Prof. Tulio Chavez-Gil, PhD Catedrático Asociado, Química

Marquis Science Hall
Prof. Tulio Chavez-Gil, PhD
Catedrático Asociado, Química.
Universidad InterAmericana de Puerto Rico.
Dept of Biología, Química y Ciencias del Ambiente
San German Campus
What makes a great mentor?
Teaching is one of the most complicated jobs today.
It demands broad knowledge of subject matter, curriculum and standards;
enthusiasm, a caring attitude and a love of learning; knowledge of discipline and
classroom management techniques; and a genuine desire to make a difference
in the lives of young people.
Here are some characteristics of great mentores:
Great mentors set high expectations for all students.
They expect that all students can and will achieve high education standards
as well as best professional success.
Great mentors have clear, written-out objectives.
Effective mentors have lesson plans that give students a clear idea of what they
will be learning, what the assignments are and what the responsibility policy are.
Assignments have learning goals and give students ample opportunity to
practice new skills. The mentor is consistent in grading and returns work in a
timely manner.
Great mentors are prepared and organized.
They are in their classrooms or laboratory early and ready to teach. They
present knowledge in a clear and structured way. Their are organized in such a
way as to minimize distractions during learning and work time.
Laboratorio de Investigacion Subgraduada
M-205
Areas:
1.  Bio-Inorgánica: Química Medicinal de compuestos de Vanadio (V)
2.  Organometálica: Síntesis de nuevos Escorpionatos de Cu, Ni, W, Cr, Mo
3.  Bio-Materiales: Crecimiento de Cristales de Sn, Sb usando Matricex Sol-Gel
4.  Moléculas Quirales: Síntesis de Ligantes con propiedades ópticas
para estudios de ADN
5. Fitoquímica: Extracción de componentes activos de Plantas Medicinales
Estudiantes subgraduados que han formado parte del grupo*
Mr. Michael Rodriguez (2006-2007)*
Ms. Maria Vega (2006-2008)*
Ms. Jessica Rodriguez (2006-2008)*
Mr. Juan Bonilla (2007)*
Mr. Alexis Pacheco (2007-2010)*
Mr. Joel Lugo (2007-2010)*
Mr. Manuel Rosario-Alomar (2008-2009)*
Mr. Eric Alicea (2009-2010)*
Ms. Alexandra Lopez (2009-2010)*
Ms. Yamairy Ramos (2009-2010)*
Ms. Ana Nuñez-Lapeña (Estud. Intercambio, Uni-Complutense, Madrid (España)
(2007-2008)
Mr. Angel Vega (2008-2009)
Mr. Peter Rosado (2007-2009)
Mr. Ernie Cordero (2008)
Mr. Kevin Chaparro (2009)
Ms. Mae Lyn Acobis (2010)
Ms. Mary-C Quiles (2010)
* Estudiantes del Programa Ronald E. McNair In plants, ethylene binds to protein receptors which sends a signal that starts seed
germination, plant growth, fruit ripening, flower abscission and senescence.
Plant puberty the top six ripening hormones
Sn crystals
Sb crystals
CuI crystal
VOx crystal
Fotos de cristales obtenidos de
[Cu-K (3,5- Ph2Pz)2(3,5- Ph2Pz)]0
Microscopio – Mo*c 2000 – B3 – Pro Series – 4X Microscopio – Mo*c 2000 – B3 – Pro Series – 10X Microscopio – Mo*c 2000 – B3 – Pro Series – 40X Síntesis de VO(acac)2
1
2
3
Reacción Química
O
O
NH4+
-
2
2,4-pentadione
5.21 mL (25.4 mmol)
O
Sol.H2SO4
V
Sol. NaHCO3
Ammonium Metavanadate ~ 0 oC, 18 hs
(Z)
HC
C
O
O
O
C
O
CH
C (Z)
V
C
O
3.00 g (25.6 mmol)
CH3
H3C
O
O
CH3
H3C
VO(acac)2
Reacción y síntesis
(3,5-­‐Ph2Pz)3 HBK [Cu-­‐K (3,5-­‐ Ph2Pz)2(3,5-­‐ Ph2Pz)]0 Espectro de Infrarrojo en KBr
Espectro 1H-NMR (500
MHz, CD3CN)
3
H
H
H4
H3
5
1,5 2,4 H2
N
N
H
H1
pz pz 1,5(2H, 7.85 ppm), 3(H, 7.5 ppm), 2,4(2H, 7.35 ppm) Pz(H, 7.2 ppm) Diagrama ORTEP del compuesto
[Cu-(3,5-Ph2Pz)2K(3,5-Ph2pz)]0
Synthesis and Crystal Structure of [CuII (BH2-(3, 5-Ph-pz))2] (Ph = Phenyl, pz = pyrazole).
