The adult human testis produces millions of sperm

Transcription

2012
ANNUAL REPORT
2012
ANNUAL REPORT
2013 – Istituto Pasteur-Fondazione Cenci Bolognetti
Edited by Lucia Ugo
P.le Aldo Moro 5 – 00185 Roma
[email protected]
www.istitutopasteur.it
Contents
Contents
y Foreword
7
y Boards and Staff
8
y Fellowships awarded
9
y Conferences, Seminars and Bookshop talks
13
y Scientific Reports
15
Research Area 1: Molecular biology of microorganism and viruses
Andrea Bellelli, Structural biology of the thiol-dependent redox systems of Schistosoma
mansoni and Plasmodium falciparum
Alberto Boffi, Bacterial globins as regulators of thiol redox equilibrium in bacteria
Daniela De Biase, The acid resistance genes of Escherichia coli: does their complex
transcriptional control hide novel biological roles?
Alberto Faggioni, Epstein-Barr virus interactions with cellular microRNAs
Paola Londei, From leaderless to leadered mRNAs: mRNA features modulating ribosome
binding and translation initiation in Archaea
Cristina Mazzoni, Study of RNA damage in yeast as a model for age-related degenerative
diseases
17
19
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25
27
Research Area 2: Pathogenic mechanisms of microbially associated diseases
Guido Antonelli, Molecular characterization of viruses causing bronchiolitis and study of
viral and host factors affecting Type I IFN antiviral response induced by respiratory viruses
Maria Lina Bernardini, Lipopolysaccharide and peptidoglycan adaptation to host as an
immune evasion strategy of gram-negative pathogens
Bianca Colonna, Involvement of sRNA molecules in the complex regulatory circuits of
virulence gene expression in Shigella flexneri and in enteroinvasive E.coli
Francesca Cutruzzolà, Inhibition of Pseudomonas aeruginosa biofilms: new molecular
strategies targeting cyclic-di-GMP metabolism
Paolo Sarti, Defense mechanisms against oxidative and nitrosative stress in pathogenic
protozoa
Vincenzo Vullo, Immunopathogenesis of HIV infection: study of innate immunity and
dendritic cells
31
33
35
37
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41
Research Area 3: Molecular genetics of eukaryotes
Fiorentina Ascenzioni, Assembly and functional analysis of genomic context vectors
containing the human CFTR locus
Paola Ballario, Light control of filamentous fungi life cycle: from system models to
applications
Silvia Bonaccorsi, Functional analysis of morgana, a gene involved in the control of
centrosome duplication
Irene Bozzoni, RNA-RNA and RNA-protein interactions: role of small non-coding RNAs in
gene expression control
45
47
49
51
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Contents
Emanuele Cacci, Role of metalloproteinases and their tissue inhibitors in the regulation of
neurogenesis and gliogenesis from neural stem/progenitor cells
Paola Caiafa, Does PARylated PARP-1 introduce an epigenetic mark on chromatin?
Giorgio Camilloni, DNA recombination of repeated sequences and genome instability:
epigenetic implications
Antonella De Jaco, Processing of the Neuroligins proteins and autism-related mutations
Patrizio Dimitri, Functional analysis of CG40218, a Drosophila melanogaster gene
encoding a BCNT-like protein required for chromosome organization
Maurizio Gatti, Genetic and molecular analysis of the mechanisms of Drosophila telomere
protection
Alessandro Giacomello, Role of epicardium and EMT/MET processes in cardiac progenitor
cells generation and differentiation
Alberto Gulino, Hedgehog signaling regulatory networks in brain cancer stem cells
Marco Lucarelli, The interplay between epigenetics, cell cycle and homologous
recombination in gene therapy by Small Fragment Homologous Replacement (SFHR)
Rossella Maione, Interplay between myogenic factors and cell cycle control: regulation and
role of the cdk inhibitor p57kip2
María Elena Miranda Banos, Handling and toxicity of mutant serpins underlying human
disease
Antonio Musarò, Characterization of the muscle-nerve crosstalk in a neuromuscular
disease
Rodolfo Negri, Role of the COP9 signalosome in transcription modulation and chromatin
organization in yeast and plants
Sergio Pimpinelli, The biogenesis of piRNAs and their involvement in transposon silencing
and heterochromatin formation in Drosophila
Sabrina Sabatini, To the root of organ growth: the control of root meristem activity in
Arabidopsis
Mario Stefanini, Biological characterization and in vitro culture of spermatogonial stem cells
Marco Tripodi, Molecular mechanisms driving liver stem cell fate
Giuseppe Zardo, Profiling the Polycomb/Trythorax target genes in normal and leukemic
hematopoiesis
53
55
57
59
61
63
65
67
69
71
73
75
77
79
81
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Research Area 4: Molecular recognition in biomolecules
Enzo Agostinelli, Toxic effects induced by polyamine metabolites on melanoma cells: a
new therapeutic approach
Fabio Altieri, Cellular response to oxidative stress: involvement of protein disulfide
isomerases
Paolo Bianco, Role of Gsα signalling in the bone marrow hematolymphopoietic
microenvironment as revealed by novel transgenic models
Maurizio Brunori, The dark side of protein folding: denatured states and misfolded species
in molecular recognition and pathological processes
Stefano Cacchione, The role of nucleosomes in the stability of human telomeres
Felice Cervone, Plant innate immunity: signalling and recognition of Damage-Associated
Molecular Patterns (DAMPs)
Ernesto Di Mauro, Spontaneous generation and evolution of genetic information
Cristina Limatola, Molecular and functional approaches to investigate the neuroprotective
and neuromodulatory roles of chemokines and their receptors in the central nervous
system
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Contents
Paola Paggi, Neurone response to experimental injury and lack of dystrophin: a molecular,
functional and structural study in autonomic ganglia in vivo and in vitro
Maria Rosaria Torrisi, Molecular mechanisms integrating endocytosis and signalling of
fibroblast growth factor receptors
107
109
Research area 5: Cellular and molecular immunology
Vincenzo Barnaba, Interplay amongst chronic immune activation, apoptosis, crosspresentation, immune-regulation, and autoimmunity
Rossella Paolini, Signalling events negatively regulating FceRI expression and mast cell
functional responses
Anna Riccioli, Anti-tumor pathways mediated by innate immune responses
Angela Santoni, Anti-tumor effector functions of NK cells in tumor microenviroment
Isabella Screpanti, Dissection of Notch signaling-dependent pathways involved in the
progression of T cell leukemia
Rosa Sorrentino, Predisposing factors in autoimmune diseases: correlation between
common genetic variations and function
Loretta Tuosto, CD28 co-stimulatory molecule as a key regulator of NF-κB signalling
pathway: role of cytoskeleton in coupling CD28 to NF-κB activation
Research area 6: New antimicrobial and antiviral agents
Donatella Barra, Peptide effectors of innate immunity
Mariangela Biava, New pyrrole derivatives of BM 212: a new class of antimycobacterial
agents. Design, synthesis, biological evaluation and study of their mode of action
Roberto Di Santo, New azole derivatives as antiprotozoal agents
Romano Silvestri, Drug design and synthesis of non-nucleoside inhibitors of both HIV-1
wild type and resistant mutant strains reverse transcriptase and Coxsackie B4 virus
113
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Research area 7: Biology of malaria and other vector-borne diseases
Alessandra Della Torre and Vincenzo Petrarca, Genetic and phenotypic characterization
of species and “molecular forms” of the Anopheles gambiae complex (Diptera: Culicidae),
afrotropical malaria vectors
David Modiano, Immune responses to malaria and autoimmune disorders: investigating
common gene-regulatory networks
139
141
Start-up program
Giuseppe Lupo, Specification and maintenance of retinal stem cells
145
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Annual Report 2012
Foreword
The Istituto Pasteur-Fondazione Cenci Bolognetti, the italian member of the Institute Pasteur
International Network, is a private non-profit foundation established according to the terms of the
bequest of Princess Beatrice Cenci Bolognetti. The noblewoman designated Sapienza University of
Rome to create an independent centre guided by the same mission and values of Institut Pasteur in
Paris.
At Istituto Pasteur of Rome we are devoted to studying the mechanisms that regulate the basic
processes of life, for the understanding and the treatment of severe human pathologies for which there
are no cures as yet, and for improving the existing therapies. The funding of research projects is
possible thanks to the income from donated real estate and, since 2008, thanks also to the support of
BNP Paribas BNL. While the collaboration with the roman University continues and is essential for our
progresses, the Institute is now about to open a new independent laboratory. This represents an
opportunity for scientists all over the globe to contribute to the research mission of the Pasteur
Institutes.
Our institute is a member of the Istituto Italiano di Donazione, an organization promoting and
fostering excellence, transparency, integrity, fair management practices and ethical conduct in nonprofit entities. We fund high-level research projects (selected through peer review), promote the
training of internationally competitive scientists and disseminate the scientific culture within society.
Over the past 30 years we have supported research projects in different areas: microbiology,
virology, molecular genetics, molecular recognition in proteins and nucleic acids, cellular and
molecular immunology, as well as biology of malaria and other vector-borne diseases. In 2012 our
researchers have focused on the study of pharmacological, cellular and gene therapies for the
treatment of emerging or re-emerging infectious diseases (tuberculosis, Aids, hepatitis); vector-borne
diseases (malaria, Chagas disease) and neuromuscular degenerative disorders (spinal muscular
atrophy and muscular dystrophy). Research has also focused on the molecular basis of ageing, on
developing antitumor strategies (hepatocarcinoma, leukemia) and on analysing the antitumor
pathways mediated by innate immune response. Projects have been carried out in the field of stem
cell biology for the use of cardiac, muscle, hepatic and neuronal progenitor cells to repair damaged
tissues.
As every year, we have given special attention to the new generation of researchers by awarding
a total of 90 fellowship grants to support their training in highly advanced research centres abroad,
upon their return to Italy and at the beginning of their career following a PhD degree. Last but not
least, we have also been active in promoting scientific communication by organizing seminars,
conferences and international meetings, which are important occasions for the comparison of results.
Moreover, we have promoted a school project involving a series of meetings and practical activities
with students, so they can learn to appreciate the importance of science. Lessons and courses in the
fields of genetics, biotechnology, microbiology, genomics, evolution biology, ecology and neuroscience
are carried out in the laboratories of the Sapienza University of Rome or, when possible, are
performed directly by teachers in schools. Furthermore, we organize cultural and social events that
take place in bookshops and are directed to the wider public.
This Annual Report documents the results obtained during the year 2012 thanks to the enthusiasm
and the effort of the scientists involved. It is our pleasure to invite you to follow our activity on our
website (www.istitutopasteur.it), where you may find further information.
Paolo Amati
President
Angela Santoni
Scientific Director
7
Annual Report 2012
Boards and Staff
Administrative Board
Scientific Council
The Board of Administration is presided over by
a President appointed by the Rector of
Sapienza University of Rome chosen from a list
of three names selected during a joint session
of the board and the Scientific Council.
Members of the board include a Scientific
Director, four members of the Faculties of
Pharmacy and Medicine, Natural Sciences, and
Medicine and Dentistry. Other members are a
legal expert designated by the University of
Rome Board of Administration, three auditors
nominated by the University, the Ministry of the
Treasury, and the Ministry of Universities and
Scientific Research.
The Scientific Council is a board of seven
scholars in the field of the pasteurian sciences.
Members are elected to a four-year term by the
Faculties of Natural Sciences, and Pharmacy
and Medicine. The Scientific Director, appointed
by the Scientific Council, is ex-officio a member
of the Board of Administration. The Scientific
Council attends to examine and coordinate the
research programs as well as the several
scientific activities.
President
Paolo Amati (Pharmacy and Medicine)
Members
Paolo Costantino (Natural Sciences), Alberto
Faggioni (Medicine and Dentistry), David
Modiano (Pharmacy and Medicine), Angela
Santoni (Pharmacy and Medicine), Romano
Silvestri (Pharmacy and Medicine)
Secretary
Emanuela Gloriani
Scientific Director
Angela Santoni (Pharmacy and Medicine)
Members
Ernesto Di Mauro (Natural Sciences), Laura
Frontali (Natural Sciences), Anna Teresa
Palamara (Pharmacy and Medicine), Anna
Tramontano (Pharmacy and Medicine), Marco
Tripodi (Pharmacy and Medicine), Carlo
Turano (Pharmacy and Medicine)
Staff
Caterina Cenci, Sarah Gainsforth, Maria Pia
Lorenzoni, Lynda Romani, Nicoletta Silvestri,
Lucia Ugo
Administrative Expert
Giuseppina Capaldo
Consultants
Auditors
Anna Carmela Ferrante, Simona Ranalli,
Carla Vassallo
8
Tommaso De Dominicis (legal affairs); Anna
Maria Pivetti (architectural supervision);
Barbara Hell (financial affairs)
Annual Report 2012
Fellowships awarded
Fellowships awarded for two-year
training in foreign laboratories
Fellowships awarded to students who
had a two-year experience abroad
Michele ARDOLINO, from Department of
Molecular Medicine, to Dept. of Molecular and
Cell Biology, University of California, Berkeley,
USA
Alessandro BARBARULO, from Department
of Molecular Medicine, to Institute of Child
Health, University College London, UK
Valerio BERARDI, from Department of Biology
and Biotechnologies "Charles Darwin", to State
University of New York, USA
Paola CHECCONI, from Department of Public
Health and Infectious Diseases, to Brighton &
Sussex Medical School, Brighton, UK
Sonia CONI, from Department of Molecular
Medicine, to IBDC, Universitè de Nice SophiaAntipolis, France
Daniela DIMASTROGIOVANNI, from
Department of Biochemical Sciences "A. Rossi
Fanelli", to Department of Biochemistry,
University of Cambridge, UK
Michela ESPOSITO, from Department of
Biology and Biotechnologies "Charles Darwin",
to Institut de Génétique et Microbiologie,
Université Paris XI, France
Francesca GASPARRINI, from Department of
Molecular Medicine, to Cancer Research UK.
London Research Institute, UK
Marta MORETTI, from Department of Molecular
Medicine, to Division of Immunology and
Inflammation, Imperial College, London, UK
Giuseppe SCIUMÈ, from Department of
Molecular Medicine, to NIH/NIAMS, Bethesda,
MD, USA
Matteo SPAGNOLETTI, from Department of
Biology and Biotechnologies "Charles Darwin",
to Genetics Institute, University College
London, UK
Daniela CECCARELLI, from Université de
Sherbrooke, Québec, Canada, to Department
of Biology and Biotechnologies "Charles
Darwin"
Antonio COLUCCIA, from School of
Pharmacy, University of Cardiff, UK, to
Department of Medicinal Chemistry and
Technologies
Maria Amalia DI CASTRO, from DBCM
Université de Lausanne, Switzerland, to
Department of Physiology and Pharmacology
"Vittorio Erspamer"
Elvira FORTE, from Burnham Institute for
Medical Research, La Jolla, CA, USA, to
Department of Molecular Medicine
Chiara SOLDATI, from King's College London,
UK, to Department of Biology and
Biotechnologies "Charles Darwin"
Fellowships awarded to students who
have completed their PhD and are
seeking a position
Laura ANTONUCCI, Department of Molecular
Medicine
Andrea BRENNA, Department of Biology and
Romina BURLA, Department of Biology and
Elisa CESARINI, Department of Biology and
Fabio CICCARONE, Department of Cellular
Biotechnologies and Hematology
Ramona LATTAO, Department of Biology and
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Annual Report 2012
Emanuela MICHELI, Department of Biology
and Biotechnologies "Charles Darwin"
Arianna MONTANARI, Department of Biology
Angela MORRONE, Department of
Biochemical Sciences "A. Rossi Fanelli"
Cassandra MOSTOCOTTO, Department of
Cellular Biotechnologies and Hematology
Michela MUSCOLINI, Department of Biology
Marialaura PETRONI, Department of Molecular
Medicine
Valeria PURPURA, Department of Clinical and
Molecular Medicine
Marcella VISENTINI, Department of Clinical
Medicine
Fellows working on the Institute
research programs
Angela. A. ALAGIA, Department of Biology
Francesco ANGELINI, Department of
Molecular Medicine
Simone BATTELLA, Department of Molecular
Medicine
Mariateresa CARCURO, Department of
Biology and Biotechnologies "Charles Darwin"
Nicoletta CASTIGLIONE, Department of
Roberta CASTRATARO, Department of
Physiology and Pharmacology "Vittorio
Erspamer"
Valeria CIANCARELLA, Department of
Iacopo CIARCIANELLI, Department of Biology
Fabio CICCARONE, Department of Cellular
Noemi CIFANI, Department of Biology and
Raffaela CIPRIANI, Department of Physiology
and Pharmacology "Vittorio Erspamer"
10
Rossana COCCHIOLA, Department of
Antonio COLUCCIA, Department of Medicinal
Chemistry and Technologies
Elisabetta DAMIA, Department of Biology and
Federica DE ANGELIS, Department of Biology
Cristina DE LUCA, Department of Biology and
Francesca DE VITO, Department of Biology
Enea Gino DI DOMENICO, Department of
Antonio DI GRAZIA, Department of
Maria Letizia DI MARTINO, Department of
Gabriella DOBROWOLNY, Department of
Anatomical, Histological, Forensic &
Orthopaedic Sciences
Micol FALABELLA, Department of
Flores Lietta FAVALORO, Department of
Giulia FRANCIOSA, Department of Molecular
Medicine
Maurizio GARGANO, Department of
Elisa GAUCCI, Department of Biochemical
Sciences "A. Rossi Fanelli"
Rajanish GIRI, Department of Biochemical
Sciences "A. Rossi Fanelli"
Leonardo GIUSTINI, Department of Biology
Marisa GRANATO, Department of
Experimental Medicine
Gaia GRASSINI, Department of MedicoSurgical Sciences and Biotechnologies
Naomi Antonella GUADAGNO, Department of
Maria Luisa IANNITTO, Department of
Molecular Medicine
Annual Report 2012
Vittoria IONTA, Department of Molecular
Medicine
Raffaella LA SCALEIA, Department of
Molecular Medicine
Ramona LATTAO, Department of Biology and
Dieter Matthias LAUER, Department of
Molecular Medicine
Carmen MARESCA, Department of Cellular
Ramona MARRAPODI, Department of
Molecular Medicine
Marina MATTIUSSI, Department of Biology
Angela MORRONE, Department of
Cassandra MOSTOCOTTO, Department of
Cellular Biotechnologies and Hematology
Laila MOUBAYIDIN, Department of Biology
Gaelle NOEL, Department of Biology and
Ida PACIELLO, Department of Biology and
Francesca PAGANI, Department of Physiology
and Pharmacology "Vittorio Erspamer"
Valeria Luciana PALUMBO, Department of
Chiara PAPARELLA, Department of Biology
Paola PARISI, Department of Biology and
Claudia PELLACANI, Department of Biology
Maria PELULLO, Department of Molecular
Medicine
Eugenia PENNACCHIETTI, Department of
Medico-Surgical Sciences and Biotechnologies
Serena PERILLI, Department of Biology and
Verena PICHLER, Department of Public Health
and Infectious Diseases
Silvia PIERANDREI, Department of Cellular
Samanta PINO, Department of Biology and
Michela PINZAGLIA, Department of Cellular
Francesco PISCITELLI, Department of
Medicinal Chemistry and Technologies
Dorina POLINARI, Department of Cellular
Federica SANTOLAMAZZA, Department of
Public Health and Infectious Diseases
Daniel SAVATIN, Department of Biology and
Sara VITALE, Department of Molecular
Medicine
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Annual Report 2012
Conferences
Istituto Pasteur Science Day
December 14 y L’Istituto Pasteur per i
giovani (e non solo…)
Dipartimento di Scienze di Sanità Pubblica “G.
Sanarelli” – Aula A, Sapienza-Università di
Roma
Meeting held within the week of Cyprus
culture in Italy
October 3 y HPV Papilloma Virus. L’Isola non
è più un universo chiuso.
Villa Torlonia, Roma
Seminars
December 13 y Cancer chemoprevention:
prostaglandin’s, leukotrienes and beyond
December 11 y Targeting protein kinases:
the selectivity problem
Prof. Stefan Laufer
Pharmaceutical/Medicinal Chemistry EberhardKarls-University Tuebingen, Germany
November 15 y Using high throughput
methods to study the E. coli acid stress
response
Prof. Peter Lund
School of Biosciences, University of
Birmingham, UK
November 9 y Regulation of cancer cell
proliferation and survival by NF-kappaB
Prof. Neil Perkins
Institute for Cell and Molecular Biosciences,
Medical School, Newcastle University, UK
October 19 y Shigella invasion of the
intestinal epithelium: molecular subversion
of epithelial and immune cells
October 17 y Commensals and pathogens:
War and Peace at mucosal surface
Prof. Philippe Sansonetti
Institut Pasteur Paris, France
12
September 17 y Antimicrobial peptides: role
in defence against ocular pathogens
Prof. Alison M. McDermott
University of Houston, College of Optometry,
TX, USA
June 15 y Getting to the root of pattern
formation in plant development
Emergent properties of root networks
Prof. Philip Benfey
Duke University, Durham, NC, USA
June 14 y Structural and Computational
studies of GAD65
Prof. Ashley Buckle
The Department of Biochemistry and Molecular
Biology, Monash University, Melbourne,
Australia
Roma, 14 Giugno 2012, ore 12.00
June 8 y Surprising adventures of proteins
in the cytoplasm
Prof. Eugene I. Shakhnovich
Department of Chemistry and Biochemistry,
University of Harvard, Cambridge, MA, USA
May 30 y Genome Dynamics as Motor of
Evolution and Cause of Diseases from
Yeasts to Human
May 28 y Experimental Evolution of Yeasts
illustrates the importance of Chromosomal
Dynamics
Prof. Bernard Dujon
Institut Pasteur, Paris, France
May 14 y Chromatin Boundaries and
Epigenetic Regulation of Gene Expression
Prof. Gary Felsenfeld
National Institutes of Health, Bethesda, USA
May 4 y Cyclic di-GMP Signaling and
Bacterial Persistence
Prof. Urs Jenal
The Center for Molecular Life Sciences
Biozentrum, University of Basel, Switzerland
Annual Report 2012
March 30 y What we learn about protein
folding from single molecule fluorescence
experiments
Prof. William A. Eaton
National Institutes of Health, Bethesda, USA
March 7 y How does hemoglobin create such
diverse functionality of physiological
relevance?
Prof. Takashi Yonetani
University of Pennsylvania, USA
March 1 y The gut immune response of
Drosophila
February 28 y How Drosophila has helped
understanding innate immunity? An
evolutionary perspective on Toll-like
receptors
Prof. Bruno Lemaitre
École Polytechnique Fédérale de Lausanne,
Switzerland
Bookshop talks
L’unione fa la forza: i biofilm batterici
by Francesca Cutruzzolà
Proteine sane e proteine malate
by Maurizio Brunori
Il virus dell’influenza: un vecchio (ma un po’
infido) compagno di viaggio
by Anna Teresa Palamara
Piccole e cattive: identikit della zanzara,
il nemico pubblico numero uno
by Alessandra della Torre
Le difese immunitarie: una lezione dalla
natura
by Donatella Barra and Maria Luisa Mangoni
Stress ed evoluzione
by Sergio Pimpinelli
Invecchiamento e longevità: componenti
genetiche a ambientali
by Maurizio Gatti and Giuseppe Passarino
Le nostre memorie
by Cristina Limatola and Angela Santoni
Cellule staminali e ingegneria tissutale
by Antonio Musarò
13
Area 1
Molecular biology of
microorganisms and viruses
Area 1: Molecular biology of microorganisms and viruses
Structural biology of the thiol-dependent redox systems of
Schistosoma mansoni and Plasmodium falciparum
Andrea Bellelli
Department of Biochemical Sciences "A. Rossi Fanelli"
℡: +39 06 49910824 - @: [email protected]
Malaria and schistosomiasis are among the most frequent and severe parasitic diseases of
humans. Malaria is caused by one out of four species of unicellular protozoan parasites belonging to
the genus Plasmodium. Due to their rapid life cycle (that involves two hosts, humans and Anopheles
mosquitoes), Plasmodia rapidly evolve drug resistence, a phenomenon that frustrates the attempts to
curbe the incidence of the disease. Clearly, a constant supply of new drugs is required, and to achieve
this goal a better understanding of the structural biology of the parasite is necessary.
Schistosomiasis is caused by three species of the blood-fluke Schistosoma. The disease is
debilitating and affects 200 million people in tropical areas; the death toll is estimated at two hundred
thousands people per year. Schistosomiasis is currently treated with one drug, Praziquantel, whose
precise molecular target is unknown. Several other drugs are known to kill the schistosomes in vivo
and in vitro, but these are seldom employed because of toxicity, high cost, complex administration or
other reasons. The improvement of known drugs or the development of entirely new ones is a
desirable goal, in view of the fact that strains of Schistosoma mansoni with reduced sensitivity to
Praziquantel have appeared.
In this project we are exloring known or putative macromolecular targets of drugs against malaria
or schistosomiasis; thus we focus on the biochemistry and molecular biology of these parasites. The
rationale of this approach is that drug design may become realistic if the mechanism of action of each
drug were known at atomic detail, ideally as the 3D structure of the drug in complex with its target. The
enzymes involved in the detoxification of reactive oxygen species (ROS) and other oxidants are
potential drug targets. We have already characterized from the structural and functional point of view:
Thioredoxin Reductase from P. falciparum; Glutathione S-Transferase; Thioredoxin Glutathione
Reductase; and Glutathione Peroxidase from either S. mansoni or S. haematobium. We have also
characterized a Fatty Acid Binding Protein and a Cyclophilin, both from S. mansoni, even though these
two proteins are not directly related to the ROS detoxification pathway.
Our research is focussed on enzymes that use Thioredoxin as a substrate. Thioredoxin is a small
protein involved in redox reactions of fundamental relevance for the survival of both Plasmodia and
Schistosoma, e.g. the reduction of ribonucleotides to deoxyribonucleotides and the detoxification of
peroxides. In Plasmodia it is reduced by the flavoenzyme Thioredoxin Reductase (TR), whereas in the
schistosomes the same reaction is catalyzed by the strictly related Thioredoxin Glutathione Reductase
(TGR). Given the importance of the biochemical reactions that require reduced thioredoxin, inhibition
of TR (in Plasmodia) or TGR (in Schistosoma) is lethal. Effective inhibitors of TR of TGR are
available, known to kill Plasmodia and Schistosomes: one of these is the gold containing antirheumatic
drug Auranofin (AF). We demonstrated that the molecular mechanism leading to the irreversible
inhibition of these enzymes by this drug depends on the thiolates and selenolates produced during the
catalytic cycle. These have high affinity for transition metals and combine with gold ions released by
the drug, thus becoming inactive in redox reactions. We solved the structure of the gold-TGR complex,
and found Au combined to the sulfur of Cys residues, rather than (or in addition to) the Se of the single
17
seleno-cystein residue present in the enzyme. Kinetic measurements demonstrated that the relative
velocity of the reaction, rather than the relative affinity (as previously thought), depends on the
presence of Sec residues, which appear to dictate AF selectivity. Indeed when an external source of
selenium (benzeneselenol) was added to the reaction mixture, auranofin was able to inhibit the Selacking reductases Glutathione Reductase and Sec->Cys mutated TGR with an efficiency similar to
Se-containing ones [see publication n.1].
The same experiments are under way for Thioredoxin Reductase from Plasmodium falciparum, a
validated drug target and an enzyme essential for the parasite's survival. We solved the first crystal
structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discussed its
differences with respect to the human orthologue [see publication n.2]. The most important one
resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors.
The striking conservation of this feature among the Plasmodium parasites, but not among other
Apicomplexa parasites neither in mammals, boosts its exploitability.
Another enzyme of the Thioredoxin pathway is Peroxiredoxin, a thioredoxin-dependent
peroxidase. 2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status
of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress and ATPindependent chaperones upon exposure to high peroxide concentrations. These alternative functions
have been associated with changes in the oligomerization state from low-(LMW) to high-molecularweight (HMW) species. We solved the structures of Schistosoma mansoni PrxI in both states: the
LMW decamer and the HMW 20-mer formed by two stacked decamers [see publication n.3]. The latter
is the structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the
mechanism by which chemical stressors, such as high hydrogen peroxide concentration and acidic
pH, are sensed and translated into a functional switch in this protein family. We also propose a model
to account for the in vivo formation of long filaments of stacked Prx rings. Further studies on the
molecular mechanisms respondible for the structural change of SmPrx by site-directed mutagenesis
are in progress.
Publications
Boumis G, Giardina G, Angelucci F, Bellelli A, Brunori M, Dimastrogiovanni D, Saccoccia F, Miele AE.
Crystal structure of Plasmodium falciparum thioredoxin reductase, a validated drug target. Biochem
Biophys Res Commun 2012, 425: 806-811. doi: 10.1016/j.bbrc. 2012.07.156
Saccoccia F, Angelucci F, Boumis G, Brunori M, Miele AE, Williams DL, Bellelli A. On the mechanism
and rate of gold incorporation into thiol-dependent flavoreductases. J Inorg Biochem 2012, 108: 105111. doi: 10.1016/j.jinorgbio.2011.11.005.
Saccoccia F, Di Micco P, Boumis G, Brunori M, Koutris I, Miele AE, Morea V, Sriratana P, Williams
DL, Bellelli A and Angelucci F. Moonlighting by different stressors: crystal structure of the chaperone
species of a 2-cys peroxiredoxin. Structure 2012, 20: 429-439. doi: 10.1016/j.str.2012.01.004.
Research Group
Collaborations
Adriana Erica Miele, Francesco Angelucci,
researchers; Giovanna Boumis, technician,
Fulvio Saccoccia, PhD student.
David L. Williams, Rush University, Chicago,
IL, USA.
18
Bacterial globins as regulators of thiol redox equilibrium in bacteria
Alberto Boffi
℡: +39 06 49910990 - @: [email protected]
The specific targets of the project concern the elucidation of the mechanism of thiol oxidation and
the discovery of novel pathways in thiol redox homeostasis in bacteria. Bacterial truncated
hemoglobins have been shown to play a pivotal role in thiol redox homeostasis being able to catalize
very efficiently thiol oxidation even at very low oxygen concentrations (2-10 micromolar). In particular,
the globins from Thermobifida fusca and Bacillus subtilis not only display a very fast oxygen binding
rate, but are also capable of immediately generating a reactive hydroperoxyl radical. Such radicalic
species is relatively stable but is readily resolved in the presence of sulphide containing compounds,
resulting in the production of an oxoferryl heme-iron complex and a disulphide adduct. The nature of
the heme gaseous ligand complex has been further explored in ultrafast spectroscopic experiments on
the carbon monoxide bound adduct that allowed for the first time the assignment of a picosecond
transition to a specific rotameric rearrangement of the distal residues of the heme pocket (Marcelli et
al., 2012). In parallel, molecular dynamics of carbonmonoxide motion inside the heme pocket has
been investigated and highlighted a novel mechanism of ligand stabilization within the heme pocket,
based on hydrogen bonding from a conserved tryptophan residue to the iron bound carbon monoxide
molecule. The spectroscopic characterization of the carbon monoxide complex has also been studied
in parallel both in the visible and mid-infrared regions where carbonmonoxide complexes has unveiled
a well-defined correlation between ν(C-O) vibrational frequencies and visible electronic transition
energies. For the case of Tf-trHb, we have obtained a detailed picture of H-bonding in the distal cavity
environment (Lapini et al., 2012). The interpretation of the spectroscopic data is strengthened by the
close relation with the observed carbonmonoxide photo-dissociation dynamics and molecular
dynamics simulations. All the techniques provided evidence that TrpG8 and TyrCD1 can form strong H
bonds with carbonmonoxide, whereas TyrB10 can only interact weakly. The second target of the
research concerns the identification of partner proteins involved in the complex physiological network
of thiol homeostasis. In the case of Bacillus subtilis the partner protein YjbH has been identified
already and paralogues have been found in actinomyces, including mycobacteria, and in parasitic
microorganisms, including Leishmania. On these basis, it is suggested that the globin may be able to
catalyze disulfide bonds formation and thus act as a heme based peroxiredoxin. Most interestingly, a
similar overall pathway has been identified in Leishmania species, although with a most notable
difference. Leishmania does possess a globin-like protein, similar to that of Bacillus subtilis, but its
location is outside the thiol redox homeostasis operon. The Leishmania globin is in fact fused to a
guanilate cyclase domain whose task is to activate the expression of the thiol redox pathway. The first
step towards the identification of the globin-thiol redox pathway in Leishmania sp. concerns the
demonstration of the postulated interaction between the peroxiredoxin (tryparedoxin) and its reductase
(trypareductase) located upstream within the same operon. The whole operon contaning has been
recombinantly expressed in E. coli and the structure of both proteins has been obtained by X-ray
crystallography (Fiorillo A. et al., 2012). In this framework, it appears that the thiol redox pathway can
be viewed as an important drug target, expecially for metal besed compounds (Colotti G. et al., 2012).
19
The development of novel compounds able to inhibit the thiol redox pathway in Leishmania sp. has
subsequently prompted the interest of the scientific community on the subject and a large FPVII call on
the topic has been successfully applied.
Publications
Fiorillo A, Colotti G, Boffi A, Baiocco P, Ilari A. The crystal structures of the tryparedoxin-tryparedoxin
peroxidase couple unveil the structural determinants of Leishmania detoxification pathway. PLoS
Negl Trop Dis 2012. doi: 10.1371/journal.pntd.0001781.
Lapini A, Di Donato M, Patrizi B, Marcelli A, Lima M, Righini R, Foggi P, Sciamanna N, Boffi A. Carbon
monoxide recombination dynamics in truncated hemoglobins studied with visible-pump midIR-probe
spectroscopy. J Phys Chem B 2012;116(30):8753-61. PMID: 22759230. doi: 10.1021/jp3019149.
Marcelli A, Abbruzzetti S, Bustamante JP, Feis A, Bonamore A, Boffi A, Gellini C, Salvi PR, Estrin DA,
Bruno S, Viappiani C, Foggi P. Following ligand migration pathways from picoseconds to
milliseconds in type II truncated hemoglobin from Thermobifida fusca. PLoS One 2012;7(7):e39884.
doi: 10.1371/journal.pone.0039884.
Research Group
Collaborations
Alessandra Bonamore, Alberto Macone,
research fellows; Paola Baiocco, post-doc
fellow; Andrea Ilari, CNR researcher.
Giulietta Smulevich Dipartimento di Chimica,
Università di Firenze; Leonardo Boechi
Departamento de Química Inorgánica, Analítica
y Química Física, Universidad de Buenos Aires.
20
The acid resistance genes of Escherichia coli: does their complex
transcriptional control hide novel biological roles?
Daniela De Biase
Department of Medical-Surgical Sciences and Biotechnologies
℡: +39 0773 1757212 - @: [email protected]
For successful colonization of the gut, enteric bacteria must overcome the extreme acid stress
(pH<2.5) encountered during transit through the host stomach. The glutamate-dependent acid
resistance (GDAR) system is by far the most potent acid resistance (AR) system present in orallyacquired bacteria, such as E. coli, in S. flexneri, in L. monocytogenes and in L. lactis. GDAR requires
the intracellular proton-consuming activity of two glutamate decarboxylase isoforms, GadA and GadB,
and the antiport activity of an integral membrane protein, GadC, which exchanges glutamate for γaminobutyrate (GABA), the decarboxylation product. The gadB and gadC structural genes are 2.1 Mb
from gadA, which belongs to the so-called acid fitness island (AFI), a 14-kb genome region repressed
by H-NS and controlled by RpoS. The AFI contains 14 genes that contribute to E. coli AR in different
ways.
The gadA and gadBC promoter regions are under the control of a very complex regulatory network
that integrates multiple environmental signals and physiological adaptations. The essential regulator
GadE is under the control of three different activation circuits. The GadX/GadW-dependent circuit, the
main object of our investigation, involves the specific transcriptional regulators GadX, GadW and
GadY, encoded by the corresponding AFI genes, and the global regulators H-NS, CRP and RpoS.
GadX and GadW, AraC-like transcriptional regulators, specifically recognize a 42-bp sequence in the
5’ regulatory regions of gadBC and the AFI operons (Tramonti et al., 2008). GadX and GadW bind to a
42-bp sequence which consists of the tandem arrangement of two 21-bp sites.
In the last year (2012) we completed the following studies.
Characterization of the GadX regulon
We analysed the position of the GadX binding sites detected by ChIP-chip and concluded that
GadX is likely to influence the expression of approx. 110 genes. Notably 42% of these genes code for
membrane/periplasmic proteins. This suggests that GadX mainly influences the protein composition of
the membrane and the periplasmic space, the cell compartments more exposed to the extracellular
acidic pH.
We improved the GadX consensus sequence and used this sequence to search for its presence in
the genomic regions encompassing the 64 peaks corresponding to the GadX binding sites. Notably,
25 GadX binding sites fully encompass the 42-bp GadX consensus, 2 sites contain only the first half of
the consensus sequence (21 bp), finally 37 binding sites encompass both halves of the consensus
sequence, though not in the typical tandem arrangement. We analysed additional targets of GadX and
found that at 37°C in rich medium GadX acts as a repressor of genes belonging to the cold-shock
stimulon.
The large number of GadX binding sites suggests that GadX is a global regulator modulating the
response of E. coli to many stressful environments, not just acidic.
21
Role of the gadBC system in Brucella microti
In collaboration with Dr. A. Occhialini (Montpellier, France) we concluded the work carried out in
2011 on the contribution of gadBC operon in the adaptation of B. microti to acid environments. B.
microti was found to be glutamate decarboxylase-positive, able to export GABA to the extracellular
medium and to exhibit GDAR. Three acid sensitive E. coli mutants of MG1655 K12 strain were used
for heterologous in trans complementations with the B. microti gadBC operon. All complemented strain
recovered GDAR, exported GABA and decarboxylated glutamate, thereby confirming conserved
functions of the gadBC system in both bacterial species. A B. microti gadBC mutant was not
attenuated during macrophage infection, where Brucella resides in an acidified vacuole at pH 4-4.5
during the early phase of intracellular infection, but it was affected in survival in a murine model
following oral infection. This work provides first evidence that GDAR might play an essential role in the
resistance of an environment-borne, pathogenic Brucella species to extreme acid shock as during
passage of the host stomach following oral infection (Occhialini et al., 2012).
Publications
Occhialini A, de Bagüés MP, Saadeh B, Bastianelli D, Hanna N, De Biase D, Köhler S. The glutamic
acid decarboxylase system of the new species Brucella microti contributes to its acid resistance and
to oral infection of mice. J Infect Dis 2012, 206: 1424-1432. doi: 10.1093/infdis/jis522.
