olga P. Matyshevska
Kyiv Taras Shevchenko University, Department of Biochemistry, Kyiv,
Ukraine
e-mail: Olga Matyshevska <[email protected]>
Changes in the level of intracellular Ca2+ ([Ca2+]i) provide
calcium signals of different kinetics, amplitude and subcellular localization (derived either from external medium or
from internal stores) which in integration with other signaltransduction cascades are shown to control not only cell
proliferation, but apoptosis, cell transformation and tumorigenesis. Remodelling of Ca2+-signalling and Ca2+ buffering
is supposed to reduce Ca2+-dependent ROS production, to
diminish resultant toxicity and to avoid Ca2+-dependent apoptotic pathways in cells with sustained transformed phenotype. The recent data concerning apoptotic signals that
induce endoplasmic reticulum (ER ) — mitochondrial Ca2+
flux and their modification during cancer are analysed.
In the present study the comparative evaluation of the resting [Ca2+]i and Ca2+-release from ER and mitochondrial
pools in both normal T-cells, derived from Wistar rat thymus, and transformed T-cell lines (Jurkat, L1210, MT-4)
was done using indo-1, thapsigargin, CCCP in normal and
Ca2+-free medium in order to identify the capacity of Ca2+
buffering system components and the possible targets for
controlling Ca2+-dependent pathways of trasformed cells
resistance to apoptosis.
Recent progress in nanobiotechnology have arised interest
in biomedical application of fullerenes C60 — spherical carbon nanostructures, able to penetrate into the cells, to accommodate inside hydrophobic regions of cell membranes
and to generate ROS with almost 100% quantum yield after
UV/Vis irradiation. We have demonstrated that combined
action of fullerenes C60 and irradiation in λ=320–600 nm
diapason is followed by cell death due to activation of apoptotic mitochondrial pathway in transformed, but not in
normal T-cells. The effects of fullerenes C60 in nonexcited
state and after photoexcitation on Ca2+-permeability of
plasma and ER membranes, intracellular ROS production
and Ca2+-buffering are analysed.
Ca2+- and Mg2+-induced changes
of membrane potential of smooth
muscle mitochondria
Natalia Kandaurova, Lidiya Babich, Sergiy
Shlykov, Sergiy Kosterin
O.V. Palladin Institute of Biochemistry, National Academy of Sciences of
Ukraine, Kyiv, Ukraine
e-mail: Kandaurova Natalia <[email protected]>
Mitochondrial membrane potential is important factor for
calcium accumulation in mitochondria. On the other hand
cations are able to regulate the accumulation of Ca2+ in mitochondria. There can be no doubt about the importance
of investigation of mitochondrial membrane potential
regulation by the physiologically important cations — Ca2+
and Mg2+. Myometrium mitochondria were isolated by differential centrifugation. To determine purity, preparations
of mitochondria were stained using NAO (Ex 488 nm,
Em 525 nm, 100 nM). In all preparations analyzed, more
than 95% of the events were NAO-positive. The changes
of membrane potential of isolated smooth muscle mitochondria were studied using flow cytometry analysis and
potentialsensitive fluorescent dye TMRM (Ex 488 nm, Em
590 nm, 100 nM). Registration of the membrane potential was done in the incubation medium containing (mM):
20 Hepes (рН=7.4), 250 sucrose, 0.1 Pi (K+-phosphate
buffer, рН=7.4), 0.5 MgCl2, and 5 sodium succinate. It
was shown, that Ca2+ (100 µM) addition to the incubation
medium induced mitochondrial membrane depolarization.
The preliminary presence in the incubation medium of the
Ca2+-uniporter inhibitor – ruthenium red (10 µM) – partly
prevented Ca2+-induce depolarization. Ca2+ accumulation
in the mitochondria matrix activates Ca2+ efflux through
the H+/Ca2+ exchanger and led to the elevation of H+
concentration in the matrix, that is, to the mitochondrial
membrane potential dissipation. So, Ca2+-induce depolarization could be explained by Са2+/Н+exchanger activation
which functioning lead to membrane potential dissipation.
At the presence of Mg2+ (3 mM) and ATP (3 mM), Ca2+
addition did not cause depolarization. Suggestion has been
made that in this case ATP-synthase acts in the opposite
direction as Н+-pump and prevents mitochondrial membrane potential dissipation. Mg2+ (7 mM) addition to the
incubation medium induced mitochondrial membrane hyperpolarization. In the case of protonophore CCCP (1 µM)
preliminary presence in incubation medium, Mg2+ addition
did not lead to membrane potential hyperpolarization.
Also it was shown that Mg2+ (7 mM) preliminary presence
in incubation medium didn’t protect mitochondria from
Ca2+-induced depolarization. Thus it was shown that Ca2+
and Mg2+ are able to change the polarization level of the
smooth muscle mitochondria inner membrane.
O8.2
Inhibitory properties of calixarenes
relative to ion-transporting АТРase of
myometrium cells plasma membrane.
Kinetic and structural analysis
Alexandr A. Shkrabak, Tatiana O. Veklich, Alexandr V. Bevza
Palladin Institute of Biochemistry of NASU, Muscle Biochemistry
Department, Ukraine
e-mail: Alexandr Shkrabak <[email protected]>
By now a great attention of scientists is paid to calixarenes
as original molecular platforms which are perspective for
designing of biologically-active compounds. Calixarenes
are macrocyclic oligophenolic compounds and some of
them posses bactericidal, antiviral, antitumoral, antithrombotic activity, they practically have no toxic action on cells.
In our previous investigation carried out on the suspension
of myometrium cell plasma membranes we found that calixarenes such as С-107, C-99 and C-97 effectively inhibited
Na+,K+-ATPase and did not influence on activity of other
membrane-bound АТРases. Considering further development conception about mechanisms of inhibition of the
plasma membrane Na+,K+-ATPase of uterus cells by this
calixarenes we have more thoroughly investigated the influence of these compounds and their structural analogs on
the Nа+,K+-АТРase activity.
It was shown that calixarene C-107, inhibiting Na+,K+-ATPase, did not considerably change the kinetic parameters
(Km, nH) of reaction velocity dependence on concentration
of substrate and activation ions (Mg, Na+, K+. Therefore,
we can conclude that inhibitory action of calixarene C-107
on Na+,K+-ATPase has uncompetitive character relative to
ATP, Mg, Na+, K+ and, perhaps, interaction of calixarene
C-107 with enzyme leads to decrease of enzyme turnover
number. It was also shown that this calixarene increased
twice the affinity of the enzyme towards the sodium pump
conventional inhibitor - ouabain.
It was carried out the computer modeling of the calixarene
C-107 interaction with Na+,K+-ATPase and shown the
probable site of calixarene association with enzyme.
The influence on Na+,K+-АТРase of calixarenes which
differ from the calixarenes C-97 and C-99 only some substituents on the upper and lower rim of macrocycle was
analyzed. This investigation let us establish functional
groups which are important for inhibitory properties of
calixarenes.
The inhibition of Ca2+,Mg2+-ATPase activity of uterus
cell plasma membrane by calixarene C-90 has been shown.
This influence is selective relative to other ATPases of plasma membrane and quite effective — the apparent constant
of inhibition was 20.2 ± 0.5 µM.
We consider that these calixarenes are perspective for creation of pharmaceuticals in order to regulate muscle contractility.
Acknowledgements
We are thankful to corresponding members of NASU V.I. Kalchenko and
S.O. Kosterin for helpful discussion and scientific cooperation.
85
Part II
Lectures
L9.1
Neuronal store-operated calcium entry
pathway as a novel therapeutic target
for Huntington’s disease treatment
Ilya B. Bezprozvanny
University of Texas, Southwestern Medical Center, Dallas, Texas, USA
e-mail: Ilya Bezprozvanny <[email protected]>
Huntington’s disease (HD) is a neurodegenerative disorder
caused by a polyglutamine expansion within Huntingtin
(Htt) protein. In the phenotypic screen we identified a class
of quinazoline-derived compounds which delayed a progression of a motor phenotype in transgenic Drosophila
HD flies. We found that the store-operated calcium (Ca2+)
entry (SOC) pathway activity is enhanced in neuronal cells
expressing mutant Htt and that the identified compounds
inhibit SOC pathway in HD neurons. The same compounds
exerted neuroprotective effects in glutamate-toxicity assays
with YAC128 MSN primary cultures. We demonstrated a
key role of TRPC1 channels in supporting SOC pathway
in HD neurons. We concluded that the TRPC1-mediated
neuronal SOC pathway constitutes a novel target for HD
treatment and that the identified compounds represent a
novel class of therapeutic agents for treatment of HD and
possibly other neurodegenerative disorders.