Chavez-Gil. Tulio 1*, Hamaker Christopher G.2, Cedeño. David L2 and Gonzalez Angel3.
1 Department of Biology, Chemistry & Env. Sc. Inter-American University of Puerto Rico. PO. Box
5100-PMB- 29. San German, PR 00683-9801.
2 Department of Chemistry, Illinois State University. 214 Julian Hall. Campus Box 4160. Normal, IL
61790-4160.
3Illinois State University, NIH-Bridges Summer Fellow, Farragut High School Academy, Chicago, IL
*[email protected]
Abstract
The title compound [CuII(BH2-(3,5-Ph-Pz)2] contains a four-coordinate Cu2+ ion lying on a
crystallographic inversion center, giving rise to a near-regular square-planar stereochemistry. The
complex was obtained indirectly during the reaction of [Cu(CO)HB-(3,5-Ph-pz)3] and cyclohexene in
dried methylene chloride (CH2Cl2) in a 9:1 (v/v) ratio. Recrystallization afforded brown monocrystals,
which present an axial contact of 2.508 Å between the Cu ion and the ligand B-H group. This
interaction is, to our knowledge, the shortest Cu-H “agostic” value found until date in a copper(II)
complex of this kind.1 The FT-IR (KBr) spectra shows the characteristic stretching modes at 3072 (C-H, Ph),
2530 (C-H, Pz), and 2530 (B-H) cm-1. The complex has good solubility in several solvents turning them suitable
for a broad variety of physical and chemistry studies.Density functional Theory (PWP91/LACVP**) studies
reveal that the extent of the Cu-H “agostic” interaction is proportional to the bulkiness of the substituent in the 3
and 5 positions of the pyrazole moiety.
1. Dias, H.V.R; Gorden, J. D., Inorg. Chem., 1996, 35, 318-324.
Aknowledgements: ACS-PRF # 39604.01-GB3 (T.Ch-G, D L. C) and NIH-Bridges Fellowship (A. G).
Title: Synthesis, Spectroscopy, Crystal Structure & DFT Evidence of a B-H-Cu Agostic interaction in a
[CuII (BH
2-(3, 5-Ph-pz))2] (Ph = Phenyl, pz = pyrazole) Complex.
Christopher G. Hamaker,1 David L. Cedeño,1* Jessica M. Rodriguez(U),2 Maria L. Vega(U),2 and Tulio ChavezGil.2* 1Department of Chemistry, Illinois State University.214 Julian Hall. Normal, IL 61790-4160. 2Department of
Biology, Chemistry and Environmental Sciences, Interamerican University of Puerto Rico. PO.Box 5100, PMB 29, San
German, PR 00683-9801.
Abstract
In the title compound [CuII (BH2-(3,5-Ph-pz))2]0, the copper atom is in a pseudooctahedral environment
comprising a square planar arrangement relative to the nitrogen atoms of the pyrazole rings, while one of the
hydrogens of each boron sits in a pseudo axial position forming the agostic bond coordination. The IR absorption
spectrum of the ligand shows two intense bands in the region 2200-2400 cm-1 (with maximum at 2207 and 2270 cm-1)
due to the B-H stretching and the 1460 and 1110 cm-1 intense bands attributed to the C-N and N-N stretching. For the
complex, the BH stretching bands are shifted to higher frequency (2529 and 2326 cm-1) and decrease in intensity and
shape, as a result of the perturbation of the H-B-H moiety associated to the geometrical change upon chelation with
the copper ion. Density functional Theory (PWP91/LACVP**) studies reveal that the extent of the Cu••H–B “agostic”
interaction is proportional to the bulkiness of the substituent in the 3 and 5 positions of the pyrazole moiety following
the relationship: H(3.121 Å) > CH3(2.799 Å) > CF3(2.579 Å) > Ph(2.508Å)
ORTEP plot of the complex [Cu(BH2-(3, 5-Ph-pz))2]0. Drawn with 50% ellipsoids and hydrogen atoms omitted for clarity.