De Biase D, Pennacchietti E. Glutamate decarboxylase-dependent acid resistance in orally acquired
bacteria: function, distribution and biomedical implications of the gadBC operon. Mol Microbiol 2012,
86: 770–786. doi: 10.1111/mmi.12020
Research Group
Collaborations
Angela Tramonti, CNR researcher; Eugenia
Pennacchietti,
post-doc
fellow;
Daniela
Bastianelli, Gaia Grassini, PhD students.
Guido Capitani, Paul Scherrer Institute, Villigen
PSI, Switzerland; Maurice CR Franssen,
Wageningen University and Research Centre, The
Netherlands; Alessandra Occhialini, Université
Montpellier I, CPBS, UMR 5236, France.
22
Epstein-Barr virus interactions with cellular microRNAs
Alberto Faggioni
Department of Experimental Medicine
℡: +39 06 4461500 - @: [email protected]
Epstein-Barr Virus (EBV) is an oncogenic human herpesvirus that is involved in the pathogenesis
of various tumors of B-cell origin such as Burkitt’s and Hodgkin’s lymphoma, diffuse large B-cell
lymphoma (DLBCL). Its oncogenic property is highlighted by the ability of EBV to growth-transform Blymphocytes; these so-called lymphoblastoid cell lines (LCL’s) are the in vitro counterparts of B-cell
lymphoproliferative disorders, the insurgence of which is often related to immunosuppression. In the
various EBV-associated tumor entities, the virus expresses different sets of transformation-associated
proteins as well as non-coding RNAs.
miRNAs are small, non-coding RNAs with a length of ~22 nucleotides that are encoded by virtually
all multicellular organisms and some viruses. miRNAs bind to the 3′UTR of target mRNA transcripts
with partial or perfect sequence complementarity, resulting in translational repression and/or mRNA
destabilization. Although the precise parameters that govern target recognition remain incompletely
understood, the great majority of functional target sites minimally exhibit perfect Watson-Crick pairing
with the so-called seed region (nts. 2–7) of the miRNA.
Given the important role that miRs play in cancer pathogenesis, we have focused our efforts to
understand how EBV might affect cellular miR expression to potentiate B cell transformation and thus
may contribute to lymphoma development.
The EBV genome possesses DNA sequences for at least nine latent proteins. All these virally
encoded proteins are indeed expressed in LCLs and in tumors which display viral latency III. In
particular, LMP1 and EBNA2 are considered the chief transformation associated proteins. Our
laboratory has been busy in the past few years to figure out how these two viral proteins influence
cellular miRs.
At this regard, we have discovered the following in the last couple of years. We found that LMP1
upregulates miR-29b and downregulates TCL1 through this miR. This is an exciting finding as for the
first time the molecular mechanism involved behind the B cell inhibitory growth potential of EBV has
been unravelled. Our findings provide rational for developing miR-29b based therapeutical approaches
in those tumors which are driven by high expression of TCL1 oncogene (Anastasiadou et al,
Oncogene 2010).
More recently, we established global miR profiling of EBNA2 transfected DLBCL cells. We found
that EBNA2 can strongly induce miR-21 expression (Rosato et al, Leukemia 2012). This miR is
considered an oncomiR and its deregulation is hallmark of many tumors. It is thus quite satisfying to
have found that EBNA2 might influence the B cell transformation by upregulating miR-21. Interestingly,
a tumor suppressor miR namely, miR-146a was downregulated. This miR is endowed with tumor
suppressive capabilities. Thus its fascinating to envisage that EBNA2 contributes to B cell
transformation by simultaneously increasing an oncomiR and downregulating a tumor suppressive
miRs. Clearly, more experiments are required to directly implicate these miRs in EBV induced B cell
transformation and one such possibility is currently being tested in our laboratory. We have infected
primary B cells with a recombinant EBV in the presence of a locked nucleic acid anti-sense
23
oligonucleotide miR-21 (LNA miR-21) to block its expression. The idea was to see if EBV induced
transformation and thus the generation of LCLs is hampered in the presence of LNA miR-21. A recent
study has shown that B cells can successfully uptake LNA oligos without addition of any lipid reagent
(gymnosis) and the silencing effect can last as long as six months in the continuous presence of the
silencing LNA oligonucleotide. Our preliminary data show that the onset of LCLs formation is delayed
when LNA miR-21 is present in B-cell EBV transformation assay in comparison to scrambled
oligonucleotide controls.
Further investigations are on way how EBNA2 turns off miR-146 a expression. Specifically, we are
trying to understand the molecular mechanisms which might involve lin-28B because this protein is
involved in negative regulation of miR-146a and it is our hypothesis that EBNA2 might downregulate
miR-146a by upregulating lin-28b. We are also investigating alteration in miR expression in EBV
infected primary effusion lymphoma and multiple myeloma cells.
Publications
Cirone M, Conte V, Farina A, Valia S, Trivedi P, Granato M, Santarelli R, Frati L, Faggioni A. HHV-8
reduces dendritic cell migration through down-regulation of cell-surface CCR6 and CCR7 and
cytoskeleton reorganization. Virol J 2012, 9: 92. doi: 10.1186/1743-422X-9-92.
Cirone M, Di Renzo L, Lotti LV, Conte V, Trivedi P, Santarelli R, Gonnella R, Frati L, Faggioni A.
Primary effusion lymphoma cell death induced by Bortezomib and AG490 activates dendritic cells
through CD91. PLoS One 2012, 7:e31732. doi:10.1371/journal.pone.0031732.
Rosato P, Anastasiadou E, Garg N, Lenze D, Boccellato F, Vincenti S, Severa M, Coccia EM, Bigi R,
Cirone M, Ferretti E, Campese AF, Hummel M, Frati L, Presutti C, Faggioni A, Trivedi P. Differential
regulation of miR-21 and miR-146a by Epstein-Barr virus encoded EBNA-2. Leukemia 2012, 26:
2343-52. doi: 10.1038/leu.2012.108.
Research Group
Antonio Angeloni, Pankaj Trivedi, professors;
Roberta Santarelli, Mara Cirone, Antonella
Farina, Roberta Gonnella, researchers; Marisa
Granato, Eleni Anastasiadou, post-doc
fellows; Rachele Bigi, PhD student; Claudia
Zompetta, technician.
24
From leaderless to leadered mRNAs: mRNA features modulating
ribosome binding and translation initiation in Archaea
Paola Londei
Department of Cellular Biotechnologies and Hematology
℡: +39 06 4940463 - @: [email protected]
Background and aims
Initiation of protein synthesis, entailing ribosomal recognition of the mRNA start codon and the
setting of the correct reading frame, is the rate-limiting step of translation and the main target of
translation regulation. However, the mechanism and molecular machinery for initiation have diverged
in the primary domains of life: Bacteria, Archaea and Eukarya. In Bacteria, translation initiation is
relatively simple, while in Eukarya is complex and requires more components. The Archaea, despite
their prokaryotic phenotype, have a complex initiation process, requiring at least six initiation factors.
Archaeal mRNAs differ widely in the structure of their translation initiation regions (TIR). Only a
minority of TIRs are endowed with Shine-Dalgarno (SD) sequences for ribosome binding. In most
cases there is no SD motif, and often a 5’ untranslated region (5’UTR) may lack altogether. In the
latter case the mRNA is called leaderless. Leaderless mRNAs can constitute up to 80% of all
transcripts in certain archaeal species. Archaeal SD-less and leaderless mRNAs have been proposed
to be translated using initiation mechanisms alternative to those employed for canonical mRNAs
having SD motifs, but experimental data in support of this hypothesis are still scarce. The aim of the
project is to investigate the leaderless and leadered modes of initiation in the thermophilic archaeon
Sulfolobus solfataricus, defining the respective mechanisms and eventually shedding light on the
evolution of translational initiation.
Results and perspectives
Initially, we analyzed how the in vitro translational efficiency of a model leaderless mRNA was
affected by the addition at its 5’ end of short UTRs of monotonous sequence (oligo-A) and different
lengths, up to 15 nucleotides. The mRNAs were obtained by in vitro transcription of the cloning
plasmids, and their translatability was assayed in a cell-free system for protein synthesis derived from
lysates of S.solfataricus.
The results obtained so far indicate that a leaderless mRNA is still efficiently translated if its start
codon is preceded by no more than 4-5 additional nucleotides. Longer sequences severely impaired or
altogether abolished translation. Dephosphorylation of the mRNA 5’ end was also found to inhibit
translation. These data suggest that leaderless translation entails a direct binding of the ribosome to
the mRNA 5’ end, probably aided by the recognition of the terminal tri-phosphate. More experiments
with other model mRNAs are in progress.
In addition, a system for in vivo translational analysis is being developed, using the uracil
auxotrophic PBL2025 strain of S. solfataricus. The strain was transformed with the plasmid
pCMalLacS which contains the selectable marker pyrEF and the leaderless lacS gene. A number of
positive transformants have been selected on uracil-deficient plates and lacS expression has been
detected by the colorimetric β-galactosidase assay. The starting construct has been engineered to
lengthen its 5’ end, as described above. Translational analysis is in progress; the results so far
obtained confirm the in vitro findings, namely that even short 5’UTRs are highly detrimental to
translational efficiency of leaderless mRNAs.
25
Subsequently, we addressed the question of whether leaderless and leadered translation require
different sets of initiation factors. So far, we have analysed the role of the factors termed aIF1-A and
aSUI1. These proteins are essential in the early phases of translational initiation on leadered mRNAs,
as they help the small ribosomal subunit to locate the initiation codon. The SUI1 and the aIF1-A gene
were cloned and inserted in the pBS plasmid, ensuring that in vitro transcription of the constructs with
T7 polymerase yielded mRNAs having intact natural TIRs and producing proteins of the expected
molecular weights upon in vitro translation. Increasing amounts of these mRNAs were co-translated in
vitro with a fixed amount of either a model leaderless mRNA or a model leadered mRNA, to determine
the effect of increasing concentrations of aIF1A and aSUI1 on the two modes of translation.
counts
As shown in the figure, both aIF1A and SUI1 selectively stimulated translation only of the leadered
mRNA (black symbols). The same results were obtained by adding the recombinant proteins to the
translation system.
On the whole, the data indicate that ribosome binding to leaderless mRNAs relies on a simpler
mechanism than that employed for
leadered messages, possibly entailing a
Stimulation of leadered and leaderless mRNA translation in
presence of increased amounts of translation initiation factors
direct, factor-free recognition of the start
1800
codon by the ribosome. Importantly, this
1600
surmise strongly supports the proposal that
leaderless mRNAs are the ancestral type
1400
of message, universally readable by all
1200
types of ribosomes.
leadered mRNA +
1000
aIF1A
Further analyses with other leadered
leaderless mRNA+
aIF1A
and leaderless mRNAs and other initiation
800
leadered mRNA +
aSUI1
factors are in progress. Eventually, we
600
expect to identify precisely the set of
400
factors involved in the different types of
initiation. This will be essential to unravel
200
the underlying mechanisms and to trace a
0
meaningful pattern of the evolution of the
0
10 pmol
20 pmol
40 pmol
initiation step of translation.
Publications
Benelli D, Cialfi S, Pinzaglia M, Talora C, Londei P. The translation factor eIF6 is a Notch-dependent
regulator of cell migration and invasion. PLOS One 2012, 7:e32047. doi: 10.1371/
journal.pone.0032047.
Research Group
Collaborations
Dario Benelli, researcher; Michela Pinzaglia,
PhD student; Dorina Polinari, graduated fellow.
Anna La Teana, Università Politecnica delle
Marche; Sonja-Verena Albers, Max Plank
Institute for Terrestrial Microbiology, Marburg,
Germany.
26
Study of RNA damage in yeast as a model for age-related
degenerative diseases
Cristina Mazzoni
Department of Biology and Biotechnologies "Charles Darwin"
℡: +39 06 49912257 - @: [email protected]
The aim of the present project is to elucidate how cells can handle and respond to the
accumulation of oxidized RNAs.
Recently, a growing body of studies suggests that RNA oxidation is an early event in a wide
variety of neurological diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis
(ALS), and in the progressive loss of muscle mass and strength which occur during aging
(sarcopenia). Uncovering on the consequences and cellular handling of the oxidized damaged RNA
may provide significant insights into the pathogenesis of neurodegenerative diseases. Due to the
complexity of studying these processes in mammalian model organisms, yeast could represent,
because of its easy handling and the high conservation of fundamental cell pathways, a convenient
model to trace the basal root for determining the effects of RNA damage on cell physiology. We
previously demonstrated that mutants in mRNA degradation, beside the accumulation of mRNAs,
show premature aging, high level of intracellular ROS and apoptotic cell death features.
These mutants represent a convenient tool to study the effects of mRNA modifications during
aging.
In preliminary experiments, we observed that in mRNA degradation mutants (Kllsm4∆1) the
reversion rate of mutation hugely increased with aging, compared to the wild type.
High reversion rate, as that measured in mRNA degradation mutants, was also observed in
extremely aged wild type cells after 20 days of cultivation, suggesting that such phenomenon is
physiologically related to aging.
Interestingly, we found that most of the reversions observed in both wild type and Kllsm4∆1
mutant, were not due to adaptive DNA mutations since cells, after repeated cultivation, showed again
the tryptophan auxotrophy.
This fact suggests that such mutations were not at the DNA level but, rather, at the RNA level
(mRNA and/or tRNA). We hypothesize that the mechanism underlying the suppression of the
auxotrophic mutations under selective pressure might result from translational errors induced by
oxidized mRNAs.
According to this, our data concerning the mutation rate might indicate that, in yeast cells, the
capability to handle oxidized RNAs drops with aging. In this respect, the Kllsm4∆1 mutant, which
accumulates mRNAs and ages much faster that the wild type strain, represents a useful tool for
deciphering the molecular mechanisms of cell response to mRNA oxidation.
First, we verified the possibility that oxidized mRNAs might impair the formation of polysomes. In
this respect, we compared the polysome profiles of the Kllsm4∆1 and wild type strains and we could
not find evident differences (manuscript in preparation)
Nevertheless, the fact that Kllsm4∆1 mutant cells are markedly more sensitive to the proteasome
inhibitor MG132 and to the presence of the arginine-analog L-canavanine, which increases the amount
of misfolded proteins, indicates the accumulation of aberrant proteins that could be generated by an
incorrect translation of mRNA codons. In fact, the oxidized ribonucleotides could induce premature
27
termination and/or generation of modified full-length proteins with reduced or missing enzymatic
activity. This suggests that, as demonstrated in HEK293 cells, oxidized mRNAs can induce translation
errors.
It is well known that many foods of plant origin contain, at different levels, antioxidant compounds.
We used the Kllsm4∆1 mutant to study the capability of different apple’s fractions to increase
viability and oxidative stress resistance in yeast cells. We showed that whole apple extracts, closely
followed by the polyphenol fraction, were able to reduce the levels of intracellular ROS and extend
lifespan, suggesting that the treatments with antioxidants compounds represents a strategy to alleviate
the phenotypes caused by the accumulation of oxidized RNA.
To make a correlation between mutation frequency and RNA oxidation, we are also developing a
protocol to measure the oxidized fraction of total RNAs from wild type and Kllsm4∆1 mutant cells
during aging by using 8-oxoguanosine antibodies (15A3). In fact, the high-performance liquid
chromatography-electrochemical detection (HPLC-ECD) technique suggested by us at the beginning
of the project, proved to be very tricky and not applicable for our purposes.
In parallel to the studies in liquid cultures, we developed a procedure, named clonal life span
(ClLS) that can represent a third way of studying aging together with the replicative life span (RLS)
and chronological life span (CLS).
By the ClLS approach we can follow the growth and aging of yeast colonies derived from single
cells isolated by micromanipulation and seeded one by one on separated plates, to avoid growth
interference by surrounding colonies. We observed that over time the formation of cell mass similar to
the human “senile warts” (seborrheic keratoses, SK), the skin lesions that often appear after 30 years
of life and increase in number and size over the years. We observed that similar signs of aging appear
in yeast colonies after about 30 days of growth and increase during aging. In this respect we
hypothesize to use yeast as a clock to study the onset of human aging phenotypes.
Cells coming from these “warts” can show higher mutation frequency and some signs of cancer
cells. Up to now, the patho-mechanisms of SK are not fully understood. Gene expression analysis
study by DNA microarray identified several up-regulated genes, including the oncogenic form ∆Np63
of the transcriptional regulator p63. Recently, a yeast gene related to p63, NDT80, has been identified
in controlling the aging process. In fact, this gene is involved in rejuvenilation of yeast cells during
sporulation and yeast cells over-expressing this gene are able to double their lifespan.
We are planning to study the role of RNA oxidation in promoting the appearance of these agerelated “warts” and possible mechanisms promoting or preventing their appearance.
Publications
Palermo V, Mattivi F, Silvestri R, La Regina G, Falcone C, Mazzoni C. Apple can act as anti-aging on
yeast cells. Oxid Med Cell Longev 2012, 2012:491759. doi: 10.1155/2012/491759.
Mazzoni C, Mangiapelo E, Palermo V, Falcone C. Hypothesis: is yeast a clock model to study the
onset of humans aging phenotypes? Front Oncol 2012, 2: 203. doi: 10.3389/fonc.2012.00203.
Research Group
Collaborations
Claudio Falcone, professor; Vanessa Palermo,
post-doc fellow; Michele Saliola, graduated
technician.
Frank Madeo, Institute of Molecular Biosciences,
University of Graz, Austria; Joanna Kufel,
University of Warsaw, Poland
28
Area 2
Pathogenic mechanisms of microbially
associated diseases
Area 2: Pathogenetic mechanisms of microbially associated diseases
Molecular characterization of viruses causing bronchiolitis and
study of viral and host factors affecting Type I IFN antiviral
response induced by respiratory viruses
Guido Antonelli
℡: +39 06 4474122 - @: [email protected]
We proposed to characterize viral factors associated with the severity of bronchiolitis and to study
the role of innate immunity in antiviral response.
First, we characterized viral infections, with a comprehensive panel of PCR-based reactions, in
respiratory samples from patients seeking medical care in Emergency Departments, Policlinico
Umberto I, Sapienza University. Respiratory syncytial virus (RSV) was the major pathogen in
bronchiolitis, followed by rhinovirus (RV), metapneumovirus and Bocavirus (Pierangeli et al., 2012).
We evaluated the association between RSV load and disease severity or between clinical
parameters and mRNA-levels of the interferon stimulated gene (ISG) 56 in 132 infants hospitalized for
bronchiolitis and infected with RSV. Viral load was positively related to the clinical severity of
bronchiolitis, the length of hospital stay, the levels of glycaemia and the number of ISG56-mRNA
copies, whereas an inverse correlation was observed with levels of haemoglobin. RSV load
significantly decreased between the first and second sample in most single and RSV coinfections.
Indeed, infants with high RSV load on hospital admission are more likely to have both more severe
bronchiolitis and a higher airway activation of antiviral immune response (Scagnolari et al., 2012).
Given the importance of IFN lambda activation during RSV infection, we determined the
frequencies of the IL-28B single nucleotide polymorphisms (SNPs) near the interleukin (IL) 28B gene,
also known as IFN lambda3 [namely rs12979860 (C/T) and rs8099917 (T/G)], strongly associated with
clearance of hepatitis C virus. We then evaluated whether the IL28B rs12979860 and rs8099917
SNPs were associated with RSV susceptibility, RSV load, disease severity and other clinical
parameters in 138 bronchiolitis cases, comparing RSV positive and negative. IL28B rs12979860 and
rs8099917 SNPs had no effects on the RSV susceptibility and on viral load. We then evaluated
infant characteristics independently of RSV infection; IL28B rs12979860 SNP only increased the risk
of hospitalization at early age (Scagnolari et al., 2012).
We performed also genotyping of RSV strains over the entire study period; a dominance of RSV A
during 2009-2010 and a co-circulation of RSV A and B during 2010-2011 and 2011-2012 seasons was
revealed. We then performed a phylogenetic analysis of RSV A and B circulating strains targeting the
C-terminal portion of the attachment glycoprotein (G), the major antigenic protein, that demonstrates
considerable genetic variability. Besides point mutations, major genetic changes occur in the Cterminal portion of the G gene more frequently than in other genomic regions. We found for the first
time since its identification in Ontario (Canada) in December 2010, a novel RSV-A strains with a 72base duplication in the G hypervariable region. Diffusion of strains with the duplicated segment could
be enhanced by the absence of herd immunity. Compared with an ~ 10% prevalence of the ON1 strain
in Ontario in the 2010/11 winter season, we calculated an infection rate of about 25% in 2011/12.
Moreover, we found other RSV A strains bearing novel amino acid changes in the conserved portion
of the G protein never reported before. Patient data of RSV cases were then analyzed; but no
significant differences between strains with or without the insert were found. The lack of significant
31
differences in clinical severity between the dominating strains of the RSV A GA2 genotype and the
emerging ON1 genotype may be due to the limited number of ON1 samples (Pierangeli et al.,
submitted).
To clarify the important issue of the proposed association of RV first infection with development of
paediatric asthma, the rate of recurrent wheezing was analyzed 12 months after hospitalization for
bronchiolitis: multivariate analysis identified as major independent risk factors for recurrent wheezing,
RV infection and a positive family history for asthma (Midulla et al., 2012).
RV 5’ UTR and VP4/VP2 sequencing and phylogenetic analysis was performed in patient strains:
the 5’ proximity of the initiator AUG, the VP4-VP2 cleavage sites and the position of the cis-acting
replication element were highly conserved. Differently, we found for the first time that the 24 amino
acids (aa) motif exposed during the so-called breathing of the internal protein VP4, was conserved in
RV A and B (21/24 aa), but not in RV C isolates (only 9 aa out of 24). Patient and clinical data of RV
A and C cases were then analyzed revealing a tendency of RV C strains to be associated with
hospitalization of older children and with wheezing, with respect to RV A (Pierangeli et al., Med
Microbiol Immunol 2013).
Publications
Pierangeli A, Scagnolari C, Selvaggi C, Monteleone K, Verzaro S, Nenna R, Cangiano G, Moretti C,
Papoff P, Antonelli G, Midulla F. Virological and clinical characterization of respiratory infections in
children attending an emergency department during the first autumn-winter circulation of pandemic
A (H1N1) 2009 influenza virus. Clin Microbiol Infect 2012, 18: 366-73. doi: 10.1111/j.14690691.2011.03590.x.
Scagnolari C, Midulla F, Selvaggi C, Monteleone K, Bonci E, Papoff P, Cangiano G, Di Marco P,
Moretti C, Pierangeli A, Antonelli G. Evaluation of viral load in infants hospitalized with bronchiolitis
caused by respiratory syncytial virus. Med Microbiol Immunol 2012, 201: 311-7. doi: 10.1007/
s00430-012-0233-6.
Scagnolari C, Midulla F, Riva E, Monteleone K, Solimini A, Bonci E, Cangiano G, Papoff P, Moretti C,
Pierangeli A, Antonelli G. Evaluation of interleukin 28B single nucleotide polymorphisms in infants
suffering from bronchiolitis. Virus Res 2012, 165: 236-40. doi: 10.1016/j.virusres.2012.02.018.
Midulla F, Pierangeli A, Cangiano G, Bonci E, Salvadei S, Scagnolari C, Moretti C, Antonelli G, Ferro
V, Papoff P. Rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up. Eur Respir J 2012,
39: 396-402. doi: 10.1183/09031936.00188210.
Research Group
Ombretta
Turriziani,
professor;
Laura
Antonelli, Fabio Midulla, Paola Papoff,
Alessandra Pierangeli, Carolina Scagnolari,
researchers; Carla Selvaggi, research fellow;
Katia Monteleone, PhD student.
32
Lipopolysaccharide and peptidoglycan adaptation to host as an
immune evasion strategy of gram-negative pathogens
Maria Lina Bernardini
℡: +39 06 49917855 - @: [email protected]
Successful microbial pathogens have evolved complex and efficient methods to overcome innate
and adaptive immune mechanisms, which can result in acute disease or chronic infections. Shigella is
an enteroinvasive pathogen provoking bacillary dysentery in humans. This disease is based on an
acute and severe inflammation of colonic and rectal mucosa induced by the invasive process of
Shigella. Shigella is able to drive the immune response mainly by manipulating the host machinery
involved in cell death and by subverting the signaling leading to the inflammatory reaction. However,
poor information is available on the adaptation to host of Shigella PAMPs (Pathogens Associated
Molecular Patterns) recognized by the Pattern Recognition Receptors, (PRRs), which are the sentinels
of the innate immunity, during the invasion process.
Pseudomonas aeruginosa (PA) is an opportunistic pathogen causing acute and chronic infection
in the airways of Cystic Fibrosis (CF) patients. Strains establishing life-long chronic infection are
usually pathogenic variants distinguished from the initially acquired strains, often differing for the
presence/absence of virulence factors and harboring PAMPs modifications. The aim of our study
focused on the impact of PAMPs on the host response during the invasive process of Shigella flexneri
(SF) and lung colonization of PA.
Pathogenic bacteria may modify their surface to evade the host innate immune response. We
found that SF modulates its lipopolysaccharide (LPS) lipid A structure according with cues
encountered by bacteria during the intracellular residence in epithelial cells. The Shigella lipid A
purified by intracellular bacteria mainly contains variants with a reduced number of acyl chains while in
Lipid A of conventionally grown bacteria the hexa-acylated lipid A form is prevalent. Purified LPS of
intracellular bacteria show a low immunostimulatory potential comparing to LPS of bacteria grown in
laboratory medium. Moreover, the hypo-acylated LPS poorly stimulate macrophages to produce
inflammatory cytokines and neutrophils to trigger ROS production. The SF modifications of lipid A
moiety to a less acylated form may be considered as a strategy to evade the host innate immunity,
thereby enhancing bacterial pathogenicity. Similarly, SF peptidoglycan (PGN) is remodeled through a
set of enzymes including the plasmid encoded gene SfpgdA, which acts as a PGN deacetylase
producing a PGN more resistant to the lisozyme activity.
Environmental PA strains possess suitable PRR ligands, and these ligands are used during
infections to elicit efficient responses in the lung. Therefore, in the healthy human respiratory tract,
innate mechanisms could be sufficient to manage infection by PA. However, these mechanisms
become disabled in the CF patient, permitting chronic infection, which is exacerbated further by
dysfunctional cellular killing mechanisms and direct effects of the mutated CFTR (cystic fibrosis
transmembrane conductance regulator) in these patients. By using the sequential clinical isolates we
found that the process of PA adaptation to host selects bacterial pathogenic variants equipped with
molecules and /or molecular complexes, such as flagellin and flagella, and rod proteins and Type III
secretion system, that deeply influences the recognition and the sensing of PRRs. Especially PRRs
involved in inflammasome assembly and activity are differently stimulated by the ligands that are
33
carried by strains isolated from initial or ongoing stages of the infection, thereby highlighting the
relevance of this process in the establishment of PA chronic residence in lungs. In conclusion, our
findings indicate that, SF and PA exploit similar immune evasion strategies despite the different style
of life in the host.
Publications
Kaoukab-Raji A, Biskri L, Bernardini ML, Allaoui A. Characterization of SfPgdA, a Shigella flexneri
peptidoglycan deacetylase required for bacterial persistence within polymorphonuclear neutrophils.
Microbes Infect 2012, 14: 619-27. doi: 10.1016/j.micinf.2012.01.009.
Research Group
Collaborations
Luigi Lembo Fazio, Valeria Ciancarella,
Gaëlle Noël, post-doc fellows; Ida Paciello,
Anna-Karin Hermansson PhD students.
Abdel Allaoui, Lab. Bactériologie Moléculaire,
Université Libre de Bruxelles, Belgium;
Alessandra Bragonzi, Divisione di Immunologia,
Trapianti e Malattie Infettive, Istituto Scientifico
San Raffaele, Milano; Antonio Molinaro,
Dipartimento di Chimica Organica e Biochimica,
Università di Napoli Federico II; Maria Rescigno,
Dipartimento di Oncologia Sperimentale, Istituto
Europeo di Oncologia, Milano.
34
Involvement of sRNA molecules in the complex regulatory circuits
of virulence gene expression in Shigella flexneri and in
enteroinvasive E. coli
Bianca Colonna
℡: +39 06 49917582 - @: [email protected]
Shigella is an intracellular pathogen causing bacillary dissentery (shigellosis), a severe enteric
syndrome, in humans. The disease is endemic throughout the world and millions of cases occur every
year, mostly in the developing countries. Shigellosis is transmitted by the fecal-oral route and is
characterized by an acute intestinal inflammation resulting in strong abdominal cramps, fever and
bloody diarrhoea. Recently Shigella spp. have been shown to belong to extremely diverse E.coli
species rather than forming a distinct genus. The analysis of the evolutionary relationship among
Shigella and E. coli indicates that Shigella strains have been derived repeatedly from different
branches of the E. coli tree by convergent evolution involving both, gain and loss of genes. While the
acquisition of the large virulence plasmid (pINV) by horizontal gene transfer has induced, in a single
step, the capability to enter and multiply inside the highly specialized intracellular environment of the
human intestinal mucosa, pathoadaptive mutations have acted progressively to increase the
pathogenic potential of Shigella. In this evolutionary scenario acquisition and loss of genetic regions
likely entail the introduction or the disappearance of regulators. This might have contributed to alter the
trascriptional profile of the ancestor cell, favouring the coordinated expression of virulence factors in
novel niches.
The actin based motility of Shigella, depends on IcsA, an outer membrane protein that
accumulates at one pole of the bacterial cell. IcsA is capable of directly recruiting and activating host
cell factors, thus catalyzing the assembly of an actin tail which provides the propulsive force for
intracellular movement and intercellular dissemination of bacteria through the human intestinal
epithelium. Over the years evidence has built up indicating that the regulation of icsA depends on the
nucleoid protein H-NS which acts as a repressor, and on the AraC-like protein VirF, the major activator
of the Shigella invasivity regulon. We have recently shown that an additional player is involved: RnaG,
a non coding sRNA which acts as an antisense regulator on the icsA transcript.
Small noncoding RNAs (sRNA) have emerged in recent years as crucial elements in bacterial cell
regulation. In particular, it is becoming increasingly evident that sRNAs, besides acting in gene
regulation in general, can also play a key role in controlling the expression of virulence genes involved
in the virulence or in the survival within the host.
In particular we have found that RnaG is able to negatively interfere with icsA transcription by
means of two independent mechanisms: transcriptional interference and transcriptional attenuation. In
this context we have shown that silencing of the RNAG promoter results in higher activity of the
convergent icsA promoter. On the other hand, RnaG can also cause a direct repression of icsA
transcription by acting as antisense RNA, provoking a premature termination of the icsA messenger. In
order to evaluate regulatory mechanism controlling the expression of the icsA gene, we have analysed
the synergistic and antagonistic interplay among the regulatory proteins and the RnaG molecule. The
results obtained show that both proteins, H-NS and VirF, recognize the icsA and RnaG promoter
35
regions in vivo and in vitro and that the temperature-dependent expression of icsA depends on a
delicate balance among H-NS repression,
VirF activation and RnaG transcription.
These data stress how complex
regulatory circuits are functioning in
Shigella to obtain a coordinated and
efficient
expression
of
virulence
determinants inside the host and shed light
on how they have evolved by integrating
environmental signals, nucleoid associated
proteins, sRNAs and transcriptional
regulators.
Fig.1 - Regulatory circuit of the master regulator
and icsA genes in Shigella.
Publications
Prosseda G, Di Martino ML, Campilongo R, Fioravanti R, Micheli G, Casalino M, Colonna B. Shedding
of genes that interfere with the pathogenic lifestyle: the Shigella model. Res Microbiol 2012, 163:
399-406. doi:10.1016/j.resmic.2012.07.004.
Research Group
Collaborations
Gianni Prosseda, researcher; Maria Letizia Di
Martino, post-doc fellow; Rosaria Campilongo,
PhD student.
Gioacchino Micheli, Istituto di Biologia e
Patologia Molecolari, CNR, Roma; Maurizio
Falconi, Dipartimento di Biologia Molecolare,
Cellulare e Animale, Università di Camerino;
Mariassunta Casalino, Dipartimento di Biologia,
Università di Roma Tre.
36
Inhibition of Pseudomonas aeruginosa biofilms: new molecular
strategies targeting cyclic-di-GMP metabolism
Francesca Cutruzzolà
℡: +39 06 49910955 - @: [email protected]
Biofilms formed by bacterial pathogens are responsible of more than 70% of all infections in
developed countries; the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP) controls the switch
to the biofilm lifestyle. We study the signals controlling biofilm development in the opportunistic human
pathogen Pseudomonas aeruginosa in order to find new targets for effective anti-biofilm drugs. We
characterize strategic P. aeruginosa proteins involved in c-di-GMP metabolism (diguanylate cyclasesDGCs and phosphodiesterases-PDEs) and study the connection between the c-diGMP pathway and
other pathways relevant for pathogenesis, including the metabolism of nitric oxide. P. aeruginosa
survives in the low oxygen (nitrate-rich) environment of cystic fibrosis patients by forming robust
biofilms and using anaerobic metabolism; in P. aeruginosa, pathogenesis, biofilm formation and
anaerobic reduction of nitrite, are closely related. By studying the transcriptional regulator DNR
(Rinaldo et al., 2012) and the enzyme responsible of nitric oxide production (Radoul et al., 2012;
Castiglione et al., 2012) we have shown that the nitrite reductase activity (and its regulation) is crucial
for energy production in anarobiosis, thus contributing to biofilm formation.
In order to study the activity and inhibition of DGCs and PDEs, a novel approach was devised,
based on the sigmoidal CD signal displayed by the intercalated dimer of c-di-GMP and on the ability of
manganese ions (but not Mg2+ ions) to promote
dimerization of the cyclic dinucleotide (Stelitano et al.
Nucleic Acid Research 2013, 41:e79). The CD signal is
specific of the dimeric form of c-di-GMP; other
nucleotides (GTP, pGpG or GMP, c-di-AMP) do not show
the same spectroscopic feature, suggesting that the
sigmoidal CD signal arises from the stacking of the
guanine bases in the c-di-GMP dimer. We have
determined the crystallographic structure of the complex
of c-di-GMP with Mn2+, demostrating that c-di-GMP binds
to the two metals (Mn and Mg) with a similar geometry
(Fig.1).
Fig. 1 - Structure of the c-di-GMP-Mn complex.
Using this novel approach, c-di-GMP production and degradation has been measured in real-time
on two reference enzymes, i.e. PleD DGC and RocR PDE. The method will allow conceiving presteady state kinetic experiments, to date unexplored for these enzymes, but of crucial value to
determine the reaction mechanism of both DGCs and PDEs and to design inhibitors.
37
In parallel, we have characterized selected P.aeruginosa DGCs or PDEs. In P.aeruginosa, 2 HDGYP PDEs are found, able to decrease c-di-GMP levels in vivo; these proteins contain the PDE
catalytic domain and a regulatory domain, either of the CheY-type (PA4781) or unknown (PA4108).
Both proteins were purified and tested for their PDE activity in vitro, monitoring c-di-GMP hydrolysis to
GMP. Since PA4108 and PA4781 show very low (or none) enzymatic activity in vitro, and in order to
bypass a possible activation step, a truncated version of PA4781 was also analyzed (PA4781-G). This
protein is inactive in vitro, but active in cell extracts or in intact E.coli cells, showing that for PA4781 to
be active, interaction with an intracellular partner (currently being identified) is required.
The PA1120 protein (TpbB) is a DGC involved in biofilm development and Small Colony Variant
(SCV) (a persister morphotype) formation. We have crystallised and determined the structure of the
DGC catalytic domain of TpbB. Previous kinetic and binding experiments (by ITC, see also Chi et al,
2012 for the method) suggested that TpbB is not significantly inhibited by c-di-GMP. The structure of
the catalytic domain not only confirms that the inhibitory site (I-site) of TpbB lacks an Arg residue
necessary for the binding of dimeric c-di-GMP but also allowed us to propose an allosteric model for
TpbB regulation. In future, the TpbB structure will be employed as a starting point to design selective
inhibitors.
Publications
Castiglione N, Rinaldo S, Giardina G, Stelitano V, Cutruzzolà F. Nitrite and nitrite reductases: from
molecular mechanisms to significance in human health and disease. Antioxid Redox Signal 2012,
17: 684-716. doi:10.1089/ars.2011.4196.
Chi C, Haq S, Rinaldo S, Dogan J, Cutruzzolà F, Engström Å, Gianni S, Lundström P, Jemth P.
Interactions outside the boundaries of the canonical binding groove of a PDZ domain influence
ligand binding. Biochemistry 2012, 51: 8971-9. doi: 10.1021/bi300792h.
Radoul M, Barak Y, Rinaldo S, Cutruzzolà F, Pecht I, Goldfarb D. Solvent Accessibility in the Distal
Heme Pocket of the Nitrosyl d1-Heme Complex of Pseudomonas Stutzeri cd1 Nitrite Reductase.
Biochemistry 2012, 51: 9192–9201. doi: 10.1021/bi3011237.
Rinaldo S, Castiglione N, Giardina G, Caruso M, Arcovito A, Della Longa S, D’Angelo P, Cutruzzolà F.
Unusual heme binding properties of the Dissimilative Nitrate respiration Regulator (DNR), a bacterial
nitric oxide sensor. Antioxid Redox Signal 2012, 17: 1178-89. doi:10.1089/ars.2011.4226.
Research Group
Collaborations
Serena
Rinaldo,
researcher;
Nicoletta
Castiglione, Stefano Franceschini, Giorgio
Giardina, post-doc fellows; Silvia Fernicola,
Valentina Stelitano, PhD students; Manuela
Caruso, technician.
A. Paiardini, Università di Roma; Davide
Antoniani, Paolo Landini, Università di Milano;
Livia Leoni, Giordano Rampioni, Università di
Roma Tre; Loredana Cappellacci, Università di
Camerino.
38
Defense mechanisms against oxidative and nitrosative stress in
pathogenic protozoa
Paolo Sarti
℡: +39 06 49910944 - @: [email protected]
Pathogenic protozoa, such as Giardia intestinalis, Entamoeba histolytica, Trichomonas vaginalis
etc. are O2-sensitive microorganisms. Some of them, typically Giardia, though lacking conventional
ROS-scavenging enzymes, such as catalase and superoxide dismutase, keep colonizing the fairly
oxygenated small intestine. Our research has been focused on the structural and functional
characterization of those enzymes that allow those parasites to cope with human natural defenses
based on oxy-nitrosative stress reactions. Major target of these defense reactions is the cell (parasitic)
bioenergetics machinery.
These reactions, indeed, are better studied in eukaryotes equipped with mitochondria. Over the
2012 we have focussed our attention on the Terminal Oxidases (TOX), and their interaction with nitric
oxide (NO). The chemistry occurs at four redox centres of the active O2-binding site, where also NO
binds and inhibits the parasitic growth. These oxidases contain in the active site one heme iron and a
copper (CuB); in bacterial cytochrome bd the copper ion is substituted by one heme d, providing the
enzyme of peculiar activity properties far from being understood.