Abstracts
86
L9.2
L9.3
Intracellular calcium dysregulation
in Alzheimer’s disease
Neuronal calcium signaling
mediated by calmyrin proteins
Brian J. Bacskai
Urszula Wojda
MassGeneral Instituted for Neurodegenerative Diseases, Massachusetts
General Hospital/Harvard Medical School, USA
International Institute of Molecular and Cell Biology in Warsaw,
Laboratory of Neurodegeneration, Warsaw, Poland
e-mail: Brian Bacskai <[email protected]>
e-mail: Urszula Wojda <[email protected]>
Alzheimer’s disease is characterized by the deposition of
senile plaques in the brain resulting in toxicity that ultimately leads to neural network disruption. Intracellular calcium
is a tightly regulated second messenger whose activation
leads to numerous downstream events, including cell death.
It has long been postulated that dysregulation of calcium
homeostasis plays a role in Alzheimer’s disease, however,
this has not been demonstrated directly. We used multiphoton microscopy of a genetically encoded calcium reporter
in the intact brains of living mouse models of AD. With
single cell, and single spine resolution, we measured calcium concentrations quantitatively. We found that resting
calcium in wildtype mice was tightly regulated at a value
of ~80nM. These levels were increased in neurons in APP
mice with cortical plaques. The elevated calcium levels in
neurons (calcium overload) depended to some extent on
the proximity to individual senile plaques. However, even
far from plaques, calcium overload was detected in ~20%
of neurites, revealing a selectively vulnerable population
of cells in the cortex. The neuronal calcium overload was
not observed in transgenic mice expressing mutant human presenilin alone. Likewise, the calcium overload was
correlated both with the loss of spino-dendritic compartmentalization important for synaptic coordination and the
morphological correlates of neurodegeneration. These
data reveal for the first time disruptions in calcium homeostasis in neurons in the living brain of mouse models of
AD. Together, the results demonstrate that the aberrant intracellular calcium levels in the brain provide insight into
the pathophysiology of AD and suggest that specific manipulation of calcium levels may be a productive avenue for
Alzheimer’s disease therapeutics.
Ca2+ signals are sensed and transmitted to cellular effectors
by multiple Ca2+-binding proteins, including such EF-hand
family members as calmodulin, calcineurin B, and a group
of neuronal calcium sensors (NCSs). The EF-hand Ca2+sensors transduce various Ca2+-encoded information based
on differences in their localization, Ca2+ affinity, and specific interactions with protein ligands. Calmyrins sustain
a novel subfamily of the EF-hand Ca2+-sensors, mostly
similar to the NCSs. Genes encoding calmyrins were identified from invertebrates to human by genomic research
and structural comparisons. In human four genes encode
calmyrins (CaMy1–CaMy4). We have characterized CaMy1
and its closest homolog CaMy2 proteins in human and rat
brains and found that Ca2+-binding, localization in brain
neurons, and interaction partners differentiate these two
members of the CaMy subfamily [1] CaMy1 seems to be involved in Alzheimer’s disease (AD) [2, 3]. We found CaMy1
in senile plaques in brains of AD patients and characterized
CaMy1 interaction with AD-associated presenilin 2 [3, 4].
Moreover, we identified a novel ligand of CaMy1 in human
brain, microtubule-destabilizing factor SCG10 (stathmin2),
which is involved in neuronal growth and regeneration. Our
results indicate that CaMy1 via SCG10 couples Ca2+ signals
with the dynamics of microtubules during neuronal outgrowth [5]. Search for protein ligands of CaMy2 resulted in
identification of SNARE proteins that are essential for the
vesicular traffic between the cell membrane and the Golgi
compartment. Colocalization, coimmunoprecipitation, and
functional assays point to CaMy2 involvement in SNAREmediated intracellular trafficking in neurons. These studies
reveal novel, distinctive Ca2+-signaling functions of CaMy1
and CaMy2 in neurons and point to CaMy1 role in AD
pathogenesis.
References
1. Blazejczyk et al. (2009) Arch Biochem Biophys 487.
2. Sutcliffe et al. (2011) J Neuroscie Res 89.
3. Bernstein et al. (2005) Neuropathol Appl Neurobiol.
4. Blazejczyk et al. (2006) Biochim Biophys Acta – Mol Basis Dis 1762.
5. Sobczak et al. (2011) Biochim Biophys Acta – Mol Cell Res 1813.
Oral presentations
O9.2
O9.1
Presynaptic ionotropic glutamate
receptors serve as a trigger of ROS
production in axon terminals
FBPase stabilizes mitochondrial
function in cardiomiocytes Michal Pirog, Agnieszka Gizak, Dariusz Rakus Department of Animal Molecular Physiology, Wroclaw University,
Wroclaw, Poland e-mail: Michał Piróg <[email protected]>
A growing body of evidence suggests that muscle fructose 1,6-bisphosphatase (FBPase) is involved in regulation
of cell mortality and survival. However, its precise role in
these processes remained unknown. We show that, in HL-1
cardiomyocytes, inhibition of glycogen synthase kinase 3
(GSK3) or elevation of cellular Ca2+results in translocation
of muscle FBPase to mitochondria, which is correlated
with morphological changes of the organelles. In vitro experiments demonstrate that FBPase reduces the rate of calcium-induced mitochondrial swelling and affects ATP synthesis. Mass spectrometry reveals that interaction partners
for FBPase are a set of mitochondrial proteins involved
in regulation of volume and energy homeostasis, such as
ATP synthase subunit alpha, ADP/ATP translocase and
voltage-dependent anion channel 2.
Taken together, we show that muscle FBPase plays a role
in cellular stress response and might constitute a novel
link between cardiac hypertrophic signalling and cell survival. We propose a mechanism in which GSK3-inhibition
and calcium-stimulated association of FBPase with mitochondria helps to adapt cardiomyocytes to Ca2+overload
during prolonged, high-intensity exercise training without
progression to cardiac failure and death. Our results might
provide new prospects for cardiac failure treatment. 87
Olga Krupko, Alla Tarasenko, Nina Himmelreich
Palladin Institute of Biochemistry of the National Academy of Sciences
of Ukraine Department of Neurochemistry, Ukraine
e-mail: Olga Krupko <[email protected]>
There is a large body of evidence that reactive oxygen
species (ROS), such as superoxide and hydrogen peroxide (H2O2), are important signaling molecules underlying
mammalian learning and memory. Specifically, ROS production has been shown to be required for a form of synaptic plasticity called long-term potentiation (LTP) and for
biochemical signal transduction cascades that are believed
to underlie LTP and memory formation. We are particularly interested in presynaptic modulatory systems that selectively alter the release of glutamate as well as of inhibitory
neurotransmitters such as GABA.
We studied whether cortical and hippocampal axon terminals would respond to activation of presynaptic ionotropic
glutamate receptors by increasing the intensity of ROS
production. Glutamate receptors activation was triggered
by specific agonists of glutamate receptors: glutamate,
NMDA and kainate. Evaluation of ROS production was
performed with fluorogenic compound H2DCF-DA. We
have revealed an augmented ROS generation by nerve
terminals after the application of glutamate, NMDA and
kainate, and this process could be significantly attenuated
by antagonists of NMDA and kainate receptors, MK-801
and CNQX respectively. The intensity of ROS production as spontaneous as evoked by the glutamate receptor
activation depended on a type of energetic fuel and was
higher in conditions favoring to generation of NADPH,
e.g. when axon terminals consumed glucose. Consumption
of pyruvate led to a significant decrease of ROS production, and this effect of pyruvate appeared to be only partially due to its scavenger activity. Kainate as a stimulant
of ROS production was much more potent than NMDA,
especially when pyruvate served as a fuel. DPI, an inhibitor
of NADPH oxidase, effectively blocked the production of
ROS. Together, these findings suggest the important role
of NADPH oxidase in ROS production evoked by activation of presynaptic glutamate receptors. With regard to
data from the astrocyte-neuron lactate shuttle hypothesis,
presynaptic kainate receptors could be considered as a
main trigger of ROS production in axon terminals, when
they preferably consume lactate/pyruvate.
Abstracts
88
Posters
P8_9.2
P8_9.1
Potassium channels as major
determinants of neuronal excitability
Neuritogenesis in PMCA2 or PMCA3reduced PC12 cell lines is accompanied by
alterations in CaM, GAP43 and MAP kinases
Tomasz Boczek1, Bozena Ferenc1, Michalina Kosiorek2, Slawomir Pikula2, Ludmila Zylinska1
1Medical University of Lodz, Department of Molecular Neurochemistry,
Lodz, Poland; 2Nencki Institute of Experimental Biology, Department of
Biochemistry, Warsaw, Poland
e-mail: Tomasz Boczek <[email protected]>
Ca2+ is a potent second messenger triggering nearly all cellular processes. However, in order to sustain proper functioning of the cell, Ca2+ homeostasis must be tightly controlled.
The fundamental element of Ca2+ extruding systems is
Ca2+-ATPase (PMCA), maintaining the cytosolic concentration of Ca2+ at nanomolar range. From 4 main isoforms
of PMCA, PMCA2 and PMCA3 are found in excitable
cells including PC12 cell line. These isoforms differ by affinity to calmodulin (CaM), a strong activator of PMCA.