Acknowledgements. The authors want to acknowledge Illinois State University for partial financial support of this
research. TCG and DLC acknowledge the support of the donors of the Petroleum Research Fund administered by the
American Chemical Society. Finally, TCG, MLV and JMR acknowledge financial support from the Ronald E. McNair
Program (undergraduate scholarship-IUPR-SG).
Role of kaempferol on deacetylation of mitochondrial proteins
Eliezer Romeu, Min-Joon Han, Huseyin Cimen, Hasan Koc and Emine C. Koc
Department of Biochemistry and Molecular Biology Pennsylvania State University, University Park, PA 16802
Introduction
Mitochondrion is an intracellular organelle found in
most eukaryotic cells. These organelles are involved in
many processes, such as cellular differentiation and
signaling, and supply over 90% of the energy used by
mammalian cells through the process of oxidative
phosphorylation. Reversible acetylation of mitochondrial
proteins at e-amino groups of lysines is implicated in the
regulation of mitochondrial function (Figure 1).
The
mitochondrial NAD+-dependent deacetylase SIRT3, a
mammalian homolog of the yeast Sir2gene, is critical for
deacetylation of many of these mitochondrial proteins.
Our previous studies show that kaempferol treatment of
mammalian cells changes acetylation status of the proteins
by increasing SIRT3 expression. The objective of our
research is to investigate the changes in acetylation status of
mitochondrial proteins by nicotinamide and kaemferol
treatment. In this study, we will perform various proteomic
tools to separate and analyze acetylated/deacetylated
proteins by 1D- or 2D-gels and LC-MS/MS analyses.
Acetylati
on
Deacatyl
ation
Figure 1. Reversible acetyla*on of proteins at e-­‐ amino groups of proteins.
Methodology
Western Blot Analysis- Protein samples for western blot
analysis were prepared from cells lysed in a buffer containing
50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1mM
EGTA, 0.5% NP-40, 0.1% SDS, and protease inhibitor
cocktail. After incubating the whole cell lysates 10min on the
ice, soluble protein fractions were collected by centrifugation
for 15 min at 14,000 X g at 4°C. Protein concentrations will be
determined by BCA assay (Pierce, Rockford, USA) using
bovine serum albumin (BSA) as a standard. Samples (10 µg)
were electrophoresed in 10-16% SDS-PAGE and transfer onto
PVDF membranes (Bio-Rad, Richmond, USA). After the
transfer, membranes were blocked for 2 h in Tris-buffered
saline (TBS) containing 0.1% Tween-20 and 5% (w/v) dry
skim milk powder. The membranes were washed with TBST
(TBS containing 0.1% Tween-20) three times and then
incubated overnight at 4°C with primary antibody. Following
the primary antibody incubation and 2h incubation with
appropriate secondary antibody (Pierce, Rockford, USA),
bound antibody signal was detected by chemiluminescence,
SuperSignal® West Pico kit (Pierce). Antibody for acetylatedLysine was purchased from Cell Signaling (Danvers, USA),
antibody for DAP3 was purchased from BD Biosciences (San
Jose, USA), and antibody for MRPS18-2, MRPL10 and
MRPL47 were generated by Covance (Denver, USA).
Results (cont’d)
A)
B)
KP Con NAM
95
72
KP Con NAM
Separation
V
III
43
34
26
pH 3
pH 10
95 kDa
55
II
IV
I
Proteolysis
In-gel
digestion
Mitochondria
Isolation
72 kDa
K562 cell treatment
1D- or 2D-gel
55kDa
16
Hsp60
Protein identification
LC-MS/MS analysis
MRPL47
Database
search
Figure 2. Role of kaempferol and nicotinamide treatment on acetylation of
mitochondrial proteins. A) Western blot analysis of untreated K562 cells (Con)
and treated with kaempferol (KP) and nicotinamide (NAM).