Nitric oxide (NO) cytochrome oxidase (CcOX) and cellular respiration
Control experiments carried out in parallel using mammalian cells in culture proved that NO has a
key role in the control of cell energetics. During hypoxia and at high reducing pressure, the reaction of
NO with CcOX occurs in competition with O2 inhibiting the aerobic ATP production. In addition, NO
reacts with the superoxide anion (O2-), a reactive O2 species (ROS), forming peroxynitrite (ONOO-),
which controls the intracellular concentration of ROS and reactive nitrogen species (RONS) [38, 39].
These observations provide a molecular basis for the reported anti-microbial activity of NO produced
by the NO synthase of macrophages, explaining also the evolution of NO degrading molecular
systems in pathogenic microorganisms and protozoa. Interestingly, some bacterial TOX can degrade
NO, oxidatively to nitrite NO2-, or reductively to N2O.
Reaction mechanism between NO and CcOX
Depending on the experimental conditions, a rapid (millisecond) formation of two different CcOX
inhibited adducts are observed: the nitrosylated (Fe2+NO) and the nitrite bound forms (Fe3+NO2-) [3].
Relevant to cellular metabolism, the two adducts have a well different cell destiny and hence
pathophysiological relevance. The nitrite dissolved in blood and tissues has been suggested to
represent a potential reservoir of NO [47] with potential antimicrobial function but also with
pathophysiological backfalls, depending on its actual concentration. The heme proteins, hemoglobin
and myoglobin, as well as the xantine oxidase are able to reduce nitrite to NO. Peroxidases and
globins in the oxo-ferryl state (FeIV) can be reduced by nitrite to (FeIII). The reaction is potentially toxic,
again for both the parasite and the host, as it leads to accumulation of nitrite-radical, responsible of a
prompt tyrosine nitration.
39
Cytochrome bd
The cytochrome bd 3D structure is now available. The enzyme is expressed at low O2 tension and
/or under stress condition by several microorganisms including pathogens, such as E.coli, A.
vinelendii, B. abortus, M.tubercolosis). We have found that Cytochrome bd catalyses the reduction of
O2 to water by using quinols as substrates; differently from the canonical oxidases, it contains two
haems in the active site. Cytochrome bd has a high affinity for O2 and favors the survival and infectivity
of some microaerophilic bacteria. We showed that bd-type oxidase, as the haem-copper ones, are
potently and reversibly inhibited by
NO. Our experiments have shown
also, however, that the recovery of
cytochrome bd activity after inhibition
is fast, due to a very high NO off rate
from the enzyme (koff= 0.13 s-1, T =
20°C).
This
observation
has
pathophysiological relevance, since
NO is produced by the human
immune system to fight microbial
infections, and provides suggestions
for new pharmacological approaches.
Publications
Giuffrè A, Borisov VB, Mastronicola D, Sarti P, Forte E. Cytochrome bd oxidase and nitric oxide: from
reaction mechanisms to bacterial physiology. FEBS Lett 2012, 586: 622–9. doi:10.1016/j.febslet.
2011.07.035.
Sarti P, Forte E, Giuffrè A, Mastronicola D, Magnifico MC, Arese M. The chemical interplay between
nitric oxide and mitochondrial cytochrome c oxidase: reactions, effectors and pathophysiology. Int J
Cell Biol 2012, 2012: 571067. doi:10.1155/2012/571067.
Sarti P, Forte E, Mastronicola D, Giuffrè A, Arese M. Cytochrome c oxidase and nitric oxide in action:
molecular mechanisms and pathophysiological implications. Biochim Biophys Acta 2012, 1817:
610–19. doi:10.1016/j.bbabio.2011.09.002.
Research Group
Collaborations
Elena Forte, researcher, Daniela Mastronicola,
Fabrizio Testa, post-doc fellows, Alessandro
Giuffrè, CNR researcher.
Pier Luigi Fiori, Università di Sassari; Brajendra
K. Singh, Department of Chemistry, University of
New Delhi, India; Miguel Teixera, Instituto de
Tecnologia Química e Biológica, Universidade
Nova de Lisboa, Portugal; João B. Vicente,
Universidade de Lisboa, Portugal.
40
Immunopathogenesis of HIV infection: study of innate immunity
and dendritic cells
Vincenzo Vullo
Department of Public Health and Infectious Diseases
℡: +39 06 49970313 - @: [email protected]
The study of dendritic cells (DCs) as a link among innate and specify immunity is very important
for pathogenesis, clinical and therapeutic approaches in HIV infection. In this study we investigate the
role of dendritic cells and immune activated CD4 and CD8 T lymphocytes in bone disease, a common
co-morbidities among HIV infected people. Both antiretroviral drugs and HIV related immune
dysfunction have been shown to be implicated in this process.
50 HIV-infected (24 M, 26 F) patients were enrolled for this study. They received stable effective
classical triple ART regimen and they were consecutive submitted to DEXA analysis and
immunological test including plasmocytoid (pDCs) and myeloid dendritic cells (mDCs) count, activated
CD4+ and CD8+ T cells expressing HLA-DR and CD38. Vitamin D plasma levels were also tested A
group of age and sex matched (2:1) healthy subjects were used as control and the statistical analysis
was done by the Mann-Whitney U test and the Spearman rank correlation test.
Our results shown that all treated patients had a complete virological response with HIV-RNA <20
cp/ml with a mean CD4+ of 655 cells/mmc. Only 14% of patients presented a normal bone mineral
density (NBMD) with a T score <-1, 86% were osteopenic or osteoporotic (LBMD). A significant
augmentation of DR+38+CD4 was found in LBMD (p=0,005). No significant differences were found in
term of pDCs and mDCs count between NBMD and LBMD. However a positive correlation was found
between T score and mDCs in 48yrs younger subjects (p=0,02, R=0,51, fig.1). Both mDCs and pDCs
were inverse correlated with Delta of CD4 (p=0,04 and p=0,001 respectively) and with DR+CD4+
(p=0,01 R=-0,34) and DR+CD8+ (p=0,05 R=-0,27). pDCs was also correlated with vitamin D level,
especially in 48yrs older subjects and in men. Immune activated CD4+ was inverse correlated with T
score only in men (p=0,06 R=0.38) (Fig.1). Vitamin D levels were deficient in both HIV and healthy
subjects.
Therefore, a complex interrelations between different factors was determinant for bone disease
genesis in HIV infected subjects. Immune activation and dendritic cells seem to play a role in particular
settings such us younger age and male gender in which classical risk factors are less important.
On the other hand, another factor in sustaining immune activation in HIV patients was CMV coinfection, which could play a role in determining immune senescence. In this study, we evaluated the
prevalence and predictors of CMV co-infection in a cohort of HIV+ patients and assessed the impact of
CMV co-infection on the risk of AIDS and non-AIDS events. We included 6053 patients in the ICONA
study with<1 month follow-up and<1 CMVIgG (CMV) test available without active CMV disease.
Characteristics of patients at baseline (first CMV test) were compared in those tested positive (CMV+)
and negative (CMV-) using X2/Wilcoxon tests. Factors associated with CMV+ were identified by logistic
regression. A prospective analysis was also performed with endpoints AIDS/AIDS-related death and
severe non-AIDS (SNA: cardio-cerebrovascular, neurologic disease, renal failure, non-AIDS
tumours)/death due to SNA. Time to event was estimated by Kaplan-Meier curves and Cox regression
(multivariable model included: age, gender, ethnicity, risk factor for HIV, HCVAb and HBsAg, AIDS
and CD4 at baseline, initiation of ART prior to baseline). 83.7% of patients were tested CMV+ a
41
median of 17 (IQR 6-45) months after enrolment. As compared to CMV-, CMV+ were older (adjusted
odds ratio (AOR) 1.03 per 1 year older [95% CI 1.02-1.04]), HIV infected by homosexual route (MSM)
(AOR 1.39 [95% CI 1.06-1.82]), less frequently Caucasian (AOR 0.56 [95% CI 0.42-0.76]), with higher
CD4 count at baseline (AOR per 1 cell higher 1.035 [95% CI 1.00-1.06] By 10 years from first CMV
test, 402 (12.6% [95% CI 11.1-13.6]) CMV+ and 74 (10.1% [95% CI 7.7-12.5]) CMV- developed
AIDS/AIDS-related death (log-rank p=0.43). After adjustment for potential confounders, CMV+ was still
not associated with the risk of AIDS/AIDS-related death (adjusted hazard ratio (AHR) 1.23 [95% CI
0.96-1.60]). By 10 years, 339 (10.6% [95% CI 9.4-11.9]) CMV+ and 41 (6.4% [95% CI 6.1-6.6]) CMVpts experienced a non-AIDS event/non-AIDS death (p=0.0006): 151 cancers, 128 CVD, 33
neurological, 1 renal. The association was still significant after controlling for a number of potential
confounders: AHR 1.77 [95% CI 1.25-2.51] p=0.001). In our study population, CMV/HIV co-infection
was associated with the risk of non-AIDS events/deaths independently of other prognostic factors,
supporting a potential role of CMV infection in vascular/degenerative organ disorders commonly
associated with chronic immune activation and aging.
Fig. 1 - An inverse correlation was found between Tscore/mDC and immune activated CD4+/Tscore in young
men HIV+ subjects.
Research Group
Collaborations
Claudio M. Mastroianni, professor; Miriam
Lichtner, researcher; Raffaella Rossi, Ilaria
Sauzullo, post-doc fellows; Gabriella d’Ettorre,
Claudia D’Agostino, hospital physicians; Fabio
Mengoni, graduated technician.
Anne Hosmalin, Inserm
Cochin, Paris, France.
42
U1016,
Institut
Area 3
Molecular genetics
of eukaryotes
Area 3: Molecular genetics of eukaryotes
Assembly and functional analysis of genomic context vectors
containing the human CFTR locus
Fiorentina Ascenzioni
℡: +39 06 49917577/7614 - @: [email protected]
We have previously demonstrated the functional activity of a BAC-encoded CFTR in model cells of
by molecular approaches and electrophysiology. Additionally, we have study the interaction of the
epithelial cells expressing the BAC-encode CFTR with Pseudomonas aeruginosa, which represent the
most important pathogen for Cystic Fibrosis patients. In the last year we focused on the improvement of the BAC-CFTR with two principal aims: reduce
its size to make it more amenable to the standard protocols of BAC preparations; to introduce/remove
relevant sequences to improve CFTR expression into target cells. Additionally we analysed the
functional activity of a CFTR-containg episome in CF bronchial epithelial cells.
CFTR-BAC engineering
The most used approaches to engineer inserts cloned into BAC vectors make use of homologous
recombination or site-specific homologous recombination in bacterial cells. We have set up both
procedures by using the Cre/loxP system and homologous recombination. By expressing the Cre
recombinase, the selectable markers phleomycin and Kanamycin were deleted from the BAC
cCFTRD12. Different clones that acquired sensitivity to phleomycin and/or G418 were isolated and
analyzed by PCR. This analysis confirmed deletion of the markers in the phleoS and G418S clones,
suggesting that the Cre promoted the excision of the loxP bracketed fragments. We are confirming
this result by amplification of the regions flanking the deleted regions and subsequent sequencing.
After extensive analysis of the 3’ and 5’ ends of the CFTR locus we have identified the regions
that, according to the literature, can be eliminated without affecting CFTR expression.
Using the multiple rounds of amplification with hybrid primers, we have assembled 3’-end and 5’end tagging fragments containing the marker phleomycin and kanamycin, respectively. These
fragments will be introduced in yeast cells containing the cCFTRD12 to reduce both the up- and downstream regions of the BAC vector.
pEPI-CFTR episome
In order to test non-viral vectors, other than artificial chromosomes, that have the capacity to be
stably retained within the target cells, we focused on the episomal vector pEPI that, by the presence of
a S/MAR (scaffold/matrix attached region) fragment, appears to be stably retained in vitro, in different
cell lines, and in vivo in animal models. We have assembled a pEPI-CFTR vector that was delivered to
epithelial cells homozygous for the F508del CFTR mutation (CFBE, cystic fibrosis bronchial epithelial
cells). The episome within the transfected cells was detected by different methods and the results
showed that it was retained at least up the 15 days after transfection. Based on the consideration that
the transfected cells continued to divide and in 15 days made approximately 13 mean population
doublings, we conclude that the episome is stably maintained in CFBE cells. By using allele-specific
PCR we have demonstrated the presence of wt CFTR mRNA in the transfected cell. Overall, although
other important aspects need to be analyzed such as the transgene localization and CFTR channel
45
activity, we conclude that the S/MAR based episomes are good vector for stably retention of the CFTR
in bronchial epithelial cells without interfering with the host genome.
Nanodelivery of nucleic acids
Non-viral vectors require transfecting agents to be delivered within the cells. We focused on
chitosan (Cs)-based nanoparticles that have been successfully used to transfect a variety of cell types.
Cs is a biocompatible cationic polysaccharide, obtained from the crustaceans and insects
exoskeletons, that is able to compact DNA into nanoparticles. Our results showed that Cs/pDNA
nanoparticles efficiently delivered the plasmid to the cells and that, differently from the Escort-pDNA
complexes, transfection efficiency peaked 4-5 days following transfection. Afterwards, the fraction of
plasmid-containing cells slowly declined over the next 15 days. Overall, we observed an efficient and
time sustained delivery of plasmid DNA by chitosan-based nanoparticles.
Research Group
Collaborations
Enea Gino Di Domenico, post-doc fellow,
Cifani Noemi, PhD student; Paola, Parisi,
graduated student; Barbara Pompili, student.
Dr. Cleofe Palocci Dipartimento di Chimica,
Sapienza Università di Roma.
46
Light control of filamentous fungi life cycle: from system models to
applications
Paola Ballario
℡: +39 06 49912392/2318 - @: [email protected]
The long term aim of our laboratory investigation is to elucidate light transduction pathways in
filamentous fungi. The specific finalities of this project are two:
A) To clarify the mechanism of light control of gene expression in filamentous fungi (Neurospora
crassa and Tuber melanosporum), In particular we are interested in the role of White collar-1. a
multitask large regulator of light-dependent genes ( i.e. albino: carotenoid biosynthesis and Frq:
circadian conidiation) and its function with epigenetic regulation of chromatin.
B) Characterization of the sensor LOV domains of epigeous and hypogeous fungi with the
purpose of identifying variants of LOV differing in the timing of light reaction, in the threshold of light,
in the time required for LOV recovery in the dark. LOV domain can be in fact subjected to genetic
manipulations in order to construct chimeric protein that directly respond to light stimulation. These
constructs deriving from photosensors are promising tools for obtaining molecules leading to an
optical control of cellular behavior, we are only beginning to understand how to couple these light
detectors to effectors of choice .
Results of aim A have been published (MBoC 2012). We showed that in N.crassa lightdependent–phenomena are regulated by WC-1 and WC-2. In addition to its transcriptional activity,
WC-1 is, in fact, able to sense light through a LOV sensor domain. Its location in the nucleus and
heterodimerization with WC-2, together with the presence of a zinc-finger DNA-binding domain and an
environmental sensor domain, all resemble the functional evolutionary architecture adopted by
vertebrate nuclear receptors (NRs). Here we describe a scenario in which WC-1 represents a
functional orthologue of NRs and acts through association with the chromatin modifier NGF-1,
homolog of yeast HAT Gcn5. We accordingly show a direct association between WC-1 and NGF-1
that depends on a WC-1 region containing a conserved LXXLL motif, previously described as being an
exclusive signature of NR/coactivator interaction. Our data suggest that a WC1/NGF1 complex is
preassembled in the dark on light-inducible promoters and that, after light stimulation, NGF1–
associated HAT activity acetylates histone H3 and activates transcription (Fig 1). Finally, we provide
evidence for a NGF-1–independent
acetylation of WC-1. Overall, our
data indicate that Neurospora and
higher eukaryotes share a common
mechanism for signal transduction of
environmental stimuli.
Fig.1 - Model of light signal transduction
in Neurospora.
47
During the second year of the project we have studied a Neurospora wc-1 mutant in the Zn-finger
domain (from Yi Liu) in vitro and in vivo . We have preliminary data demonstrating the role of WC-1
DNA binding domain as repressor of light dependent gene transcription in the dark. This finding
indicates that WC-1 is not only an activator but also a repressor and its role might depend on the
association with coactivators/ coorepressors. We will attempt to identify the WC-1 corepressor.
B) The other aspect we have developed is the characterization of the light sensor domain (LOV:
Light, Oxigen, Voltage) of Tuber melanosporum. The LOV domain is a region (~150aa), whose
photoexcitation generates a covalent adduct between flavin C4(a) carbon and an invariant cysteine,
that broadly absorbs blue light. LOV domains are ubiquitous in Prokaryotes and Eukaryotes and
respond to the stimuli with a change of conformation that is propagated to the rest of the molecule, the
effector. There is now a strong interest in characterizing or mutagenizing LOV from different sources
in order to utilize such “new” LOVs fused to effector domains to obtain active molecules under
dark/light control.
Putative photoreceptors were identified in Tuber melanosporum by genomic sequencing, on the
basis of the in silico analysis we made two hypothesis. The first was that the putative photoreceptors
were evolutively adapted to dark environment thus acquiring or losing functions or, alternatively, that
the threshold for sensing blue light in Tuber was very low due to the limited amount of photons
penetrating the soil. To clarify this we transformed a Neurospora wc-1 ko mutant with a Neurospora
WC-1 bearing a LOV domain from N.crassa or from the hypogeous T. melanosporum. By real time
PCR we have been able to show that the chimeric construct carrying the T. melanosporum LOV
domain is able to completely activate a light dependent gene with a faster kinetic (15’ instead of 30’ as
in Neurospora).
The Neurospora strain containing the chimeric WC-1 (T. melanosporum LOV) will be further
analysed (carotenoids content, response to light and dark cycles, production of conidia...) in
comparison with wild type. In addition we aim to produce high amount of LOV domain to perform
further photochemical investigation. We have so far expressed Neurospora and Tuber LOV domains in
bacteria (pET24 and Maltose binding protein), however we obtained a highly insoluble (80%) fusion
protein only partially bound to the flavin chromophore (20%). We plan to express the protein in other
systems (Pichia pastoris , baculovirus).
Publications
Brenna A, Grimaldi B, Filetici P, Ballario P. Physical association of the WC-1 photoreceptor and the
histone acetyltransferase NGF-1 is required for blue light signal transduction in Neurospora crassa.
Mol Biol Cell 2012, 23: 3863-72. doi: 10.1091/mbc.E12-02-0142.
Research Group
Collaborations
Patrizia Filetici, CNR researcher; Claudia
Canzonetta, Cristina De Luca, research
fellows; Andrea Brenna, post-doc fellow;
Raffaele Gerace, PhD student.
Simone Ottonello, Dipartimento di Biochimica,
Università di Parma; Leonardo Baciarelli
Falini, Dipartimento di Biologia Vegetale e
Biotecnologie Agroambientali e Zootecniche,
Università di Perugia; Benedetto Grimaldi, IIT,
Genova.
48
Functional analysis of morgana, a gene involved in the control of
centrosome duplication
Silvia Bonaccorsi
℡: +39 06 49912473 - @: [email protected]
We have recently demonstrated that Morgana/chp1, a conserved CHORD (cysteine and histidine
rich domains) containing protein, is required for proper centrosome duplication and genome stability in
both Drosophila and mouse. Null mutations in Drosophila morgana (mora) cause lethality and elicit a
complex mitotic phenotype. Larval brains of mora mutants exhibit a high mitotic index with cells
arrested in a prophase/prometaphase-like state, a high frequency of polyploid cells and most diploid
cells displaying multiple centrosomes. Notably, the fly mitotic phenotype is fully rescued by the human
orthologue of mora, highlighting a conserved function of this protein. morgana -/- mice die at the
perimplantation stage, while morgana +/- primary cells and mice display a high frequency of
supernumerary centrosomes and increased susceptibility to neoplastic transformation. In addition,
tumor tissue array histochemical analysis revealed that Morgana is underexpressed in a large fraction
of breast and lung human cancers. Thus, Morgana/chp-1 appears to prevent both centrosome
amplification and tumorigenesis.
Our research programme is aimed at elucidating the role of Morgana (Mora) in the Drosophila
model system. Given the striking evolutionary conservation of the Mora function, these studies will
provide information that can potentially be applied to humans, specifically in the field of cancer biology.
Based on the complex phenotype elicited by mora mutations, we hypothesized that Mora plays
multiple roles during the cell cycle, and that these roles are mediated by different protein domains. In
plants, CHORD containing proteins (CHPs) have only 2 tandemly repeated CHORD domains. In
animals, in addition to the 2 CHORD domains, CHPs contain a C-terminal extension, indicated as CS
domain, homologous to yeast Stg, an essential component of the ubiquitin ligation machinery. To
obtain insight into the functional role of the individual domains of Drosophila Mora, we transformed
mutant flies with a transgene carrying only the two CHORD domains. This truncated form of Mora did
not rescue the lethal phenotype associated with mora mutation. However, in brains from mutant larvae
carrying this transgene, although the majority of cells was arrested in a prophase/prometaphase-like
state, most mitotic spindles had a normal bipolar morphology and did not display multiple
centrosomes. These results strongly suggest that the CHORD domains are primarily required for the
control of centriole duplication, while the CS domain is likely to be involved in the control of cell cycle
progression. To confirm this hypothesis we will characterize the cytological phenotype of mora mutant
flies carrying a transgene containing only the Mora CS domain.
We have also continued our in vivo functional analysis of mora in the Drosophila embryo, using
video time-lapse microscopy. To characterize the effect of Mora depletion on centrosome behaviour,
we injected with an anti-Mora antibody wild type embryos expressing the centriolar protein Asterless
fused with YFP (Asl-YFP). As shown in Fig.1, at each divison cycle cells from control embryos
invariably exhibit two fluorescent spots located at the opposite poles of the mitotic spindle. Conversely,
in injected embryos cells progressively accumulate multiple and irregularly localized Asl-GFP signals,
indicating that Mora plays a crucial role in the control of centrosome duplication also in the rapidly
dividing cells of syncitial embryos.
49
Finally, we have constructed flies carrying
the V34 GAL4 maternal driver and the PUASpmora transgene to overexpress wild type and
GFP-tagged Mora. In both cases overexpression of the mora transgene did not induce
any visible phenotype. We are currently using
extracts from embryos overexpressing GFPtagged Mora to isolate Mora-interacting proteins
by
co-immunoprecipitation
and
mass
spectroscopy.
Fig. 1 - Selected frames from time lapse sequences
of embryos expressing Asterless-YFP. (A) Bufferinjected embryo (control); (B) embryo injected with an
anti-Mora antibody.
(A) Control embryo exhibits an evenly spaced
monolayer of dividing cells at the surface of the
blastoderm; each dividing cell (encircled by dashed
lines) contains a couple of unduplicated or duplicated
cetrioles.
(B) In Mora-immunodepleted embryo nuclei are not
uniformly distributed at the cortex and the divisions
appear asynchronous. In addition, each dividing
nucleus displays multiple YFP signals that are
irregularly localized. The variable sizes of these
signals strongly suggest that they correspond to both
mature and immature centrioles.
Numbers indicate time (min.) of recording after
injection.
Publications
Lattao R, Bonaccorsi S, Gatti M. Giant meiotic spindles in males from Drosophila species with giant
sperm tails. J Cell Sci 2012, 125: 584-8. doi: 10.1242/jcs.101469.
Gatti M, Bucciarelli E, Lattao R, Pellacani C, Mottier-Pavie V, Giansanti MG, Somma MP, Bonaccorsi
S. The relative roles of centrosomal and kinetochore-driven microtubules in Drosophila spindle
formation. Exp Cell Res 2012, 318: 1375-80. doi: 10.1016/j.yexcr.2012.05.001.
Research Group
Collaborations
Maria Grazia Giansanti, CNR researcher;
Valeria Palumbo, post-doc fellow; Ramona
Lattao, PhD student.
James Wakefield, College of Life and
Environmental Sciences, University of Exeter,
UK.
50
RNA-RNA and RNA-protein interactions: role of small non-coding
RNAs in gene expression control
Irene Bozzoni
℡: +39 06 49912202 - @: [email protected]
Non coding RNAs (ncRNAs) have provided in the last decades a very exciting research field with
the discovery that a largely unexplored fraction of our genome encodes for complex families of RNAs
with peculiar biosynthetic pathways and unusual functions in gene expression control. Several ncRNA
molecules associate with chromatin structures to regulate transcription, others exert their control at the
post-transcriptional level and still others perform key structural or catalytic functions.
In 2012 we have continued our studies on non-coding RNAs with specific interest in their
biogenesis in muscle and neural differentiation and related diseases.
miRNA biogenesis in neuronal cells
microRNA abundance has been shown to depend on the amount of the microprocessor
components or, in some cases, on specific auxiliary cofactors. In our recent work we have shown that
the FUS/TLS protein, associated with familial forms of Amyotrophic Lateral Sclerosis (ALS),
contributes to the biogenesis of a specific subset of microRNAs. Among the large repertoire of nuclear
functions, we focused on the observation that FUS was described as a Drosha interactor. We
demonstrated that the FUS protein has a dual function of interacting with specific pri-miRNA
sequences and with Drosha. Moreover, we have shown that FUS binds to nascent pri-miRNA
molecules
and
helps
Drosha
recruitment on the chromatin allowing
efficient miRNA processing (Fig. 1 and
Morlando et al., 2012).
Fig. 1 - Schematic representation on how
FUS/TLS
cooperates
with
the
microprocessor complex for pri-miRNA
processing
We have also shown that, among the others, FUS affects the biogenesis of miRNAs with a
relevant role in neuronal function, differentiation and synaptogenesis such as miR-9, miR-125b and
miR-132. Notably, we observed that the accumulation levels of these miRNAs were lowered even
when the residual amount of FUS was half with respect to control. These data could explain why
mutations affecting FUS nuclear dosage could have a remarkable negative effect on miRNA
homeostasis, thus providing a possible correlation with the ALS pathogenesis.
miRNA biogenesis in muscle cells
In muscle cells the synthesis of specific miRNAs was shown to depend on the DYS-NOS-HDAC2
pathway. We discovered that the poor HDAC2 nitrosylation state in Duchenne conditions downregulated the expression of a specific subset of microRNAs.
51
Interestingly, these miRNAs (including muscle miR-1 and miR-133 and the ubiquitous miR-29 and
miR-30), poorly expressed in mdx, recovered to almost wild type levels when dystrophin was rescued
at 10% of wt value. Moreover, while their transcriptional activation correlated with HDAC2 release from
their promoters, the low miRNA expression in the absence of dystrophin, paralleled persistent HDAC2
binding.
The use of mutant HDAC2 and treatment of mdx mice with NO-donors indicated that release of
HDAC2 from the chromatin was due to its nitrosylation. Altogether, these data allowed us to conclude
that Dystrophin, nNOS, HDAC2 nitrosylation and specific miRNA expression are on the same
regulatory pathway.
Previous work demonstrated that nNOS localization depends on the spectrin-like repeats 16 and
17 of dystrophin, encoded by exons 41-46. In order to demonstrate the importance of the
Dystrophin/nNOS/HDAC2 pathway on the Duchenne phenotype, we have tested whether this circuitry
is altered in Becker individuals that have deletions of these domains. Since it is known that different
Becker mutations have different degrees of severity we have analysed whether this could be due to
altered nNOS localization and subsequent control of gene expression.
Comparing different Becker deletions, we have shown (Cazzella et al., 2012) that those lacking
nNOS localization display molecular and clinico-pathological phenotypes closer to Duchenne. In
particular, they are unable to sustain the expression of those miRNAs that are controlled by the DYSNOS pathway.
These results have allowed us to conclude that the ability to rescue nNOS localization is a crucial
feature that can differentially affect the outcome of exon skipping on different DMD mutations.
Publications
Cazzella V, Martone J, Pinnarò C, Santini T, Twayana SS, Sthandier O, D’Amico A, Ricotti V, Bertini
E, Muntoni F, Bozzoni I. Exon 45 skipping through U1-snRNA antisense molecules recovers the
Dys-nNOS pathway and muscle differentiation in human DMD myoblasts. Mol Ther 2012, 20: 213442. doi: 10.1038/mt.2012.178.
Morlando M, Dini Modigliani S, Torrelli G, Rosa A, Di Carlo V, Caffarelli E, Bozzoni I. FUS stimulates
microRNA biogenesis by facilitating co-transcriptional Drosha recruitment. EMBO J 2012, 31: 450210. doi: 10.1177/1947601911416357.
Salvatori B, Iosue I, Mangiavacchi A, Loddo G, Padula F, Chiaretti S, Peragine N, Bozzoni I, Fazi F,
Fatica A. The microRNA-26a target E2F7 sustains cell proliferation and inhibits monocytic
differentiation of acute myeloid leukemia cells. Cell Death Dis 2012, 3:e413. doi: 10.1038/
cddis.2012.151.
Research Group
Collaborations
Fatica Alessandro, professor; Alessandro
Rosa, researcher; Elisa Caffarelli, CNR
researcher; Julie Martone, Beatrice Salvatori,
research fellows; Mariangela Morlando,
technical
manager;
Valentina
Gazzella,
Stefano Dini Modigliani, Shyam Twayana,
Valerio Di Carlo, Chiara Pinnarò, PhD
students.
Francesco Muntoni, Dubowitz Neuromuscular
Centre, London; Enrico Bertini, Ospedale
Bambino Gesù, Roma.
52
Role of metalloproteinases and their tissue inhibitors in the
regulation of neurogenesis and gliogenesis from neural
stem/progenitor cells
Emanuele Cacci
℡: +39 06 49912206 - @: [email protected]
Neural progenitor (NP) cells generate different neural cell types during development and their
differentiation is temporarily and spatially controlled by means of both cell autonomous and external
signals. In addition NPs continuously generate new neurons and glia in the adult brain. Our studies
are aimed at clarifying the interplay between extrinsic and intrinsic cellular signals and the molecular
mechanisms underlying the regulation of NP properties.
NPs can be isolated from the fetal and adult brain and maintained in vitro as self-renewing,
multipotent cell lines. In collaboration with the group headed by prof Gabriella Tocco we demonstrated
that NP lines isolated from different regions of the brain of E13.5 mouse embryos retain their positional
identity in vitro, as shown by preservation of regulated expression profiles of region-specific
transcription factors. In addition we found that these NPs respond to the over-expression of a
transcription factor crucial for the generation of specific midbrain neurons (namely Nurr-1) by giving
rise to a small number of dopaminergic neurons, although this ability is critically dependent on their
area of origin.
Taking advantage of NP cell cultures we have been studying how cellular and diffusible
component of the niche, the specialized microenvironment in which NP reside, can affect their
properties. Specifically, we demonstrated, in collaboration with the group headed by Dr. Minghetti at
ISS, that microglia is either beneficial or detrimental to neurogenesis depending on their activation
state (Cacci et al. 2008; Ajmone Cat et al., 2010), and that the anti-neurogenic effects of proinflammatory microglia are reverted by their treatment with long-chain polyunsatured fatty acid (LPUFA), a family of essential fatty acids obtained from oily fish, fish oils, and seafood (Ajmone-Cat et
al., 2011).
Microglia exert their modulatory function in part by releasing soluble factors. Among released
factors, matrix metalloproteinases (MMPs) are emerging as contributors to neurogenesis modulation.
We have now clarified several aspects of MMPs/TIMPs-regulated neurogenesis. In particular we
observed that the pharmacological inhibition of MMPs (obtained by using the inhibitor Marimastat)
profoundly affected NP differentiation fate. Inhibition of MMPs affected Notch signalling activation and
triggered NP differentiation, strongly promoting neuron differentiation. Our data exclude a major
involvement of the MMPs -2 and -9, while highlight the ability of TIMP-2 to act as a key effector of the
pro-neurogenic response to an inducing stimulus, such as Marimastat.
The effects of the wide spectrum of extracellular signals acting in the niche on the regulation of NP
properties are relayed and integrated within cells by cell-intrinsic regulators such as transcription
factors and micro RNAs. We focused our attention on the role of RE1-silencing transcription factor
(REST), a master regulator of neuronal genes, in regulating NPs properties. REST, is implicated in the
regulation of many genes although it is present at distinct sites in different cell types (Johnson et al.,
2008). Combining microarray and Chip sequencing analysis we identified several REST target genes,
including MMPs, and a member of the bone morphogenic protein (BMP) family.
53
In the last year of the project we obtained strong experimental evidences that BMPs, depending
on the experimental culture conditions, inhibit cell proliferation and induce NPs to either terminally
differentiate into astrocytes or to entry in a quiescent state. As expected, quiescent NPs re-enter cell
cycle and behave as multipontent, self-renewing cells after BMP withdrawal and exposure to fresh
medium containing growth factors. We have been using this in vitro model to study the molecular
mechanisms controlling NP quiescence, activation and differentiation.
In collaboration with Dr. Caffarelli group (CNR), we have been studying the expression profile of
miRNAs during NP differentiation into astrocytes. Among the most upregulated miRNAs, we focused
on miR-23 and miR-125, which regulate Msi1, an RNA binding protein critical for NP self-renewal. We
are currently investigating the effect of these two miRNA on NP proliferation and fate commitment.
Publications
Sinno M, Biagioni S, Ajmone-Cat MA, Pafumi I, Caramanica P, Medda V, Tonti G, Minghetti L,
Mannello F, Cacci E. The Matrix Metalloproteinase Inhibitor Marimastat Promotes Neural Progenitor
Cell Differentiation into Neurons by Gelatinase-Independent TIMP-2-Dependent Mechanisms. Stem
Cells Dev 2012 Epub. doi: 10.1089/scd.2012.0299.
Soldati C, Cacci E, Biagioni S, Carucci N, Lupo G, Perrone-Capano C, Saggio I, Augusti-Tocco G.
Restriction of neural precursor ability to respond to nurr1 by early regional specification. PLoS One
2012, 7:e51798. doi: 10.1371/journal.pone.0051798.
Research Group
Stefano Biagioni, professor;
Caramanica, PhD student.
54
Collaborations
Pasquale
Ferdinando Mannello, Gaetana A. Tonti,
Università Carlo Bo, Urbino; Luisa Minghetti,
Maria
Antonietta
Ajmone-Cat,
Istituto
Superiore di Sanità, Roma; Bukley Noel,
King’s College, London, UK.
Does PARylated PARP-1 introduce an epigenetic mark on
chromatin?
Paola Caiafa
℡: +39 06 49976530 - @: [email protected]
During the last decade, it has been shown that PARylation plays important roles in physiological
processes as DNA damage repair, protein localization and transcription. It has been recently
demonstrated the involvement of PARylated PARP-1 in the regulation of chromatin function by acting
on histones and DNA methylation (Krishnakumar and Kraus, 2010). Thus, PARylation can be now
considered an essential chromatin epigenetic mark. Our contribution to this research field has
demonstrated the importance of PARylated PARP-1 in the protection of methylation-free regions of
genome from the action of DNA methyltransferase activity (Caiafa et al., 2009). In fact, PARylated
PARP-1 localized on specific loci is able to inhibit Dnmt1 enzymatic activity and in absence of PARP
activity DNA undergoes hypermethylation as demonstrated for Dnmt1 (Zampieri et al., 2009) and
thromobomodulin promoters (Nocchi et al., 2011). This epigenetic role of PARP-1 can be played in
association with CTCF, a multifunctional factor involved in transcriptional regulation and insulation.
Moreover, CTCF interacts with PARP-1 and induces the allosteric activation of the enzyme. Thus, the
co-localization of both proteins on CTCF target sequences can allow the preservation of their
unmethylated state necessary for CTCF binding (Zampieri et al., 2012). This relevant interplay
between PARylation, CTCF and DNA methylation has been more deeply investigated analyzing the
effect of PAR depletion on CTCF sub-cellular localization and chromatin state. In fact, we and others
have recently demonstrated that in absence of PARylation CTCF loses its binding to specific target
sequences which can then become methylated (Zampieri et al., 2012; Witcher and Emerson, 2009).
We showed by confocal microscopy that after PAR depletion CTCF accumulated at the periphery of
the nucleus where it was linked with nuclear pore complex proteins remaining external to the
perinuclear Lamin B1 ring. The use of time-lapse videomicroscopy clarified that this perinuclear
localization of Ctcf was due to its blockage from entering the nucleus. Besides CTCF nuclear
delocalization, the outcome of PAR depletion led to changes in chromatin architecture.
Immunofluorescence analyses indicated DNA redistribution, a generalized hypermethylation and an
increase of inactive compared with active chromatin marks in PAR depleted cells. Notably, the same
effects were obtained after CTCF silencing. These results underline the importance of PARP-1 and
CTCF cross-talk in the maintenance of nuclear organization (Guastafierro et al., 2013).
Besides the role of PARylation in the maintaining of methylation-free regions on DNA, we also
demonstrated that hyperactivation of PARP-1 can induce genome-wide demethylation (Guastafierro et
al., 2008). Demethylation events occur in pathological and physiological conditions. As concerns
normal mammalian development, primordial germ cells (PGCs), the embryonic precursors of mature
germ cells, undergo a wave of DNA demethylation. This process is necessary for the generation of
totipotent gametes with proper sex-specific imprints and for the erasure of epimutations (De Felici,
2011). We analyzed the role of PARylation in this moment of germline specification suggesting that
PARP-1 participates in the erasure of PGC DNA methylation with several roles. In fact, PARP-1
activity and expression were noticeably high when the bulk of DNA demethylation occurs even before
55
the DNA damage response
associated with active DNA
demethylation. We showed
that PARP inhibition impairs
both genome-wide and locusspecific DNA demethylation in
PGCs. Despite its role as
sensor of DNA breaks, an
important function has been
suggested in the inhibition of
the activity of Dnmt1, which is
still
present
in
PGCs.
Moreover, PARylation has
been shown to coordinate the
transcription of the gene that
codifies for TET1 protein,
which is involved in the active
DNA demethylation process
(Ciccarone et al., 2012) (Fig.1).
Fig. 1 - Multiple involvement of PARylation during the epigenetic reprogramming of germline.
Publications
Ciccarone F, Klinger FG, Catizone A, Calabrese R, Zampieri M, Bacalini MG, De Felici M,Caiafa P.
Poly(ADP-ribosyl)ation acts in the DNA demethylation of mouse primordial germ cells also with DNA
damage-independent roles. PLoS One 2012, 7:e46927. doi: 10.1371/journal.pone.0046927.
Zampieri M, Guastafierro T, Calabrese R, Ciccarone F, Bacalini MG, Reale A, Perilli M, Passananti
C,Caiafa P. ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of
Ctcf target sites. Biochem J 2012, 441: 645-52. doi: 10.1042/BJ20111417.
Research Group
Anna Reale, professor, Michele Zampieri,
researcher, Maria Giulia Bacalini, Tiziana
Guastafierro, Fabio Ciccarone, post-doc
fellows, Roberta Calabrese, graduated student.