Function of CaM is regulated by GAP43 that serves as
a reservoir of CaM, but not CaM/Ca2+ complex. Thus,
changes in Ca2+ concentration constitute a signal stimulating the release of CaM from CaM/GAP43 complex,
making CaM available for its intracellular targets. We have
previously shown that suppression of PMCA2 or PMCA3
in PC12 cell line induced morphological changes similar to
the early stages of neuritogenesis. Here we present some
aspects of a molecular background for this phenomenon.
Using qPCR and immunodetection techniques we detected
that in both PMCA2 or PMCA3-reduced lines a raise in
total CaM was associated with stimulation of expression of
calmI and calmII genes. We also found a higher amount of
mRNA and GAP43 protein, however its phosphorylation
level declined in relation to the control cells. Double staining revealed significant changes in distribution of GAP43
and CaM between control, PMCA2- and PMCA3-reduced
cell lines. Since a rise in [Ca2+]c can also activate MAP kinase family members, participating in the transmission of
signals, as a next step we examined changes in their content
by immunoblotting technique. PMCA2 or PMCA3 reduction did not affect p38 MAPK amount, but p-p38 MAPK
was diminished. In contrast, a total amount of JNK declined without changes in its phosphorylation. Whereas
ERK1/2 proteins increased in both PMCA-reduced cell
lines, the p-ERK1 decreased to a half in comparison to the
control cells, but p-ERK2 amount was similar in all examined lines. Our data point to a functional importance of
PMCA2 and PMCA3 isoforms in differentiation of PC12
cells and highlight a relation between PMCA composition
and regulation of various signaling pathways.
Acknowledgements
Supported by grants 502-03/6-086/502-64-003 and 503/6-086-02/50301 from the Medical University of Lodz, Poland.
N. A. Bogdanova1, E. V. Dolgaya2, I. S. Magura3 ,
N. Kh. Pogorelaya4, V. A. Yavorsky5, E. A. Lukyanets6
Bogomoletz Institute of Physiology, Department of General Physiology,
Ukraine
e-mail: Nataliia Bogdanova <[email protected]>
Potassium (K+) channels are the most diverse class of ion
channels, and are important for regulating neuronal excitability and signaling activity in a variety of ways. They are
major determinants of membrane excitability, influencing
the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation.
Potassium channels fulfill important function in many signal transduction pathways in the nervous system. Complex
processing and integration of the signals observed in neurons are facilitated by a diverse range of the gating properties of the ion channels in this cell type, particularly of the
voltage-gated K+ channels (Kv). A distinctive combination
the of K+ channels endows neurons with a broad repertoire of the excitable properties and allows each neuron
to respond in a specific manner to a given input at a given
time. The properties of many K+ channels can be modulated by second messenger pathways activated by neurotransmitters and other stimuli. K+ channels are among the
most frequent targets of the actions of several signaling
system. Voltage-gated K+ cannels exist as macromolecular
complexes able to integrate a plethora of cellular signals
that fine tune channel activities. Specificity of information
is generally encoded by the kinetics of action potential frequency, duration, bursting, and summation. If longer term
modifications in firing patterns are required, the cell may
alter the transcriptional expression of ion channel genes
for diverse functions. The number of K+ channel genes is
relatively large; however, the diversity of endogenous K+
current phenotypes observed from various excitable cells
is much greater. Additional processes such as alternative
splicing, posttranslational modification, and heterologus
assembly of pore-forming subunits in tetramers contribute
to extend the functional diversity of the limited repertoire
of K+ channel gene products. Even greater diversity can be
achieved through interactions between K+ channel proteins
and accessory proteins.
Keywords: potassium channels, neuronal excitability, signaling activity.
P8_9.3
P8_9.4
Altered glutamate transport and the
development of synaptopathy
Mitochondria express alpha 7 nicotinic
acetylcholine receptors involved in
Ca2+ buffering and apoptosis
Tatiana Borisova, Roman Sivko, Natalia Krisanova
Palladin Institute of Biochemistry NAS of Ukraine, Department of
Neurochemistry, Ukraine
e-mail: Tatiana Borisova <[email protected]>
The study was focused on the analysis of the changes in
the key characteristics of glutamatergic neurotransmission:
(1) Na+-dependent glutamate uptake by rat brain nerve terminals; (2) the extracellular level and tonic release of glutamate, i.e., parameters, which are important for spontaneous
activity of the brain; and (3) stimulated by depolarization
of the plasma membrane exocytotic (Ca2+-dependent)
and transporter-mediated (Ca2+-independent) release of
glutamate under different experimental conditions. Two
model systems were used to investigate the alterations in
glutamate transport and the experiments were carried out
on: (*) nerve terminals of rats subjected to hypergravity
in the cranio-caudal direction and (**) cholesterol-deficient
nerve terminals that were obtained after the treatment with
cholesterol-depleting agent, methyl-b-cyclodextrin (MCD).
The changes in uptake, unstimulated/stimulated release
and the ambient level of L-[14C]glutamate were found in
both experimental models. Interrelations between uptake,
tonic release and the ambient level of the neurotransmitter were determined under conditions of hypergravity and
cholesterol deficiency. Analysing stimulated by depolarization release of glutamate under hypergravity, it was shown
a decrease in Ca2+-dependent and an increase in Ca2+-independent release of glutamate that was a result of partial decrease in the proton gradient of synaptic vesicles, and thus
the redistribution of glutamate between the vesicular and
cytosolic pools. In the model of cholesterol deficiency, different mechanisms underlying changes in glutamate transport were found in the presence and after washing of MCD
from synaptosomes. Dissipation of the proton gradient of
synaptic vesicles was a cause for additional suppression of
glutamate transporter activity, an increase in tonic, Са2+independent release and ambient level as well as dramatic
decrease in exocytotic release of glutamate from nerve terminals in the presence of MCD in the incubation media.
The treatment of isolated synaptic vesicles by complex
MCD-cholesterol, which enriched membranes with cholesterol, increased the proton gradient of synaptic vesicles
that augmented their ability to accumulate neurotransmitter
and efficiency of synaptic transmission. Based on the experimental data obtained from the models of hypergravity
and cholesterol-deficiency, different mechanisms and conceptual scheme of the development of synaptopathy, i.e.
synaptic dysfunction and impairment of synaptic transmission, were proposed.
89
Galyna L. Gergalova1, Olena Yu. Lykhmus1,
Olena M. Kalashnyk1, Lyudmyla M. Koval1,
Victor I. Tsetlin2, Sergiy V. Komisarenko1, Maryna V. Skok1
1Palladin Institutre of Biochemistry, Department of Molecular
Immunology, Ukraine; 2Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry, Molecular Bases of Neurosignalization Department, Russia
e-mail: Galina Gergalova <[email protected]>
Nicotinic acetylcholine receptors composed of α7 subunits (α7 nAChRs) are expressed in both neurons and nonexcitable cells to mediate fast synaptic transmission, survival and anti-inflammatory signaling. Here we show for
the first time that functional α7 nAChRs are present in
mitochondria. Purified mouse liver mitochondria bound
α7 nAChR-specific antibodies and α-cobratoxin; the binding was decreased in mitochondria of α7-/- mice. In U373
cell line the bound α7-specific antibody was co-localized
with mitochondria outer membrane translocase-specific
antibody, while in liver mitochondria lysate α7 nAChR was
co-captured with the voltage-dependent anion channel
(VDAC). Both the VDAC inhibitor and α7 nAChR agonists
acetylcholine, choline and PNU-282987 impaired intramitochondrial Ca2+ accumulation. Similarly to VDAC inhibitor, choline decreased mitochondria cytochrome c release
stimulated with CaCl2 and its effect was attenuated with
α7-specific antagonist methyllicaconitine (MLA). Choline
effective dose (10 μM) was in the range of its inhibitory
concentrations, while MLA stimulated cytochrome c release in the absence of nAChR agonists or Ca2+. These
data demonstrate that α7 nAChRs expressed in mitochondria outer membrane regulate the VDAC-mediated Ca2+
transport and mitochondrial permeability transition in ion
channel-independent manner. This finding discovers a novel mechanism regulating mitochondria-driven apoptosis by
non-canonical mode of α7 nAChR functioning.
Abstracts
90
P8_9.5
P8_9.6
Localization and role of S100A6,
a calcium binding protein, in
differentiating epidermis
Effect of mutations in presenilin-1 gene on
ER calcium release in Alzheimer’s disease
Agnieszka Graczyk, Wiesława Leśniak
Nencki Institute of Experimental Biology PAS, Laboratory of Calcium
Binding Proteins, Warsaw, Poland
e-mail: Agnieszka Graczyk <[email protected]>
S100A6 is a small (10 kDa), Ca2+-binding protein belonging to the S100 protein family characterized by the presence
of two EF-hand structures. S100A6 is expressed mainly in
fibroblasts and epithelial cells including keratinocytes that
build the epidermis. Furthermore, its gene is located on
chromosome 1q21 in a gene cluster known as EDC (Epidermal Differentiation Complex) which is formed by genes
expressed at different stages of epidermal differentiation
[1]. For those reasons we decided to investigate the localization and role of S100A6 during epidermal differentiation.
