Prof. Tulio Chavez-Gil, PhD Catedrático Asociado, Química.
Universidad InterAmericana de Puerto Rico. Dept of Biología, Química y Ciencias del Ambiente
San German Campus
Marquis Science Hall
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- Ph
2Pz)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
2
NH4+
V
O
O
-O
Ammonium Metavanadate
V
O
OO
O O C
CC
C
HC(Z)
CH(Z)
H3C
H3C CH3
CH3
VO(acac)2
OO
2,4-pentadione
Reacción Química
3.00 g (25.6 mmol) 5.21 mL (25.4 mmol)
Sol.H2SO4
Sol. NaHCO3 ~ 0 oC, 18 hs
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)
N NH
HH
HH
HH1
2
3
4
5
1,5(2H, 7.85 ppm), 3(H, 7.5 ppm), 2,4(2H, 7.35 ppm) Pz(H, 7.2 ppm)
pz
1,5
3
2,4
pz
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 (BH2-(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 Chavez-Gil.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).
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.
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 downce-homogenizer. 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. 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).
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
Results (cont’d)
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).
Acetylation
Deacatylation
Figure 1. Reversible acetyla*on of proteins at e-‐ amino groups of proteins.
KP Con NAM KP Con NAM
MRPL47
V III II I IV
Hsp60 16 26 34 43 55 72 95
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).
A) B)
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 over-expression 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.
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 acetylated-Lysine 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).
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.
Figure 3. 2D-gel separation of mitochondrial proteins isolated from 10 mM nicotinamide treated K562 cells. The gel is stained with Coomassie Blue stain.
In-gel digestion
Database search
Figure 4. Flow chart of experimental approach to identify acetylated proteins of mitochondria by 2D-gel analysis and mass spectrometry. LC-MS/MS: Liquid chromatography tandem mass spectrometry.
Proteolysis Separation
1D- or 2D-gel
LC-MS/MS analysis
LPSLALAQGELVGGLTLLTAR, 1048.2, 2+
K562 cell treatment
Mitochondria
Isolation
Protein identification
Methodology (cont’d)
Mass spectrometric analysis of mitochondrial proteins – Identification of mitochondrial proteins and mapping of post-translational 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.
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).
pH 3 pH 10
95 kDa
72 kDa
55kDa
• 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.
Recent peer reviewed papers
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 = bis-pyrazol; 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.