56
DNA recombination of repeated sequences and genome instability:
epigenetic implications
Giorgio Camilloni
℡: +39 06 49912808 - @: [email protected]
The purpose of our study, centered on Saccharomyces cerevisiae, is to investigate the basic
mechanisms of genome stability, to be translated to mammal model systems. The ribosomal DNA
(rDNA) locus is one of the few repeated regions in the genome of the yeast S. cerevisiae. Because of
its repetitive nature, rDNA may undergo unequal recombination among the units, producing
Extrachromosomal Ribosomal Circles (ERCS) or variation of the units copy number. These events are
normally suppressed to avoid genomic instability and aging. In addition, we focused our attention on
the ribosomal rDNA because of the high and peculiar concentration of basic DNA transactions such as
RNA transcription (by Pol I, II and III), DNA replication and DNA recombination. These processes
meet, at rDNA, both physically and functionally. Thus, it is conceivable that these activities must be
tightly regulated to avoid rDNA instability and common regulatory mechanisms should be active.
We explored the role of epigenetics in connecting recombination and transcription. This aspect is
still poorly investigated and its detailed characterization could reveal epigenetics as further control
element. To gain informations on these kind of regulations, it may be useful to manipulate similar
transactions in diseases raising from genome instability.
During this second year of activity we identified different processes affecting genome stability at
rDNA mainly conncted with the hyperacetylation of the H4K16 residue.
Unequal recombination among the rDNA units, due the repetitive nature of the locus, triggers the
production of ERCs species that we have measured in different mutants linked to epigenetic control of
this region. We considered Sir2p, Hst1-4p, Rpd3p (histone deacetylases) and Nhp6 A and B
(architectural chromatin proteins). We have demonstrated that the ERC production is controlled by
several chromatin regulators, especially Sir2p, Hst3 and 4 and Nhp6 A/B.
Genome instability at rDNA has been also connected with transcriptional silencing of ncRNAs by
RNA polymerase II. Our investigations demonstrated that an altered ERCs production corresponds to
changes in ncRNA transcription. Considering that histone deacetylases were implicated in silencing
and stability of the rDNA, we studied the acetylation level of the rDNA chromatin. By Chromatin
immunoprecipitation analyses we highlighted that histone H4 acetylation correlates with ERCs and
ncRNAs production and in addition, H4K16 acetylation overlaps with H4 global acetylation in
chromatin mutants. Thus, all these data, taken together, demonstrate that H4K16 acetylation controls
two basic DNA transactions such as recombination (ERCs accumulation) and RNA transcription
(ncRNAs silencing).
While our conclusions are coherent with the involvement of histone deacetylases in stability and
transcription of rDNA, the Nhp6A/B influence in altering histone acetylation is not easily justifiable. We
thus hypothesized that NHP6 may, by a direct or indirect mechanism, affect Sir2p presence at rDNA.
By studying SIR2 expression (RNA and protein) we concluded that the nhp6ab mutation affects SIR2
expression. In addition, we demonstrated that SIR2 overexpression restores most of the WT
phenotypes in the nhp6ab mutant.
57
Fig. - rDNA analysis by ChIP seq
confirms the regular ChIP data.
INPUTS profiles (green) are
identical between Wt and nhp6ab
mutant. When IP profiles are
considered (red), nhp6 shows
more acetylation, particularly at
the ARS region (arrow). Note the
different scale among the graphs:
nhp6 from 700 to 40.000; WT from
300 to 13.000.
Our study has provided the experimental evidences allowing us to propose a control model
concerning the Transcription Associated Recombination (TAR), based on epigenetic modification and
recruitment/exclusion of epigenetic modifiers. In addition, we have provided evidences that a single,
rapid, and reversible posttranslational modification, the acetylation of the H4K16 residue, is involved in
the coordination of transcription and recombination at rDNA.
Previously (Celona et al., 2011), we have demonstrated that in nhp6ab mutants nucleosomes are
genome wide reduced in the chromosomes. This reduction causes genome instability both in yeast
and mouse cells in HMGB1 mutants (homologue to Nhp6). ChIP-seq experiments on H4K16 are now
in progress (Figure), comparing WT and nhp6 cells, in order to further shed light on this important
epigenetic histone modification.
Publications
Cesarini E, D'Alfonso A, Camilloni G. H4K16 acetylation affects recombination and ncRNA
transcription at rDNA in Saccharomyces cerevisiae. Mol Biol Cell 2012, 23: 2770-81. doi:
10.1091/mbc.E12-02-0095.
Research Group
Collaborations
Anna D’Alfonso, Francesca Di Felice,
research fellows; Elisa Cesarini, post-doc
fellow,
Davide
Gaglio,
PhD
student;
Alessandro Angerilli, Diletta Durano, Andrea
Lukacs, students.
Marco Bianchi, Università Vita-Salute San
Raffaele, Milano; Bastia Deepak, Department
of Biochemistry & Molecular Biology; University
of Charleston, USA; Giulio Pavesi, Università
degli Studi di Milano.
58
Processing of the Neuroligins proteins and autism-related
mutations
Antonella De Jaco
℡: +39 06 49912310 - @: [email protected]
The autism spectrum disorders (ASDs), commonly called autism, are neurodevelopmental
conditions that may affect as much as 1% of the population. Despite an important role of hereditary
factors, the genetics of the ASDs is complex, making the identification of susceptibility genes
challenging. Candidate genes are found in the cell adhesion proteins localized at the synapse. In
support of this are the rare mutations identified, in autistic patients, in the genes encoding for synaptic
cell adhesion molecules such as Neuroligins, Neurexins and contactin-associated protein-like 2.
Neuroligins (NLGNs) are transmembrane cell adhesion proteins that play a key role in the
formation, maturation, activity-dependent validation, and maintenance of synapses. The NLGNs
consist of a small cytoplasmic region, involved in intracellular interactions, a single transmembrane
domain and an extracellular domain responsible for trans-synaptic interactions. The extracellular
domain presents a α/β-hydrolase fold that is homologous to acetylcholinesterase,
butyrylcholinesterase and to the carboxy-terminal region of thyroglobulin. The α/β-hydrolase fold
domain of the NLGNs recognizes and interacts specifically with presynaptic ligands of the NRXNs
family. The correct folding of this domain has been shown to be crucial for proper protein localization
and function for members of the cholinesterase-like family as demonstrated by mutations found
altering the folding of this domain that are associated with congenital disorders (figure1). Several
mutations associated with the ASDs have been shown to alter NLGNs folding and trafficking. R451C
in NLGN3 causes protein misfolding and results in compromised expression of the NLGN3 protein on
the cell surface. In order to study the processing of the NLGNs and the importance of conserved
residues for the folding of the extracellular α/β-hydrolase domain, we have studied mutations found
naturally in the α/β-hydrolase domain of thyroglobulin, where they alter protein secretion. Our results
show that homologous mutations transfered from thyroglobulin to the AChE and the NLGNs proteins
cause identical defects with respect to the folding of the common domain and result in blocking protein
transit along the secretory pathway. Our studies show that mutations at conserved residues may be
transferred amongst homologous proteins to produce common trafficking defects, despite very
different functions and cellular dispositions of the proteins (De Jaco et al., 2012).
Among the candidate genes, most frequently linked to autism is the contactin-associated proteinlike 2 (CNTNAP2) also called CASPR2, a neuronal cell adhesion molecule known to be necessary in
the clustering of the Kv1 potassium channels at juxtaparanodes. In particular, the CASPR2-D1129H
mutation was found in monozygotic twins, both affected with autism. We have studied processing of
the wild type and D1129H mutant proteins and shown that the mutation is causing retention of the
protein in the endoplasmic reticulum (ER) indicating a folding impairment of CASPR2. Our results
show that CASPR2-D1129H, but not wild type, is preferentially degraded by the proteosome and its
accumulation in the ER causes a significant activation of ATF6, indicating that overexpression of the
mutant protein promotes activation of a protective cellular response possibly through binding with BiP,
Calnexin and ERp57 (Falivelli et al., 2012).
59
Overall our results outline the
importance of studying the processing of
synaptic cell adhesion proteins in relation
to ASDs and suggest that gene
alterations leading to protein misfolding
and subsequent ER retention, could
activate an ER stress response.
Fig. 1 - Structures of neuroligin, acetylcholinesterase, butyrylcholinesterase and the
α/β-hydrolase domain of thyroglobulin.
Mutations affecting folding of the α/βhydrolase domain in the different proteins are
associated to diseases.
Publications
De Jaco A, Dubi N, Camp S, Taylor P. Congenital hypothyroidism mutations affect common folding
and trafficking in the α/β-hydrolase fold proteins. FEBS J 2012, 279: 4293-305. doi:
10.1111/febs.12019.
Falivelli G, De Jaco A, Favaloro FL, Kim H, Wilson J, Dubi N, Ellisman MH, Abrahams BS, Taylor P,
Comoletti D. Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and
ATF6 activation. Hum Mol Genet 2012, 21: 4761-73. doi: 10.1093/hmg/dds320.
Research Group
Collaborations
Federica De Angelis, Lisa Ulbrich, post-doc
fellows ; Flores Lietta Favaloro, PhD student.
Davide Comoletti, Palmer Taylor, , University
of California, San Diego, USA; Michael Lin,
Stanford University, San Francisco, USA,
Stefan Marciniak, University of Cambridge,
UK.
60
Functional analysis of CG40218, a Drosophila melanogaster gene
encoding a BCNT-like protein required for chromosome
organization
Patrizio Dimitri
℡: +39 06 49917948 - @: [email protected]
Production of antibodies against YETI (GC40218) protein
Polyclonal antibodies directed against a specific peptide of the YETI protein were raised in rabbit
by Genescript. Total protein extracts from Drosophila melanogaster larvae were tested with anti-YETI
to assess its specificity. A band of the expected MW (32kDa) was detected in protein extracts of
LP1/SM1,Cy heterozyogous larvae and of LP1/LP1; UAS-Yeti-M17/tub-gal4 larvae that express the
wild-type YETI protein, while it is absent in LP1/LP1, LP1/Df mutants that not express YETI.
Immunostaining of salivary gland polytene chromosomes with anti-YETI failed to reveal a significant
staining.
Transgenic lines expressing YETI-tagged fusion protein
In the first year we shown that the YETI-GFP fusion protein is present in all nuclei and is recruited
at hundreds of sites along the chromosome arms. Now, we have found that YETI-GFP expression,
under the tub-GAL4 driver, produces a clear rescue of polytene chromosome morphology of LP1/LP1
mutant larvae, albeit such rescue does not ameliorate the lethal phenotype of LP1 null allele. Thus, in
absence of endogenous YETI wild-type protein, the YETI-GFP fusion protein maintains its binding
specificity to chromatin. We also prepared and tested several transgenic lines carrying YETI tagged
with 3xFLAG. The immunostaining confirms the results obtained with YETI-GFP in that the YETI
3xFLAG protein binds to polytene chromosome. We also tested deletions of the Yeti coding region
(DYETI) tagged with 3xFLAG. Preliminary observations indicate that a chromatin-binding domain of
YETI is located in the C-terminal portion of the protein (last 90 amino acid residues).
Identification of YETI interactors
The results of our immunofluorescence experiments carried out with anti-H2Av on polytene
chromosomes suggested that YETI is responsible for the proper deposition of H2Av into chromatin.
These results lead us to investigate whether YETI is able to interact with H2Av in vivo. Using the GFPTRAP sistem, H2Av-GFP fusion protein was immunoprecipitated from protein extracts of H2Av-GFP
larval brains and salivary glands. Immunoprecipitates were analyzed by western blotting using
antibodies against YETI, GFP or Actin. As negative control we used the Oregor-R strain. The results
show that YETI is found only in the immunoprecipitate from H2Av-GFP estracts, while it was absent in
the immunoprecipitates from the Oregor-R control.
CFDP1 in HeLa cells
In order to study the localization of CFDP1, the human ortholog of YETI, we carried out
immunostaining experiments in HeLa cells using polyclonal antibodies to CFDP1. All nuclei of Hela
cells were stained by the anti-CFDP1, while no significant signals were observed on metaphase
61
chromosomes (Fig. 1A). After RNAi mediated inactivation of CFDP1 in HeLa cells ((Fig. 1B, C),
metaphase chromosomes with aberrant morphology and dramatic condensation defects were seen.
Fig. 1 - CFDP1 in HeLa cells. A) DAPI
staining of nuclei is shown in blue, antiCFDP1 immunostaining is shown in red (left
and middle panels); DAPI staining of Hela
metaphase chromosomes is shown blue, antiCFDP1 immunostaining is shown in red (right
panels); B) Western blotting with anti-CFDP1
of protein estracts from HeLa cells, after RNAi
mediated inactivation. The intensity of the of
the two CFDP1 bands (42kDa and 35kDa) in
RNAi treated cells (2) is about 40% compared
to non RNAi control cells (1); C) Cytological
analysis of RNAi treated HeLA cells reveals
the presence of cearly aberrant chromosome
methaphases that fail to undergo proper
condensation (middle and right panels)
compared to the controls (left panel).
In a total of 115 metaphases scored, we found 45% of affected figures (Fig. 1C), while only 3% of
methaphases with an abnormally condensed phenotype were seen in the controls. Notably, the
nuclear staining obtained with anti-CFDP1 disappears in RNAi treated HeLa cells. We also
investigated whether CFDP1 interacts in vivo with members of the human SRCAP remodeling
complex, that catalyzes ATP-dependent exchange of histone H2A/H2B dimers for H2A.Z/H2B dimers.
HeLA cells cells were transfected with V5-CFDP1 and Myc-Arp6 expression vectors and the lysate
was immunoprecipitated (IP) with V5 antibodies. Immunoprecipitates were analyzed by western
blotting using antibodies against Myc. Myc-Arp6 was found only in the immunoprecipitate from V5CFDP1 lisate, while it was absent in the immunoprecipitates from Ms IgG negative control.
Together, these results provide evidence in favour of a conserved epigenetic role of YETI and
CFDP1 proteins in chromatin remodeling and open the possibility for a role of these proteins in gene
expression and development.
Publications
Yeh SD, Do T, Chan C, Cordova A, Carranza F, Yamamoto EA, Abbassi M, Gandasetiawan KA,
Librado P, Damia E, Dimitri P, Rozas J, Hartl DL, Roote J, Ranz JM. Functional evidence that a
newly evolved Drosophila sperm-specific gene boost sperm competition. Proc Natl Acad Sci USA
2012, 109: 2043-8. doi/10.1073/pnas.1121327109 IF 9.771
Research Group
Collaborations
Francesca Romana Mariotti, post-doc fellow,
Elisabetta Damia, Giovanni Messina, PhD
students, Emanuele Celauro, student.
Ruggiero Caizzi, Università di Bari; Ennio
Giordano, Università di Napoli “Federico II”.
62
Genetic and molecular analysis of the mechanisms of Drosophila
telomere protection
Maurizio Gatti
℡: +39 06 49912842 - @: [email protected]
Drosophila telomeres are epigenetically determined, sequence-independent structures that are not
maintained by telomerase, but by transposition to chromosome ends of specialized retroelements.
Genetic and biochemical analyses have recently shown that fly telomeres are capped by terminin, a
complex that includes at least four proteins: HOAP, HipHop, Modigliani (Moi) and Verrocchio (Ver).
With the exception of Ver, which exhibits a structural homology with Stn1, the terminin proteins are not
conserved outside the Drosophilidae and are all encoded by fast-evolving genes. Terminin localizes
and appears to function only at telomeres just like shelterin, the complex that protects human
telomeres. Mutants in genes encoding the terminin components cause high frequencies of telomeric
fusions (TFs) in larval brain cells. Collectively our data strongly suggest that terminin is a functional
analogue of shelterin. We proposed that after telomerase loss Drosophila evolved terminin to bind
telomeres in a sequence-independent manner.
We have now analyzed the structure of terminin using suitable protein truncations and DNA
binding assays. HOAP binds double stranded (ds) and Ver single stranded (ss) DNA; Moi does not
bind DNA but interacts directly with HOAP and Ver forming a bridge between the two proteins. Thus,
the architecture of terminin is similar to that found in other telomere capping complexes including
shelterin, where the ss DNA-binding protein Pot1 is connected to the TRF1/TRF2 ds DNA-associated
proteins by the non-DNA-binding factor TPP1. Our data further suggest that Ver and Moi mask ss
DNA at Drosophila telomeres, just like TPP1-Pot1 at human telomeres. When chromosome ends lack
either Moi or Ver, telomeres form DNA repair foci that contain the phosphorylated form of the H2Av
histone. Collectively, these results reinforce the idea that the basic mechanisms of telomere capping
are conserved from yeast to flies and humans, and support a unifying model for telomere protection.
We have also continued our genetic and molecular analyses of pendolino (peo). Null mutations in
peo cause a very high frequency of TFs (~ 6 TFs per cell). We have recently isolated a hypomorphic
peo mutant that exhibits a low frequency (0.5/cell) of TFs, most of which involve the telomeres of
heterochromatic chromosome arms (the Y, XR and 4L telomeres). This finding suggests that in cells
with slightly reduced peo activity heterochromatic telomeres have a higher fusion capacity than
euchromatic telomeres; this capacity is masked in cells with multiple TFs caused by null peo
mutations. The Peo protein shares homology with the E2 variant ubiquitin-conjugating enzymes,
which lack the catalytic cysteine residue in the Ub-binding domain. GST pulldown experiments showed
that Peo directly interacts with HOAP, Moi and Ver but not with HP1. However, Peo is not a terminin
component, as it is associated with numerous polytene chromosome bands. In addition, Peo has a
conserved human homologue (AKTIP) and is not encoded by a fast-evolving gene. Finally, we have
shown that the wild type function of peo is not required for HOAP, Moi or Ver localization at telomeres.
peo is instead required for replication of polytene chromosomes; in peo mutants, polytene
63
chromosomes fail to bind the DNA replication
factor PCNA. Collectively, our results strongly
suggest that peo plays an essential role in
telomere replication, and that late replicating
heterochromatic telomeres have a higher
requirement of peo function compared to
euchromatic telomeres. This interpretation implies
that defects in Drosophila telomere replication lead
to telomere fusion.
Fig. - Examples of telomeric fusions (TFs) observed in
Drosophila neuroblasts. (a) Wild type cell with 4
metacentric and 2 dot autosomes, and 2 sex
chromosomes. (b, c and d) Mutant cells showing
telomeric fusions.
Publications
Lattao R, Bonaccorsi S, Gatti M (2012). Giant meiotic spindles in males from Drosophila species with
giant sperm tails. J Cell Sci 2012, 125: 584-88. doi: 10.1242/jcs.101469.
Gatti M, Bucciarelli E, Lattao R, Pellacani C, Mottier-Pavie V, Giansanti MG, Somma MP, Bonaccorsi
S. The relative roles of centrosomal and kinetochore-driven microtubules in Drosophila spindle
formation. Exp Cell Res 2012, 318: 1375-80; doi: 10.1016/j.yexcr.2012.05.001.
Wainman A, Giansanti MG, Goldberg ML, Gatti M. The Drosophila RZZ complex: roles in membrane
traffic and cytokinesis. J Cell Sci 2012, 125: 4014-25; doi: 10.1242/jcs.099820.
Research Group
Collaborations
Laura Ciapponi; Giovanni Cenci, professors;
Grazia Daniela Raffa, Fiammetta Vernì,
researchers; Maria Patrizia Somma, CNR
researcher.
Stefano Cacchione, Dipartimento di Biologia e
Biotecnologie “Charles Darwin”. Sapienza
Università di Roma; Domenico Raimondo,
Dipartimento di Scienze Biochimiche “A. Rossi
Fanelli”, Sapienza Università di Roma.
64
Role of epicardium and EMT/MET processes in cardiac progenitor
cells generation and differentiation
Alessandro Giacomello
℡: +39 06 4461481 - @: [email protected]
Aim of the study. Cardiac progenitor cells (CPCs) isolated as Cardiospheres (CSps), represent a
promising candidate for autologous cardiac cell therapy. CSps can be easily obtained from cells
spontaneously migrating out of primary cardiac explants (explant-derived cells, EDCs) and re-create in
vitro a niche-like microtissue, which seems to favour the maintenance of a “stemness” status and
confers resistance to oxidative stress, thus enhancing in vivo engraftment (Messina et al., 2004, Li et
al., 2011). CSp-derived cells (CDCs) can be expanded in monolayers and retain the ability to form
secondary cardiospheres (IICSps) when plated back in the appropriate conditions. Thus, this method
yields a significant number of adult autologous CPCs suitable for clinical application, which can
contribute in vivo to all the three main cell lineages of the heart. A better understanding of the
mechanism underlying CSps formation and differentiative potential is required to control their fate in
vivo and potentiate a cardiogenic outcome rather than a fibrogenic one, even in the hostile ischemic
environment of an infarcted heart. Epithelial-to-mesenchymal transitions (EMT) play a central role in
embryogenesis. Four distinct waves of EMT occur at different stages of heart morphogenesis. EMT
has also been associated with the acquisition of stem cells properties both in adult tissues and cancer
and with cardiac post-ischemic remodelling. The aim of this study was to analyze the possible role of
EMT in CSp generation and development, by means of gene expression analysis at different culture
stages and in vitro treatments with TGFβ, which is a key EMT inducer, and its antagonist SB431452.
Results. Gene expression profiling and modulation of TGFβ signaling in vitro showed that EMT
has a crucial role in CSps formation (Forte et al. 2012), and may be involved in the acquisition of stemcell like properties by cardiac-explant derived cells as demonstrated in other adult tissues (Mani et al.,
2008; Caja et al., 2011). Furthermore, the significant upregulation of WT1 upon CSp formation
suggests that developmental epicardial genes are reactivated, possibly as part of the EMT process.
This might resemble the response of epicardial cells to injury, which stimulates them to undergo EMT
and generate multipotent mesenchymal progenitors (Bax et al 2011), even in adult hearts (Di Meglio et
al 2010).
Our studies implicate TGFβ-dependent EMT as an essential feature of CSp formation, and define
a molecular signature for CSps and subsequent in vitro culture stages. In particular we have observed
that CSps express higher levels of important cardiac progenitor transcription factors such as MEF2c,
TBX5, GATA4 (which are per se sufficient to induce transdifferentiation of fibroblasts in induced
cardiomyocytes), compared to the EDCs from which they derived and to CDCs expanded in
monolayer; while they express significantly lower levels of Baf60c, Mesp1 early markers of pre-cardiac
mesoderm, and of c-myc (reprogramming factor, TGFβ1 and TGFβ1R inhibitor), compared to EDCs
and CDCs. On the basis of developmental studies, these results suggest the presence of an atypical,
incomplete form of cardiac mesoderm intermediate in the CSps, resembling that which is transiently
65
expressed in embryos and embryonic stem cell (ESC)-derived embroyid bodies (EBs) (Willems E,
2011).
Future perspectives. Recent studies have shown that is possible to obtain fully differentiated
cardiomyocytes from activated epicardial-derived cells in vivo (Qi et al. 2012), by introducing specific
cardiac transcription factors or miRNA. The molecular analysis confirms that CSps are prone to
cardiac differentiation. Our aim is to overcome the apparent hurdle which prevents the obtainment of
fully differentiated cardiomyocytes. In particular small molecules and miRNA, previously screened on
embryonic stem cells, are going to be tested, in vitro and in vivo, for their cardiogenic potential on
CSps, labelled with the appropriate lentiviruses for lineage tracing.
Publications
Chimenti I, Forte E, Angelini F, Giacomello A, Messina E. From ontogenesis to regeneration: learning
how to instruct adult cardiac progenitor cells. Prog Mol Biol Transl Sci 2012, 111: 109-37. doi:
10.1016/B978-0-12-398459-3.00005-8.
Forte E, Miraldi F, Chimenti I, Angelini F, Zeuner A, Giacomello A, Mercola M, Messina E. TGFβdependent epithelial-to-mesenchymal transition is required to generate cardiospheres from human
adult heart biopsies. Stem Cells Dev 2012, 21: 3081-90. doi: 10.1089/scd.2012.0277.
Gaetani R, Doevendans PA, Metz CH, Alblas J, Messina E, Giacomello A, Sluijter JP. Cardiac tissue
engineering using tissue printing technology and human cardiac progenitor cells. Biomaterials 2012,
33: 1782-90. doi: 10.1016/j.biomaterials.2011.11.003.
Research Group
Collaborations
Elisa Messina, medical manager; Francesco
Angelini, research fellow; Isotta Chimenti,
Elvira Forte, Roberto Gaetani, post-doc
fellows; Vittoria Ionta, PhD student.
Luigi Aurisicchio, Takis Srl, Biogem, Rome;
Mark Mercola, Sanford-Burnham Medical
Research Institute, San Diego, USA; Pilar
Ruiz-Lozano, Stanford School of medicine,
Stanford, CA, USA.
66
Hedgehog signaling regulatory networks in brain cancer stem cells
Alberto Gulino
℡: +39 06 4464021 - @: [email protected]
Hedgehog pathway plays a pivotal role in development and tumorigenesis (including
medulloblastoma), processes sustained by stem cells.
We have observed that HDAC1 and HDAC2 cause enhanced Gli1 transcriptional activity, whereas
ectopic expression of HAT/p300 has an opposite inhibitory effect. Hedgehog leads to Gli1
deacetylation and Hedgehog-target gene activation through HDAC upregulation. Acetylation of Gli1
and Gli2 proteins (human Gli1 K518 and Gli2 K757) is a transcriptional checkpoint of Hedgehog
signaling, having acetylated Gli reduced transcriptional function (Canettieri et al., 2010). HDAC1
degradation is in turn controlled by the tumor suppressor RENKCTD11, a putative tumor suppressor that
we have isolated and that is monoallelically deleted and silenced by hypermethylation in
medulloblastoma (Mancarelli et al., 2010). REN is a member of a novel family of Cul3 adaptors that
includes two additional members (termed KCASH2 and KCASH3 - KCTD Cullin3 Adaptors and
Suppressors of Hedgehog), that form homo- and heteromers. These proteins forms a complex with
Cul3 E3 ligase, via their BTB domain (Correale et al., 2011), bind and degrade HDAC1 thus inhibiting
Gli function. (Canettieri et al., 2010; De Smaele et al., 2011).
Our findings suggest that HDAC inhibitors may be a promising and novel therapeutic strategy for
medulloblastoma.
We have also observed that Hedgehog signaling may be silenced in medulloblastoma cells by E3
ubiquitin ligase Itch-dependent Gli1 degradation (Di Marcotullio et al., Nature Cell Biol 2006). The
developmental protein Numb enhances the activity of Itch against Gli1, through activation of the
catalytic activity of Itch, by releasing it from an inhibitory intramolecular interaction between its
homologous to E6-AP (HECT) C-terminus and WW domains. As a consequence, Gli1 is ubiquitinated
and degraded via a novel Itch-dependent degron composed of a combination of two PPXYs and a
phospho-serine/proline motifs. Loss of this process determines enhanced Gli1-dependent
medulloblastoma growth, migration and invasion abilities, as well as in vitro transforming activity,
providing a novel rationale for therapeutic targeting of Hedgehog-dependent tumors.
Research Group
Gianluca Canettieri, Enrico De Smaele, Lucia
Di Marcotullio, Elisabetta Ferretti, professors;
Paola Infante, Evelina Miele, Marta Moretti,
Agnese Po, researchers.
67
The interplay between epigenetics, cell cycle and homologous
recombination in gene therapy by Small Fragment Homologous
Replacement (SFHR)
Marco Lucarelli
℡: +39 06 4451784 - @: [email protected]
This project is aimed to the clarification of molecular mechanisms underlying cell invasion by
exogenous DNA and to the optimization of an in situ gene targeting approach called Small Fragment
Homologous Replacement (SFHR). The therapeutic use of this approach is currently limited by an
inadequate comprehension of its molecular mechanisms. This project focuses on the functional
interconnection between SFHR, DNA methylation and chromatin structure, as well as mechanisms of
DNA damage repair and cell cycle control.
The experimental system mainly used during the 2012 year of project is made up of mouse
embryonic fibroblast (MEF) stably integrating a nonsense mutated enhanced green fluorescent protein
(mEGFP). A small DNA fragment (SDF) is used as a corrector to restore the wild-type EGFP function.
The correction efficiency is evaluated at DNA and RNA levels, as well as by FACS analysis. The best
correction efficiencies we have obtained were of 0.5% when cells were synchronized in G2/M cell
cycle phase and of 2.5% with an additional simultaneous inhibition of DNA methylation and poly-(ADPribose) polymerase. This highlighted synergistic effects of several different pathways.
During the last year of project, the study of the expression of 84 genes involved in the response to
DNA damage was completed and that of the expression of 84 genes of cell cycle regulation was
performed. This was achieved by using quantitative real time expression arrays after treatment of
mEGFP MEF with low and high dosage of SDF. Although the expression modulation of both DNA
repair and cell cycle genes appeared to be influenced from the nucleofection protocol also in the
absence of SDF (mainly up to 8 h experimental time), several SDF-specific, dose-dependent and timeregulated inductions were evidenced. With some differences in temporal patterns, for most DNA repair
and cell cycle genes, an early (8 h) and intermediate (24 h) phase of upregulation followed by a
progressive late (72 h) dowregulation (to control level or lower) were evidenced. For example, after
nucleofection with high dosage of SDF, at 8 h the 69.0 % of DNA repair genes and the 83.3 % of cell
cycle genes appeared upregulated in respect to untransfected control; proportion that at 24 h became,
respectively, 83.3 % and 61.9 %. On the contrary to current opinions, the DNA repair modulated genes
belong to different DNA repair pathways, not only to homologous recombination (the main mechanism
supposed to be involved in SFHR). Also cell cycle genes resulted related to different phases and
functional roles, evidencing a wide impact of SFHR on the cell cycle regulation. Their overall early
upregulation, probably pointed to a coordinated induction of cell cycle arrest and DNA repair, may
constitute the molecular basis for the correction, whereas their late downregulation may be
responsible for the reduced correction efficiency.
To select the best SFHR-modulated and specific gene targets, a combination of statistical (mainly
a significance of Student’s t test after Bonferroni’s correction, p<0.0006) and biological criteria (mainly
a dose-dependent modulation and at least a 3 fold change in respect to control) were used. According
to these criteria, 5 genes more specific of the DNA repair pathway (Trex1, Neil2, Parp3, Pms2,
Rad51l3), 7 genes more specific of the cell cycle pathway (Slfn1, Cdkn1a, Prm1, Mdm2, Gpr132,
69
Ddit3, Chek1) and 6 genes directly involved in both pathways (Sesn2, Rad9, Ppm1d, Atm, Brca1,
Brca2) were selected. Interestingly, in addition to their role in DNA repair and cell cycle regulation,
possibly favorable for SFHR, Trex1 and Rad9 also have a 5’->3’ exonuclease activity on double strand
DNA. This raised the working hypothesis that this activity may degrade the SDF with consequent
unfavorable effects on SFHR efficiency.
Cell cycle, DNA methylation and DNA repair pathways appeared to be interconnected to mediate
the cellular response to the invasion by exogenous DNA and its genomic integration. Their interplay
and timing appear to influence the correction efficiency of SFHR. Several specific molecular effectors
were selected. Their manipulation may increase the gene repair efficiency to allow a practical SFHR
application in ex vivo therapeutic protocols.
Publications
Luchetti A, Filareto A, Sanchez M, Ferraguti G, Lucarelli M, Novelli G, Sangiuolo F, Malgieri A. Small
Fragment Homologous Replacement: evaluation of factors influencing modification efficiency in an
eukaryotic assay system. PLOS One 2012, 7:e30851. doi: 10.1371/journal.pone.0030851.
Research Group
Collaborations
Roberto Strom, professor; Fabrizio Ceci,
Giampiero Ferraguti, researchers; Silvia
Pierandrei, PhD student; Sabina Maria Bruno,
graduated student.
Paola Borgiani, Annalisa Botta, Andrea
Luchetti, Arianna Malgieri, Giuseppe Novelli,
Federica Sangiuolo, Dip. di Biopatologia e
Diagnostica per Immagini, Università di Roma
Tor Vergata.
70
Interplay between myogenic factors and cell cycle control:
regulation and role of the cdk inhibitor p57kip2
Rossella Maione
℡: +39 06 4457737 - @: [email protected]
The interdependence of cell proliferation and differentiation has been extensively investigated in
the skeletal muscle system, in which the whole differentiation program can be initiated by the MyoD
family of muscle-specific transcription factors, even when ectopically expressed in non muscle cell
types. MyoD directs a complex program of gene expression through the cooperation with other
transcription factors and chromatin remodeling enzymes. In addition to regulate muscle-specific
genes, MyoD is also involved in coupling the onset of differentiation with cell cycle withdrawal. The
regulated expression of cdk inhibitors (CKIs) plays a fundamental role in muscle differentiation.
The function and the regulation of the CKI p57kip2 (p57) are quite peculiar. p57 is the only CKI
which deficiency causes severe developmental defects both in humans and mice. Moreover,
compared to other CKIs, p57 shows a more restricted tissue and cell-type distribution. Finally, p57
belongs to a network of imprinted genes involved in growth regulation and is subject to a complex
epigenetic control. Despite the importance of this CKI, the specific functions of p57 and the molecular
mechanisms regulating its expression during differentiation processes are still poorly understood.
Our research project has been focused on two related topics. One concerns the specific roles that
p57 plays in the establishment and in the maintenance of terminal differentiation in muscle cells. The
other one concerns the transcriptional mechanisms controlling p57 expression during myogenesis.
During the last year we were primarily concerned with the second subject.
As reported in a recent publication (Busanello et al., 2012), we have identified an unexpected
mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We found that
the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting
control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We
also found that differentiation-dependent regulation of p57kip2, while involving a region implicated in the
imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings
highlighted a novel mechanism, involving the modification of higher order chromatin structures, by
which MyoD regulates gene expression. Our results also suggested that chromatin folding mediated
by KvDMR1 could account for the highly restricted expression of p57kip2 during development and,
possibly, for its aberrant silencing in some pathologies.
In a more recent manuscript, that we are going to submit for publication, we have addressed the
molecular mechanisms involved in the formation, maintenance and disruption of the chromatin loop
associated with p57 silencing in muscle cells (C. Battistelli, A. Busanello and R. Maione, 2013.
Functional interaction between MyoD and CTCF in the regulation of long-range chromatin interactions
during muscle differentiation). In this work we reported that the CCCTC-binding factor (CTCF), a
structural organizer of chromatin, is located at the base of the higher-order chromatin structure that
places together the two regulatory elements. Moreover, using RNA interference, we demonstrated that
CTCF is required both for the maintenance of the chromatin loop and for the repression of p57
transcription. Finally, we showed that MyoD physically interacts with CTCF within KvDMR1, interfering
71
with its ability to maintain chromatin looping. These findings not only reveal a critical role of CTCFmediated looping in p57 silencing, but also suggest that MyoD can regulate chromatin looping at
CTCF binding sites, thus providing new insights into the epigenetics of muscle differentiation.
Publications
Busanello A, Battistelli C, Carbone M, Mostocotto C, Maione R. MyoD regulates p57kip2 expression
by interacting with a distant cis-element and modifying a higher order chromatin structure. Nucleic
Acids Res 2012, 40: 8266-75. doi: 10.1093/nar/gks619.
Research Group
Collaborations
Anna Busanello, Rosaria Carbone, post-doc
fellows; Cecilia Battistelli, Agnese Ciotti,
Cassandra Mostocotto, PhD students.
Maurizia Caruso, Istituto di Neurobiologia e
Medicina Molecolare, CNR, Roma.
72
Handling and toxicity of mutant serpins underlying human disease
María Elena Miranda Banos
℡: +39 06 49912310 - @: [email protected]
The serpinopathies are human pathologies caused by mutations that promote polymerisation and
intracellular deposition of proteins of the serpin (serin protease inhibitors) superfamily, leading to cell
toxicity and death. The dementia FENIB is caused by polymerisation of the neuronal serpin
neuroserpin (NS), while polymerisation of alpha 1 antitrypsin within hepatocytes leads to liver disease
and emphysema associated to alpha 1 antitrypsin deficiency. Research on the molecular bases of
polymer accumulation and toxicity using in vitro model systems is important for understanding these
conditions and for developing therapeutic interventions.
Our aim is to understand how NS polymers accumulate within the endoplasmic reticulum (ER) of
neurons and what is the mechanism of their cellular toxicity. More specifically, we focus on two
aspects:
- The role of N-linked glycosylation on the ER handling of NS.
- Understanding the cell toxicity of NS polymers, using a new neuronal model of the dementia FENIB.
Human NS has three consensus sites for N-linked glycosylation, at N157, N321 and N401. We
have shown that: i) the N401 site is not used in wild type NS, while it is partially glycosylated on
Gly392Glu NS, a mutant variant that causes severe dementia; ii) removing the N321 site enhances NS
polymerisation, demonstrating that glycosylation is important for the structural stability of this protein;
and iii) removing the N401 glycosylation site in Gly392Glu
NS leads to increased accumulation of the mutant protein
within the cells, probably by interfering with its
degradation by the proteasome. We have confirmed all
our results by western blot and by sandwich ELISA,
analysing both the cell lysates and culture medium
supernatants of COS-7 cells transiently transfected with
plasmids expressing the different glycosylation variants,
created during the first year of the grant by site-directed
mutagenesis. We will now use pulse-chase analysis to
investigate the intracellular processing of our
glycosylation mutants in more detail. In collaboration with
Prof. Lomas and Dr. Marciniak, in the UK, we will also
look into levels of ER stress associated to the expression
of each glycosylation mutant, in order to understand the
relationship between glycosylation, ER retention and
polymerisation of NS mutants that cause dementia.
73
In order to study the cell toxicity of NS polymers, we have created a new cell model of FENIB. In
collaboration with Dr. Giuseppe Lupo and Dr. Emanuele Cacci, we have generated stably transfected
neural progenitor cell lines from mouse brain, expressing human wild type, Gly392Glu and delta
(control truncated version) NS. We have measured the expression levels of each NS variant, at the
mRNA level by RT-PCR and at the protein level by SDS-PAGE and western blot and by ELISA. Using
non-denaturing PAGE and western blot, we have shown that Gly392Glu NS forms polymers, which
are mostly retained within the ER, while the wild type NS is normally secreted as a monomeric protein
into the culture medium. These results have also been confirmed by ELISA, using different monoclonal
antibodies to quantify total NS or polymerised NS only. We are currently performing
immunocytochemistry to study the intracellular distribution of retained NS. This new model system will
now be used in long-term cultures to assess the toxicity of intracellular polymers of Gly392Glu NS, by
looking at activation of NFkB, ER stress and apoptosis.