Epidermis is a multilayered skin epithelium that provides
the external impermeable and resistant skin barrier. Epidermal keratinocytes undergo a well defined differentiation
process which is to a great extent regulated by calcium ions
[2]. In our studies we used a spontaneously immortalized,
non-transformed human keratinocyte cell line, HaCaT, and
employed two models of keratinocyte differentiation: 1)
HaCaT cells cultured in media with different calcium concentrations and 2) organotypic 3D co-cultures in which
HaCaT cells are grown on collagen-embedded fibroblasts
that provide them with growth factors and enable to form
epidermal-like, multilayered structures and to differentiate.
Using western blot, RT-PCR, Q-RT-PCR and immunocyto- or immunohistochemistry we evaluated the expression
of marker proteins (keratin 14, loricrin, involucrin) in the
two differentiation models. We have assessed the level of
S100A6 and of S100A8, another protein of the S100 family, at different stages of epidermal differentiation. Finally
we have established HaCaT cells stably transfected with
shRNA for S100A6. These cells are currently tested in keratinocyte culture models in order to check whether diminished S100A6 level can influence the process of epidermal
differentiation.
Acknowledgements
This work was supported by statutory funds from the Nencki Institute of
Experimental Biology.
References
1. Mischke BP et al. (1996) J Invest Dermatol 106: 989–992.
2. Elias PM et al. (2002) J Invest Dermatol 119: 1128-1136.
Anna Jaworska1, Joanna Gruszczyńska-Biegała1, Aleksandra
Szybińska1, Tomasz Węgierski1, Kamran Honarnejad2, Jochen Herms2, Jacek Kuźnicki1,3
1International Institute of Molecular and Cell Biology, Warsaw, Poland;
2Center of Neuropathology and Prion Research, Ludwig Maximilians
Universität, Munich, Germany; 3Nencki Institute of Experimental
Biology, Warsaw, Poland e-mail: Anna Jaworska <[email protected]>
Calcium dyshomeostasis is one of the factors involved in
Alzheimer’s disease (AD). Mutations in presenilin-1 gene
(PS1), an endoplasmic reticulum (ER) membrane integral protein are linked to the early onset, familiar form of
Alzheimer’s disease (FAD). These mutations disturb calcium release from ER store as well as store-operated calcium entry (SOCE). However, the exact mechanism of this
process remains unclear. We believe that disturbed calcium
homeostasis may be the primary cause of AD pathology.
Because of that, our goal is to find a chemical compound,
which could restore the normal calcium level and therefore
act as an AD drug.
We have investigated calcium release from different ER
calcium channels in cortical neurons from transgenic mice
overexpressing human mutant presenilin-1. To do that, we
performed calcium imaging using ratiometric calcium dye
Fura-2AM. Our preliminary data showed increased calcium
release from ER in neurons with PS1 mutation in comparison to neurons obtained from mouse overexpressing wild
type PS1. We have also checked calcium response of immortalized lymphocyte B lines derived from FAD patients
bearing PS1 mutations by stimulating them with different
ER calcium mobilizing agents. We were able to see calcium
response to 4-Cmc, which in higher concentrations acts as
SERCA-pump inhibitor and RyRs agonist. We have found
increased calcium release from ER in lymphocytes from
two family members bearing PS1 S170F mutation.
After the initial screening of the library of chemical compounds, we were able to select the most promising hits and
we plan to check their effectiveness on above mentioned
AD models.
He hope, that our findings will take us one step forward in
establishing an effective AD treatment. Parnas Conference Warsaw 2011
91
P8_9.7
P8_9.8
Alternative signaling pathways compensate
Rho-kinase inhibition in glioma C6 cells
New role of neuron-specific PMCA isoforms?
Jaroslaw Korczynski1, Anna Wasik2, Katarzyna Sobierajska3,
Wojciech Brutkowski1, Pawel Pomorski1, Wanda Klopocka1
1Nencki Institute of Experimental Biology in Warsaw, Department of
Biochemistry; 2Department of Cell Biology, Warsaw, Poland; 3Medical
University of Lodz, Department of Molecular and Medical Biophysics,
Lodz, Poland
e-mail: Jarosław Korczyński <[email protected]>
Signaling pathways related to Rho family proteins control
actin cytoskeleton dynamics mainly by regulating the activity of two actin binding proteins: cofilin and myosin II. We
have found that the effect of Rho-kinase activity inhibition
can be reversed by activation of P2Y2 receptor in glioma
C6 cells. In current study we attempt to answer the following question: what signaling pathways induced by P2Y2 receptor activation are involved in compensation of changes
caused by Rho-kinase inhibition?
To evaluate molecular mechanism of this compensation,
activity of two actin-binding proteins: cofilin and myosin
II, was studied in glioma C6 cells. We have observed that
ROCK inhibition decreases cofilin and myosin II phosphorylation and causes changes in the distribution of examined
proteins. We revealed, however that stimulation of P2Y2
nucleotide receptor in such cells induces cofilin and myosin
II phosphorylation. The myosin light chain phosphorylation stimulates actomyosin activity and cell contractility
while cofilin phosphorylation inactivates this protein and
stabilizes actin network. We have observed that complete
renormalization of glioma C6 cells with blocked Rho-kinase required increasing the level of phosphorylated both:
cofilin and myosin II.
We confirmed that myosin light chain phosphorylation is
increased by calcium dependent MLCK activated by calcium signaling mediated by Gq-protein. We suggest also that
Go-mediated Rac activation and Rac/PAK/LIMK pathway
is responsible for cofilin phosphorylation. Activation of
this pathway requires however interactions between P2Y2
receptors and αVβ5 integrins what is impossible in nonadherent cells or in calcium-free environment.
Antoni Kowalski, Ludmiła Żylińska, Elżbieta Rębas
Medical University of Lodz, Department of Molecular Neurochemistry,
Lodz, Poland
e-mail: Elżbieta Rębas <[email protected]>
GABA-shunt is a metabolic pathway that consists of three
enzymes. Glutamate decarboxylase (present in two isoforms: GAD65 and GAD67) is found in cytosol and catalyzes
the synthesis of neurotransmitter GABA from glutamate.
Remaining two enzymes — GABA-aminotransferase
(GABA-T) and succinic semialdehyde dehydrogenase
(SSADH) are present in mitochondria and responsible for
conversion of GABA to TCA-cycle intermediate — succinate. Thus, GABA can be used by cells as a support
for energetic metabolism. Apart from neurons, GABA is
synthesized in endocrine cells, in which it regulates both
the production and secretion of hormones. In previous
study we had examined if GABA-shunt enzymes activity
and expression is dependent on calcium homeostasis in
rat anterior pituitary GH3 cell line. Now we focused on
PC12 (pheochromocytoma) pseudoneuronal cells, derived
from rat adrenal medulla. We obtained stably transfected
cell lines with suppressed expression of plasma membrane
calcium ATPase (PMCA) isoforms 2 or 3, which are specific primarily for excitable cells. Enzymes activities were
measured with spectrofluorimetric and spectrophotometric methods. Expression was measured using on mRNA
and protein level. Our results confirm these previously obtained from GH3 cells. In both examined types of cells
PMCA2 can play a great role in regulation of GABA-shunt.
Reduced expression of this calcium pump isoform resulted
in significant decrease of GAD and mitochondrial part of
the pathway. Moreover, suppression of PMCA3 caused significant increase in GAD65 isoform, which is known for
synthesizing GABA for neurotransmisson.
Acknowledgements
This work was supported by the Ministry of Science and Higher Education grant N N401 076337 and Medical University of Lodz grants 50216-810 and 503-6086-2.
Abstracts
92
P8_9.9
P8_9.10
Epothilone B — mechanism of action in
human ovarian cancer cells
Matrix metalloproteinases secrecion
without affecting the intracellular Ca2+ level
in HT29 cell line activated by thymosin β4
Aneta Rogalska, Agnieszka Marczak,
Arkadiusz Gajek, Marzena Szwed, Zofia Jóźwiak
1Institute
of Biophysics, Department of Thermobiology, Lodz, Poland
e-mail: Aneta Rogalska <[email protected]>
Epothilones are a new group of compounds with mechanisms of action similar to taxanes. In the present study, we
investigated the anticancer activity of epothilone B (Epo B)
in SKOV-3 ovarian cancer cells. Paclitaxel, from group of
taxanes was used for comparison.
We measured the level of apoptotic and necrotic cells by
double staining with Hoechst 33258 and propidium iodide
(PI). The production of reactive oxygen species (ROS)
and content of intracellular calcium in cells exposed to
Epo B were studied using specific probes: DCFH2-DA
(2’,7’-dichlorodihydrofluorescein diacetate) and Fluo-4
NW Calcium Assay Kits, respectively. The experiments
were performed in the presence and absence of an antioxidant, N‑acetylcysteine (NAC). The cytotoxic activity of the
drugs was determined by the MTT (3-(4,5-dimethylthiazol2-yl)-2,5 diphenylotetrazolium bromide) test.