Figure 3. 2D-gel separation of mitochondrial proteins isolated from 10 mM
nicotinamide treated K562 cells. The gel is stained with Coomassie Blue
stain.
LPSLALAQGELVGGLTLLTAR, 1048.2, 2+
Figure 4. Flow chart of experimental approach to identify acetylated
proteins of mitochondria by 2D-gel analysis and mass spectrometry. LCMS/MS: Liquid chromatography tandem mass spectrometry.
Methodology (cont’d)
Methodology (cont’d)
Mitochondria Isolation- For analysis of mitochondrial proteins,
mitochondria were isolated from K562 cells. K562 cells were
collected and washed with PBS. The whole cell pellets were
resuspended with 500uL of homogenizer buffer containing (10mM
Tris-HCl(pH 7.6), 10mM KCl, 0.15mM MgCl2, 70mM Sucrose,
220mM Mannitol, 1mM EDTA,1mM DTT, 1mM PMSF) and
incubated for 10 min on ice. cells were lysized by downcehomogenizer. Lysates were centrifuged at 700g for 10 minutes to
remove nuclear fraction. After centrifugation, the pellets were
corresponded to nucleus were discarded and supernatant were
centrifuged at 10000g for 15 min to get mitochondrial pellet.
Collected mitochodrial pellet was washed with suspension buffer,
(10mM Tris- HCl(pH 7.6), 0.15 mM MgCl2, 0.25mM Sucrose,
1mM PMSF, 1mM DTT) and centrifuged at 12000g for 5 min.
Mass spectrometric analysis of mitochondrial proteins –
Identification of mitochondrial proteins and mapping of posttranslational modification (PTM) sites was achieved by database
searching of tandem mass spectra of proteolytic peptides searched
against protein databases. The modification of acetylation at lysine
residues increased +42 dalton. Tandem MS spectra obtained by
fragmenting a peptide by collision-induced dissociation (CID)
were acquired using a capillary liquid chromatography nanoelectrospray ionization - tandem mass spectrometry (LC/MS/
MS) system that consisted of a Surveyor HPLC pump, a Surveyor
Micro AS autosampler, and an LTQ linear ion trap mass
spectrometer (ThermoFinnigan). The raw CID tandem MS spectra
were converted to Mascot generic files (.mgf) using the extract
msn software (ThermoFinnigan). Both, the .mgf and .raw files
were submitted to site-licensed Mascot (version 2.2) and Sequest
search engines, respectively. Tandem MS spectra were manually
evaluated at the raw data level with the consideration of overall
data quality, signal-to-noise of matched peaks, and the presence of
dominant peaks that did not match to any theoretical m/z value.
2D-gel Separation- Approximately, 200 mg of mitochondrial
proteins resuspended in Destreak Rehydration Buffer (Amersham
Biosciences Inc.) and loaded into the rehydration tray dropwise.
The ReadyStrip IPG Strip ampholytes pI 3-10 (Bio-Rad
Laboratories, Inc.) and 11cm in length covered the sample and
rehydration occurred overnight. After running the first dimension
the strips were equilibrated in 6 M urea, 0.375 M Tris-HCl pH 8.8,
2% SDS, 20% glycerol, and 2% (w/v) DTT for 10 min. Once
removed, the strips were equilibrated in a second buffer with the
same components described above except with 2.5% (w/v)
iodoacetamide. The strips were loaded onto the second dimension
SDS-PAGE gel and either stained with Coomassie Blue or
transferred to a PVDF membrane probing with anti acetyl-Lys
antibody at a 1:500 dilution (Cell Signaling).