Publications
Fra AM, Gooptu B, Ferrarotti I, Miranda E, Scabini R, Ronzoni R, Benini F, Corda L, Medicina D,
Luisetti M, Schiaffonati L (2012). Three new alpha1-antitrypsin deficiency variants help to define a
C-terminal region regulating conformational change and polymerization. Plos One 2012, 7:e38405.
doi: 10.1371/journal.pone.0038405.
Schmid S, Koepke J, Dresel M, Hattesohl A, Frenzel E, Perez J, Lomas DA, Miranda E, Greulich T,
Noeske S, Wencker M, Teschler H, Vogelmeier C, Janciauskiene S, Koczulla A. The effects of
weekly augmentation therapy in patients with PiZZ α1-antitrypsin deficiency. Int J Chron Obstruct
Pulmon Dis 2012, 7: 687-96. doi: 10.2147/COPD.S34560.
Research Group
Collaborations
Claudia Moriconi, post-doc fellow; Noemi
Guadagno, PhD student; Francesca Cosmi,
student.
Emanuele
Cacci,
Nicoletta
Carucci,
Antonella De Jaco, Giuseppe Lupo, Sapienza
Università di Roma; David A. Lomas, Stefan
Marciniak, University of Cambridge, UK; Juan
Pérez, University of Málaga, Spain; Nigel
Birch, University of Auckland, New Zealand;
Martino
Bolognesi,
Stefano
Ricagno,
Università di Milano, Mauro Manno, Vincenzo
Martorana, Rosetta Noto, CNR, Istituto di
Biofisica, Palermo; Luisa Schiaffonatti, Anna
Fra, Università di Brescia.
74
Characterization of the muscle-nerve crosstalk in a neuromuscular
disease
Antonio Musarò
Department of Anatomy, Histology, Forensic Medicine and Orthopaedics
℡: +39 06 49766956 - @: [email protected]
The goals of the project are to define the molecular signature of the retrograde muscle-nerve
interaction, using appropriate experimental models in which either muscle or nerve is severely
compromised. The functional interplay between muscle and nerve is crucial for both partners to
survive and function adeguately through the life. In several pathologic conditions, including ALS, SMA,
aging etc this interaction is severely compromised. Thus, the characterization of molecular
mechansims of the physio-pathologic interaction between muscle and nerve will help to design more
appropriate strategy to treat neuromuscular diseases.
In a previous work we demonstrated that muscle-restricted expression of IGF-1 supports muscle
integrity, stabilizes neuromuscular junctions and enhances motor neuron survival in SOD1G93A mouse
model of ALS, and improves muscle mass and extends the survival of severe SMA mice. At first, we
extended these previous works demonstrating that perinatal administration of IPLEX (rhIGF-1
complexed with rhIGFBP-3), even if does not influence survival and body weight of mice, results in
reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in
SMA mice.
During the second year of activity, we specifically studied how oxidative stress, accumulated in
MLC/SOD1G93A muscle, triggers pathological alterations in skeletal muscle.
Of note, increased oxidative stress was associated with pathologic alteration in mitochondria
distribution and morphology. To verify whether altered morphology of mitochondria was also
associated with altered activity, we evaluated the capability of both wild type and MLC/SOD1G93A
muscle mitochondria to maintain a membrane potential. We observed a decay in the ability of
mitochondria of MLC/SOD1G93A muscle to maintain the difference in potential across their inner
membrane. It has been reported that skeletal muscle fibers of the classical animal model of ALS,
namely the SOD1G93A mice, display localized loss of mitochondrial inner membrane potential in fiber
segments near the neuromuscular junction (NMJ). Interestingly, these defects occur in young G93A
mice prior to disease onset.
To demonstrate whether selective accumulation of SOD1G93A in skeletal muscle is sufficient to
trigger NMJ alteration, in absence of motor neuron degeneration, and to determine whether the
observed mitochondrial defect in MLC/SOD1G93A fibers are associated with NMJ, we merged
mitochondrial lesion and the NMJ by staining live MLC/SOD1G93A muscle fibers with TMRE and abungarotoxin (BTX), which stains the subunit of acetylcholine receptors (AChRs).
Immunofluorescence analysis revealed that the fiber segments with depolarized mitochondria included
the muscle side of the NMJ, suggesting that oxidative stress and mitochondrial dysfunction induce
alteration in NMJ.
To substantiate this hypothesis, we analysed the morphological complexity of NMJ in both wild
type and MLC/SOD1G93A mice. Histological examination of NMJs in MLC/SOD1G93A muscle
demonstrates marked alterations in NMJs compared to age-matched wild type. In particular, while wild
75
type endplates display the classical pretzel-like shape, the MLC/SOD1G93A endplates are dispersed
and extensively fragmented. Electron microscopy analysis corroborated the histological and
morphometric observations, revealing marked alteration and fragmentation of postsynaptic folds. The
normal pretzel-shaped synaptic contact area is replaced by myriad small contact areas spread over an
extended length of the fiber.
To final prove the instability of NMJ in MLC/SOD1G93A mice we performed a pulse chase
experiment on wild type and MLC/SOD1G93A mice, demonstrating an higher turnover of AChR in the
muscle of MLC/SOD1G93A mice, compared to that of wild type littermates. Altogether these
experiments demonstrate that altered muscle might play a key role in initiating the NMJ dismantlement
and therefore alteration with the nerve.
Further work will define the molecular mechanisms by which oxidative stress trigger NMJ
alteration.
Publications
Kuraitis D, Ebadi D, Zhang P, Rizzuto E, Vulesevic B, Padavan DT, Al Madhoun A, McEwan KA,
Sofrenovic T, Nicholson K, Whitman SC, Mesana TG, Skerjanc IS, Musarò A, Ruel M, Suuronen EJ.
Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic
injury. Eur Cell Mater 2012, 24: 175-95.
Murdocca M, Malgieri A, Luchetti A, Saieva L, Dobrowolny G, De Leonibus E, Filareto A, Quitadamo
MC, Novelli G, Musarò A, Sangiuolo F. IPLEX administration improves motor neuron survival and
ameliorates motor functions in a severe mouse model of SMA. Mol Med 2012, 18: 1076-85. doi:
10.2119/molmed.2012.00056.
Musarò A. Understanding ALS: new therapeutic approaches. FEBS J 2012 Epub. doi: 10.1111/
febs.12087.
Nucera E, Nicoletti C, Chiapparino C, Pacello ML, D'Alessio V, Musarò A, De Santis R. AvidinOX for
tissue targeted delivery of biotinylated cells. Int J Immunopathol Pharmacol 2012; 25:239-46.
Research Group
Collaborations
Gabriella Dobrowolny, Emanuele Rizzuto,
post-doc fellows; Carmine Nicoletti, technician;
Martina Martini, graduate student.
Marco
Sandri,
Dipartimento
Scienze
Biomediche- VIMM, Università di Padova;
Feliciano Protasi, Ce.S.I., Università degli
Studi G. d’Annunzio, Chieti; Rüdiger Rudolf,
Univ. of the State of Baden-Wuerttemberg and
National
Laboratory
of
the
Helmholtz
Association- Germany; Federica Sangiuolo,
Giuseppe Novelli, Department of Biomedicine
and Prevention, University of Rome Tor
Vergata, Rome, Italy.
76
Role of the COP9 signalosome in transcription modulation and
chromatin organization in yeast and plants
Rodolfo Negri
℡: +39 06 49917790 - @: [email protected]
Accurate regulation of transcription in time and space is critical for the establishment and
maintenance of gene expression patterns in eukaryotes. In the past years, several components of the
ubiquitin/proteasome system (UPS) have been shown to be necessary for the tight regulation of gene
expression with possible important implications for cellular homeostasis. A key component of the UPS
is the COP9 signalosome (CSN), a protein complex conserved in all eukaryotes. CSN removes the
small peptide NEDD8 (an ubiquitin like modifier) from the cullin-RING family of E3 ubiquitin ligases. E3
ligases are the enzymes responsible for poly-ubiquitination of specific substrates, which can be
subsequently recognized and degraded by the proteasome. The reaction catalyzed by CSN is
necessary for the tight regulation of the assembly/disassembly cycles of these ligases and is essential
for all higher eukaryotes, since null CSN mutants from different organisms ranging from plants to
mammals are lethal at very early developmental stages.
At the cellular level, CSN mutants from different organisms display de-repression and, more in
general, miss-regulation of several sets of genes. This could be due simply to the fact that CSN is
necessary for the proper degradation of several transcription factors. However, recent evidence
suggests that CSN might perform specific tasks directly on the genomic chromatin.
The CSN from budding yeast S.cerevisiae has been characterized and, like in higher eukaryotes,
is responsible for the de-neddylation of cullin-RING ubiquitin ligases. However, while the CSN in most
higher eukaryotes is composed of 8 subunits and is a paralog of the lid of the proteasome, the CSN
from budding yeast consists of six subunits. In addition, in contrast to other eukaryotes, all the CSN
subunits from S.cerevisiae are non-essential. The non essentiality of CSN components and the
availability of powerful genetic tools make S.cerevisiae a very promising model system to elucidate
some aspects of the nuclear role of this complex.
We previously performed a transcriptomic analysis of a S.cerevisiae strain deleted in CSN5 (the
de-neddylating subunit of the CSN), as compared with its isogenic wild type strain.
The results obtained suggested a role for Csn5 in the regulation of genes involved in zinc uptake
and metabolism, and in ergosterol biosynthesis. We have further characterized the results of our
transcriptome profiling. We first confirmed by qRT-PCR that genes involved in zinc uptake and
metabolism and ergosterol biosynthesis are indeed down-regulated in Csn5 mutant (∆csn5) cells. We
have also confirmed that their expression levels are down-regulated also in the deletion mutants of
other CSN subunits, suggesting that their misexpression is caused by a defect in the function of the
whole complex, rather than being linked to a missing function of the single Csn5 protein.
Second, we assessed whether the observed expression changes could be reflected at the
phenotypical level by alterations in ergosterol or zinc metabolism and/or uptake and we were able to
observe that ∆csn5 cells contain less ergosterol than their wild-type counterpart and are more
sensitive to low Zinc levels in the medium.
77
Third, since the transcriptomic modulations observed in the ∆csn5 appeared reproducible and
coherent, we verified if they corresponded to similar variations in protein abundance by analyzing the
genome-wide modulations of the proteome. To this purpose we used a label-free shotgun approach. In
order to be reliable, this approach requires multiple independent protein extract preparations. We
analyzed four independent samples for each strain, run simultaneously. We obtained reliable data for
approximately 750 proteins. Among them, 106 showed a quantitative modulation at a FDR cut-off of
20%. An in-depth analysis of these modulated proteins showed that the enzymes involved in
ergosterol biosynthesis, such as Erg2 and Erg9, that were already shown to be down-regulated at the
mRNA level (see Fig.1), are also less abundant at the protein level. Most of the other proteins found,
however, show a different regulation as compared with that observed at the mRNA level. This is not
surprising, since it is possible that these proteins are directly regulated by the CSN at the protein level.
Further experiments are required to address this issue.
We
are
currently
in
process of analyzing if proteins
are
differentially
modified
(ubiquitylation or neddylation)
in the ∆csn5 strain. This will
facilitate the identification of
regulators which could be
responsible for the quantitative
changes observed at the
mRNA level.
Fig. 1 - Ergosterol biosynthesis
is down-regulated both at the
mRNA and at the protein level in
∆csn5 cells.
Publications
Serino G, Pick E. Duplication and familial promiscuity within the proteasome lid and COP9
signalosomekin complexes. Plant Sci 2012 Epub. http://dx.doi.org/10.1016/j.plantsci.2012.12.018.
Research Group
Collaborations
Giovanna Serino, professor; Teresa Rinaldi,
researcher; Valerio Licursi, post-doc fellow;
Chiara Salvi, PhD student.
Martin Bard, Biology Department, Indiana
University, Indianapolis, USA; Elah Pick, Haifa
University, Israel.
78
The biogenesis of piRNAs and their involvement in transposon
silencing and heterochromatin formation in Drosophila
Sergio Pimpinelli
℡: +39 06 49912876 - @: [email protected]
The aim of our research during the last three years was to systematically search, by a combined
genetic, cytological and molecular approach, genes involved in piRNAs biogenesis, heterochromatin
formation and transposons silencing.
To this end we collected mutants that induce the expression of the repetitive Stellate sequences of
Drosophila melanogaster normally repressed by piRNAs interference mechanism. The analysis of
most of them is still ongoing.
Among these mutants, however, we identified mutations of Hsp90 heat shock factor. We focused
our experimental efforts to it because this factor appears involved in stress and in molecular
mechanisms underlying the canalization and assimilation processes.
The "canalization" concept (Waddington, 1959) describes the resistance of a developmental
process to phenotypic variation, regardless of genetic and environmental perturbations, owing to the
existence of buffering mechanisms. Severe perturbations, which overcome such buffering
mechanisms, produce altered phenotypes that can be heritable and can themselves be canalized by a
genetic "assimilation" process.
Our recent study (Specchia et al., 2010) has suggested a mechanism underlying canalization. We
have shown that, in Drosophila, functional alterations of Hsp90 affect the Piwi interacting RNA (piRNA;
a class of germ-line-specific small RNAs) silencing mechanism leading to transposon activation and
the induction of morphological mutants.
This indicates that Hsp90 mutations can
generate new variation by transposonmediated ‘canonical’ mutagenesis.
We propose that, in general, stress
causes the activation of transposons that
induce de novo gene mutations affecting
development pathways. The transposon
induced mutations can be expressed and
fixed across subsequent generations by
a process consisting in a co-selection of
two independent somatic and germinal
events giving the same phenotype.
Fig. - (left) above, wild type eyes, and below
a phenocopy, without eyes, induced by heat
shock; (right) larval activation of two
transposons by heat shock.
79
Study the effect of stress on transposon activity, we applied discrete of continuous heat shocks to
larvae and adults and analyzed, in their germ and somatic cells, the transcription and mobilization of
different classes of transposons also assessing the presence of their corresponding piRNAs or endosiRNAs. The results sofar obtained, have shown that stress induces phenocopies and activates
transposons trancription (Figure).
To obtain e global view of transposons genomic redistribution, we performed an analysis by New
Generation Sequencing methodology whose results are under bioinformatic analysis. We are also
performing a a global analysis of piRNAs by cloning and deep sequencing.
Part of these were included in a paper in preparation.
Research Group
Collaborations
Laura Fanti, professor; Lucia Piacentini,
researcher; Enzo Marchetti, research fellow;
Marcella Marchetti, post-doc fellow.
Maria Pia Bozzetti, Università di Lecce; Maria
Berloco; Università di Bari.
80
To the root of organ growth: the control of root meristem activity in
Arabidopsis
Sabrina Sabatini
℡: +39 06 4991 7916 - @: [email protected]
Plant post-embryonic development takes place in the meristems. In the root of the model plant
Arabidopsis thaliana, stem cells organized in a stem cell niche in the apex of the root meristem
generate transit-amplifying cells, which undergo additional division in the proximal meristem, and
differentiate in the distal meristem transition zone that encompasses the boundary between dividing
and expanding (differentiating) cells in the different cell files (Dello Ioio et al, 2007). For meristem
maintenance, and therefore continuous root growth, the rate of cell differentiation must equal the rate
of generation of new cells: how this balance is achieved is a central question in plant development. By
means of a genetic and molecular analysis, we have shown that in the Arabidopsis root meristem the
balance between cell differentiation and cell division necessary for controlling root meristem size and
root growth is the result of the interaction between two plant hormones, cytokinin and auxin, through a
simple regulatory circuit converging on a single gene, the SHY2 gene (Dello Ioio et al, 2008). In
particular, in the vascular tissue of the transition zone, a primary cytokinin-response transcription
factor, ARR1 (Sakai et al, 2001), activates the gene SHY2 (Kepinsky et al, 2006), a repressor of auxin
signaling that negatively regulates the PIN genes that encode auxin transport facilitators (Blilou et al,
2005). Thus, cytokinin causes redistribution of auxin, prompting cell differentiation (Dello Ioio et al,
2008). Conversely, auxin mediates degradation of the SHY2 protein (Kepinsky et al, 2006), sustaining
the activity of the PIN genes and prompting cell division (Dello Ioio et al, 2008).
However, two important questions concerning the ARR1/SHY2/PIN regulatory network remain to
be answered:
- First, since cytokinin affects PIN activity in the vascular tissue of the transition zone, how is this
change perceived by the neighbouring cell files in the transition zone in order for them to shift from the
meristem into the differentiation zone (radial cell non-autonomous effect),
- Second, in maintaining a balance between cell differentiation and cell division, how does the
mechanism that controls the former (which takes place in the distal meristem transition zone) influence
the division of stem cells (which takes place in the apical stem cell niche).
Objectives. The work proposed addresses the two above-mentioned questions, as part of a larger
effort of our laboratory - which includes elucidating how the basic ARR1/SHY2/PIN regulatory circuit
interacts with other (hormonal) signals and regulatory networks that affect root growth - aiming to
clarify the molecular mechanisms through which cytokinin and auxin control Arabidopsis root growth,
and ensure the robustness of such a complex developmental process.
As of this proposal, the work consist of two main lines of research aimed to:
1) Identify and characterize the molecular components that ensure perception by all cell files in the
transition zone of the cytokinin-dependent redistribution of auxin that occurs in the vascular tissue
(radial cell non-autonomous effect).
81
2) Identify and characterize the molecular components that allow the cytokinin/auxin interaction to
influence stem cell activities.
Results. Point 1 of this proposal has extensively addressed in the past two years. Progress in
point 2 of the Objectives shows that ARR1 expression in the stem cell niche is suppressed by the
transcription factor SCARECROW (SCR, Sabatini et al, 2003) corroborating the idea that SCR
maintains stem cell division by preventing cytokinin action in the stem cell niche. We provide
mechanistic evidence that the SCR gene maintains the activity of stem cell niche by suppressing
cytokinin signaling in the QC, the stem cell organizer, and that it presides over the spatial coordination
of stem cell division with cell differentiation in transition zone at the opposite end of the root meristem.
SCR achieves this coordination by positioning the cytokinin response regulator ARR1. SCR binds
directly to the promoter of ARR1 in the QC, repressing its expression and allowing stem cell division
(Fig1). In the transition zone, SCR controls expression of ARR1 via the DELLA protein RGA, thus
controlling the rate of cell differentiation. This modulation of the cytokinin input by SCR allows a fine
tuning of auxin concentration along the meristem, acting both on its synthesis via auxin biosynthesis
gene ASB1, and on signalling and transport via SHY2, thus ensuring coherent root growth. The results
of this work are under preparation and will be soon send out for reviewing in high impact international
journal.
Fig. 1 - Root stem cell niche of, from left
to right, Wt, scr-1 and arr1-4;scr-1.
Double labeling of QC (in blue) and
differentiated columella cells (in purple)
visualized by QC specific marker and
amyloplast staining, respectively, reveal
columella stem cells activity restoration
(black arrows) in the arr1-4;scr-1 double
mutant. Asterisk indicates the preumptive position of QC cells in scr-1.
References:
Blilou et al., Nature 2005, 433: 39-44.
Dello Ioio et al., Curr Biol 2007, 17: 678-682.
Dello Ioio et al., Science 2008, 322: 1380-1384.
Kepinski et al., Curr Opin Plant Biol 2006, 9: 28-34
Sabatini et al., Genes & Development 2003, 17: 354-358
Sakai et al., Science 2001, 294: 1519-1521
Research Group
Laila Moubayidin, Serena Perilli, post-doc
fellows; Riccardo Di Mambro, Phd student.
82
Biological characterization and in vitro culture of spermatogonial
stem cells
Mario Stefanini
℡: +39 06 49766570 - @: [email protected]
In the male gonads of mammals, the germline stem cells are identified as a subpopulation of
undifferentiated spermatogonia localized in the basal layer of the seminiferous epithelium. The daily
production of millions of sperm cells through spermatogenesis depends on their biological activity. In
recent years, evidence has emerged showing the importance of GDNF for proper spermatogenesis. In
mice, GDNF is secreted by Sertoli cells and promotes spermatogonial stem cell self-renewal
(Hofmann, Mol Cell Endo 2008). Over-expression of GDNF in adult transgenic mice induces
proliferation of clusters of undifferentiated spermatogonia within seminiferous tubules. Additionally,
older animals develop testicular tumors that mimic human seminoma, suggesting that over-activation
of this pathway may lead to the development of germ cell tumors (Meng et al., Science 2001). These
data suggest that deregulation of the GDNF pathway may be implicated in germ cell carcinogenesis.
To explore the involvement of the GDNF pathway in the onset and progression of testicular germ cell
tumors, we analyzed GFRA1 and Ret expression patterns in seminoma samples. GFRA1 and Ret are
the two co-receptors for GDNF. We found that in human seminiferous tubules displaying qualitative
normal spermatogenesis GFRA1 expression was detected in subsets of dark and pale type A
spermatogonia and in interstitial Leydig cells. Interestingly, GFRA1 but not Ret was up-regulated in
both CIS, intratubular seminoma cells, and in invasive seminoma cells (Ferranti et al., Intern Journ
Androl, 2012). Functional analysis of the GDNF biological activity was performed on TCam-2
seminoma cell-line. RT-PCR and immunohistochemical analyses demonstrate that TCam-2 cells
express both GFRA1 and Ret mRNA, but only GFRA1 was detected at the protein level.
Unexpectedly, we found that GDNF does not modulate Tcam-2 proliferation and survival. Since in
other cellular systems, GDNF has been implicated in the migration and invasion of normal and tumor
cells, we tested the hypothesis that GDNF may chemoattract TCam-2 cells. We found GDNF is able
to induce migration of TCam-2 cells, possibly through the Src and MEK pathways. Moreover, GDNF
promotes invasive behavior, an effect dependent on pericellular protease activity, possibly through the
activity of matrix metalloproteinases (MMPs). In normal human testis, GDNF is produced by the
Sertoli and peritubular cells, which form the wall of the seminiferous tubules (Spinnler et al. Hum Repr,
2010). At present, data are not available regarding the expression level and regulation of GDNF in
normal human testis or those affected by pathological conditions. However, our preliminary results
indicate that GDNF mRNA levels are heterogeneous in normal testis and seminoma samples. In
mouse models, GDNF levels are inversely correlated with the germ cell content of the testis,
(Tadokoro et al., Mech Dev, 2002), and are induced by inflammatory cytokines (Simon et al., Exp Cell
Res, 2007). Therefore, it could be speculated that, as described in some breast cancers, local levels of
GDNF in the tumor microenvironment may be increased as a consequence of leukocyte infiltration
and/or spermatogenic arrest, which are commonly found in germ cell tumors. Our data support the
idea that deregulation of pathways implicated in stem cell maintenance, may be implicated in germ cell
carcinogenesis.
83
Publications
Ferranti F, Muciaccia B, Ricci G, Dovere L, Canipari R, Magliocca F, Stefanini M, Catizone A, Vicini E.
Glial cell line-derived neurotrophic factor promotes invasive behavior in testicular seminoma cells.
Int J Androl 2012, 35: 758-6. doi: 10.1111/j.1365-2605.2012.01267.x.
Research Group
Elena Vicini, professor; Barbara Muciaccia,
post-doc fellow; Dante Lamberti, Anna Maria
Lustri, PhD students; Stefania Fera, Tiziana
Menna, technicians.
84
Molecular mechanisms driving liver stem cell fate
Marco Tripodi
℡: +39 06 4461387 - @: [email protected]
Our work focused on the study of mechanisms driving liver stem cell maintenance and
differentiation. To this aim we made use of a number of liver stem cell lines named RLSCs (from
Resident Liver Stem Cells) that we previously established and characterized.
RLSCs immunophenotype (Sca1+, CD34-, CD45-, Alpha-fetoprotein+, Albumin-), together with
their expression profile and the wide differentiation potentiality, permitted us to locate them in a prehepatoblast/liver precursor cells hierarchical position (Conigliaro et al., 2008).
RLSCs plasticity is coherent with their molecular phenotype, typical of embryonic and adult stem
cells and characterized by co-expression of epithelial and mesenchymal markers as well as of a
variety of chromatin remodeling genes. These features underline a highly dynamic cell state, named
“metastability”, thought to be the major determinant of stem cell fate and considered as the result of
continuous oscillations between mesenchymal to epithelial and epithelial to mesenchymal transitions
(EMT/METs).
In the last year, we demonstrated that RLSCs are able to give rise, at the clonal level, to both
epithelial and mesenchymal liver cell types, including hepatocytes, cholangiocytes and hepatic stellate
cells (HSC). Orthotopic cell transplantations showed the RLSC ability to give rise a progeny able to
efficiently colonize and properly integrate in the liver architecture; heterotopic cell transplantations and
cell culture procedures documented as this differentiation ability is in part based on cell-autonomous
mechanisms. Our data, although based on the use of an immortalized cell line, provided a first
intriguing evidences for an adult stem/precursor cell capable to differentiate in both
epithelial/parenchymal and mesenchymal/non-parenchymal liver specific components and candidate
the liver as the instructive site for the as yet anatomically un-located adult reservoir of HSC
(Conigliaro et al., Cell Death Differ, in press). Being HSC the main fibrogenic cells involved in the
onset and progression of liver fibrosis, our data open new perspectives on the study of pathogenesis
and on the therapy of this severe human pathology.
We previously characterized the EMT/MET dynamics in hepatocyte, attributing to these
phenomena a key role both in differentiation and maintenance of epithelial identity. In particular, we
unveiled a simple cross-regulatory circuit between the EMT master gene Snail and the hepatocyte
differentiation master gene HNF4α in which these transcriptional factors repress each other with a
consequent their mutually exclusive expression: Snail/HNF4α dynamics define the outcome of the cell
habit between the mesenchymal/undifferentiated and the epithelial/differentiated phenotype. This more
or less balanced mini-circuitry could play a key role also in pathological situations such as the
progression of hepatocellular carcinoma. The loss of transcriptional factor HNF4α, in fact, together
with high level of Snail, is frequently observed in less differentiated/more aggressive HCC; on the
other hand its re-expression, repressing the EMT program Snail-mediated, is able to restore in cell
culture a more differentiated epithelial phenotype. These and other observations suggested HNF4α as
a new therapeutic tool for HCC gene therapy.
85
However, recent data showed that in a TGFβ-containing environment, such as the tumor niche,
the restoration of HNF4α function does not suppress TGFβ-induced tumor promoting effects; the
cytokine appears, indeed, dominant on HNF4α activity, thus indicating that the therapeutic use of
HNF4α gene delivery is limited by the presence of TGFβ in the tumor environment. We investigated
the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity demonstrating that TGFβ
induces HNF4α PTMs that correlate with the early loss of its DNA binding activity on target gene
promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α
functional inactivation: the GSK3β chemical inhibition results in HNF4α DNA binding impairment while
a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor
suppressive activity (Cozzolino et al., 2013).
We also provided evidences that human cord blood haematopoietic progenitors (CD34+45+144-),
triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating
endothelial precursors (CD144+CD105+CD146+CD31+CD45-) capable of functioning as haemogenic
endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by
haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic
cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life.
Furthermore, highly enriched populations of human post-fetal haemogenic endothelium may pave the
way for projects at possibly clinical level (Pelosi et al., 2012).
Finally, combining quantitative proteomics, computational biology and molecular analysis, we
demonstrated that the Ferritin heavy chain is the host factor responsible for HCV-induced inhibition of
apoB-100 production and is required for efficient viral infection (Mancone et al., 2012).
Publications
Mancone C, Montaldo C, Santangelo L, Di Giacomo C, Costa V, Amicone L, Ippolito G, Pucillo LP,
Alonzi T, Tripodi M. Ferritin Heavy Chain Is the Host Factor Responsible for HCV-Induced Inhibition
of apoB-100 Production and Is Required for Efficient Viral Infection. J Proteome Res 2012, 11:
2786-97. doi: 10.1021/pr201128s.
Pelosi E, Castelli G, Martin-Padura I, Bordoni V, Santoro S, Cerio AM, Conigliaro A, Marighetti P,
Desantis Puzzonia M, Biffoni M, Testa U, Amicone L, Alonzi T, Bertolini F, Peschle C, Tripodi M.
Human Haemato-endothelial Precursors: Cord blood CD34+ Cells Produce Haemogenic
Endothelium. PLoS One 2012, 7:e51109. doi: 10.1371/journal.pone.0051109.
Research Group
Laura Amicone, professor, Carla Cicchini,
Alessandra Marchetti, researchers, Carmine
Mancone, Alice Conigliaro, post-doc fellows;
Laura Santangelo, Angela Cozzolino, AIRC
fellows; Valeria De Nonno, Marco De Santis
Puzzonia, Viviana Costa, PhD students;
Claudio Cavallari, technician.
86
Profiling the Polycomb/Trythorax target genes in normal and
leukemic hematopoiesis
Giuseppe Zardo
℡: +39 06 4469843 - @: [email protected]
In human embryonic stem cells (HESCs), the promoter regions of the genes coding for lineage
specific transcription factors show a chromatin pattern known as “bivalent domain” where repressive
H3K27me3 and activating H3K4me3 marks are overlapped. Bivalent domains keep developmentally
regulated and tissue-specific genes transcriptionally poised and progenitor cells to be kept primed for
an alternate lineage fate. Chromatin bivalent domains resolution is required in order to allow
expression or repression of the lineage specific genes and to address the progenitor cells toward a
specific lineage fate. Polycomb (PcGs) and trithorax (TrxGs) proteins are responsible for the
trimethylation of H3K27 and of H3K4, respectively.
Our research is focused on the epigenetic events occurring at promoter region of the Nfi-a gene
during normal granulopoiesis, and on the regulation of chromatin bivalent domains during leukemic
transformation.
NFI-A is a repressor of granulopoiesis and a post-transcriptional target of miR-223. Nfi-a promoter
region presents a bivalent domain in HESC and in human hematopoietic stem cells
(CD34+HSC/HPCs). ChIP experiments identified increased H3K27me3, decreased H3K4me3 and
PolII promoter binding after induction of granulopoiesis of myeloid cell lines by RA. PcGs (YY1 and
Suz12) and components of the RNAi machinery, Dicer1 and Ago1, also interacted with the Nfi-a
promoter region under these conditions. These events were miR-223 dependent. MiR-223 localized to
the nucleus after the induction of granulopoiesis, directly interacted with complementary binding sites
in the promoter of Nfi-a and served as a guide for the localization of a PcGs/RNAi complex, allowing
the resolution of the Nfi-a promoter bivalent domain, epigenetic silencing of the Nfi-a gene and
differentiation of myeloid cell lines.
A role for miR-223 in driving the transcriptional gene silencing by PcGs-RNAi complexes was
suggested by the presence on the Nfi-a promoter region of two DNA sequences complementary to the
miR-223 seed sequence. We identified nuclear localization and Nfi-a promoter targeting of miR-223 by
confocal microscopy of transfected fluorescent miR-mimics and in situ hybridization to the endogenous
miRNA with fluorescent oligonucleotides complementary to the sequence of endogenous miR-223. In
agreement, by combining transfection of a Cy5-labelled miR-223 with anti-Cy5 chromatin
immunoprecipitation we were able to demonstrate the presence of miR-223 on the Nfi-a promoter
DNA. Different types of ncRNAs are known to modulate target gene transcription. Our research
demonstrates that endogenous miRNAs can now be added to this group of transcription-regulating
ncRNAs.
It is known that the presence of H3K27me3 chromatin mark at gene promoter regions in HESCs
makes these genes sensitive to aberrant DNA methylation during tumor transformation. By a genome
wide approach and gene specific ChIPs we confirmed that aberrantly methylated genes in leukemic
samples were H3K27me3 labeled in HESC and CD34+HSC/HPCs. Interestingly, we identified also a
relevant number of aberrantly methylated genes presenting at their promoter region the H3K4me3
87
olII
Suz12
activating mark in HESC and/or in HSCs/HPCs and in CD34- cells. Indeed, in leukemic samples,
H3K4me3 mark was aberrantly replaced by H3K27me3 mark, proving that leukemic transformation is
also associated with an anomalous PcG/TrxG activity. Our data show that a possible explanation
might be represented, among others, by altered expression of chromatin modifiers. In leukemic
samples the H3K4me1/2/3 demethylases Lsd1, Jarid1a, Jarid1b, and the H3K27me methylase Ezh2
were aberrantly overexpressed in respect to normal CD34+HSC/HPCs and CD34- cells, whereas
Jmjd3 an H3K27me3 demethylase was downregulated.
Our research will enable a
Granulocytic differentiation
more comprehensive knowledge of
epigenetic mechanisms regulating
K4me3
K4me3
K27me3
the activity of hematopoietic master
K27me3
Transcriptional
Transcriptional
Ac H3K9
regulators and unveil new targets
activation
silencing
Dicer1
for leukemia therapy.
+1
-1400
-1400
+1
CpGs
YY1
CpGs
Ago1
-A p
NF I
YY1
o
rom
YY1
ter
Suz12
RNAP
YY1
H3K9 Ac
K4me3
+ RA
NFI
-A p
o
rom
ter
K4me3
K27me3
K27me3
RISC
Ago1
Dicer1
RISC
miR-223
miR-223
Fig. - Schematic model for the hetrochromatic silencing of Nfi-a gene by
PcG.miR-223 complexes in RAinduced granulocytic differentiation.
Publications
Zardo G, Ciolfi A, Vian L, Starnes LM, Billi M, Racanicchi S, Maresca C, Fazi F, Travaglini L, Noguera
N, Mancini M, Nanni M, Cimino G, Lo-Coco F, Grignani F, Nervi C. Polycombs and microRNA-223
regulate human granulopoiesis by transcriptional control of target gene expression. Blood 2012,
119: 4034-46. doi: 10.1182/blood-2011-08-371344.
Zardo G, Ciolfi A, Vian L, Billi M, Racanicchi S, Grignani F, Nervi C. Transcriptional targeting by
microRNA-polycomb complexes: a novel route in cell fate determination. Cell Cycle 2012, 11: 35439. doi: 10.4161/cc.21468.
Research Group
Alberto Ciolfi, post-doc
Maresca, technician.
88
Collaborations
fellow;
Carmen
Clara Nervi, Dip. di Scienze e Biotecnologie
Medico-Chirurgiche, Sapienza Università di
Roma; Robin Foa’, Sabina Chiaretti, Dip. di
Biotecnologie
Cellulari
ed
Ematologia,
Sapienza Università di Roma; Joseph F.
Costello, Comprehensive Cancer Center,
University of California, San Francisco, USA.
Area 4
Molecular recognition
in biomolecules
Area 4: Molecular recognition in biomolecules
Toxic effects induced by polyamine metabolites on melanoma cells:
a new therapeutic approach
Enzo Agostinelli
℡: +39 06 49910838 - @: [email protected]
The metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine,
has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found
essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at
a cell type–specific set point through the coordinated and highly regulated interplay between
biosynthesis, transport, and catabolism. In particular, polyamine catabolism involves copper–
containing amine oxidases. Several studies showed an important role of these enzymes in several
developmental and disease–related processes in animals through a control on polyamine homeostasis
in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The
production of toxic aldehydes and reactive oxygen species (ROS), H2O2 in particular, by these
oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by
which the oxidases can be exploited as antineoplastic drug targets.
The purpose of this research is to develop a new anticancer therapy against melanoma (M14),
taking advantage of the high polyamine content of tumor cells, since such polycations may become a
source of cytotoxic metabolites. For their generation an enzyme, bovine serum amine oxidase (BSAO)
will be employed. It is expected that by delivering BSAO into cancer cells, toxic enzymatic oxidation
products could be produced intracellularly for selective killing in situ.
During the second year of the project,
a novel superparamagnetic maghemite
nanoparticles (SAMNs, surface active
maghemite nanoparticles), characterized
by a diameter of 10 ± 2 nanometers, were
modified with bovine serum amine oxidase,
using rhodamine-isothiocyanate adduct as
fluorescent spacer arm. A fluorescent and
magnetically drivable adduct comprising
bovine, copper containing, amine oxidase
immobilized on the surface of specifically
functionalized magnetic nanoparticles was
developed (SAMN@RITC-BSAO) Fig. 1.
Fig.1 - Enzyme binding: production of
SAMN@RITC-BSAO. Rhodamine B Isothiocyanate (RITC) is able to bind to nanoparticle
surface (SAMN@RITC) suggesting a multiple
point binding recognition process between
nanoparticle and the fluorophore (Sinigallia et
al., 2012).
91
The multifunctional nanomaterial was characterized by transmission electron microscopy, infrared
spectroscopy, mass spectrometry and activity measurements. Results showed that bare magnetic
nanoparticles form stable colloidal suspension in aqueous solutions. The maximum binding capacity of
bovine serum amine oxidase was about 6.4 mg g-1 nanoparticles. The immobilization procedure
reduced the catalytic activity to 30 ± 10 % with respect to the native enzyme and the Michaelis
constant was increased by a factor of 2. We suggest that SAMN@RITC-BSAO complex, characterized
by a specific activity of 0.81 IU g-1, could be used, in the presence of polyamines, as fluorescent
magnetically drivable H2O2 and aldehydes producing system. Future applications in the selective
tumor cells destruction might be suggested.
Publications
Battaglia V, Tibaldi E, Grancara S, Zonta F, Brunati AM, Martinis P, Bragadin M, Grillo MA, Tempera
G, Agostinelli E, Toninello A. Effect of peroxides on spermine transport in rat brain and liver
mitochondria. Amino Acids 2012, 42: 741-9. doi: 10.1007/s00726-011-0990-3.
Grancara S, Battaglia V, Martinis P, Viceconte N, Agostinelli E, Toninello A, Deana R. Mitochondrial
oxidative stress induced by Ca2+ and monoamines: different behaviour of liver and brain
mitochondria in undergoing permeability transition. Amino Acids 2012, 42: 751-9. doi:
10.1007/s00726-011-0991-2.
Sinigaglia G, Magro M, Miotto G, Cardillo S, Agostinelli E, Zboril R , Bidollari E, Vianello F.
Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable
nanoparticles. Inter J of Nanomedicine 2012:7 1-11. doi: 10.2147/IJN.S28237.
Tavladoraki P, Cona A, Federico R, Tempera G, Viceconte N, Saccoccio S, Battaglia V, Toninello A,
Agostinelli E. Polyamine catabolism: target for antiproliferative therapies in animals and stress
tolerance strategies in plants. Amino Acids 2012, 42:411-26. doi: 10.1007/s00726-011-1012-1.
Research Group
Collaborations
Paola Turini, professor; Giampiero Tempera,
Stefania Saccoccio, post-doc fellows; Martina
Meringolo, PhD student.
Annarica Calcabrini, Istituto Superiore di
Sanità, Roma; Diana Averill-Bates, Université
du Québec à Montréal, Canada; Taichi
Ueshima, Wakunaga Pharmaceutical Co.,
Hiroshima, Japan.