We idicated that epothilone B was considerably more cytotoxic towards SKOV-3 cells than paclitaxel. Its IC50 concentrations (27.5 nM) was lower four times than that of
paclitaxel (126.2 nM). Epo B, induced both apoptosis and
necrosis in the SKOV-3 cells and this process was mediated
by ROS. We were demonstrated that ROS and Ca2+ may act
as common signals to regulate apoptosis in ovarian cancer
cells. The increased intracellular levels of free Ca2+ initiated
a cascade of events leading to cell injury and apoptosis.
Drugs induced in the tested cells an increase in the intracellular free calcium during the first 48 h following the treatment. Maximal increase in the level of calcium in SKOV-3
cells, was observed 24 h after treatment with EpoB (20%)
and 48 h upon incubation with PTX (56%). Free radical
scavenger, NAC, markedly reduced drugs-induced changes.
The ROS production was observed after 2h of incubation
with Epo B, but the highest level (118±3.1% in comparison
to the control) was noted after 24h.
Our results suggest that Epo B manifest higher antitumor activity against SKOV-3 cells than paclitaxel, but their
mechanisms of cell death induction are similar.
Anna Selmi1, Mariusz Malinowski2,
Radoslaw Bednarek1, Czeslaw S. Cierniewski1,2 1Department of Molecular and Medical Biophysics, 2Medical University
of Lodz, Institute of Medical Biology, Polish Academy of Science, Lodz,
Poland
e-mail: Anna Magdalena Selmi <[email protected]>
Thymosin β4 (Tβ4), the most ubiquitously expressed
member of b-thymosins family, induces diverse pleiotropic
extracellular effects, and is now recognized as the potent
regenerative peptide in a variety of tissues. It acts as an effective angiogenic factor with anti-inflammatory properties
that promotes both endothelial and keratinocyte migration
into cutaneous wounds. However, it is still uncertain which
functions of the Tβ4, the intracellular and/or extracellular,
has greater importance for its biological activity. Therefore,
the aim of this study was to compare the stimulatory effect
of Tβ4 on cancer cells tested in HT29 cells transfected with
pEGFP-N1-Tb4 with those treated with Tβ4 added extracellularly. Our data show that in both experimental settings,
Tβ4 induces synthesis and secretion of matrix metalloproteinases such MMP-2, MMP-7, and MMP-9. Interestingly,
in contrast to other agonists, Tβ4 added extracellularly does
not affect the intracellular Ca2+ level indicating that its effect does not involve activation of the cell membrane receptors which facilitate entry of Ca2+ in response to stimulation. As the major G-actin-sequestering protein in cells,
Tβ4 regulates and controls polymerization of actin and
thus can affect the motility of cells. However, transfection
of HT29 cells with the Tβ4 mutant deprived of the actin
binding sequence (pEGFP-N1-Tß4(KLKKTET/7A)) resulted
in the same extent of HT29 stimulation as that produced
by the wild type Tβ4 indicating that interference with actin
polymerization is not involved in this mechanism. In conclusion, our data suggest that stimulation of HT29 cells by
Tβ4 is mediated predominantly by the intracellular receptors that do not involve actin polymerization or the Ca2+
influx and mobilization.
93
P8_9.11
P8_9.12
NARP mutation and lack of mtDNA trigger
mitochondrial biogenesis which can be
modulated by selenite supplementation
Large-conductance potassium channel
opener NS1619 affects calcium homeostasis
in muscle and non-muscle cells
Marta Wojewoda, Jerzy Duszyński, Joanna Szczepanowska
Antoni Wrzosek1, Agnieszka Łukasiak2, Adam Szewczyk1
Nencki Institute of Experimental Biology, Department of Biochemistry,
Warsaw, Poland
1Laboratory of Intracellular Ion Channels, Nencki Institute of
Experimental Biology, Warsaw, Poland; 2Department of Biophysics,
University Warsaw of Life Sciences-SGGW, Warsaw, Poland
e-mail: Marta Wojewoda <[email protected]>
Mutation within the mitochondrial genome such as the
T8993G substitution associated with a severe disease (Neuropathy, Ataxia and Retinitis Pigmentosa, NARP) and loss
of mtDNA (Rho0 cells) lead to mitochondrial dysfunction
and subject cells to chronic mitochondrial stress associated with elevation of intracellular calcium levels and ROS
overproduction. Under such conditions, mitochondria-tonucleus (retrograde) signaling triggers adaptive changes in
the expression of multiple nuclear genes what can alter the
program of mitochondrial biogenesis. Since we found that
ROS and calcium levels were altered in human osteosarcoma NARP and Rho0 cells, we hypothesised that ROS
and calcium could be implicated in the retrograde signaling
from the mitochondria to the nucleus in these cells. We
previously found that selenite lowered ROS and calcium
levels in NARP cybrids and Rho0 cells. Therefore, we suggested that selenite could also exert the modulatory effect
on mitochondrial biogenesis in these cells.
e-mail: Antoni Wrzosek <[email protected]>
It is well documented that potassium channels openers have
cytoprotective properties in models of cell injury especially
in protection of ischemia-reperfusion induced cell damage.
It is suggested that usage of the potassium channel openers
also could be a protective pharmacological strategy against
cell dysfunction. The large-conductance Ca2+-activated potassium channels (BKCa channels) opener NS1619 is one
of the widely considered compounds to have a protective
effect in neurons and cardiomyocytes during the ischemiareperfusion events. It was shown that NS1619 acts on
coronary circulation, mitochondrial function, especially
on mitochondrial membrane potential, and mitochondrial
respiration in cultured EA.hy 926, H9C2 and C2C12 cell
lines. We have found that NS1619 caused changes in intracellular Ca2+ concentration in cultured H9C2, C2C12 and
EA.hy926 cell lines as measured using fura-2 fluorescence
ratiometric method. The main aim of the study was to find
the mechanisms and sources of Ca2+ elevation caused by
NS1619 in different type of the cells. The main source of
intracellular calcium is sarco/endoplasmic reticulum (SR/
ER). To test the effect of NS1619 on SR the rat cardiac and
skeletal muscle SR vesicles were isolated and fluorimetric
methods were used to measure the SERCA pump activity
and Ca2+ uptake by the isolated SR vesicles. We have found
that SR Ca2+ uptake was inhibited by NS1619 at the concentrations as low as 10 μM with half-maximal inhibition
seen at approximately 40 μM. The BKCa channels opener
NS1619 has also a profound effect on SERCA pump activity in the concentration-dependent manner which correlates with decreasing in Ca2+ uptake into SR vesicles. The
NS1619 inhibition of SERCA pump caused shift in the enzyme affinity for calcium and ATP to the higher concentration. The results are consistent with the hypothesis that the
large-conductance potassium channels opener NS1619 affects intracellular calcium homeostasis by interaction with
SR. Our results showed that NS1619 pleiotropic actions,
at least in part, may be triggered by the NS1619-induced
modulation of intracellular Ca2+ homeostasis caused by inhibition of SERCA pump in studied cells lines.
Acknowledgements
This study was supported by the Nencki Institute of Experimental Biology
and Ministry of Sciences and Higher Education (793/N-DAAD/2010/0).
Abstracts
94
P8_9.13
P8_9.14
The effect of PMCA2 or PMCA3
suppression on MAPK signaling pathways
during PC12 cells differentiation
Influence of a Fenugreek extract on Ca755
mouse mammary carcinoma growth,
polyamines level and expression of protein
products of some genes in tumor cells
Tomasz Boczek, Monika Sobczak, Bozena
Ferenc, Ludmila Zylinska
Medical University of Lodz, Department of Molecular Neurochemistry,
Lodz, Poland
e-mail: Ludmila Zylinska <[email protected]>
Transient increase in intracellular Ca2+ and subsequent
activation of downstream signaling cascades trigger neuronal differentiation. The plasma membrane Ca2+-ATPase
(PMCA) plays an important role in maintaining low cytosolic Ca2+ by extruding it from the cytoplasm across
the plasma membrane. Four isoforms of PMCA have
been detected. PMCA2 and PMCA3 appear to be neuronspecific while PMCA1 and PMCA4 are ubiquitously represented. We have previously shown that dibutyryl-cAMP
differentiated PC12 cell with reduced level of PMCA2
or PMCA3 exhibited mild but sustained increase in basal
[Ca2+]c. Because Ca2+ is known to be a regulator of differentiation process, we investigated the effect of disturbed
Ca2+ homeostasis on MAPK signaling cascades aswell as
the expression of MAPK-targeted transcription factors.
Using antibodies recognizing total protein content or its
phosphorylated form, we found that diminished level of
neuro-specific PMCAs contributed to decrease in p38
MAPK activation. Interestingly, in line with reduced level
of PMCA2, ERK2 was highly active during the differentiation process whereas reduction of PMCA3 caused late
activation of this kinase. PMCA3 suppression also affected
ERK1 activation. No dramatic alteration between modified
and control line were detected for JNK. Modifications in
MAPK signaling pathways activity were accompanied by
diverse changes in mRNA level of MAPK-targeted transcription factors. Using microarray approach, we found
that expression of kcnh8, myc, cebpa, jun and mef2c was
stimulated in both modified lines. Except jun, an increase
in mRNA level of these genes was much more remarkable
in line with reduced PMCA3 than PMCA2. Suppression of
neuro-specific PMCAs differentially affected max expression. While PMCA3-depleted cells exhibited significant
increase in mRNA level of this gene, PMCA2 reduction
resulted in nearly 4-time decline in max expression. Moreover, the expression of 2 unique genes (sp1and stat1) was
detected in PMCA2-reduced cells and also 2 genes (nfatc2
and creb1) in PMCA3-suppressed line. Our data indicate
on relationship between PMCAs composition and regulation of signaling cascades and possible PMCAs role in regulation of transcription during PC12 cells differentiation.