Results
In our initial analysis, we have observed changes in acetylation
status of proteins obtained from treatment of K562 cell lines with
nicotinamide and kaempferol treatment. To determine effect of
kaempherol and nicotinamide treatments on acetylation of
mitochondrial proteins, we have isolated mitochondria from K562
cell lines and performed Western blot analysis using N-acetyl Lys
antibody. As detected in Western blot analysis, We have also
observed increased acetylation of mitochondrial proteins isolated
from nicotinamide (NAM) treated K562 cells with N-acetyl Lys
antibody (Figure 2A). In addition to changes in acetylation levels,
we also observed changes in oxidative phosphorylation
complexes (Figure 2B).
As shown by arrows, expression of complex II and IV proteins
decreased in kaempferol (KP) treated mitochondria. We have
also probed the same westerns using Hsp60 and MRPL47
antibodies as equal loading control.
After observing the changes in acetylation status of proteins in
NAM treated K562 cells, we have isolated mitochondria from
NAM treated K562 and control cells and separated proteins on
2D-gels using IPG strips (Figure 3). As shown in the
experimental flowchart, proteins those were detected to be
acetylated are being processed to be analyzed in the LC-MS/MS
for protein identification (Figure 4).
Discussion
Our results confirm that there is an increase in acetylation
of mitochondrial proteins isolated from nicotinamide treated
human cells and kaemferol treatment could induce
deacetylation of mitochondrial proteins. Nicotinamide is well
known inhibitor for SIRT3. The kaempferol induces the overexpression of the SIRT3 that is localized in the mitochondrial
matrix. The over-expression of the SIRT3 contributes to the
deacetylation of mitochodrial proteins on the cell. Our
observation showed that regulation of SIRT3 by using
nicotinamide and kaempferol could regulate overall acetylation
level in mitochondria. We also observed changes in oxidative
phosphorylation complexes. Especially the expression of
complexes II and IV proteins decreased in kaempferol treated
mitochondria. In contrast, the expression of the complexes II
and IV proteins were the same in the control group and the
nicotinamide treated mitochondria. Kaempferol induces the
less ribosomal activity for translation of 13 proteins, because
kaempferol might be deacetylating some of the key proteins in
the mitochondria. In contrast, nicotinamide induces acetylation
of mitochondrial proteins by inhibiting SIRT3 and active
mitochondrial translation. References
Ball Haydn, Chen Yue, Cheng Tzuling, Grishin Nick, Kim Sung
Chan, Kho Yoonjung, Pei Jimin, Sprung Robert, White Michael,
Xiao Hao, Xiao Lin, Xu Yingda, Yang Xiang-Jiao, Zhao Yingming.
2006. Substrate and Functional Diversity of Lysine Acetylation
Revealed by a Proteomics Survey. Molecular Cell 23, 607-618.
Blader Gil, Guarente Leonard. 2004. The SIR2 Family of Protein
Deacetylases. Annu. Rev. Biochem. 2004.73:417-435
Fini Massimo, Indelicato Manuela, Marfe Gabriella, Pucci Bruna,
Reali Valentina, Russo Antonio, Sinibaldi-Salimei Paola, Tafani
Marco. 2009. Kaempferol Induces Apoptosis in Two Different
Cell Lines Via Akt Inactivation, Bax and SIRT3 Activation, and
Mitochondrial Dysfunction. Journal of Cellular Biochemistry
106:643-650.
Recent peer reviewed papers
•  T. Chavez-Gil, C. G. Hamaker, D L. Cedeño, J. Lugo(U). “Crystal Structure of
[(3, 5-Ph2Pz)-Cu-(3, 5-Ph2Pz)2-Cl]+ an Unusual Trigonal Planar Complex.”. Acta
Crystallographica, Section C, 2011. Final form to be submitted.
•  T. Chavez-Gil; D. L. Cedeño; C. G. Hamaker; M. Vega(U); J. Rodríguez(U),
“Synthesis, Characterization, and Crystal Structure of [Cu{(3,5-Ph2Pz)2BH2}2]0:
Evidence of a B-H-Cu Agostic Interaction” Journal of Molecular Structure, 2008,
888, 168-172.
•  H. Kurosaki, K. Matsuda(U), N. Yamakawa, Y. Yamaguchi, and T. Chavez-Gil.
“Crystal Structure of Chlorobis(R-1-pyridine-2-ylethylamine)copper(II) chloride”.