92
Cellular response to oxidative stress: involvement of protein
disulfide isomerases
Fabio Altieri
℡: +39 06 49910880 - @: [email protected]
The aim of this research project is to elucidate the involvement of protein disulfide isomerases in
the cellular response to oxidative stress and we focused the attention on ERp57/PDIA3. This protein,
mainly involved in the correct folding of newly synthesized glycoproteins, has been implicated in
cellular stress and various diseases. ERp57 participates in the unfolded proteins response and is
known to be differently expressed in stress conditions and neoplastic transformation.
Considering that ERp57 is able to interact with a number of macromolecules or small ligands,
although the specific binding sites and target substrates have not been fully identified yet, it has the
potential to be a pharmacological target. We started analyzing the binding of several natural
substances to ERp57 and their effects on protein activities, to find molecules able to specifically inhibit
the binding between ERp57 and selected interactors and modulate its biological functions.
Fluorescence quenching measurements and surface plasmon resonance (SPR) analysis showed that
green tea catechins (GTCs) can bind ERp57, but only the galloylated ones, epigallocatechin (ECG)
and epigallocatechin-gallate (EGCG), display a significant affinity, quenching the fluorescence of
tryptophan residues, and are able to modify ERp57 properties. Quenching analysis was extended to
isolated redox domains of ERp57, confirming the binding of catechins near the ERp57 redox-active
sites and that the interaction require the presence of the galloyl moiety. This structural feature is also
responsible for the effects on protein activities. The galloylated catechins are able to weakly inhibit
reductase activity of ERp57 comparably with what was previously observed for vancomycin, but they
can strongly inhibit, expecially EGCG, the DNA binding capability of ERp57. In fact, we showed that at
EGCG concentration in the range 10-100 µM there is a 20% inhibition of ERp57 redox activity,
measured using oxidized glutathione as substrate, and greater than 90% inhibition of the DNA binding
activity, assayed by EMSA using an AT-rich DNA fragment. Considering the high affinity of galloylated
catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands,
some effects of catechins interaction with this protein on eukaryotic cells may be expected.
The in vitro/in vivo binding of ERp57 with known interactors and the effect these interactions on
cellular functions was further analyzed. The interaction of ERp57 with calreticulin (CRT) has been
studied by SPR. Kinetic analysis estimated a KD of 2.6 µM and the obtained binding curves suggest a
conformational change in the initial protein complex that increase its stability. This result confirms the
great flexibility in the ERp57 structure that allows, through specific conformational changes, to handle
the interaction with so many different protein partners. Vancomycin can bind to ERp57 and inhibit
ERp57-CRT interaction in vitro and in vivo. Vancomycin KD obtained by SPR analysis is similar to that
observed for CRT. Moreover, vancomycin administration to HeLa cells prevents the ERp57-mediated
translocation of calreticulin to the plasma membrane. This result confirms a role for ERp57 in the
trafficking of proteins from endoplasmic reticulum to plasma membrane.
Studies on EGFR activation and internalization conducted on breast cancer cells, MDA-MB-468,
highlighted the involvement of ERp57 in the mechanism of action of epithelial growth factor receptor
93
(EGFR). Breast cancer cells, where ERp57 was silenced by RNA interference, have been subjected to
EGF stimulation. Preliminary studies suggest that ERp57 can interact with EGFR and is implicated in
EGFR endocytosis and autophosphorylation, as well as the activation of downstream signaling
pathways. Although ERp57 silencing has no effect on EGFR expression or plasma membrane
exposure, internalization of the complex EGF-EGFR by endocytic vesicles is hampered in silenced
cells and most of the ligand-receptor complex remained on the plasma membrane. The ERp57
silencing has been found to reduce the EGFR autophosphorylation, and this reduction leads to a
decreased activation of STAT3 and PLCγ1, which are directly dependent on EGFR phosphorylation.
Moreover, preliminary co-immunoprecipitation experiments have shown a direct interaction between
ERp57 and EGFR in EGF stimulated cells. All these results suggest that ERp57 interacts with EGFR
modulating its internalization and activation, thus leading to changes in the activation of downstream
pathways.
The binding of vitamin D3 active form (calcitriol) with ERp57 was analyzed by fluorescence
quenching measurements confirming the high affinity of this molecule toward the protein especially
when is in the reduced state. Preliminary experimental data and bioinformatics analysis suggest a
binding site in the C-terminal domain within a pocket positioned far from the active site.
Publications
Trnková L, Ricci D, Grillo C, Colotti G, Altieri F. Green tea catechins can bind and modify
ERp57/PDIA3 activity. Biochim Biophys Acta 2012, pii: S0304-4165(12)00331-5. doi: 10.1016/
j.bbagen.2012.11.011.
Frasconi M, Chichiarelli S, Gaucci E, Mazzei F, Grillo C, Chinazzi A, Altieri F. Interaction of ERp57
with calreticulin: Analysis of complex formation and effects of vancomycin. Biophys Chem 2012,
160: 46-53. doi: 10.1016/j.bpc.2011.09.003.
Research Group
Collaborations
Margherita Eufemi, professor; Caterina Grillo,
post-doc fellow; Rossana Cocchiola, PhD
student.
Silvia Chichiarelli, Daniela Ricci, Elisa
Gaucci, Dipartimento di Scienze Biochimiche
“A. Rossi Fanelli”, Sapienza Università di
Roma.
94
Role of Gsα signalling in the bone marrow hematolymphopoietic
microenvironment as revealed by novel transgenic models
Paolo Bianco
℡: +39 06 4441049 - @: [email protected]
Cells of osteogenic lineage maintain, organize and transfer the hematopoietic microenvironment
(HME)/niche in the bone marrow. In search for the specific cell type conveying these effects, attention
over the past few years has been directed first to osteoblast proper (i.e., the mature bone forming
cells) and later to osteoprogenitors (aka “mesenchymal stem cells”). Studies using osteoblast-targeted
KO have implicated Gsa signaling within cells of osteogenic lineage in the regulation of the HME/niche
and B lymphopoiesis. We have generated mice in which GsaR201C (causing Fibrous Dyplasia of
bone in humans) is either targeted to osteoblasts through the COL1A1 2.3kb promoter, or expressed
widely in the HME as directed by costitutive promoters (PGK or EF1a). The general aim of the project
is to elucidate a) the changes in the organization of the HME/niche b) the changes in the hematolymphopoietic progenitor compartments brought about by overactivity of Gsa signaling. Functional effects of GsaR201C in different osteogenic cell/HME compartments. We have now conclusively elucidated the key changes induced in the HME in FD mice, and how
they lead to the FD phenotype. We have identified two specific molecular effects of mutated Gsa that
occurs in osteoprogenitors only: 1) the overexpression of RANK-L, leading to excess
osteoclastogenesis; 2) induction of ectopic Matrix-Gla protein (MGP) expression. The latter effect,
unique to FD mice and human FD bone cells, leads to osteomalacia in FD. We have also defined
where in the HME/osteolineage do Gsa mutations specifically determine a perturbation of lineage
allocation/differentiation. Surprisingly, the target of GsaR201C is a differentiating marrow adipocyte,
which appears at defined times in postnatal development. This cell (Sca-1-, PLIN+ MGP+) is
downstream of the adipocyte progenitor (Sca-1+ PLIN-), does not express the osteogenic master
regulator Osx/SP7, and is converted to an abnormal osteoblast in FD (mice). This effect on marrow
adipocytes is part of a global effect of mutated Gsa on WAT and BAT adipocytes in our mice, which
mimics a b-adrenergic effect of enhanced lipolysis, thermogenesis, and generation of ‘beige’ fat via
UCP-1, which we have now documented. Marrow adipocytes, long-known members of the HME
downstream of a progenitor shared with osteoblasts, are the key culprits of FD development, and key
targets of Gsa regulation in the HME. These data are incorporated in a paper being completed at the
time of this report.
Functional effects of GsaR201C expressed in the osteogenic lineage on hemato-lymphoid
compartments. Earlier published data based on analysis of either mice with constitutively active PTH1R targeted
to osteoblasts, or mice with ostoeblast-targeted Gsa KO, had suggested the notion that Gsa in
osteoblasts would contribute to regulate HSCs and B-lymphopoiesis, representing a major determinant
of the niche effect. Our data do not confirm this early suggestion; 2.3COL1A1-GsaR201C mice did not
show changes in the size of HSC, CMP, CLP, pre-B, pro-B, or B cell compartments. In sharp contrast,
95
mice expressing the same transgene in osteoprogenitors, do show a change in composition, but not
size, of the LSK compartment: a significant increase in cycling LSK cells occurs in these mice. This
could represent a “mobilizing” effect, which would be consistent with the reported effects of badrenergic stimulation (Gsa-mediated) in the niche; to test this hypothesis, we analyzed the PB of
mice of different age, but could not detect an increase in circulating HSCs. We have now determined
that the LSK cells that are in fact activated to divide in mice expressing constitutively active Gsa in
osteoprogenitors are a specific subset of LSK cells noted for expression of Flt3.
Next directions The LSK-Flt3 cells may represent an early compartment of MPP able to generate DCs,
macrophages and osteoclasts. If so, our data would suggest that constitutively active Gsa in
osteoprogenitors, but not in osteoblasts, would cue osteoclastogenesis not only by promoting
maturation of proximal progenitors to osteoclasts (via RANK-L), but also by regulating an early
compartment of HSPCs. Enhanced osteoclastogenesis in FD and murine models thereof would then
reveal not only a novel role of Gsa signaling in the HSC niche, but also its specific functional
significance. In addition, it would make FD a paradigm of a hitherto poorly recognized mechanism of
normal and abnormal osteoclastogenesis operating at the HSPC compartment. This hypothesis will be
tested conclusively in the last year of the project.
Research Group
Collaborations
Mara Riminucci, Isabella Saggio, professors;
Alessandro Corsi, Benedetto Sacchetti,
researchers; Stefania Cersosimo, Rossella
Costa, Cristina Remoli, post-doc fellows;
Letizia Astrologo, Paola Comite, PhD
students; Emanuela Spica, technician. Ana Cumano, Pasteur Institut, Paris; Enzo
Marinari, Dipartimento di Fisica, SapienzaUniversità di Roma; Margherita Fiani,
Dipartimento di Ingegneria Civile, Università di
Salerno.
96
The dark side of protein folding: denatured states and misfolded
species in molecular recognition and pathological processes
Maurizio Brunori
℡: +39 06 4991 0579 - 06 49910544 - @: [email protected]
The protein folding problem. The ultimate goal of biomolecular science is to discover general rules
by analyzing the behavior of simple systems, a particularly challenging task in the case of protein
folding. In fact, when considering different globular proteins, complexity stems by-and-large from the
differences in sequence but also the multiplicity of 3D structures of the native and denatured states.
Much experimental work has been focused on comparing the folding behavior of proteins belonging to
the same “functional family”, which differ in amino acid sequence but share the same fold. The design
of heteromorphic proteins with very high sequence identity (up to the extraordinary limit of 95%) but
different structure and function affords the unique opportunity to address the protein folding problem
from a complementary perspective.
To unveil the relative role of overall topology vs. sequence composition in dictating the folding
pathway(s), we have characterized in considerable detail the folding mechanism of different
heteromorphic pairs, displaying increasingly high sequence identity (namely 30, 77 and 88%), but
different structures, called Ga (a 3-α helix fold) and Gb (an α⁄β fold) (see Figure 1). The problem was
tackled by a combination of mutagenesis, structural analysis and kinetics, a combined experimental
approach that proved powerful in obtaining structural information about transition states. In this
methodology, known as φ−value analysis, residue-specific structural information is inferred by
comparing the kinetics of folding of the wild-type protein with that of a series of conservative single
mutants selected by analysis of the 3D structure of the protein.
Our results are fully consistent with the idea that protein topology is committed very early along the
pathway of folding. Furthermore, data reveals that when folding follows a simple two-state model, as in
the case of the Ga domains, the structural features of the transition state are almost independent from
changes in sequence composition. On the other hand when folding is more complex and multistate, as
for the Gb domains, the formation of different nuclei and multiple pathways can be alternatively
selected for by perturbation of the primary structure.
Addressing the role of disorder in protein recognition. Much of our current understanding of
proteins is based on the structure-function dogma, ‘sequence determines structure determines
function’. From these premises, the recent discovery that a significant fraction of proteins or protein
domains lack a well-defined structure and are intrinsically disordered (known as Intrinsically Denatured
Proteins, IDPs) is set to revolutionize our understanding of protein function and molecular recognition.
Despite being disordered, IDPs are functional and often involved in complex mechanisms of proteinprotein or protein-DNA interaction. IDPs can recognize a target ligand and undergo a structural
transition to a folded state only once bound. To date, convincing experimental data on the binding and
recognition of IDPs to their physiological partners is relatively scarce, limiting our understanding of the
mechanism and biological role of disordered systems.
The CREB-binding protein (CBP) is a co-activator that mediates the interaction between DNAbound activator proteins and the components of the basal transcription complex. A small globular
domain of CBP, known as KIX, modulates such interactions via binding to different IDP systems.
Whilst the structural features of the interaction between KIX and different IDPs have been
97
investigated, the mechanism by which such recognition occurs is still to be unveiled. More to the point,
it is unclear whether IDPs recognize KIX in their ordered or disordered conformation – a critical
question in clarifying the biological role of disorder in IDP systems.
We have analyzed in detail the mechanism of binding and recognition between KIX and the
transactivation domain of c-Myb, an IDP protein that folds upon binding into a helical structure. Our
results clearly demonstrate that c-Myb recognizes KIX in an unstructured conformation and that
complex formation precedes folding. Furthermore, by studying the pH dependence of the binding
kinetics, we have proposed a plausible mechanism for complex formation. Our results pave the way to
future work aimed at clarifying the role of disorder in molecular recognition and the control of
transcription activation.
Ga88
Gb88
TTYKLILNLK QAKEEAIKEL VDAGIAEKYI KLIANAKTVE GVWTLKDEIL TFTVTE
TTYKLILNLK QAKEEAIKEL VDAATAEKYF KLYANAKTVE GVWTYKDETK TFTVTE
Fig. 1 - Three-dimensional
structures of Ga88 and Gb88
along with their amino acidic
sequence: identical residues
are shown in blue.
Publications
Brunori M, Gianni S, Giri R, Morrone A, Travaglini-Allocatelli C. Morphogenesis of a protein: folding
pathways and the energy landscape. Bioch Soc Trans 2012, 40: 429-32. doi:
10.1042/BST20110683.
Giri R, Morrone A, Travaglini-Allocatelli C, Jemth P, Brunori M, Gianni S. Folding pathways of proteins
with increasing degree of sequence identities but different structure and function. Proc Natl Acad Sci
USA 2012, 109: 17772-6. doi: 10.1073/pnas.1201794109.
Morrone A, Giri R, Brunori M, Gianni S. Reassessing the folding of the KIX domain: Evidence for a
two-state mechanism. Protein Sci 2012, 21: 1775-9. doi: 10.1002/pro.2159.
Gianni S, Morrone A, Giri R, Brunori M. A folding-after-binding mechanism describes the recognition
between the transactivation domain of c-Myb and the KIX domain of the CREB-binding protein.
Biochem Biophys Res Comm 2012, 428: 205-9. doi: 10.1016/j.bbrc.2012.09.112.
Research Group
Collaborations
Luca Federici, Stefano Gianni, Carlo
Travaglini Allocatelli, professors; Adele Di
Matteo, CNR researcher; Angela Morrone,
post-doc fellow; Rajanish Giri, PhD student.
Per Jemth, University of Uppsala, Sweden;
Michele
Vendruscolo,
University
of
Cambridge, UK.
98
The role of nucleosomes in the stability of human telomeres
Stefano Cacchione
℡: +39 06 49912238/40 - @: [email protected]
The establishment of a specific nucleoprotein structure, the telomere, protects chromosome ends
from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a
DNA damage response that leads to replicative senescence. In normal human somatic cells,
characterized by telomere shortening with each cell division, telomere uncapping is a regulated
process associated with cell turnover. Nevertheless, it could also be the consequence of a
pathological pathway leading to genomic instability and to cancer. Despite the essential role telomeres
play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure
involved in telomere maintenance is still limited.
Theoretical analysis of the higher-order organization of telomeric chromatin
The telomeric chromatin is characterized by a shorter nucleosome repeat length than bulk nuclear
chromatin. The compaction of nucleosomes in chromatin is directed by their positioning along DNA
and therefore by the length of the DNA linkers that bridge and mutually orient the adjacent
nucleosomes in the space. Due to their peculiar DNA sequence features, telomeric nucleosomes
occupy isoenergetic positions having the periodicity of the telomere repeat (Scipioni and De Santis, in
press). The systematic search of the possible nucleosome packing allows selecting different and
unique structures of the telomeric chromatin due to the peculiar nucleosome repeat lengths that are
multiples of the vertebrate telomeric repeat.
Crosstalk between TRF proteins and nucleosomes
The interplay between nucleosomes and telomeric proteins is a relevant issue in telomere
protection. We found that TRF2 affects chromatin organization in a concentration-dependent way. As
a consequence of TRF2 overexpression, histone density at telomeres diminishes whereas
nucleosome spacing increases. This remodeling is a cell cycle-regulated event occurring at the end of
the S-phase (Galati et al., 2012). We propose that TRF2 can alter chromatin organization at telomeres
either by directly inducing nucleosome sliding (Fig. 1A) or by recruiting chromatin remodeling
complexes (Fig. 1B).
Fig. 1 - Models of how TRF2 remodels telomeric
chromatin. (A) TRF2 may bind between
nucleosomes and increase their spacing by
enhancing their intrinsic mobility. (B) TRF2 could
act by recruiting ATP-dependent remodeling
complexes to telomeres.
99
In addition, by means of in vitro assembly model systems we found that i) the short nucleosome
spacing is an intrinsic feature of telomeric sequences ii) differently from TRF2, TRF1 is not able to
remodel in vitro chromatin organization and iii) the different interplay of TRF1 and TRF2 with
nucleosomes is mediated by their N-terminal domain and by histone tails (Galati et al., in preparation).
Overall, our data suggest that the crosstalk between TRF proteins and nucleosomes is likely to play a
relevant role in the dynamic change from a protected to a deprotected telomeric state (Galati, Micheli
and Cacchione, in press).
Perspectives
To get deeper into this issue, we realized a cell line derived from C33A cells containing an
engineered telomere in which a strong nucleosome positioning sequence – the 601 DNA - is placed
directly upstream of telomeric DNA. Preliminary results obtained by means of ligation-mediated PCR
(LM-PCR) indicate that telomeric nucleosomes are tightly spaced when proximal to the subtelomeric
601 DNA. Our aim is to map changes of the organization and the epigenetic state of telomeric
chromatin when telomeres become dysfunctional; to this purpose two different strategies to deprotect
telomeres are in progress. First, we are growing engineered C33A cells in the presence of a
telomerase inhibitor, BIBR 1532, to induce telomere shortening and consequently deprotection.
Second, telomere uncapping will be obtained by TRF1 and TRF2 shRNA depletion.
Publications
Galati A, Magdinier F, Colasanti V, Bauwens S, Pinte S, Ricordy R, Giraud-Panis MJ, Pusch MC,
Savino M, Cacchione S, Gilson E. TRF2 controls telomeric nucleosome organization in a cell cycle
phase-dependent manner. PLoS One 2012, 7:e34386. doi:10.1371/journal.pone.0034386.
Research Group
Collaborations
Stefano Morosetti, professor; Anita Scipioni,
researcher; Alessandra Galati, Emanuela
Micheli, Pasqualina Punzi, post-doc fellows.
Maurizio Gatti, Grazia Raffa, Dipartimento di
Biologia e Biotecnologie “C. Darwin”; Pasquale
De Santis, Dipartimento di Chimica, Sapienza
Università di Roma; Eric Gilson, MarieJosephe Giraud-Panis, Laboratory of Biology
and Pathology of Genomes, University of Nice,
France.
100
Plant innate immunity: signalling and recognition of DamageAssociated Molecular Patterns (DAMPs)
Felice Cervone
℡: +39 06 49912517 - @: [email protected]
Plants successfully defend themselves by detecting the presence of microbial pathogens and
activating the powerful responses of their innate immunity system. The responses are triggered upon
recognition of conserved pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) and/or
endogenous molecular patterns, which are present only when the tissue is infected or damaged
(damage-associated molecular patterns or DAMPs). Cell wall-derived oligolacturonides (OGs), typical
DAMPs formed during microbial infections, are the focus of this project. How OGs are released from
the plant cell wall, how they are perceived by membrane receptors, how their signal is transduced,
how modifications of the cell wall affect release, perception and transduction of OGs has been
investigated during 2012.
The ability of microbial pathogens to produce enzymes capable of degrading pectin and producing
OGs is related to the initiation of the infective process. Pectin is synthesized in a highly methyl
esterified form and is de-esterified in the wall by pectin methyl esterases (PMEs). The degree and
pattern of methyl esterification affect the cell wall properties with consequences on the plant
resistance to pathogens. PME activity may render pectin more susceptible to the action of the
enzymes produced by the pathogens and have an impact on the rapid release of OGs from the cell
wall at the onset of pathogenesis (Lionetti et al., 2012).
OGs are recognized by Wall-Associated Kinases, which together with other cell wall proteins
modulate the tissue response to wounding. During 2012 we have tested whether plant proteins
(polygalacturonase inhibitors, PGIPs) that favours the accumulation of OGs may be used as a tool to
protect crops against diseases caused by fungi and oomycetes. The accumulation of OGs takes place
during the degradation of pectin by microbial polygalacturonases (PGs) and is favoured when PGs
interact with specific PGIPs that are localized in the plant cell wall. In the past years we have studied
the structural characteristics of the bi-molecular complex formed by fungal PGs and plant PGIPs. The
inhibitor is a leucine-rich repeat (LRR) protein and evolves different and specific capabilities of
recognition by changing a few residues of the LRR domain. The structure of the complex may help to
perform structure-guided mutations and design PGIPs with stronger inhibitory activity and improved
recognition capabilities. These may be used to engineer transgenic crops that are naturally protected
against diseases. During 2012 we have tested whether a gene encoding a PGIP protects tobacco
against a fungal pathogen (Rhizoctonia solani) and two oomycetes (Phytophthora parasitica var.
nicotianae and Peronospora hyoscyami f. sp. tabacina).
The trials were performed by using two transgenic lines under greenhouse conditions. The main
symptoms caused by R. solani on wild type tobacco were small stem water-soaked lesions that rapidly
become brown and sunken, primarily at the level of the soil line or closely above it. Lesions
subsequently expanded throughout the stems causing the tissue to turn brown and die. Disease
symptoms (both seedling death and stem rot) were severe on wild type plants and very limited in
transgenic lines. Symptom development coincided with an increase of fungal biomass in the colonized
101
roots of the wild type plants while no significant increase of fungal biomass occurred in transgenic
plants. The two transgenic tobacco lines were also remarkably resistant to the oomycete pathogen P.
parasitica var. nicotianae. At 5-week post-inoculation severe leaf wilting and stem rot symptoms were
observed in the wild type plants, which all died 5 days later. Instead, both transgenic lines remained
healthy and showed a high level of resistance. Transgenic plants also displayed a high level of
resistance to P. hyoscyami f. sp. tabacina, which was comparable to that of Nicotiana species that are
naturally resistant to this oomycete. We concluded that expression of PGIP is a powerful way of
engineering a broad-spectrum disease resistance (Borras-Hidalgo et al., 2012).
Publications
Lionetti V, Cervone F, Bellincampi D. Methyl esterification of pectin plays a role during plant-pathogen
interactions and affects plant resistance to diseases. J Plant Physiol 2012, 169: 1623-30. doi:
10.1016/j.jplph.2012.05.006.
Borras-Hidalgo O, Caprari C, Hernandez-Estevez I, De Lorenzo G, Cervone F. A gene for plant
protection: expression of a bean polygalacturonase inhibitor in tobacco confers a strong resistance
against Rhizoctonia solani and two oomycetes. Front Plant Sci 2012, 3: 268-73. doi:
10.3389/fpls.2012.00268.
Research Group
Giulia De Lorenzo, Daniela Bellincampi,
professors; Simone Ferrari, Benedetta Mattei,
researchers;
Giovanni
Salvi,
Daniela
Pontiggia, Isabel Santori, research fellows;
Manuel Benedetti, Claudia Fabbri, Fedra
Francocci, Vincenzo Lionetti, Daniel Savatin,
Francesca Sicilia, Francesco Spinelli, postdoc fellows; Federico Andreani, Elisa
Bastianelli, Nora Gigli Bisceglia, Giovanna
Gramegna,
Matteo
Gravino,
Vanessa
Modesti, Chiara Paparella, PhD students.
102
Spontaneous generation and evolution of genetic information
Ernesto Di Mauro
℡: +39 06 49912880 - @: [email protected]
Which were the initial reactions that lead to the generation of genetic information? We have
extended the observations that have brought us to establish three principles: 1) all the nucleic bases
necessary for building extant nucleic acids can be synthesized non-enzymatically, from simple
components, with high efficiency and in plausible prebiotic conditions. 2) Nucleic bases evolve, within
the same prebiotic physical-chemical frame, into nucleosides and nucleotides. 3) Polymerization
mechanisms exist leading from spontaneously activated precursors to long nucleic polymers.
As for the spontaneous generation of pre-genetic materials, we have defined the physicalchemical conditions that extend the mechanisms identified so far, passing from the abiotic nonenzymatic synthesis of homogenous polymers to the generation of complex sequences.We have
experimentally established that it is possible to generate spontaneously RNA sequence complexity,
based on intrinsic properties of the RNA structure. The general question that we now ask: Is it possible
to correlate the generation of RNA complexity to the origin of the protein coding process? And: is it
possible to correlate the development of (proto)genetic systems with the development of
(proto)metabolic systems?
The data obtained provide the following answers:
it is actually possible to generate purine RNA sequences in abiotic conditions. We have reported
the spontaneous polymerization of 3’,5’ cyclic GMP in water, in formamide, in dimethylformamide,
and in water in the presence of a Brönsted base such as 1,8-diazabicycloundec-7-ene. The
reaction is untemplated, does not require enzymatic activities, is thermodynamically favoured and
selectively yields 3’,5’-bonded ribopolymers as long as 25 nucleotides.
A reaction pathway is proposed, based on (i) the measured stacking of the 3’,5’-cyclic monomers,
(ii) the activation by Brönsted bases, (iii) the determination by MALDI ToF Mass Spectrometry, by 31P
NMR, and by specific Ribonucleases of the molecular species produced. The reaction pathway meets
several of the attributes of a click-like reaction. The products undergo RNA sequence
complementarity-driven nonenzymatic ligation, thus leading to RNA chains as long as 100 units.
We have previously reported two reactions of RNA G:C sequences occurring non-enzymatically in
water in the absence of any added cofactor or metal ion: (a) sequence complementarity-driven
terminal ligation; (b) complementary sequence adaptor-driven multiple tandemization. The two abiotic
reactions increase the chemical complexity of the resulting pool of RNA molecules and change the
Shannon information of the initial population of sequences.
Taken together, these studies show how spontaneously generated short oligonucleotides may
progress along the path of generation of longer sequences. The possibility of introducing “errors” in the
abiotic replication of RNA sequences has been shown, suggesting evolutionary mechanisms in the
absence of complex phenotypes.
Relevant results pertain to the identification of the products of prebiotic synthesis of organic
compounds from formamide in the presence of specific classes of catalysts like boron-containing
minerals and minerals from the Murchison meteorite. In addition to nucleic bases, complex population
of carboxylic acids were observed, showing the possibility that in the same test-tube the precursors
formed of both RNA and key metabolic cycles. The rational of these studies has been published.
103
The
picture
shows
the
spontaneous
polymerization of 3′,5′-cyclic GMP occurring in
water, in formamide, in dimethylformamide, and
(in water) in the presence of a Brønsted base
such as 1,8-diazabicycloundec-7-ene. The
polymerization
occurs
among
stacked
monomers, is thermodynamically favoured and
selectively yields 3′,5′-bonded ribopolymers. For
further details see Costanzo et al., 2012.
Publications
Costanzo G, Saladino R, Botta G, Giorgi A, Scipioni A, Pino S, Di Mauro E. Generation of RNA
molecules by a base-catalysed click-like reaction. Chembiochem 2012, 13: 999-1008. doi:
10.1002/cbic.201200068.
Saladino R, Botta G, Pino S, Costanzo G, Di Mauro E. From the one-carbon amide formamide to RNA
all the steps are prebiotically possible. Biochimie 2012, 94: 1451-56. doi: org/10.1016/
j.biochi.2012.02.018.
Saladino R, Botta G, Pino S, Costanzo G, Di Mauro E. Genetics first or metabolism first? The
formamide clue. Chem Soc Rev 2012, 41: 5526-65. doi: 10.1039/c2cs35066a.
Saladino R, Crestini C, Pino S, Costanzo G, Di Mauro E. Formamide and the origin of life. Phys Life
Rev 2012, 9: 84-104. doi:10.1016/j.plrev.2011.12.002.
Research Group
Collaborations
Giovanna
Costanzo,
CNR
researcher,
Samanta Pino, post-doc fellow; Silvia Lopizzo,
technician.
Raffaele Saladino, Dip. ABAC, Università della
Tuscia, Viterbo; Claudia Crestini, Università
Tor Vergata, Roma; Edward N. Trifonov,
University of Haifa, Israel.
104
Molecular and functional approaches to investigate the
neuroprotective and neuromodulatory roles of chemokines and
their receptors in the central nervous system
Cristina Limatola
Department of Physiology and Pharmacology "Vittorio Erspamer"
℡: +39 06 49690243 - @: [email protected]
The main aim of our research project is the description of the molecular mechanisms involved in
the neuroprotective activity of chemokines in the central nervous system, with particular interest in the
investigation of the role of glial cells and their communication with neuronal cells. The main results
obtained in the last year can be summarized as follows:
Neuroprotective effects of CXCL16: role of astrocytic A3R
We completed our study on the neuroprotective effect of CXCL16 in in vitro model of glutamateinduced excitotoxicity (Rosito et al., 2012). In this work we describe the mechanism of neuroprotection
induced by CXCL16 demonstrating that CXCL16-stimulated astrocytes release the chemokine
monocyte chemoattractant protein-1 MCP-1/CCL2, with a mechanism requiring adenosine receptor
type 3 (A3R) activation. We are now continuing these experiments in vivo, in a mouse model of
cerebral ischemia (permanent mean cerebral occlusion, pMCAO), and preliminary experiments
demonstrate that intracerebroventricular infusion of CXCL16 reduced the total ischemic brain volume
in mice upon pMCAO, see figure.
Contribution of astocytes to the neuroprotective effects of
fractalkine.
In 2012 we continued the study of the neuroprotective
activity of fractalkine in models of excitotoxicity in hippocampal
mixed cultures, demonstrating that fractalkine increased both
the expression level and the activity of two subtypes of
excitatory aminoacid transporters (EAATs), GLT-1 and GLAST,
but that only GLT-1 activation is important for the
neuroprotective activity. In particular we described that
fractalkine activates CX3CR1 on microglia, releasing
adenosine, with the consequent activation of A1R on astrocytes
and increased uptake of extracellular glutamate by astrocytes.
These results are now under second revision in the scientific
journal “Glia”.
We also investigate the effect of fractalkine on NMDA
receptor activity in acute brain slices obtained from the
hippocampal region of mice, with electrophysiological
recordings of field excitatory postsynaptic potential (fEPSP). We
demonstrated in the past that AMPAR-mediated currents are
depressed by fractalkine treatment through the specific
dephosphorylation of Ser 845 on the GluR1 subunit of AMPAR
(Ragozzino et al. 2006). Few information are available on
chemokine-mediated modulation of another important Glu
105
receptor, the NMDAR, a very important mediator of synaptic transmission in the brain. We obtained
data demonstrating that fractalkine induces the release of D-serine by glia (measured by HPLC-MS
analysis), and that this D-serine is involved in the potentiation of NMDAR-mediated current, acting at a
Glu-co-agonist site. The potentiating effects of fractalkine are indeed abolished by serine racemase,
an enzyme that degrades extracellular D-Ser. In addition fractalkine effects are mimicked by A2AR
agonist VT7 (synthesized by Gloria Cristialli, at Camerino University), abolished by specific A2AR
antagonist, and absent in A2AR-/- KO mice, demonstrating that also for this effect the action of
fractalkine does require the presence of extracellular adenosine and the activation of specific
adenosine receptors. These results have just been submitted for publication in the “Journal of
Neuroinflammation”.
We also participate to an international study aimed at investigating the role of adenosine kinase in
modulating synaptic activity. In particular we investigated the effect of modulating extracellular level of
adenosine (in different transgenic mice which either over-express (fb-tg ADK) or have a reduced
(ADK-def) expression of ADK in specific forebrain regions (generated by Detlev Boison, Portland
University), demonstrating that both GABAergic transmission (run down of GABAAR-mediated
currents) and glutamatergic transmission (fEPSP) are modulated by local alteration of extracellular
adenosine content. These results have been published in “Cerebral Cortex” (Diógenes et al., 2012).
Publications
Bertollini C, Murana E, Mosca L, D'Erme M, Scala F, Francioso A, Catalano M, Limatola C,
Bregestovski P, Di Angelantonio S, Ragozzino D. Transient increase in neuronal chloride
concentration by neuroactive aminoacids released from glioma cells. Front Mol Neurosci 2012, 5:
100. doi: 10.3389/fnmol.2012.00100.
Diógenes MJ, Neves-Tomé R, Fucile S, Martinello K, Scianni M, Theofilas P, Lopatár J, Ribeiro JA,
Maggi L, Frenguelli BG, Limatola C, Boison D, Sebastião AM. Homeostatic Control of Synaptic
Activity by Endogenous Adenosine is Mediated by Adenosine Kinase. Cereb Cortex 2012, Sep 20.
doi: 10.1093/cercor/bhs284.
Palma E, Conti L, Roseti C, Limatola C. Novel approaches to study the involvement of a7-nAChR in
human diseases. Curr Drug Targets 2012, 13; 579-63. doi: 10.2174/138945012800398838.
Rosito M, Deflorio C, Limatola C, Trettel F 2012 CXCL16 orchestrates adenosine A3 receptor and
MCP- 1/CCL2 activity to protect neurons from excitotoxic cell death in the CNS. J Neurosci 2012,
32: 3154-63. doi: 10.1523/JNEUROSCI.4046-11.2012.
Research Group
Collaborations
Francesca Grassi, Sergio Fucile, Eleonora
Palma, Davide Ragozzino, professors; Myriam
Catalano, Flavia Trettel, researchers; Raffaela
Cipriani, Clotilde Lauro, post-doc fellows;
Grimaldi Alfonso, Stefano Garofalo, PhD
students; Giuseppina Chece, technician.
Maria Grazia De Simoni, Pia Villa, Istituto
Mario Negri, Milano; Letizia Antonilli,
Dipartimento di Fisiologia e Farmacologia,
Sapienza Università di Roma; Bertil Fredholm,
Karolinska Institute, Stockholm, Sweden;
Angelo Spinedi, Università di Roma Tor
Vergata; Heike Wulff, Davis University,
California, USA; Gloria Cristalli, Università di
Camerino; Detlev Boison, Portland University,
USA.
106
Neurone response to experimental injury and lack of dystrophin: a
molecular, functional and structural study in autonomic ganglia in
vivo and in vitro
Paola Paggi
℡: +39 06 49912323 - @: [email protected]
This research project analyzes the molecular mechanisms involved in the establishment,
maintenance and plasticity of neural connections and circuitry by using, as main experimental model,
the superior cervical ganglion (SCG) of wild-type (WT) and genetically dystrophic mdx mice, an animal
model of Duchenne muscular dystrophy (DMD). In the year 2012 we focused on some of the relevant
pathological changes produced by lack of full-length dystrophin (Dp427), a protein of the cortical
cytoskeleton absent in DMD patients and mdx mice, by investigating the following aspects:
a) analysis of gene expression in the SCG of WT and mdx mice of different postnatal dates. Based
on our previous observations, describing development-related alterations in mdx mouse ganglia, we
verified whether lack of Dp427 affects transcript levels of genes related to aspects of neuron
development and differentiation. Ontological Analysis of more than 500 modulated genes showed
significant differences in genetic class enrichment at each postnatal date. Up-regulated genes mainly
fell in the categories of vesicular trafficking, cytoskeletal and synaptic organization, whereas downregulated genes were associated with axon development, growth factors, intracellular signal
transduction, gene expression regulation, synapse morphogenesis and nicotinic receptor clustering. In
particular, down-regulation of genes related to trophic factor signalling pathways and involved in axon
growth and differentiation suggest that lack of Dp427 could affect trophic factor sensitivity and axon
growth dynamics.
b) neuron-target organ interplay and analysis of axon growth and regeneration, both in vivo and in
vitro. To unravel the question whether Dp427 played a role in axon growth dynamics, WT and mdx
mice SCG neurons were axotomized to reactivate development-related mechanisms of axon
elongation. Levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis
localized along noradrenergic neuron axons and nerve terminals, were analyzed in the iris and
submandibular gland (SCG muscular and non muscular target, respectively), as an indicator of
peripheral axon degeneration/regeneration. TH levels decreased soon after axonal damage in both
target organs. However, while those in the mdx mouse iris never recovered, at least up to 60 days
after crush, suggesting lack of reinnervation, those in the submandibular gland, which is particularly
rich in nerve growth factor (NGF), similarly recovered in both genotipes. To define whether NGF
modulation of axon growth and regeneration was different between WT and mdx mice, we examined
neurite elongation of SCG neurons grown in vitro in the presence of different concentrations of NGF
(10 ng/ml to 100 ng/ml). Neurite number and length positively correlated with NGF concentration in
both genotypes; however, both parameters were always significantly lower for mdx mouse neurons
respect to WT. Accordingly, levels of phosphorylated TrkA and kinases downstream to NGF signalling
were also reduced. A parallel study of SCG neuron axotomy in vitro, confirmed a reduction in the
number of regenerated axons in mdx mouse neuron cultures, compared to WT, in the presence of
limiting NGF concentrations (10 ng/ml). Our data suggest that lack of Dp427, which mediates
cytoskeleton-extracellular matrix linkage, interferes with NGF signal transduction, affecting axon
107
growth and regeneration. These results, presented to national and international meetings, are object of
a manuscript in preparation.
A study was also conducted on an other model of neuronal degeneration/regeneration: i.e. mice
and non-human primates injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MTPT), elective
experimental models of Parkinsonism, in which degeneration of the nigro-striatal dopaminergic
pathway is associated with prominent neuroinflammation in both substantia nigra and striatum. The
study, besides having validity on the specific topic of Parkinson’s disease, poses the ground for future
studies concerning the possibility of axonal regeneration in central neural circuits following
oligodendrocyte (OL) activation. We performed a qualitative and quantitative analysis at the confocal
microscope of OL-associated changes induced by MPTP treatment, demonstrating an increase in OL
cell number and average size, which parallels the reduction in striatal dopaminergic innervation. This
event may result in an important, although neglected, aspect in the onset and progression of
Parkinsonism.