Acknowledgements
Supported by the grants 502-03/6-086/502-64-003 and 503/6-08602/503-01 from Medical University of Lodz.
Veronika Zhylenko1,2, Oleg Klenov2, Sophia Zaletok2
1Taras Shevchenko Kyiv National University of Kyiv, Educational and
Scientific Centre “Institute of Biology”, Department of Biochemistry,
Kyiv, Ukraine; 2R. E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Department of Tumor
Growth Biochemistry, Ukraine
email: Veronika Zhylenko <[email protected]>
Search for plant agents with anticancer properties is of current importance. Literary data and our own experiments
show that Fenugreek (Trigonella foenum graecum) has
growth-inhibiting activity as to some kinds of experimental tumors. It is known that polyamines (PA) — putrescine
(Put), spermidine (Spd) and spermine (Spn) — are required
for growth, proliferation and differentiation of normal and
tumor cells. High PA level have been reported in many
kinds of cancer and, because of this, it was proposed as a
biomarker of cancer growth.
The aim: to study antitumor effect of a Fenugreek extract
on the Ca755 mammary carcinoma in mice, PA content,
expression of the NF-κB transcription factor and protein
products of the NF-κB-dependent genes in the tumor cells.
Materials and methods: Experiments were carried out
on the mature C57Bl/6 female mice with subcutaneously
grafted Ca755 mammary carcinoma. The animals in experimental group were administered with the mix of Fenugreek extract powder (250 mg/kg of body mass) and standard mash from the day of tumor grafting up to sacrifice.
PA content in the tumor tissue was evaluated by the high
pressure liquid chromatography, expression of proteins –
by Western-blotting.
Results: Consumption of the Fenugreek extract led to
significant inhibition of Ca755 carcinoma growth in mice,
essential decrease of Put content, decreased levels of Spn
and Spd and weak increase of Spn acetylated forms in cancer tissue versus the control animals. Also, consumption of
the Fenugreek extract led to decreased level of expression
of the p50-subunit of the NF-κB transcription factor in
the nuclei of the tumor cells. Level of the p65 subunits was
also decreased but inessentially. Dramatic (almost 3-fold)
increase of level of p53 protein expression and decreased
c-myc protein expression were revealed in tumor cells.
Conclusions: Thus, these results show that extract of the
Fenugreek can affect different pathways of proliferation
control, including activation of the NF-κB transcription
factor and the regulation of PA metabolism.
Index
A
Abrosimovaa L.A.
Adamchuk-Chala N.
Adir N.
Airapetov L.G.
Akabayov B.
Amborska R.
Ambrozkiewicz M.
Arkin I.T.
Armon L.
Ashani Y.
Ashery-Padan R.
Assia Y.
Aviner R.
Avraham S.
Azem A.
Azoulay I.
P7.26
P5.12
P.3.7
P7.54
O7.1
P4.17
P4.12
L6.2
P5.1
P7.49
P1_2.3
P1_2.12
P1_2.4
P1_2.10
P7.1, P7.54
P7.1
B
Babich L.
Bacova B.
Bacskai B.J.
Bakurova E.M.
Balsewicz J.
Bańkowski E.
Baranova A.
Baranova A.V.
Barbash S.
Basaliy A.
Bąkowska-Żywicka K.
Bednarek R.
Bejtka M.
Bem J.
Ben-David M.
Ben-Meir D.
Ben-Yishay R.
Ben-Zvi S.
Berson A.
Bevza A.V.
Bezprozvanny I.B.
Bialopiotrowicz E.
Bieganowski P.
Bielczyk E.
Biernacka M.
Bigay J.
Bochtler M.
Boczek T.
Boesch-Saadatmandi C.
Bogdanova N.A.
Boiko N.
Boretska M.
Borisova L.
Borisova T.
Borowczyk K.
Borzenko B.G.
Borzęcka K.
Boyarshin K.
Bragoszewski P.
Brody Y.
Brodyak I.
Brosh R.
Brutkowski W.
Bura M.
Burlova-Vasilieva N.
Byts N.
O8.1
P5.16
L9.2
P7.24
P7.32
P7.10
P1_2.1, P1_2.9
P1_2.17, P7.41
L4.1
P7.34
P1_2.19
P8_9.10
P7.20
P4.1
P7.49
P1_2.3
O1.2
P3.2
L4.1
O8.2
L9.1
P4.2
P7.35
P5.13
P5.13
P7.18
L7.1, P7.42
P5.17, P8_9.1, P8_9.13
P1_2.14, P1_2.8
P8_9.2
P5.6
P7.2
L8.2
P6.1, P8_9.3
P4.7
P7.24
P5.9
L3.1
P7.3
O1.2
O5.2
P1_2.12
P8_9.7
P5.5
P7.4
P7.34, P7.37, P7.5
C
Caplan S.R.
Cassel D.
Cecchini G.
Chacinska A.
Chaturvedi M.
O5.1
P7.18
O5.1
P7.3
P4.3
Chazin W.J.
Chełstowska A.
Cherkas V.
Chumak V.V.
Ciarkowski J.
Ciechanover A.
Ciechomska I.
Cierniewski C.S.
Cieśla M.
Cieślar-Pobuda A.
Cusack S.
Czapinska H.
Czeredys M.
Czuryło E.A.
P7.43
P5.8
P7.27
P3.5
P7.53
K.1
P4.4
P8_9.10
P1_2.2
P5.19
L3.1
L7.1, P7.42
O6.2
P7.16
D
Dabrowski M.
Dankevich L.
Darzynkiewicz E.
Denysova O.
Dergai M.
Dergai O.
Derlacz R.
Dezube B.J.
Dolgaya E.V.
Domińska K.
Dor Y.
Dosenko V.E.
Dragovoz I.
Dreazen A.
Drobot L.
Dubey .I.Y.
Dulak J.
Duszyński J.
Dvashi Z.
Dyka M.
Dzbek J.
Dziembowska M.
Dżugaj A.
O4.2, P3.1, P3.4, P3.6, P4.9
P5.2
P7.29
P6.2, P6.3
L4.3, P7.31, P7.6, P7.7
L4.3, P7.31, P7.6, P7.7
P7.14
P7.21
P8_9.2
P5.3
P1_2.10
P5.16, P4.11
P5.2
P1_2.10
P7.34, P7.37, P7.5
P7.57
P1_2.14, P1_2.2, P1_2.8, P7.14
P7.17, P7.20, P8_9.11
P1_2.3
P5.14
L3.3
P1_2.6
O.1, P7.51
E
Eisenbach M.
El’skaya A.
Ellert-Miklaszewska A.
Elroy-Stein O.
Enyenihi A.A.
O5.1, P5.1, P7.39
P7.47
P4.10, P4.9
P1_2.4
P7.39
F
Fedorenko V.
Fedoseenko A.
Felder C.
Ferenc B.
Fiedorowicz E.
Filatova A.Y.
Filipek A.
Filipek S.
Filipowicz W.
Filonenko V.V.
Fiszer A.
Fraiberg M.
Friedman A.
Fyodorova S.V.
O3.2, P1_2.7
P7.34
P7.49
P8_9.1, P8_9.13
P5.4
P1_2.1
P7.10, P7.12, P7.35, P7.43, P7.48
P7.12
L1.2
O6.1
L4.2
P7.39
L4.1
P5.18
G
Gabrusiewicz K.
Gajek A.
Gajos-Michniewicz A.
P4.9, P4.10
P8_9.9
P5.3
Abstracts
96
Gamian A.
Ganguly K.
Garmanchuk L.
Gendaszewska-Darmach E.
Gergalova G.L.
Givati A.
Gizak A.
Gnatek Y.
Goedhart J.
Gokhman I.
Goldfinger N.
Goll Y.
Gornicka A.
Gośliński M.
Graczyk A.
Grochot-Przęczek A.
Groholska O.B.
Groopman J.E.
Gruszczyńska-Biegała J.
Grygalewicz B.
Gryshkova V.S.
Gumenyuk V.
P7.15
P4.5
P5.10
O7.2
P8_9.4
P3.2
O9.1
L4.1
P7.9
L5.2
P1_2.12
L4.1
P7.3
P1_2.16
P8_9.5
P7.14
P7.40
P7.21
P8_9.6
P5.13
O6.1
P4.6
H
Haklai-Topper L.
Halytskiy V.A.
Hanin G.
Havrylyuk D.Y.
Heneha A.