Journal of Analytical Science (JP). X-ray Structure Analysis Online, 2008, 24
(13), x305.
•  M I. Rodriguez, T. Chavez-Gil, Y. Colón(U), N. Díaz(U), E. Melendez.
“Molybdenocene-DNA Interaction Studies using Electrochemical Analysis”.
Journal of Electroanalytical Chemistry. 2005. 576 (2), 315-322.
International and National Congresses Presentations (undergraduates are underlined)
9. “Phytochemistry Comparison of Extraction Techniques for Medicinally important Caribbean Folk Plants”. Edith Torres,
Tulio Chavez-Gil. 43th IUPAC World Chemistry Congress. San Juan, PR. July-August, 2011. Accepted.
8. “Synthesis and characterization of stable organometallic complexes of [M-(Bpz)2(olefin)]0, M = Ni, Cu, Mn, Cr; Bpz = bispyrazol; olefin = cyclic-, linear- olefin”. T. Chavez-Gil, J. Lugo, M. Quiles, D L. Cedeño. 43rd IUPAC World Chemistry
Congress. July 30-August 4, 2011. San Juan PR. Accepted.
7. “Motif D of RdRp from HCV: Determinant of Nucleotide Incorporation Fidelity and Drug Sensitivity”. M F. Rosario-Alomar, C
E. Cameron, J J. Arnold, A. Lugo and T Chavez-Gil. 61st ACS Southeastern Regional Meeting (SERMACS). Oct 21-24, 2009
- San Juan, PR. Paper # 713
6. “Phythochemistry studies and pharmacological screening of anti-ulcerae activity of chiranthodendron pentadactylon flowers
(Devil's Hand Tree)”. T. Chavez-Gil, K. Slowing, and A. Nuñez-Lapeña. 61st ACS Southeastern Regional Meeting
(SERMACS). Oct 21-24, 2009 - San Juan, PR. Paper # 653
5. “The interaction of cyclic olefins and copper: eta-2 bonding vs. carbene formation”. David L. Cedeño.; Nathaniel Smart.;
Kyle Holder.; Tulio Chavez-Gil. ACS-Fall meeting. Washington DC. August 16-20, 2009. Organometallic Chemistry, Paper #
239.
4. “Phytochemistry Studies of D-chiro-Inositol from Mormodica Charantia. Isolation, Purification and Chemical
Characterization”. M Rosario-Alomar and Dr. T. Chavez-Gil. Ronald E. McNair Post- Baccalaureate Achievement
Symposium. San Juan, PR. Medicinal Chemistry. May 22. 2009.
3. “Synthesis of Sn-, Sb-TTA (TTA= tartaric acid) crystals by sol-gel growth methods”. Peter J. Rosado and Tulio Chavez-Gil.
3rd international symposium on Partnership for research & Education in Bio-materials. UPR-Humacao. Humacao, PR. May 8,
2009.
2. “Synthesis and Characterization of Oxovanadium (IV) Bio-mimetic Complexes”. A. Pacheco-Laracuente, T. Chavez-Gil, C
G. Hamaker, D L. Cedeño. ACS-Spring meeting. Salt Lake City, UT. March 22-26, 2009, paper # 203.
1. “Synthesis, Characterization and Crystal Structure of [Cu-(3, 5-Ph2Pz)3]Cl, an Unusual Trigonal Planar Complex”. T.
Chavez-Gil, J. Lugo, C G. Hamaker, D L. Cedeño. ACS spring meeting. Salt Lake City, UT. March 22-26, 2009, Inorg. Chem
paper # 212.
Agradecimientos
•  Al Programa Ronald E. McNair por su incondicional apoyo financiero con
los reactivos, supplies, participación en congresos y ayuda a los estudiantes
• A los (as) estudiantes de los bachilleratos de Biología y Química
•  A los colegas del Departamento de Química en Illinois State University
Normal, Ill por la colaboracion con los X-rays y análisis elemental.
•  Al Prof. Enrique Melendez (UPR-M) por permitirme el uso del NMR
•  A los colegas de la UPR-Humacao por incluir mi grupo en los workshops
de bio-materiales durante los últimos tres años.