Publications
Annese V, Barcia C, Ros-Bernal F, Gómez A, Ros CM, De Pablo V, Fernández-Villalba E, De Stefano
ME, Herrero MT. Evidence of oligodendrogliosis in 1-methyl-a-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-induced Parkinsonism. Neuropathol Appl Neurobiol 2012 Epub. doi: 10.1111/j.13652990.2012.01271.x.
Licursi V, Caiello I, Lombardi L, De Stefano ME, Negri R, Paggi P. Lack of dystrophin in mdx mice
modulates the expression of genes involved in neuron survival and differentiation. Eur J Neurosci
2012, 35: 691-701. doi:10.1111/j.1460-9568.2011.07984.x.
Research Group
Collaborations
Maria Egle De Stefano, professor, Silvia Di
Angelantonio, researcher, Valerio Licursi,
Loredana Lombardi, post-doc fellows.
Tamara Petrucci, Istituto Superiore di Sanità,
Laboratorio di Biologia Cellulare, Roma; Cecilia
Gotti, CNR, Istituto di Neuroscienze, Centro di
Farmacologia Cellulare e Molecolare, Dip. di
Farmacologia Medica, Università di Milano;
Irene Bozzoni, Ernesto Di Mauro, Andrea
Mele, Rodolfo Negri, Alberto Oliverio,
Dipartimento di Biologia e Biotecnologie “C.
Darwin”, Sapienza Università di Roma; Carla
Perrone Capano, Dipartimento di Scienze
Biologiche, Università di Napoli “Federico II”;
Casper Hoogenraad, Erasmus Medical
Center,
Department
of
Neuroscience,
Rotterdam, The Netherlands; Falk Schroedl,
Paracelsus University of Salzsburg, Austria.
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Molecular mechanisms integrating endocytosis and signalling of
fibroblast growth factor receptors
Maria Rosaria Torrisi
Department of Clinical and Molecular Medicine
℡: +39 06 33775502 - @: [email protected]
Endocytosis represents one of the main mechanisms of attenuation of the signal transduction and
the endocytic machinery and signalling of receptor tyrosine kinases (RTKs) appear to be intimately
linked. In contrast to most of the RTKs, the keratinocyte growth factor receptor (KGFR/FGFR2b)
appears to play an unique role in epithelial homeostasis, controlling proliferation and differentiation
and exerting a tumor suppressive function in vitro and in vivo. KGFR endocytosis is clathrindependent, but once internalized the receptors may follow distinct endocytic pathways. Therefore,
KGFR/FGFR2b represents a model system to shed light on the molecular mechanisms which control
endocytic trafficking and receptor signaling. A consistent effort of the second year of the project was
aimed to analyze the possible role of KGFR/FGFR2b expression, ligand-induced activation and
receptor signaling in the control of the autophagic process in human keratinocytes, strongly
encouraged by the results obtained during the first year on the contribution of the receptor in
regulating phagocytosis (Belleudi et al. FASEB J. 2011). We found that KGFR signaling triggers the
formation of LC3-positive autophagosomes and stimulates their fusion with lysosomes for degradation.
In addition, because autophagy appears to be involved in keratinocyte differentiation as a crucial selfdegrading mechanism necessary for the phenotypic switch from basal to suprabasal cells and we
have demonstrated that KGFR/ FGFR2b expression represents a key event regulating keratinocyte
early differentiation (Belleudi, Purpura et al., PLoS One 2001), we investigated the receptor-mediated
triggering of autophagy also in differentiating keratinocytes: our findings show that both autophagy and
differentiation are increased upon cell stratification, suggesting the existence of a tight molecular
crosstalk among the two processes and the key role played by KGFR/FGFR2b in such interplay
(Belleudi et al. in preparation). Finally, since KGFR/FGFR2b is altered in its signaling and endocytic
traffic in undifferentiated keratinocytes expressing the E5 protein encoded by high-risk human
papillomavirus (HPV) type 16 (Belleudi et al, Oncogene 2011), we focused on the possible interplay of
16E5 with the receptor forcing the overexpression or depletion of KGFR in E5 expressing human
keratinocytes under synchronous waves of differentiation. We found that KGFR expression as well as
triggering of its kinase activity and signaling are able to efficiently counteract, through a decrease of
p63, the E5-induced impairment of differentiation, suggesting that the opposite tumor-suppressive
action of KGFR and oncogenic role of E5 might both involve p63 (Purpura, Belleudi submitted).
RTKs are targets of a growing number of monoclonal antibodies (mAbs) used for the treatment of
different types of cancer and many of them are directed against members of the ErbB receptor family:
because the anti-tumor effect of such antibodies is mostly due to their ability to down-regulate receptor
expression through the triggering of antibody-mediated endocytosis and degradation, in collaboration
with the group of Prof. Ciliberto (University of Catanzaro "Magna Grecia") we have evaluated the
potential therapeutic efficacy and the mechanisms of action of a set of three recently generated antihuman ErbB3 monoclonals (mAbs) in human melanoma cells. We demonstrated that, while receptor
ligand activates the PI3K/AKT pathway leading to increased cell proliferation and migration, two of the
109
antibodies are able to potently inhibit ligand-induced signalling, proliferation and migration and that
their efficacy strongly correlates with antibody-induced receptor internalization, degradation and
inhibition of receptor recycling to the cell surface (Belleudi et al., Cell Cycle 2012). We are currently
testing the combination of these mAbs on melanoma cell growth and receptor signalling/endocytosis:
preliminary experiments indicate that their combination induces a surprisingly efficient receptor
ubiquitination, fast internalization and durable degradation as well as inhibition of signal transduction.
These findings have been presented to recent meetings (Fattore et al. FISV 2012, Roma; SIC 2012,
Bologna) and further studies will be aimed to identify the molecular mechanisms responsible for mAbinduced ErbB3 intracellular trafficking and degradation.
Publications
Belleudi F, Marra E, Mazzetta F, Fattore L, Giovagnoli MR, Mancini R, Aurisicchio L, Torrisi MR,
Ciliberto G. Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits
growth and migration of human melanoma cells. Cell Cycle 2012, 11: 1455-67. doi:
10.4161/cc.19861.
Leone L, Vetrano M, Ranieri D, Raffa S, Vulpiani MC, Ferretti A, Torrisi MR, Visco V. Extracorporeal
Shock Wave Treatment (ESWT) Improves In Vitro Functional Activities of Ruptured Human TendonDerived Tenocytes. PLoS One 2012, 7:e49759. doi: 10.1371/journal.pone.0049759.
Raffa S, Leone L, Scrofani C, Monini S, Torrisi MR, Barbara M. Cholesteatoma-associated fibroblasts
modulate epithelial growth and differentiation through KGF/FGF7 secretion. Histochem Cell Biol
2012, 138: 251-69. doi: 10.1007/s00418-012-0947-y.
Research Group
Patrizia Mancini, Maurizio Alimandi, Lavinia
Lotti, Vincenzo Visco, professors, Francesca
Belleudi, Salvatore Raffa, researchers; Valeria
Purpura, Danilo Ranieri, post-doc fellows;
Luigi
Fattore,
PhD
student;
Antonio
Sabatucci, technician.
110
Area 5
Cellular and molecular
immunology
Area 5: Cellular and molecular immunology
Interplay amongst chronic immune activation, apoptosis, crosspresentation, immune-regulation, and autoimmunity
Vincenzo Barnaba
Department of Internal Medicine and Medical Specialties
℡: +39 06 491268 - @: [email protected]
Apoptotic antigen-specific T cell responses in HCV infection
Chronic inflammatory diseases are characterized by continuous cycles of T cell activation,
subsequent T cell apoptosis and self apoptotic antigen-specific T cell responses, whose role in
maintaining chronic inflammation has been recently proposed (Moroni Rawson et al, Nat Med, 2007).
We have already identified a huge number of self-peptides derived from caspase-cleaved proteins that
in turn had been previously identified by proteomic technologies. Recently, we demonstrated the high
avidity CD8 responses against epitopes derived from caspase-cleaved proteins in individuals acutely
infected with HCV: these patients show high frequencies of polyfunctional auto-reactive CD8 cells
producing IFN-γ, IL-17, IL-4, IL-2 that are correlated with progression towards chronic infection. The
responses were directly correlated with the plasma viral load or the serum ALT levels, and were then
sustained over time in relation to the viral persistence. Our parallel study indicates that similar
autoreactive CD8+ T cell responses in chronically infected patients are recruited in the inflamed livers,
are related with the signs of hepatic damage, and decrease in relation with the decline or the
disappearance of the viral load upon antiviral therapy (interferon plus ribavirin@).
Apoptotic antigen-specific T cell responses in multiple sclerosis
In this project, we evaluated if the autoreactive responses described above sustains autoimmune
diseases such as multiple sclerosis (MS), whether they play a role in the outcome of this disease, and
if they may be used as biomarkers to monitor the efficacy and safety of biological drugs or other antiinflammatory drugs in course of clinical development. Our preliminary data demonstrated that selfspecific CD8+ T cells as detected by pentamers of HLA-A*0201 molecules complexed to apoptotic
(MYH9478-486, MYH9741-749, VIME78-87, VIME225-234, LAM388-397) epitopes that had been
previously identified as the most immunogenic among all patients tested, were significantly higher in
HLA-A2+ MS patients than in HLA-A2+ healthy donors. In addition, we observed that apoptotic
epitope-specific CD8+pentamer+ T effector memory (EM) cells promptly produced moderate amounts
of all the cytokines tested (IL-17, IFN-γ, GM-CSF, IL-4, IL-2, IL10), in MS patients. In particular, we
found that CD8+ TEM cell responses directed to apoptotic self-epitopes decreased after one month of
therapy, in relation with the disease activity reduction. Therefore, the detection of these autoreactive
CD8+ T cells may be relevant in determining whether the contraction or the quality variation of the
related polyfunctional responses can be used as predictive biomarkers to verify the protective effects
of conventional or innovative biological therapies.
Cytokines and costimulatory receptors in regulatory T cell plasticity in HCV-related liver inflammation
and tumor
Several evidences suggest that the pool of regulatory T cells (Treg) is functionally far more
heterogeneous and plastic than so far perceived. Treg are highly susceptible to microenvironmental
113
cues, which may finely tune their in vivo behavior. Our results indicate Treg adaptation to different
microenvironments. Indeed, few Treg can be detected in tumor-free poorly cirrhotic liver tissue,
expressing Foxp3 at low levels, but significantly expressing PD-1 and secreting IFN-γ ex vivo. On the
contrary, HCC and tumor-free highly cirrhotic liver tissue are infiltrated by abundant Treg that express
Foxp3 at higher levels and poorly secrete IFN-γ, despite a high expression of the Th1-related factor Tbet. Interestingly, these Treg upregulate OX40 expression, which is not constitutive in human Treg
cells. OX40 expression and IFN-γ secretion are mutually exclusive, suggesting that OX40 correlates
with protection from Treg deprogramming into a Th1-direction. OX40L, the ligand for OX40, can be
detected on a variety of immune cells, including T cells and plasmacytoid dendritic cells, at the HCC
site. These observations have been recapitulated in experiments in vitro. Indeed, Treg enriched from
peripheral blood of healthy donors upregulate OX40 when stimulated with IL-2 and/or TNF-α in vitro.
Importantly, these OX40+ Treg are protected from the acquisition of IFN-γ production when cultured
under a Th1 cytokine cocktail. Moreover, OX40 stimulation in vitro boosts Treg suppressive function
and proliferation. Overall these results support the notion that Treg can reprogram their functions
toward suppression or inflammation in different contexts in HCV-infected individuals and that HCVrelated cirrhosis may orchestrate a network of immunosuppressive signals fostering Treg expansion
and favoring tumorigenesis.
Publications
Spadaro F, Lapenta C, Donati S, Abalsamo L, Barnaba V, Belardelli F, Santini SM, Ferrantini M.
Interferon-alpha enhances cross-presentation in human dendritic cells by modulating antigen
survival, endocytic routing and processing. Blood 2012, 119: 1407-17. doi: 10.1182/blood-2011-06363564.
Franceschini D, Del Porto P, Piconese S, Trella E, Accapezzato D, Paroli M, Morrone S, Piccolella E,
Spada E, Mele A, Sidney J, Sette A, Barnaba V. Polyfunctional type-1, -2, and -17 CD8+ T cell
responses to apoptotic self-antigens Correlate with the Chronic Evolution of Hepatitis C Virus
Infection. Plos Pathog 2012, 8:e1002, 759. doi: 10.1371/journal.ppat.1002759.
Magnacca A, Persiconi I, Nurzia E, Caristi S, Meloni F, Barnaba V, Paladini F, Raimondo D, Fiorillo
MT, Sorrentino R. Characterization of a Proteasome and TAP-independent Presentation of
Intracellular Epitopes by HLA-B27 Molecules. J Biol Chem 2012, 287: 30358-67. doi:
10.1074/jbc.M112.384339.
Research Group
Daniele Accapezzato, Silvia Piconese,
researchers;
Alessandra
Citro,
Chiara
Focaccetti, Helene Martini, Valeria Schinzari,
post-doc fellows; Carmela Martire, Eleonora
Timperi, PhD students; Fabio Palombo, visiting
scientist.
114
Signalling events negatively regulating FcεRI expression and mast
cell functional responses
Rossella Paolini
℡: +39 06 49255670 - @: [email protected]
Mast cells are key effectors in allergic diseases: upon ligation of the high affinity receptor for IgE
(FcεRI) they release preformed and newly synthesized pro-inflammatory mediators.
FcεRI is composed of an IgE-binding α chain, and the ITAM-containing β and γ subunits. Upon
FcεRI cross-linking, the β chain-associated Src family PTK Lyn, phosphorylates β and γ-chain ITAMs
allowing the recruitment and consequent activation of Syk, responsible for signal propagation (Nadler
et al., Adv immunology 2000).
Besides these positive signals, FcεRI engagement has recently been understood to generate
negative intracellular signals capable of limiting the rate and the extent of mast cell functional
responses (Molfetta et al., Arch Immunol Ther Exp 2007). Relevant to this, others and we have
demonstrated that the Cbl family proteins control the amplitude of FcεRI-generated signals by specific
ubiquitin modification of activated receptor subunits and associated protein tyrosine kinases (Paolini et
al., J Biol Chem 2002, Kio et al., Genes Cells 2003).
Concurrently, engaged receptors trigger their own endocytosis to extinguish signaling through
removal of activated FcεRI complexes from the cell surface and delivery to lysosomes for degradation
(Molfetta et al., J Immunol 2005).
The present study was aimed at identifying the molecular mechanisms ensuring the clearance of
antigen-stimulated FcεRI complexes from the cell surface, thus contributing to the termination of mast
cell functional program.
We initially demonstrated that FcεRI subunits are monoubiquitinated by c-Cbl at multiple sites
upon stimulation, and provided evidence for a role of ubiquitin as a signal regulating the initial step of
internalization (Molfetta et al., PLoS One 2009). We have then demonstrated that ubiquitination of
activated receptors controls receptor internalization and sorting along the endocytic compartments
through the action of ubiquitin-binding adapters (Molfetta et al., Mol Immunol 2010; Gasparrini et al.,
Int Arch Allergy Immunol 2011). Notably, we have envisaged a critical role for Hrs in controlling the
fate of internalized receptor complexes: Hrs depletion retains ubiquitinated receptors into early
endosomes and partially prevents their sorting into lysosomes (Molfetta et al., PLoS One 2009).
Our research activity during 2012 has been mainly focused on understanding how Hrs function is
regulated upon FcεRI engagement. To this purpose, we initially investigated whether Hrs undergoes
antigen-dependent phosphorylation and ubiquitination, since previous evidence have shown that Hrs
is subjected to covalent modifications upon ligand-stimulation of growth factor receptors (Bache et al., J
Cell Biol 2003). We demonstrated a basal level of Hrs tyrosine phosphorylation and monoubiquitination
that increase upon FcεRI engagement (Gasparrini et al., Eur J Immunol 2012).
Additionally, by RNA inteference we identified Syk as the main kinase regulating both inducible
Hrs covalent modifications, and c-Cbl as the E3 ligase required for inducible Hrs monoubiquitination.
Finally, we used biochemical approaches to demonstrate that Syk-dependent regulation of Hrs
covalent modifications, without affecting protein stability, controlled Hrs localization. Indeed, the
115
majority of phosphorylated Hrs forms are observed only in membrane compartments, whereas
ubiquitinated Hrs are predominantly cytosolic, suggesting that both modifications might impact on Hrs
function. All together our findings are compatible with the scenario depicted in Figure 1.
Upon antigen stimulation ubiquitinated FcεRI complexes are recognized by Hrs that becomes a
substrate for Syk and Cbl enzymatic activities. Phosphorylated Hrs interacts with other endocytic
adapters of the ESCRT complex in order to ensure an efficient transport of ubiquitinated cargos,
whereas monoubiquitinated Hrs is removed from endosomal sorting sites as inactive pool. In this
scenario, Hrs phosphorylation would serve to allow protein/protein interactions while Hrs
monoubiquitination to relocate the adaptor from endosomes to the cytosol, without promoting
degradative events.
Although additional experiments are required to validate our
model, we demonstrate for the first time that engagement of an
immune receptor, namely FcεRI, has the potential to trigger Hrs
phosphorylation and monoubiquitination, and that both inducible
modifications require Syk kinase activity. From a broader cell
biological perspective, this finding could be extended to include
other immune receptors, such as the TCR and BCR, providing a
novel regulatory mechanism used by the Syk family kinases to
attenuate immune responses in mammalian cells.
Fig. 1 - Model depicting how Hrs regulates endocytosis of ubiquitinated FcεRI
complexes. Upon antigen stimulation, the combined action of Syk and Cbl,
recruited to phosphorylated FcεRI as a preformed complex, leads to receptor
multiubiquitination. Ubiquitinated FcεRI complexes are then recognized by Hrs
that becomes a substrate for Syk and Cbl enzymatic activities.
Monoubiquitinated Hrs is removed from endosomal sorting sites whereas
phosphorylated Hrs interacts with other endocytic adapters of the ESCRT
complexes in order to ensure an efficient transport of ubiquitinated FcεRI
complexes to the multivesicular bodies (MVBs) and to the lysosomes for
degradation. ESCRT, endosomal sorting complex required for transport.
Publications
Capuano C, Paolini R, Molfetta R, Frati L, Santoni A, Galandrini R. PIP2-dependent regulation of
Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway. Blood 2012, 119: 2252-62.
doi: 10.1182/blood-2010-12-324160.
Gasparrini F, Molfetta R, Quatrini L, Frati L, Santoni A, Paolini R. Syk-dependent regulation of Hrs
phosphryaltion and ubiquitination upon FcεRI-engagement: impact on Hrs membrane/cytosol
localization. Eur J Immunol 2012, 42: 2744-53. doi: 10.1002/eji.201142278.
Research Group
Rosa Molfetta, researcher; Cristina Capuano;
post-doc fellow; Linda Quatrini, PhD student;
Beatrice Zitti, student.
116
Anti-tumor pathways mediated by innate immune responses
Anna Riccioli
℡: +39 06 49766585 - @: [email protected]
Toll like receptors (TLRs) are a family of transmembrane proteins recognizing structures
conserved among microbial species which are called pathogen associated molecular patterns
(PAMP). TLRs are potent activators of innate immune responses favouring pathogen elimination
(Takeuchi, Cell 2010).
TLRs are also expressed on tumour cells, where they may influence tumor growth and host
immune responses (Huang, Oncogene 2008). The use of specific TLR agonists alone or in
combination with standard chemo or radio-therapy has been shown to have a valid anti-cancer activity
in different in vitro or in vivo cancer models and several molecules have been tested in clinical trials
(clinicaltrials.gov) (Galluzzi, Oncoimmunology 2012).
We have previously reported that the TLR-3 agonist poly(I:C) elicits inhibition of proliferation
associated with a marked induction of TLR3-mediated apoptosis in LNCaP cells, an androgendependent human prostate cancer (PCa) cell line (Paone, Carcinogenesis 2008).
More recently, to confirm in in vivo anti-tumour effects obtained with poly (I:C) treatment in vitro,
we assessed tumour growth in a LNCaP-based xenograft model. 4 million LNCaP cells mixed with
Matrigel at a ratio of 1:1 were inoculated s.c. into the flank of 7-8 week-old male NOD scid gamma
mice (NSG). The NSG mouse is severely immunocompromised, and lacks mature T, B and NK cells.
NSG mice strain is the suitable xenograft model to demonstrate the crucial role of direct action of
poly(I:C) on cancer cells growth, excluding the possible antitumour immune-mediated response.
Animals were randomly divided into 3 groups when tumors reached a volume of 100-200 mm3, 10
mice each, according to tumor size, and treated one group with drug vehicle only as control, the other
2 groups with intraperitoneal (i.p.) or intratumoral (i.t.) injection of 250 microg/mouse of poly(I:C) 3
times weekly for 3 weeks. Our results show that tumour growth was severely impaired by i.p. and i.t.
poly(I:C) administration. Currently, tumour fragments will be processed for immunohistochemical and
immunobiochemical analysis of cell cycle and apoptotic marker profile.
A further research proposal is aimed at investigating autophagy modulation as a potential tool to
increase TNF-alpha dependent-apoptosis in human PCa cell lines with different aggressiveness.
TNF-alpha levels in PCa correlate with the extent of disease and are significantly elevated in the
metastatic stage. TNF receptor superfamily controls two distinct signalling cascades, leading to
opposite effects, i.e. apoptosis and survival; in PCa TNFalpha-mediated signalling induces cell survival
and resistance to therapy (Srinivasan, Apoptosis 2010). Experimental strategies aimed at improving
the proapoptotic effect of TNF-alpha in PCa are under investigation. Autophagy controls degradation
of cellular components and turnover of long-lived proteins and organelles, thus maintaining cell
homeostasis. The apoptosis of PCa cells LNCaP and PC3 was investigated upon treatment with the
autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin, in combination with
TNFalpha. Autophagy was assessed via LC3 Western blot analysis; propidium iodide and TUNEL
stainings followed by flow cytometry or caspase-8 and caspase-3 activation assays were performed to
evaluate apoptosis. TNF-alpha-induced apoptosis was enhanced by 3-methyladenine in the androgen-
117
responsive LNCaP cells, whereas no effect was observed in the androgen-insensitive PC3 cells.
Cellular-Flice-like inhibitory protein (c-Flip) plays a key role since its expression controls the resistance
to TNF-induced apoptosis. Interestingly, pro-apoptotic effect observed in LNCaP cells was associated
with reduced c-Flip levels through proteasomal degradation via increased reactive oxygen species
production and p38 activation. Conversely in PC3 but not in LNCaP cells, rapamycin stimulated TNFalpha-dependent apoptosis; such effect was associated with reduced c-Flip promoter activity. We
therefore conclude that the resistance to TNF-dependent pro-apoptotic effects may be decreased or
reversed, in PCa cells through autophagy modulators leading to c-Flip down-regulation (Giampietri,
Apoptosis 2012).
Publications
Giampietri C, Petrungaro S, Padula F, D’Alessio A, Marini ES, Facchiano A, Filippini A, Ziparo E.
Autophagy modulators sensitize prostate epithelial cancer cell lines to TNF-alpha-dependent
apoptosis. Apoptosis 2012, 17: 1210-22. doi: 10.1007/s10495-012-0752-z.
Giampietri C, Petrungaro S, Facchiano A, Filippini A, Ziparo E. Therapeutic implications of autophagy
modulation in prostate cancer. J Endocrinol Invest 2012, 35: 945.
D’Alessio A, Esposito B, Giampietri C, Ziparo E, Pober JS, Filippini A. Plasma membrane
microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells. J Cell Mol
Med 2012, 16: 627-36. doi: 10.1111/j.1582-4934.2011.01353.x.
Research Group
Collaborations
Elio Ziparo, Antonio Filippini, professors;
Claudia Giampietri, post-doc fellow; Sara
Palchetti, PdD student; Fabrizio Padula,
Simonetta Petrungaro, Donatella Starace,
technicians.
Paola De Cesaris, Dipartimento di Scienze
Cliniche Applicate e Biotecnologiche, Università
de L’Aquila.
118
Anti-tumor effector functions of NK cells in tumor microenviroment
Angela Santoni
℡: +39 06 44340632 - @: [email protected]
Our research activity is focused on the analysis of the effect of low doses of chemotherapeutic
drugs, in the regulation of NK cell recognition and eradication of multiple myeloma (MM) cells in in vitro
and in vivo models. In addition, we are focusing our attention on the analysis of the influence of the
tumor microenvironment on the chemotactic and effector function of NK cells.
We previously showed that low doses of chemotherapeutic drugs up-regulate NKG2D and DNAM1 ligands on MM cells in a ATM/Chk1/2- dependent and p53-independent manner, and this event is
triggered by ROS-dependent activation of DNA Damage Response. The chemotherapeutic drugs we
used, namely doxorubicin and melphalan, promoted the acquisition of a senescent phenotype as
demonstrated by cell cycle arrest in G2/M phase and by high level of activity of beta-galattosidase
(βGalhigh ) in the majority of MM cells.
During the last year of research we evidenced that surface NKG2DL and DNAM-1L expression
was more prominent on βGalhigh cell population. By performing RT-PCR assay we demonstrated that
ligand mRNA expression was also mainly up-regulated on senescent cells. In addition, we
demonstrated a preferential capacity of NK cells to degranulate in response to SKO-007(J3) βGalhigh
senescent cell population compared to non senescent βGallow cells, indicating that senescence
program promotes tumor cell recognition and elimination by NK cells.
We also studied the ability of chemotherapeutic drugs to enhance NK activating receptor ligand
expression in vivo in a mouse model of syngeneic orthotopic MM, the growth of which is dependent on
NK cells. We demonstrated that treatment with low doses of melphalan increased the expression of
the mouse NKG2D ligand RAE on MM cells and this was associated with increased susceptibility to
NK cells lysis ex vivo, but only with a marginal reduction of tumor growth in vivo. In order to
understand whether drug-induced induction of RAE-1 expression could promote MM-elimination by
adoptively transferred NK cells, we initially examined the factors that control the recruitment of NK
cells into BM during MM growth. By analyzing the expression of a marker of terminally differentiated
cells with reduced effector capacity, the inhibitory receptor KLRG1, we identified fully functional
populations of NK cells as KLRG1- and less functional NK cells as KLRG1+. In vivo homing
experiments were performed by transferring total splenocytes or enriched splenic NK cells (40%) from
healthy mice into control and tumor-bearing mice at three and four weeks of tumor growth, that
represent different stages of disease progression. Our results suggest that the homing of more
functional subsets of NK cells (KLRG1-) to the BM of tumor-bearing mice is impaired at three weeks of
tumor growth while all subsets are less recruited as compared to healthy control at four weeks. On the
other hand, NK cell homing capacity to spleen does not change. In order to investigate if changes of
NK cell recruitment are related to the expression of chemotactic factor into the BM, we analyzed the
expression of a panel of chemokines in the tumor microenvironment by collecting BM extracellular
fluids. Our results showed that while the chemokines IP-10, Mig, Mip-1alpha were up-regulated, SDF1 expression was down-modulated and RANTES and Fractalkine expression was not influenced by
tumor cells. In regard to SDF-1 down-modulation in the tumor microenvironment, we previously
119
demonstrated that the maintenance in the BM of selected NK cell subsets is regulated by the
CXCR4/SDF-1 axis with the exclusion of a subset of NK cells characterized by KLRG1+ expression.
These results suggest that during tumor growth selected subsets of NK cells are excluded from
BM and we are currently investigating down-modulation of CXCL12 protein levels in this event.
Experiments are also ongoing to understand whether in vivo treatment with melphalan can improve
recruitment and anti-tumor activity of NK cells in an adoptive transfer setting.
Publications
Bernardini G, Gismondi A, Santoni A. Chemokines and NK cells: regulators of development, trafficking
and functions. Immunol Lett 2012, 145: 39-46. doi: 10.1016/j.imlet.2012.04.014.
Capuano C, Paolini R, Molfetta R, Frati L, Santoni A, Galandrini R. PIP2-dependent regulation of
Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway. Blood 2012, 119: 2252-62.
doi: 10.1182/blood-2010-12-324160.
Carlino C, Trotta E, Stabile H, Morrone S, Bulla R, Soriani A, Iannitto ML, Agostinis C, Mocci C,
Minozzi M, Aragona C, Perniola G, Tedesco F, Sozzani S, Santoni A, Gismondi A. Chemerin
regulates NK cell accumulation and endothelial cell morphogenesis in the decidua during early
pregnancy. J Clin Endocrinol Metab 2012, 97: 3603-12. doi: 10.1210/jc.2012-1102.
Quinci AC, Vitale S, Parretta E, Soriani A, Iannitto ML, Cippitelli M, Fionda C, Bulfone-Paus S, Santoni
A, Di Rosa F. IL-15 inhibits IL-7Rα expression by memory-phenotype CD8+ T cells in the bone
marrow. Eur J Immunol 2012 May;42(5):1129-39. doi: 10.1002/eji.201142019.
Zingoni A, Ardolino M, Santoni A, Cerboni C. NKG2D and DNAM-1 activating receptors and their
ligands in NK-T cell interactions: role in the NK cell-mediated negative regulation of T cell
responses. Front Immunol 2012; 3:408. doi: 10.3389/fimmu.2012.00408.
Research Group
Marco
Cippitelli,
Angela
Gismondi,
professors; Giovanni Bernardini, Cristina
Cerboni, Cinzia Fionda, Alessandra Soriani,
Helena
Stabile,
Alessandra
Zingani,
researchers; Maria Luisa Iannitto, PhD student.
120
Dissection of Notch signaling-dependent pathways involved in the
progression of T cell leukemia
Isabella Screpanti
℡: +39 06 44700816 - @: [email protected]
The role of Notch signaling in the development of T cell leukemia is well established, whereas its
role in promoting and sustaining leukemia progression remains undefined. We previously showed that
the Notch3-dependent triggering of NF-kB canonical pathway, sustained by the constitutive expression
of preTCR, inducing in turn the activation of several anti-apoptotic and pro-proliferative signals (Bcl2A1, IL2, Cyclin D1), appears to exert an essential role during the expansion phase of the disease.
However, although it is widely known the role of Notch intracellular domain as a transcriptional
activator, a lot remains to be clarified on the mechanisms regulating the switch-on/off of the signalling.
This is particularly important with respect to the possibility to pharmacologically intervene on the
constitutive activation of Notch signaling in T cell leukemia. We identified a specific novel property of
Notch3, known to be overexpressed in T-ALL, that is acetylated and deacetylated by p300 and
HDAC1, respectively. Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation
of the protein. As a consequence, its transcriptional activity is decreased, thus resulting in the
impairment of downstream signaling as well as in vitro T cell proliferation and in vivo growth of Notch3induced T cell leukemia in transgenic mice.
This observation represents the first evidence highlighting the involvement of a reversible
acetylation mechanism in Notch3 protein stability and function, suggesting that acetylation may be
exploited as a therapeutic treatment, thus allowing the utilization of HDAC inhibitors in T-ALL therapy.
We previously demonstrated that specific relationships between Notch3 and canonical NF-kB
signaling pathway are crucial for the development of T cell leukemia in transgenic mice. Indeed, we
showed that the deletion of PKCtheta kinase, that plays an important role in signal transduction ending
in the triggering of NF-kB canonical pathway, significantly reduces the disease aggressiveness in the
Notch3-induced T-ALL mouse model , and induces an important delay in the progression of the
disease.
To clarify the Notch/NF-kB relationships in the onset and/or the progression of T cell leukemia, we
decided to inhibit NF-kB canonical pathway in N3-tg mice. To this purpose, we generated double
mutant mice, deleted of the NF-kB/p50 subunit in a Notch3 transgenic background (N3-tg/p50-/-). The
follow-up of N3-tg/p50-/- versus N3-tg mice was carried out to analyze the potential effects of NF-kB
inhibition on the aggressiveness and progression of T-ALL. The immunophenotypic analysis of
hematopoietic cell populations was performed at different age and in multiple tissues from the
indicated animals (thymus, spleen, bone-marrow and blood), by flow-cytometry tecniques.
As expected, p50 deletion virtually abolished the onset of T-ALL in N3-tg/p50-/- mice, as defined
primarily by the development of splenomegaly and the peripheral expansion of immature CD4+CD8+ T
cells. However, the mortality curves evidenced that double mutant mice succumb earlier than N3-tg
counterparts. Surprisingly, moribund N3-tg/p50-/- mice display the trait of a myeloproliferative disease,
with the dramatic expansion of Mac1+Gr1+ myeloid cells in both spleen, peripheral blood and Bone
Marrow. Preliminary data indicate that these cells do not express Notch3, suggesting that in the
121
absence of p50 expression, Notch3 is able to mainly influence the equilibrium of the myeloid
compartment in trans.
Our results suggest that the ablation of NF-kB canonical pathway may strongly impact on the
outcomes of a T cell specific deregulation of Notch signaling. Thus, providing a useful experimental
model to extend our understanding of Notch/NF-kB interplay and to unravel novel strategies for the
therapy of different hematological malignancies.
Publications
Palermo R, Checquolo S, Giovenco A, Grazioli P, Kumar V, Campese AF, Giorgi A, Napolitano M,
Canettieri G, Ferrara G, Schininà ME, Maroder M, Frati L, Gulino A, Vacca A, Screpanti I.
Acetylation controls Notch3 stability and function in T-cell leukemia. Oncogene 2012, 31: 3807-17.
doi: 10.1038/onc.2011.533.
Research Group
Collaborations
Maria Pia Felli, professor; Diana Bellavia,
Antonio F. Campese, researchers; Paola
Grazioli, Gaia Scafetta, post-doc fellows; Maria
Pelullo, Roberta Quaranta, PhD students.
Guido Franzoso, Department of Immunology,
Imperial College of London, UK; Antony J.
Capobianco, Molecular Oncology Research
Program, Division of Surgical Oncology,
University of Miami, USA.
122
Predisposing factors in autoimmune diseases: correlation between
common genetic variations and function
Rosa Sorrentino
℡: +39 06 49917706 - @: [email protected]
Autoimmune and several complex diseases share a common ground that is the dysregulation of
the network controlling the inflammatory response. The main interest of our research group is the
correlation between common polymorphisms and function in models of inflammatory diseases, with
particular emphasis on the Ankylosing Spondylitis (AS) and on the role played by the HLA-B27
molecules in conferring disease susceptibility. Much work has been done by us and much is still
ongoing in order to establish the effect of HLA-B27 polymorphisms in their antigen presentation
properties, in particular on the functional differences between B*2705 and B*2709 alleles differing for
a single amino acid (D116H) in the antigen presenting groove and differentially associated with AS.
During the last years, we have analyzed the effect of point mutations in the pockets of the groove
accommodating the peptides to be presented to the cytotoxic T cells. We have combined functional
experiments with extensive molecular dynamics simulations to investigate the function of the
conserved Arg62 in the pocket A and Cys67 in the pocket B. Simulations of HLA:peptide systems
suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are
metastable for both B*2705 (AS-associated) and B*2709 (non-AS-associated) subtypes under
physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR
interaction more than peptide-binding. Cys67 is also a crucial amino acid for the structure and function
of the HLA-B27 molecules. Also in this case however, the effect of mutations at this residue have a
different outcome on the two HLA-B27 subtypes. The main message stemming from these
experiments is that the polymorphism D116H in the pocket F that interacts with the peptide C-terminus
and that distinguishes the two HLA-B27 subtypes differentially associated with the disease, influences
the entire structure of the groove in which the peptides sit and furthermore, induces distinct TCR
binding modes for the B*2705 versus the B*2709 subtype (Magnacca et al., 2012; Nurzia et al., 2012).
Moreover, we have shown how the HLA-B27 molecules, differently from other HLA class I molecules,
can load the antigenic peptides, not only in the ER, as widely demonstrated for HLA-class I molecules,
but also in the Trans Golgi Network. This is a relevant finding suggesting that the HLA-B27 molecules
are more prone to present peptides possibly coming from the microenvironment through a non
canonical pathway (Magnacca et al., 2012). Other experiments have shown how the HLA-B51 allele,
the other HLA-class I molecule associated with a rheumatic disease (Behçet), likewise HLA-B27, has
a different haplotype distribution between patients and controls in the outlier Sardinian population
(Piga et al., 2012), suggesting similar evolutionary events that need to be explored further.
123
Publications
Magnacca A, Persiconi I, Nurzia E, Caristi S, Meloni F, Barnaba V, Paladini F, Raimondo D, Fiorillo
MT, Sorrentino R. Characterization of a Proteasome and TAP-independent Presentation of
Intracellular Epitopes by HLA-B27 Molecules. J Biol Chem 2012, 287: 30358-67. doi:
10.1074/jbc.M112.384339.
Nurzia E, Narzi D, Cauli A, Mathieu A, Tedeschi V, Caristi S, Sorrentino R, Böckmann RA, Fiorillo MT.
Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes
suggests distinct TCR binding modes. PLoS One 2012, 7:e32865. doi: 10.1371/journal.pone.
0032865.
Piga M, Paladini F, Lai S, Erre G, Passiu G, Carcassi C, Sorrentino R,. Mathieu A. Genetics of
Behçet’s disease in Sardinia: two distinct extended HLA haplotypes harbour the B*51 allele in the
normal population and in patients Clin Exp Rheumatol 2012; 30 (Suppl. 72): S51-S56.
Research Group
Collaborations
Maria Teresa Fiorillo, researcher; Fabiana
Paladini, post-doc fellow; Giorgio Camilli,
Valentina Tedeschi, PhD students.
Alessandro
Mathieu,
Carlo
Carcassi,
Università di Cagliari; Vincenzo Barnaba,
Domenico Raimondo, Sapienza-Università di
Roma; Rainer A. Böckmann, University of
Erlangen-Nurnberg, Erlangen, Germany.
124
CD28 co-stimulatory molecule as a key regulator of NF-κB
signalling pathway: role of cytoskeleton in coupling CD28 to NF-κB
activation
Loretta Tuosto
Department of Biology and Biotechnology "Charles Darwin"
℡: +39 06 49917595 - @: [email protected]
CD28 costimulatory receptor is a crucial determinant of the outcome of T lymphocyte activation
that following engagement by its natural ligands, B7.1/CD80 or B7.2/CD86 may act as a unique
signalling receptor and activate a non-canonical NF-κB2-like cascade leading to both the production of
pro-inflammatory cytokine/chemokines and the activation of survival genes. The intracytoplasmic
domain of CD28 contains a N-terminal YMNM motif that following phosphorylation binds the p85
subunit of phosphatidylinositol 3-kinase (PI3K). Once activated, PI3K catalyzes the conversion of
phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) and
generates the docking sites for key signalling proteins involved in T cell activation.