Herms J.
Hevkan I.I.
Himmelreich N.
Hoang V.P.
Holovchak N.
Honarnejad K.
Honchar M.
Honcharenko A.
Horbay R.
Horn D.
Horowitz M.
Horvat B.
Hubenya O.V.
Huppertz B.
L5.2
O2.1
L4.1
P3.5
P5.5
P8_9.6
P5.18
O9.2
P7.19, P7.8
P5.14
P8_9.6
P1_2.7
P7.13
P5.6
L3.2
P4.13
P4.14, P4.15
O4.1
P7.23
I
Igumentseva N.I.
Isidoro C.
Iwan M.
P7.40
P7.40
P5.4
J
Jacobi H.
Jakubowski H.
Janczarek M.
Janus P.
Janusz A.
Jarząb B.
Jaskolski M.
Jastrzebska B.
Jaworska A.
Jezierska J.
Józkowicz A.
Jóźwiak Z.
Jurewicz E.
P1_2.3
O7.2, P4.7
P1_2.5
P4.16, P4.8
P1_2.6
O3.1
L7.3, P7.51
P7.48
P8_9.6
P7.9
P1_2.2, P1_2.8, P1_2.14, P7.14
P8_9.9
P7.10
K
Kaberniyk A.
O2.2
Kachamakova-Trojanowska N.
P4.2
Kaczmarek L.
P1_2.6, P4.3, P4.5, P4.17
Kalashnyk O.M.
P7.56, P8_9.4
Kalinowska-Herok M.
P4.8
Kaminska B.
O4.2, P1_2.11, P3.1, P3.3, P3.6, P4.1, P4.10, P4.4, P4.9
Kandaurova N.
O8.1
Karkucinska-Wieckowska A.
P7.17
Kasacka I.
P7.10
Kaupp U.B.
L5.3
Kaza B.
P4.9
Kazimierczuk K.
P7.53
Ketzef M.
L4.1
Kędzierska H.
O1.1
Khalaileh A.
Kharkova A.P.
Khatib A.A.S.
Kietzmann T.
Kijewska M.
Kikulska A.
Kilanczyk E.
Kilon A.A.
Kimmel M.
Kiraga-Motoszko K.
Kiyamova R.G.
Klenov O.
Kleveta G.
Klopocka W.
Kluchivska O.
Klymyshin D.
Kobyłecki K.
Kocbach B.
Kochanek A.
Kois P.
Kolibo D.
Kolodziejczyk R.
Komisarenko S.V.
Komorowski J.
Koprowski P.
Korczynski J.
Korostynski M.
Korotkevich N.
Kosiorek .M.
Kosterin S.
Kostianets O.I.
Kotlarz M.P.A.
Kotlinowski J.
Koval L.M.
Kovalenko O.
Kovalevska L.M.
Kowalec P.
Kowalski A.
Kowalski W.
Kozakowska M.
Kozlov .O.V.
Kozlova I.
Kozlova N.
Kozminski W.
Kriklivyi I.
Krisanova N.
Kropyvko S.
Kruczyk M.
Krupko O.
Krzyzosiak W.J.
Kubarevaa E.A.
Kuchmerovska T.
Kuklin A.
Kulesza D.
Kulikova N.
Kur J.
Kurlandzka A.
Kutner A.
Kuznicki J.
Kuzniewska B.
Kuzontkoski P.M.
Kuźnicki J.
Kwiatkowska K.
Kyrylenko T.
Kоsteskii І.E.
P1_2.10
O4.1
P1_2.10
P7.37
P4.10, P4.9
P7.11, P7.25
P7.12
O4.2
P4.16, P4.8
P7.29
O6.1
P8_9.14
P5.7
P8_9.7
P5.15
P1_2.7
P7.33
P5.4
L7.2
P7.46
O2.2, P7.13
P7.51
P7.13, P7.36, P7.55, P8_9.4
P3.6
P7.22
P8_9.7
L3.3
O2.2, P7.13
P8_9.1
O8.1
O6.1
P5.13
P7.14
P7.55, P8_9.4
L3.1
P7.38
P5.8
P8_9.8
P7.15
P1_2.2, P1_2.8, P7.14
P7.57
P7.2
P7.37
P7.53
L3.1
P6.1, P8_9.3
L4.3
P3.6
O9.2
L4.2
P7.26
P4.6
O3.1
P1_2.11
P7.16
P7.32
P5.8
P5.13
P3.4, P4.12, P4.2
P4.2
P7.21
P8_9.6
P5.7, P5.9
P5.12
P5.11
L
Labyntsev A.
Lavi S.
Lavrova K.
Lebiedzinska M.
Lenart J.
Leonova N.
Lesyk R.B.
Leśniak W.
Li H.
Liberek K.
Lifshitz L.
Lipiec A.
Lipko M.
Litwińczuk N.
Loukinov D.
Lubecka E.A.
Lugassy Y.
Lugovskoy E.V.
O2.2, P7.13
P1_2.3
P5.10
P7.17
P3.1
P5.2
P3.5
P8_9.5
O5.1
L7.2
P7.18
P5.13
P4.9
L7.2
L2.2
P7.53
P4.13
P7.36
Lukash L.L.
Lukyanets E.A.
Lushnikova I.V.
Lykhmus O.Y.
P5.11
P8_9.2
P4.11
P7.55, P7.56, P8_9.4
Ł
Łoboda A.
Łuczak M.
Łukasiak A.
P1_2.2, P1_2.8, P1_2.14
O7.2
P8_9.12
M
Maciaszczyk E.
Madyagol M.
Magura .I.S.
Maistrenko A.M.
Makałowska I.
Makogon N.
Makogonenko E.M.
Makuchowski A.
Malinowski M.
Malinska D.
Malysheva T.A.
Mandzynets S.
Maor Y.
Marakhonov A.V.
Marczak A.
Marczak Ł.
Markowicz S.
Marszałek E.
Martsenyuk O.P.
Martynenko O.
Matskova .L.
Matuha I.O.
Matyshevska O.P.
Mehlman T.
Meyuhas O.
Mieczkowski J.
Milovanova G.O.
Miłek J.
Miłoszewska J.
Minchenko O.H.
Minchenko D.O.
Minis A.
Mironova K.O.
Misztal K.
Mitina N.
Mlącki M.
Mlynarski W.
Modrak-Wojcik A.
Moenner M.
Moibenko A.
Molchadsky A.
Molochkov N.V.
Mor A.
Morawiec E.
Morderer D.
Moreau J.
Moroz T.V.
Mrozińska K.
Mykowska A.
Mаcewicz L.L.
P7.51
P7.19, P7.8
P8_9.2
P4.11
P1_2.15
O3.1
P7.36
P4.16, P4.8
P8_9.10
P7.20
P4.14, P4.15
P5.5
P7.21
P1_2.1, P1_2.17, P1_2.9, P7.41
P8_9.9
O7.2
P5.13
P7.22
P7.23
P5.12
P7.7
P5.18
L8.3
P7.39
P1_2.10
O4.2, P3.3, P3.6
P5.18
P1_2.6
P5.13
O4.1
O4.1
L5.2
P7.24
P4.12
P5.6
P7.11, P7.25
L3.3
P7.29
O4.1
P5.16
P1_2.12
P7.26
O1.2
L7.2
L4.3, P7.27
P7.31
P7.24
P5.9
L4.2
P5.11
N
Nachliel E.
Nagalski A.
Najakshin A.M.
Nałęcz K.A.
Nauman A.
Navon S.P.
Negrutskii B.
Neofita O.
Nepyyvoda K.
Niedzwiecka A.
Niedźwiecka N.
Niewiadomska G.
Nikolaienko O.
Nitsche J.M.
Nocoń D.
Novikov V.
Novitskyi I.
Novokhatska O.
P7.1
P3.4
P4.15
O6.2, P7.45
O1.1
P.3.7
P7.47
P5.14
P5.10
P7.29
P7.28
P7.48
L4.3, P7.27
P6.2
P7.15
P5.5
P5.15
P7.31, P7.6
Nowak M.
97
P7.32
O
Obolenskaya M.Y.
Ochędalski T.
Ofek K.
Olszewski M.
Orlovsky M.M.
Osińska M.
Ostapchenko L.
Ostapchuk A.
Ostash B.
O3.1, P7.23
P5.3
L4.1
P7.32
P4.11
P7.33
P5.10, P7.4
P7.2
O3.2
P
Pakuła-Cis M.
Pálffy R.
Panchuk R.R.
Parnis A.
Pasichnyk G.
Perycz M.
Pevzner I.
Piastowska A.W.
Piechota M.
Piekielko-Witkowska A.
Pigłowski W.
Pikula S.
Pinton P.
Pirog M.
Piven О.О.
Plodnik D.
Pogorelaya N.K.
Polacek N.
Pomorski P.
Porycka M.
Pos K.M.
Potocki W.
Potopalsky A.
Prokhorov P.E.
Pronicki M.
Prus W.
Przanowski P.
Przewlocki R.
Pydiura N.A.