The relevance of PI3K in CD28-mediated signals regulating NF-κB activity emerges from our
recent data obtained in multiple sclerosis (MS) patients. We found that the engagement of CD28 by
either agonistic antibodies or B7 leads to the transcription and secretion of selective pro-inflammatory
cytokines in peripheral blood CD4+ T lymphocytes from MS patients compared to healthy donors (HD).
In particular, CD28 engagement stimulates IL-6, IL-17A and IL-21 production in MS patients, but not in
HD. IL-6 is associated with the clinical course of MS and plays a key role in neuroinflammation. IL-17A
is the hallmark of Th17 cells, enhances the secretion of pro-inflammatory cytokines and chemokines,
favours neutrophil infiltration and contributes to the disease pathogenesis. Pre-treatment of CD4+ T
cells with the PI3K inhibitors LY294002 or AS-605240 strongly inhibits CD28-mediated IL-6, IL-17A
and IL-21 cytokine expression. These data evidence a key role of CD28 and PI3K in amplifying the
inflammatory cascade in MS patients and suggest new therapeutic targets to dampen the
inflammatory phenotype of autoreactive T cells (manuscript in preparation).
In an attempt to characterize the molecular mechanisms of PI3K function in CD28 signalling, we
obtained interesting results evidencing a crucial role of PI3K-mediated phosphorylation of PIP2 to
PIP3. We demonstrated that CD28 regulates PIP2 turnover by recruiting and activating type I
phosphatidylinositol 4-phosphate 5-kinases α (PIP5Kα) in human primary CD4+ T lymphocytes. This
event leads to the neo-synthesis of PIP2 and to its consumption by CD28-activated PI3K. We also
evidenced that PIP5Kα activation is required for both CD28 unique signals regulating NF-κB nuclear
translocations and IL-8 gene expression as well as for CD28/TCR-induced Ca2+ mobilization, NF-AT
nuclear translocation and IL-2 gene transcription (Fig. 1). Our results highlight a previously unknown
role of CD28 in refilling the PIP2 pool necessary to ensure optimal T cell activation and elucidate a
novel mechanism that involves PIP5Kα as a key modulator of CD28 costimulatory signals (J Immunol,
in press).
In searching for specific molecules involved in coupling CD28 to both the recruitment and
activation of PIP5Kα, we identified Vav-1. Vav-1, is a GDP-GTP exchange factor for Rac-1 and Cdc42
small G proteins. Vav-1 is strongly tyrosine phosphorylated and activated by CD28, thus leading to the
recruitment and activation of the small G proteins and to actin filament nucleation. Interestingly
125
enough, we also found that Vav-1 is a key upstream regulator of the signalling cascades relating CD28
stimulation to both actin reorganization and NF-κB activation. Our data evidenced that Vav-1
constitutively associated with PIP5KIα in both primary CD4+ T lymphocytes and Jurkat T cell lines and
that this complex is recruited to the membrane following CD28 stimulation. The results obtained by
mutagenesis analyses revealed that the SH3 domains of Vav-1 are involved in PIP5Kα binding.
Experiments are in progress to identify the functional role of Vav-1/PIP5Kα association in both
CD28-mediated regulation of cytoskeleton reorganization and NF-κB signalling cascade.
Fig. 1 - PIP5Kα is required for CD28mediated transcription of both IL-8 and Il-2
+
genes. Primary CD4 T cells were transfected
with PIP5Kα siRNA or scrambled control
siRNA (scr siRNA) for 72 h and then
stimulated for 6 h with anti-CD28, or antiCD3, or anti-CD3 plus anti-CD28 Abs . IL-8
(A) and IL-2 mRNA levels (C) were measured
Real-time PCR and expressed as arbitrary
units (AU). Bars show the mean±SD of three
independent experiments. Asterisks (*) and
(**) indicate P < 0.01 and P < 0.05 calculated
by Student’s t test, compared with
unstimulated or CD28 stimulated cells
transfected with scr siRNA, respectively. (B)
The efficacy of PIP5Kα silencing of one
representative experiment is showed.
Research Group
Collaborations
Cristina Camperio, research fellow; Michela
Muscolini, post-doc fellow; Laura Muzi, PhD
student; Petra Tomassini, graduate student;
Silvana Caristi, technician.
Luca Battistini, Fondazione Santa Lucia
(I.R.C.C.S.), Roma; Ricciarda Galandrini,
Dipartimento
di
Medicina
Sperimentale,
Sapienza Università di Roma; Claudio
Gasperini Dipartimento di Neuroscienze,
Ospedale S. Camillo Forlanini, Roma.
126
Area 6
New antimicrobial and
antiviral agents
Area 6: New antimicrobial and antiviral agents
Peptide effectors of innate immunity
Donatella Barra
℡: +39 06 4456663 - @: [email protected]
Peptides with diverse biological activities are particularly abundant in skin secretions of
amphibians. Many of these compounds are pivotal components of the innate immune response, while
others are related to mammalian hormones or neurotransmitters.
We have focused the attention on the peptide components of amphibian skin secretions,
discovering a number of molecules displaying antimicrobial activity, as well as small proteins with
different properties, among which the most interesting is Bv8. Mammalian homologues of Bv8 have
been described also in humans (prokineticin 1/EG-VEGF and prokineticin 2/human Bv8), and found to
display chemokine-like activities.
We have studied in detail the mechanism of action of temporin L (TL, 13-residues long) that has a
wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its
microbicidal concentration. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly
reduced hemolytic activity and a stable helical conformation along residues 6-13. We systematically
replaced single amino acids within the α-helical domain with the corresponding D-isomers, known as
helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR
spectroscopy analyses as well as antimicrobial and hemolytic assays were performed.
Besides increasing our understanding on the structural elements that are responsible for cell
selectivity of TL, this study revealed that a single L- to D-amino acid substitution can preserve strong
anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.
Further studies have been performed on fragments of esculentin-1b, a 46-mer frog-skin peptide,
which shows an outstanding antimicrobial activity. The results obtained on the N-terminal extremity,
Esc(1-18), showed that this fragment has a clear tendency to fold in a helical conformation as
hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical
folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The
presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible
membrane-anchoring role.
Esc(1-18) has been recently studied on the dimorphic fungal pathogen Candida albicans, under
both in vitro and in vivo conditions using the simple infection model, Caenorhabditis elegans.
The results demonstrate that Esc(1-18) causes a rapid reduction in the number of viable yeast
cells and killing of the hyphal population of this microorganism, presumably by perturbing their
membrane. In addition, this peptide has been found to inhibit transition of fungal cells from their
roundish yeast shape to the more dangerous hyphal form, also in vivo (manuscript in preparation).
In parallel, by using the analogue Esc(1-21), studies on its bactericidal activity against the freeliving and sessile forms of the opportunistic pathogen Pseudomonas aeruginosa are in progress.
129
Detection of Esc(1-18) distribution and membrane perturbation of
both C. albicans ATCC 10231 yeast cells (upper panels) and
hyphae (lower panels), examined by fluorescence microscopy at
x100 magnification. Yeast cells or hyphae were treated with 16 mM
of rhodamine-labelled peptide for 30 min and then stained with
DAPI (for nuclei detection) and Sytox Green (to visualize
membrane perturbation). Controls are run in the absence of
peptide. Red, blue and green fluorescence indicate the distribution
of the rhodamine-labelled-Esc(1-18), DAPI and Sytox-Green,
respectively.
Concerning Bv8/prokineticins, we have studied their selective interaction with the G proteincoupled receptors, PKR1 and PKR2. Defects in the signalling pathways lead to various pathologies, in
particular the Kallmann syndrome and carcinogenesis. We have demonstrated the dimerization of the
PK2 receptor through in vivo experiments on a modified yeast cells strain that does not express PK
receptors. This dimeric receptor displays different properties with respect to the monomeric one,
allowing interaction with different types of α subunits of G proteins and eliciting a variety of responses.
Publications
Grieco P, Carotenuto A, Auriemma L, Saviello MR, Campiglia P, Gomez-Monterrey IM, Marcellini L,
Luca V, Barra D, Novellino E, Mangoni ML. The effect of d-amino acid substitution on the selectivity
of temporin L towards target cells: identification of a potent anti-Candida peptide. Biochim Biophys
Acta 2012 Epub. doi: 10.1016/j.bbamem.2012.08.027.
Manzo G, Sanna R, Casu M, Mignogna G, Mangoni ML, Rinaldi AC, Scorciapino MA. Toward an
improved structural model of the frog-skin antimicrobial peptide esculentin-1b(1-18) Biopolymers
2012, 97: 873-81. doi: 10.1002/bip.22086.
Research Group
Collaborations
Maurizio Simmaco, Giuseppina Mignogna,
professors; M. Luisa Mangoni, Rossella Miele,
Alessandro Paiardini, researchers; Sara
Marsango, Vincenzo Luca, PhD students;
Alessandra Franco, Alessandra Giorgi,
technicians.
Günther Kreil, Institute of Molecular Biology,
Austrian Academy od Sciences, Salzburg,
Austria; Luis Rivas, Centro de Investigaciones
Biológicas (CSIS) Madrid, Spain; Yechiel Shai,
Department of Biological Chemistry, Weizmann
Institute, Rehovot, Israel; Lorenzo Stella,
Dipartimento di Scienze e Tecnologie
Chimiche, Università di Roma Tor Vergata;
Lucia Negri, Dip. di Fisiologia e Farmacologia
“V. Erspamer”, Sapienza Università di Roma;
Paolo Grieco, Dipartimento di Chimica
Farmaceutica e Tossicologica, Università di
Napoli “Federico II”.
130
New pyrrole derivatives of BM 212: a new class of antimycobacterial
agents. Design, synthesis, biological evaluation and study of their
mode of action
Mariangela Biava
Department of Medicinal Chemistry and Technologies
℡: +39 06 49913812 - @: [email protected]
The World Health Organization (WHO) estimated that one-third of the global population has been
infected with M. tuberculosis (MTB) in 20101, therefore Tuberculosis still represents a major challenge
toward which research efforts are needed.
This project was devoted to the synthesis and characterisation of new derivatives, starting from
the chemical structures of BM2122 and BM5213, in the attempt to further improve both their biological
and drug-like parameters. These new 1,5-diphenyl pyrroles were synthesised employing a very easy
synthetic pathway consisting of three steps, allowing us to obtain them quickly and in good yields.
During the previous two years of the project we identified very active compounds such as BM650,
BM614 and BM642, as oxygen containing derivatives of the parent BM212 and BM521 hits.
Therefore, the last year of the project was focused on both synthesizing a new set of compounds
replacing the thiomorpholine with the morpholine ring and in testing all the new oxygen containing
derivatives for evaluating their drug-like parameters4.
Evaluation of the drug-like parameters, such as lipophilicity, human serum albumin (HSA) binding
and plasma protein binding (PPB) highlighted that the replacement of sulfur with oxygen gave
compounds with lower lipophilicities, which translated into lower HSA binding. The PPB also appeared
generally correlated to the lipophilicity parameters. Compounds with neutral pH CHILogD7.4 > 4.5
gave exclusively >99.9% PPB, whereas those with CHILogD7.4 < 4.0 gave 98.3 – 99.8%. More
importantly, a slower mouse microsomal clearance for the new compounds was observed, and
especially when analyzing both the fluorinated and the chlorinated derivatives, it was observed an
improvement of this parameter when changing from methyl to i-propyl substitution. This is likely due to
the fact that a longer or a branched alkyl chain could be more stable at metabolic levels.
Additionally, compounds were
tested against mycobacteria in the
physiological state of non-replicating
persistence, which is commonly
accepted as being responsible for
antimicrobial tolerance in many
bacterial infections, using a lowoxygen-recovery assay (LORA). Even
though the MICs in the LORA were
higher than the ones obtained under
aerobic conditions, we considered
these results as good, since many
known anti-TB drugs, e.g. isoniazid
(INH), are inactive in the LORA.
131
The best compound, BM635 (MIC: 0.12 µM, Tox50/MIC: >100, good microsomal stability in mice:
1.4 mL/min/g), was selected for in vivo pharmacokinetic and efficacy studies. The total exposure
reached after oral administration was compared with the total exposure reached for rifampicin (RIF),
INH, and moxifloxacin (MOX) after oral dose administration of ED99 related doses.
BM635 was then progressed to dose-response studies in an acute murine TB infection model. The
resulting ED99 of 49 mg/Kg is within the range of commonly employed tuberculosis drugs,
demonstrating the potential of this chemical series. When the areas under the curve associated with
the respective ED99 values were compared, the potency per effective concentration in vivo of BM635
was shown to be competitive with RIF, INH and MOX.
Finally, during this third year, studies regarding the identification of the molecular target of BMs
were concluded. Biological data strongly suggest that MmpL3 is the cellular target of BM2125.
References:
1. Global Tuberculosis Control: WHO report 2011, World Health Organization, Geneva
(WHO/HTM/TB/2011.16).
2. Deidda D et al, AAC 1998, 42, 3035-3037.
3. Biava M et al, JMC 2006, 49, 4946-52.
4. Poce G et al, Plos One 2013, 8, e56980.
5. La Rosa V et al, AAC 2012, 56, 324-31.
Publications
La Rosa V, Poce G, Canseco J, Buroni S, Pasca MR, Biava M, Raju R.M, Porretta GC, Alfonso S,
Battilocchio C, Javid B, Sorrentino F, Ioerger TR, Sacchettini JC, Manetti F, Botta M, De Logu A,
Rubin EJ, De Rossi E. MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212.
Antimicrob Agents Chemother 2012, 56: 324-331. doi:10.1128/AAC.05270-11.
Research Group
Collaborations
Giulio Cesare Porretta, professor, Giovanna
Poce, post-doc fellow; Claudio Battilocchio,
Salvatore Alfonso, PhD students, Roberto
Torri, Technician.
Raffaello Pompei, Alessandro De Logu, Dip.
di Scienze e Tecnologie Biomediche, Università
di Cagliari; Maurizio Botta, Fabrizio Manetti,
Dip. Farmaco Chimico Tecnologico, Università
di Siena; Edda De Rossi, Dip. di Genetica e
Microbiologia, Università di Pavia; Scott G.
Franzblau, Institute for Tuberculosis Research,
College of Pharmacy, University of Illinois at
Chicago, USA; Eric Rubin, Dep. of
Immunology and Infectious Diseases, Harvard
School of Public Health, Boston, USA.
132
New azole derivatives as antiprotozoal agents
Roberto Di Santo
Department of Medicinal Chemistry and Technologies
℡: +39 06 49913150 - @: [email protected]
Trypanosoma cruzi is the etiological agent of Chagas disease, which is recognized as one of the
world's 17 abandoned tropical diseases, and it is the most serious parasitic disease in Central and
South America. The WHO estimates that around the world, 10 million people are infected and more
than 25 million people are at risk of developing Chagas disease.
Leishmaniasis is a vector-borne complex protozoan infection caused by protozoan parasite of the
genus Leishmania. The major clinical presentations range from a simple cutaneous lesion through to
the disfiguring mucocutaneous leishmaniasis and finally to the visceralizal leishmaniasis (VL), which is
fatal if left untreated. The World Health Organization considers leishmaniasis to be one of the most
serious epidemics prone parasitic infectious diseases occurring mostly in poor and disadvantaged
resulting in substantial morbidity and mortality in an estimated 12 million people worldwide. Till now,
no vaccines are available against the above pathogens, up to day. Limitations of current drugs useful
against Chagas and VL include significant toxicity, long treatment courses as well as a lack of efficacy
due to the insorgence of drug resistance.
A few years ago we started studies on new antifungal agents characterized by imidazole and
pyrrole moieties, exemplified by the general structure 1 and 2 depicted in Figure 1, that displayed
EC50s 5-10 nM against C. albicans and Candida spp. As well known from literature the target of these
antifungal agents is the 14α-demethylase (L14DM) enzyme of fungi. Thus, taking account of the
presence of this enzyme in protozoa and our
availability of a series of compounds similar to 1
and 2, we decided to test our derivatives against
a panel of protozoa.
Figure 1. General structure of compounds found as
antiprotozoal agent, characterized by imidazole and
pyrrole moieties.
Main results
During 2012, following previous information obtained from the binding mode of RDS 2394 with T.
cruzi L14DM and the experimental results of some previously synthesized compounds we designed a
new series of azole derivatives (with a general structure like compounds 1 and 2), that present some
modification in positions R1, R2, R3 and R4 (see Figure 1). In general these residues (phenyl, electron
donor and electron withdrawing groups) are useful to increase the stability and the interaction of the
azole with the enzyme. The synthesized compounds were tested against a panel of protozoa such as
133
T. brucei, T. cruzi, L. donovani and P. falciparum. In general, the newly synthesized compounds were
good antiprotozoal agents. The potency of these compounds is high against P. falciparum and T.
cruzi, medium against L. donovani, low against T. brucei. Furthermore, from the biological analysis for
this new series of derivatives depicted in Table 1, a number of compounds were more potent than the
reference compounds, RDS 731 resulted the best hit against T. cruzi and RDS 1413 resulted the best
hit against L. donovani.
Nome
RDS 304
RDS 156
RDS 204
RDS 396
RDS 731
RDS 1255
RDS 1273
RDS 1277
RDS 1298
RDS 1314
RDS 1319
RDS 1413
RDS 2394
Melarsoprol
Benznidazole
Miltefosine
Chloroquine
T. b. rhod.
IC50 (µg/mL)
10.20
10.50
5.25
5.86
15.2
4.61
6.55
2.30
4.93
4.22
17.50
5.03
8.92
0.004
T. cruzi
IC50 (µg/mL)
3.07
1.17
0.98
0.02
0.001
3.95
0.02
1.33
0.02
1.05
5.43
0.03
0.03
L. don. axen.
IC50 (µg/mL)
4.37
4.35
9.14
2.58
1.79
3.44
2.12
3.23
5.22
3.26
2.96
0.80
0.20
P. falciparum
IC50 (µg/mL)
0.73
1.24
0.72
0.04
0.07
0.26
0.10
0.20
0.06
0.12
0.16
0.09
0.02
0.435
0.149
0.105
Table 1. In vitro potency of the newly synthesized azoles of RDS series against T. cruzi Tulahuen C4
amastigotes, L. donovani MHOM-ET-67/L82 amastigotes, as well as other protozoa like T. brucei rhodiesiense
STIB 900 trypomastygotes and Plasmodium falciparum K1 IEF, Reference compounds are Melarsoprol,
Benznidazole, Miltefosine and RDS 2394.
Perspective
The new synthesized azole derivatives show a good activity in biological tests. These results led
us to hypothesize that, in addition to L14DM, further targets could be involved in inhibition of the
growth of protozoa. As first step will be necessary the study of structure-activity relationships in these
series of compounds. These information will be useful to define the mechanism of action of these
azoles. Thus future work will be focussed to improve the potency against protozoa of this compounds
through the design and synthesis of new derivatives by the insertion of useful functionality in the azole
structure.
Research Group
Collaborations
Roberta Costi, professor; Giuliana Cuzzucoli
Crucitti, Luca Pescatori, Gaetano Miele,
Alberto Iacovo, Federica Rosi, post-doc
fellows; Antonella Messore, PhD student.
Luciana
Marinelli,
Ettore
Novellino,
Università di Napoli “Federico II”; Louis Maes,
University of Antwerpen, Belgium.
134
Drug design and synthesis of non-nucleoside inhibitors of both
HIV-1 wild type and resistant mutant strains reverse transcriptase,
and Coxsackie B4 virus
Romano Silvestri
Department of Drug Chemistry and Technologies
℡: +39 06 49913800 - @: [email protected]
UNAIDS estimates that more than 34 million people are living with acquired immuno deficiency
syndrome (AIDS)/human immunodeficiency virus (HIV) infection. In 2011, an estimated 2.5 million
people were newly infected with HIV and 1.7 million people died from AIDS. Highly active antiretroviral
therapy (HAART) based on combination of at least two or three (preferably) antiretroviral drugs from
different drug classes proved to improve dramatically the prognosis of HIV-1 infection. However, many
HAART receiving patients with undetectable RNA copies still have low HIV levels in their blood and
remain at risk of virologic rebound. Despite major improvements in antiretroviral therapies,
development of drug resistance can occur as a consequence of long period or permanent treatments,
and toxicity or incompatibility with other drugs and adverse effects may rise for various reasons.
According to the flexibility hypothesis, a powerful inhibition of the mutant variants of the HIV-1
should be correlated to the ability of the molecule to adopt variable binding conformations which would
be not affected by the interchanges of the amino acid residues into the NNBS of the mutant RTs.
Having in mind the horseshoe-like model, we designed new Indolylarylsulfone (IAS) derivatives
bearing cyclic substituents at the indole-2-carboxamide linked through a methylene or ethylene
spacer. Initially, we synthesized new IAS derivatives bearing a pirimidinylmethyl moiety. These
derivatives inhibited the NL4-3 HIV-1 strain in the low nanomolar range of concentration and were all
superior to the reference drugs AZT, NVP and EFV. When tested for their cytotoxicity (CC50 values),
they showed selectivity indexes (SI values) that were higher than those of all of the references
compounds, including IAS. The molecular docking studies carried out following our previously reported
methodology suggested the possible binding mode into the NNBS of the WT RT: (i) the indole NH
established a H-bond with the carbonyl oxygen of Lys101; (ii) the chlorine atom at position 5 of the
indole acceded to a hydrophobic pocket surrounded by Val106 and Leu234; (iii) the 3,5dimethylphenyl moiety lay in a hydrophobic cleft formed by Tyr181, Tyr188, Trp229, and Pro95
residues establishing hydrophobic interactions. Focusing on the p51/p66 interface cleft, the pyridinyl
ring was stabilized by hydrophobic interactions with the side chains of Val179 (p66), Glu138 and
Thr139 (p51). We synthesized pirimidinylethyl derivatives by joining the pirimidinyl group with an
ethylene linker. These IASs were potent inhibitors of the NL4-3 HIV-1 strain, with inhibitory
concentrations comparable to those of the corresponding pirimidinylmethyl derivatives. Finally, we
synthesized cyanophenyl derivatives by pushing out the nitrogen atom from the pirimidinyl ring while
its distance from the carboxamide nitrogen was kept fix, that were were equipotent to pyridinyl
compounds.
IAS derivative 5-chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(pyridin-4-ylmethyl)-1H-indole-2-carboxamide
was uniformly effective against the mutant Y181C, Y188L and K103N HIV-1 strains, superior to EFV
135
and equipotent to AZT. This compound was highly active against the multi-resistant mutant IRLL98
HIV-1 strain bearing the K101Q, Y181C and G190A mutations conferring resistance to NVP, DLV and
EFV, and it also potently inhibited in the higher picomolar range various HIV-1 clades, independently
of their co-receptor use, in PBMC. The carboxamide has emerged as a robust lead compound for the
development of new therapeutic agents for EFV-based HIV-1 therapies which show the emergence of
the L100I and K103N mutations. The results provide key information for further development of this
class of HIV-1 NNRTIs.
Binding mode of IAS into the HIV-1
nonnucleoside binding site of the RT.
Binding site residues of the p51 RT
subunits are reported in orange while
residues of p66 in green. H-bonds are
reported as yellow dotted lines.
Publications
La Regina G, Gatti V, Piscitelli F, Silvestri R. Venting while heating microwave-assisted synthesis of 3arylthioindoles. ACS Comb Sci 2012, 14: 258-262. doi: 10.1021/co200165j.
La Regina G, Coluccia A, Brancale A, Piscitelli F, Gatti V, Maga G, Samuele A, Gonzalez E, Clotet B,
Schols D, Esté JA, Novellino E, Silvestri R. New nitrogen containing substituents at the indole-2carboxamide yield high potent and broad spectrum indolylarylsulfone HIV-1 non-nucleoside reverse
transcriptase inhibitors. J Med Chem 2012, 5: 6634-38. doi: 10.1021/jm300477h.
Research Group
Collaborations
Giuseppe La Regina, researcher; Cesare
Giordano, CNR researcher; Antonio Coluccia,
post-doc fellow; Valeria Famiglini, PhD student.
Giovanni Maga, CNR, Istituto di Genetica
Molecolare, Pavia; Dominique Schols, Rega
Institute for Medical Research, K.U. Leuven,
Belgium; Andrea Brancale, Welsh School of
Pharmacy, Cardiff University, UK; José A.
Esté, IrsiCaixa, Hospital Universitari Germans
Trias i Pujol, Universitat Autonoma de
Barcelona, Badalona, Spain.
136
Area 7
Biology of malaria and other
vector-borne diseases
Area 7: Biology of malaria and other vector-borne diseases
Genetic and phenotypic characterization of species and “molecular
forms” of the Anopheles gambiae complex (Diptera: Culicidae),
afrotropical malaria vectors
Alessandra della Torre - Vincenzo Petrarca
Dept of Public Health and Infectious Diseases - Dept of Biology and Biotechnologies “C. Darwin”
℡: +39 06 49694268 - +39 06 49914932
@: [email protected] - [email protected]
In 2012, we focused on 2 major lines of research, as follows:
1. Analysis of the genetic and ecological differentiation of Anopheles gambiae M and S molecular
forms in areas of high hybridization. Anopheles gambiae M and S populations from the western edge
of the species range, where hybridization rates have been show to be much higher than in eastern
regions (>20% in Guinea Bissau and up to 7% in The Gambia), offer the opportunity to clarify several
aspects of the speciation process ongoing within the major Afrotropical malaria vector species. We are
carrying out several studies to analyse the genetic and phenotypic differentiation of M and S molecular
forms in this region compared to the rest of Africa. In particular we are currently analyzing field
samples collected i) along a 400 km west-to-east transect from the western coastal region of The
Gambia to south-eastern Senegal in collaboration with David Conway (MRC, The Gambia), and ii)
along a west to east transect in Guinea Bissau in collaboration with Anabela Rodrigues (INASA,
Guinea Bissau) and Joao Pinto (University of Lisbon). These samples have been molecularly identified
and karyotyped and are currently being analyzed by RFLP-PCR protocols, microsatellites, sequencing
and, also, through a wide-genome SNP analysis in collaboration with David Weetman (Liverpool
School of Tropical Medicine). So far the work is being carried out to evaluate the entity of gene-flow
dynamics, i) on a macro-geographical scale, by comparing the genome-wide divergence levels in
space and time between forms in high- and low- hybridization areas, and ii) on a micro-geographical
scale, by evaluating the habitat/ecological influence on the degree of introgression along a coastal-toinland transect across the hybridization area. Finally, the effect of gene-flow in the exchange of
adaptive alleles in genes implied in M and S form innate immunity (e.g. TEP1) is also examined in
order to evaluate the potential impact of this phenomenon on malaria transmission in the area.
2. Analysis of the genetic isolation within Anopheles melas. Anopheles melas is a brackish water–
breeding member of the Anopheles gambiae complex that is distributed along the coast of West Africa
and is a major malaria vector within its range. We participated to a multicentre study coordinated by
Michel Slotman (Texas A&M University, USA) aimed to fill the gap of knowledge on the population
structure of this species. Fifteen microsatellite markers and 1161 bp of mtDNA were analyzed in 11 A.
melas populations collected throughout its range. Compared with its sibling species A. gambiae, A.
melas populations have a high level of genetic differentiation between them, representing its patchy
distribution due to its fragmented larval habitat that is associated with mangroves and salt marsh
grass. Populations clustered into three distinct groups representing Western Africa, Southern Africa
and Bioko Island populations that appear to be mostly isolated. Fixed differences in the mtDNA are
present between all three clusters, and a Bayesian clustering analysis of the microsatellite data found
no evidence for migration from mainland to Bioko Island populations, and little migration was evident
between the Southern to the Western cluster. Surprisingly, mtDNA divergence between the three A.
139
melas clusters is on par with levels of divergence between other species of the A. gambiae complex,
and no support for monophyly was observed in a maximum-likelihood phylogenetic analysis. Finally,
an approximate Bayesian analysis of microsatellite data indicates that Bioko Island A. melas
populations were connected to the mainland populations in the past, but became isolated, presumably
when sea levels rose after the last glaciation period (10.000–11.000 BP). On the basis of the observed
level of divergence, these clusters may represent incipient species. In addition to enhancing our
understanding of the evolution of the A. gambiae complex, this work also has implications for malaria
control programs in particular on Bioko Island, as well as other locations where this species is a
dominant vector.
Publications
Deitz KC, Athrey G, Reddy MR, Overgaard HJ, Matias A, Jawara M, della Torre A, Petrarca V, Pinto J,
Kiszewski AE, Kengne P, Costantini C, Caccone A, Slotman MA. Genetic isolation within the malaria
mosquito Anopheles melas. Mol Ecol 2012, 21: 4498-513. doi: 10.1111/j.1365-294X.2012.05724.x.
Research Group
Collaborations
Marco
Pombi,
Beniamino
Caputo,
researchers, Emiliano Mancini, Federica
Santolamazza,
post-doc
fellows;
Maria
Calzetta, Paola Serini, research fellows,
Verena Pichler, graduate student.
Nora J Besansky, University of Notre Dame,
Notre Dame, IN, USA; Adalgisa Caccone, Jeff
Powell, Yale University, USA; David Conway,
Medical Research Council Laboratories, Banjul,
The Gambia; Paolo Pelosi, Università di Pisa,
Italy; Joao Pinto, Universidad de Nova Lisboa,
Lisbon, Portugal; Hilary Ranson, David
Weetman, Liverpool School of Tropical
Medicine, Liverpool, UK; N’Fale Sagnon,
CNRFP, Ouagadougou, Burkina Faso; Michel
Slotman, Texas A&M University, USA.
140
Immune responses to malaria and autoimmune disorders:
investigating common gene-regulatory networks
David Modiano
Department of Public Health and Infectious Diseases
℡: +39 06 49914933 - @: [email protected]
Background
The Fulani mount stronger immune responses to P. falciparum and are less susceptible to malaria
than sympatric populations (Modiano et al., PNAS, 1996). The Fulani also show higher responses to
other pathogens, and both their Th1 and Th2 responses are enhanced, suggesting that their
resistance to malaria could result from a generally stronger immune activation. Indeed, key genes
related to T reg cell function are down-regulated in the Fulani (Torcia et al., PNAS, 2008). This
disorder of immune homeostasis could be driven by genetic factors positively selected by P.
falciparum and may underlie the higher susceptibility of the Fulani to diseases with autoimmune
pathogenesis. To investigate this hypothesis, we are conducting a large-scale epidemiological and
immunogenetic study supported by the Malaria Genomic Epidemiology Network (MalariaGEN, Nature
2008).
Genotype data
We genotyped 363 SNPs on 2186 DNA samples from Fulani, Mossi and Rimaibé using the
Sequenom MassArray System. SNPs included polymorphisms involved in susceptibility and immune
response to malaria, as well as SNPs at autoimmunity loci.
Population genetics analysis
Principal component analysis revealed that Mossi and Rimaibé are not genetically distinct among
themselves, whereas the Fulani are a distinct group, in agreement with data on HLA class I and class
II alleles (Modiano et al., Tissue Antigens 2001; Lulli et al., Hum Immunol 2009)
Genetic association analysis
We conducted association analysis with prevalence of P. falciparum infection (yes/no) as
phenotype in individuals of all ages present at five cross-sectional surveys. We used a repeated
measure model with logistic regression adjusted for age, gender and village, both within each ethnic
group and in the all population stratifying by ethnicity. In the overall population, the strongest
association among autoimmunity loci was shown by the BLK locus. This gene encodes for B lymphoid
Tyrosine Kinase, an enzyme involved in B cell receptor signalling and development. BLK SNPs have
been previously reported to be associated with Systemic Lupus Erythematosus (Hom et al., New
England Journal of Medicine 2008) and to affect both the mRNA level of the gene (Ge et al., Nature
Genetics 2009) and protein half-life (Delgado-Vega et al., Annals of the Rheumatic Diseases 2012).
One SNP (G/T) was associated with parasite prevalence in both Fulani and Non-Fulani, where
individuals of TT genotype were more frequently infected (p-value=0.004, OR(95%CI)=5.2(1.7-16.8)).
We also conducted association analysis with incidence of clinical malaria (T>37°C and parasite
density >10000 par/µl) as phenotype in children under five years of age present at two longitudinal
141
surveys. We used a Poisson regression model adjusted for age, gender and village, both within each
ethnic group and in the all population stratifying by ethnicity. In both ethnic groups and in the overall
populations, the strongest signal of association was shown by the TGFB1 locus, encoding for Tumour
Growth Factor Beta 1 (TGF-β), a key cytokine for the development and function of T regulatory cells.
This gene was shown to be expressed at lower levels (16-fold difference) in Fulani than Non-Fulani in
a microarray experiment previously conducted by our group (Torcia et al., PNAS, 2008). Furthermore
it has been shown that TGF-β and T-reg responses are induced by P. falciparum infection and that
high TGF-β levels correlate with lower immune responses and faster parasite growth in vivo (Walther
et al., Immunity 2005) . One SNP (T/C) was associated with incidence of clinical malaria in both Fulani
and Non-Fulani, where children of CC genotype experienced more malaria episodes (p-value=0.0005,
IRR(95%CI)=2.3(1.5-3.4)).
We did not obtain so far evidence that the observed associations could be responsible of the
Fulani’s lower susceptibility to malaria, but haplotype analysis is currently ongoing to investigate this
aspect.
Research Group
Collaborations
Pamela Avellino, Valentina Mangano, Cinzia
Rizzo, post-doc fellows.
Dominic Kwiatkowski, Wellcome Trust Center
for Human Genetics, Oxford, UK; Eleanor
Riley, London School of Tropical Medicine and
Hygiene, London, UK; Bienvenu Sirima,
Centre National de Recherche et Formation sur
le Paludisme, Ouagadougou, Burkina Faso.
142
Start up program
Specification and maintenance of
retinal stem cells
Start-up Program
Specification and maintenance of retinal stem cells
Giuseppe Lupo
Department of Biology and Biotechnology "Charles Darwin"
℡: +39 06 49912206 - @: [email protected]
Our work is focused on the molecular mechanisms controlling specification and maintenance of
regional identity in progenitor/stem cells of the mammalian central nervous system (CNS). We are
using embryonic stem cells (ESCs) to model the extrinsic signalling systems directing the early
patterning of the developing CNS, and neural stem cells (NSCs) isolated from specific regions of the
foetal CNS to model the intrinsic regulatory systems that maintain positional identities of neural
progenitors following their initial specification.
Several studies have successfully produced a variety of neural cell types from hESCs, but there has
been limited systematic analysis of how different regional identities are established using well-defined
differentiation conditions. We have employed adherent, chemically-defined cultures to analyse the
roles of Activin/Nodal, Bone Morphogenetic Protein (BMP), Fibroblast Growth Factor (FGF) and
Wnt/ß-Catenin signalling in neural induction, anteroposterior patterning and eye field specification in
hESCs. We have shown that BMP inhibition or activation of FGF signalling are required for effective
neural induction but have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to
specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated
with strong posteriorization towards hindbrain/spinal cord fates. We have also demonstrated that
Wnt/ß-Catenin signalling is activated during neural induction and promotes acquisition of neural fates
posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain
specification. Finally, we have provided evidence that the levels of Activin/Nodal and BMP signalling
have a marked influence on further forebrain patterning and that constitutive inhibition of these
pathways represses expression of eye field genes. Our results show that the key mechanisms
controlling neural patterning in model vertebrate species are preserved in adherent, chemicallydefined ESC cultures and reveal new insights into the signals regulating eye field specification. These
results are described in a recently published manuscript (Lupo G, Novorol C, Smith JR, Vallier L,
Miranda E, Alexander M, Biagioni S, Pedersen RA, Harris WA. 2013. Multiple roles of Activin/Nodal,
bone morphogenetic protein, fibroblast growth factor and Wnt/ß-Catenin signalling in the anterior
neural patterning of adherent human embryonic stem cell cultures. Open Biology 3: 120167).
We have also shown that NSCs derived from different foetal CNS regions maintain in vitro
positional identities consistent with their area of derivation along the anteroposterior axis. For example,
NSCs derived from the foetal cortex, midbrain and spinal cord retain expression of transcription factors
active in the specification of the corresponding CNS regions in vivo. To address whether these
regional identities are irreversibly specified or can be influenced by molecular cues involved in CNS
patterning, we have investigated whether cells derived from different areas of the mouse CNS could
be directed to differentiate into dopaminergic neurons in vitro by forced expression of the transcription
factor Nurr1. Nurr1 plays a critical role in dopaminergic differentiation in the midbrain by controlling the
expression of key genes involved in dopamine neurotransmission. We have shown that Nurr1
overexpression can promote dopaminergic gene expression only in NPs obtained from foetal midbrain,
but not in cortex-derived NPs. These data show that Nurr1 ability to induce a dopaminergic phenotype
is critically dependent on the anteroposterior regional identity of NSCs, which is specified at an early
145
Start-up Program
phase of neural development through mechanisms that can be maintained in vitro even after exposure
to exogenous patterning signals (Soldati et al., 2012).
Proposed models of the signalling pathways controlling
neural induction, AP neural patterning and further
forebrain regionalization in adherent, chemically-defined
hESC cultures (A) or in a vertebrate embryo (B). Neural
induction is promoted by BMP antagonists (eg Chordin,
Noggin) and FGFs and is inhibited by high levels of
Activin/Nodal signalling. AP patterning is controlled by
posteriorizing factors (RA, Wnts, FGFs) and their
antagonists (eg Dkk1). Eye field specification is marked
by upregulation of eye field-specific transcription factors
(EFTFs) and, at least in hESCs, involves higher levels of
Activin/Nodal and BMP signalling compared to forebrain
specification (grey gradient in (A)). Key transcription
factors regulated during these events are also indicated.
Publications
Soldati C, Cacci E, Biagioni S, Carucci N, Lupo G, Perrone-Capano C, Saggio I, Augusti-Tocco G.
Restriction of neural precursor ability to respond to nurr1 by early regional specification. PLoS One
2012, 7:e51798. doi: 10.1371/journal.pone.0051798.
Research Group
Collaborations
Nicoletta Carucci, post-doc fellow; Valentina
Taschetta, undergraduate student.
Emanuele Cacci, Maria Elena Miranda
Banos, Dip. di Biologia e Biotecnologie “C.
Darwin”, Sapienza Università di Roma; William
Harris, Dept. of Physiology, Development and
Neuroscience, Univ. of Cambridge, UK; Roger
Pedersen, Anne McLaren Laboratory for
Regenerative Medicine, Univ. of Cambridge,
UK; Kunimasa Ohta, Dept. of Developmental
Neurobiology, Kumamoto University, Japan.
146
Istituto Pasteur-Fondazione Cenci Bolognetti
P.le Aldo Moro 5, 00185 Roma - Tel: +39 06 49255625/6/7/8 – Fax: +39 06 49255629
[email protected] - www.istitutopasteur.it

The adult human testis produces millions of sperm

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