P4.8
P7.44
P3.5
P7.18
P7.34
P1_2.6
P7.18
P5.3
L3.3
O1.1
P4.8
P8_9.1
P7.17
O9.1
P5.11
P5.12
P8_9.2
P1_2.19
P8_9.7
P4.9
L6.1
L7.2
P5.12
P1_2.9
P7.17
P7.35
O4.2, P3.6
L3.3
P7.36
R
Rabyk M.
Radice G.
Radosinska J.
Raevskyi A.
Rajewsky K.
Rakus D.
Ramji K.
Rapaport D.
Reits E.A.
Rejmak E.
Rębas E.
Richardson C.C.
Rimbach G.
Rivlin N.
Rode W.
Rodziewicz-Motowidło S.
Rogalska A.
Romanets K.L.
Romanets O.
Romaniuk M.
Romi E.
Rosner M.
Rotter V.
Rozgoni I.I.
Rudavska L.
Rukov J.L.
Ruman T.
Ryabtseva A.
Ryazanovaa A.Y.
Ryazantsev S.N.
Rylski M.
Rynditch A.
Rzepetsky Y.
Rzeszowska-Wolny J.
P1_2.7
P5.11
P5.16
L3.1
L2.1
O9.1, P7.15
P1_2.11
P4.13
P7.9
P4.5
P8_9.8
O7.1
P1_2.14, P1_2.8
P1_2.12
P7.50
L7.2
P8_9.9
P7.23
P4.14, P4.15
P5.14
L5.2
P1_2.3
P1_2.12
P5.18
P5.15
P1_2.13
P7.50
P5.6
P7.26
P7.52
P4.17
L4.3, P7.27, P7.31, P7.6, P7.7
P7.38
P5.19
98
Abstracts
S
Saenko Y.
Samluk Ł.
Samoylenko A.
Sanagurski D.
Sanahursky D.
Sarig R.
Savchuk O.
Schoenmann Z.
Schwartzman T.
Sedakov I.
Selmi A.
Serzhanova V.A.
Shabelnik M.Y.
Shainskaya A.
Shaltiel G.
Shapira T.
Shatova O.
Shav-Tal Y.
Shezer Y.
Shkrabak A.A.
Shlapatska L.M.
Shlyakhtina Y.
Shlykov S.
Shohat M.
Shomron N.
Shtapenko O.V.
Shuvayeva G.Y.
Shyian M.A.
Shysh A.
Sidorenko S.P.
Sielska M.
Silman I.
Simon G.
Sivan G.
Sivko R.
Skibo G.G.
Skoblov M.Y.
Skok M.V.
Skonieczna M.
Skorkowski E.F.
Skorupska A.
Skowronek K.
Skrypkina I.
Skrzypek K.
Slyvchuk Y.I.
Sobczak M.
Sobierajska K.
Sobota A.
Sokolowska M.
Solomon A.
Soreq .H.
Sourjik V.
Sprecher E.
Sreedhar B.
Stachurska A.
Stanek J.
Stasyk O.V.
Stawarski M.
Stefańska A.
Stepanyugin A.
Stepinski J.
Stodus K.
Stoika R.S.
Stolarski R.
Stuchlík S.
Student S.
Suliman S.
Suski J.M.
Sussman J.L.
Suszynska-Zajczyk J.
Swiatek-Machado K.
Swiezewski S.
Swoboda P.
Sybirna N.
Syrvatka V.J.
Szabadkai G.
Szczepanowska J.
Szczesniak M.
Szewczyk A.
Szołtysek K.
Szwacka M.
Szwed M.
Szybińska A.
Ś
P5.19
O6.2
P7.34, P7.37
P5.5
P5.14
P1_2.12
P7.4
L5.2
P.3.7
P7.30
P8_9.10
P1_2.17
P7.38
P7.39
L4.1
P1_2.12
P7.30
O1.2
P1_2.12
O8.2
P7.38
P5.6
O8.1
P1_2.3
L1.3, P1_2.13, P1_2.18, P3.2
P5.18
P7.40
O6.1
P5.16
P4.14, P4.15, P7.38
P4.10
P7.49
P7.39
P1_2.4
P6.1, P8_9.3
P4.11
P1_2.1, P1_2.17, P1_2.9, P7.41
P7.55, P7.56, P8_9.4
P5.19
P7.28
P1_2.5
O6.2, P7.33
L4.3, P7.27, P7.31, P7.6, P7.7
P1_2.2
P5.18
P5.17, P8_9.13
P8_9.7
P5.7, P5.9, P7.16, P7.22
P7.42
P1_2.3
L4.1
L5.1, O5.1
P4.13
P4.3
P1_2.14, P1_2.8
P7.53
P7.40
P4.5
P1_2.2
P5.12
P7.29
P7.53
O.2 , P3.5, P5.15, P5.6
P7.29
P7.19, P7.44, P7.46
P5.19
O1.2
P7.17
P7.49
O7.2
O4.2, P3.3, P3.6
L1.1
P5.13
O5.2, P5.7
P5.18
P7.17
P7.20, P8_9.11
P1_2.15
P8_9.12
P4.8, P4.16,
P1_2.16
P8_9.9
P8_9.6
Ślusarz M.
L7.2
T
Taranin A.V.
Tarasenko A.
Tawfik D.
Tischenko V.M.
Tisonczyk J.
Tkachuk L.V.
Tkachuk Z.Y.
Tokovenko B.
Topolska A.M.
Tóth C.
Trembacz H.
Tribulova N.
Trikash I.
Tsetlin V.I.
Tsfadia Y.
Tsyba L.
Tsypik O.
Tukalo M.
Tułodziecka K.
Turňa J.
Twardowska A.
Twardowski T.
Tyburczy M.
P4.14, P4.15
O9.2
P7.49
P7.52
P4.7
P7.57
P7.57
O3.1
P7.43
P7.44
P5.13
P5.16
P4.6
P8_9.4
P7.1
L4.3, P7.6, P7.7, P7.27, P7.31
O3.2
L3.1
P7.45
P7.8, P7.19, P7.44, P7.46
P1_2.16
O7.2
P4.1
U
Urbanik-Sypniewska T.
Utekal P.
P1_2.5
P7.46
V
Valkovičová L.
Vávrová S.
Veklich T.O.
Verbeek D.S.
Veremieva M.
Vinther .J.
Vodopyanova L.
Voitenko N.
P7.8
P7.8
O8.2
P7.9
P7.47
P1_2.13
P6.2, P6.3
L8.1
W
Wadelius C.
Wasik A.
Wasik U.
Weis S.
Westerink J.T.
Węgierski T.
Widłak P.
Wieckowski M.R.
Wieczorek G.
Wilanowski T.
Wilczyński G.
Wilentzik R.
Wilk P.
Winberg G.
Wisniewska M.B.
Wisniewski P.
Witalisz A.
Wlodarczyk J.
Wlodawer A.
Wojcicka A.
Wojciechowska M.
Wojciechowski M.
Wojda U.
Wojewoda M.
Wolf A.
Wrzosek A.
Wyroba E.
Wysocki R.
P3.6
P8_9.7
P7.48
P7.48
P7.9
P8_9.6
P4.16, P4.8
P7.17
P7.49
P7.11, P7.25
P7.17
P1_2.13
P7.50
P7.7
P3.4, P4.12
P4.9
P1_2.14
P7.17
K.3
O1.1
L4.2
L7.1
L9.3, P4.2
P8_9.11
O5.1
P8_9.12
P7.33
P7.51
Y
Yagensky O.
Yaremchuk G.
P1_2.2
L3.1
Yaron A.
Yavorsky V.A.
Yonath A.
Yrkiv O.J.
Yu J.
Yunger S.
Yurchenko E.
Yurchenko M.Y.
L5.2
P8_9.2
K.2
P5.18
P7.21
O1.2
O2.2
P4.14, P4.15, P7.38
Z
Zabka M.
Zaburannyy N.
Zaichenko O.
Zakcharychev V.
Zaletok S.
Zalts H.
Zamir G.
Zarbiv G.
Zarzycki M.
Zaviyalov V.P.
Zawadzka-Kazimierczuk A.
Zawirska-Wojtasiak R.
Zdioruk M.
Zhang X.
Zhegunov G.
Zhukov I.
Zhukovska A.
Zhylenko V.
Ziadeh B.
Ziegler C.
Zieliński Z.
Ziętkiewicz S.
Zinkovych I.
Zohary K.
Zozulya Y.A.
Zubarev R.
Zuba-Surma E.
Zybura-Broda K.
P7.35
O3.2
P5.6
P7.47
P8_9.14
P1_2.18
P1_2.10
O5.1
P7.51
P7.52
P7.53
P1_2.16
O5.2
P7.21
P6.2
P7.53
P5.16
P8_9.14
P7.39
L6.3
P7.50
L7.2
P7.30
P7.54
P4.15
P7.39
P7.14
P4.17
Ż
Żylińska L.
Żywicki M.
P5.17, P8_9.1, P8_9.8 , P8_9.13
P1_2.19
99

Parnas-Conference2011-abstracts - BIO

Transcription

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