John M. Denu
 |
Professor
Office: (608) 265-1859 |
Education
- B.S. 1988, University of Wisconsin-Madison;
- Ph.D. 1993, Texas A & M University;
- Postdoctoral 1993-96, University of Michigan Medical School
Honors & Awards
- Robert A. Welch Research Fellow (1992-93);
- National Research Service Award (1993-96);
- Young Investigator Award (American Cancer Association 1997-2000);
- Research Scholar Award (American Cancer Association 2001-2004)
Research Interests
The laboratory investigates the mechanism and biological function of reversible protein modifications involved in modulating signal transduction, chromatin dynamics and gene activation. Histones are one of the best examples of protein function regulated by modification, and are the target of an array of modifications including acetylation, phosphorylation and methylation. The dynamic and specific nature of these modifications has led to the proposal that there is a “Histone Code”, which when decoded would allow us to understand, for instance, what is required to maintain “silenced” or “active” chromatin. In general, histone acetylation (catalyzed by histone acetyltransferases) correlates with gene transcription, while hypo-acetylation (catalyzed by protein deacetylases) correlates with repression. To begin to understand this signaling code, we must first understand the mechanisms and regulation of the enzymes responsible for these modifications. A large component of our work is to understand the major enzyme families that catalyze these reactions.
Chromatin remodeling enzymes rely on co-enzymes derived from metabolic pathways, suggesting coordination between nuclear events and metabolic networks. A unique example of this link is the recently described NAD+-dependent protein/histone deacetylases. The founding member of this family, yeast Sir2 (silent information regulator 2), is involved in gene silencing, chromosomal stability, and ageing. Sir2-like enzymes catalyze a reaction in which the cleavage of NAD+, and histone/protein deacetylation are coupled to the formation of O-acetyl-ADP-ribose, a novel metabolite. The dependence on NAD+ and the generation of this potential second messenger offer new clues in understanding the function and regulation for nuclear, cytoplasmic and mitochondrial Sir2-like enzymes. Questions we are currently attempting to answer with this project: What is the physiological basis for the NAD+ -dependence? What are the cellular, acetylated-protein substrates for the various Sir2 homologues? What is the function of O-acetyl-ADP-ribose? What is the relationship between Sir2 enzyme function and metabolism? How are Sir2 enzymes regulated? How does Sir2 catalyze this unique reaction? To address these questions we are using a breadth of approaches that involve biochemistry, genetics, proteomics, enzymology, and use of a mammalian tissue culture system and yeast as a genetically tractable system to explore biological function.
Other projects involve understanding the mechanism of catalysis and regulation for the CBP/p300 and MYST families of histone/protein acetyltransferases, as well as the dual-specificity protein phosphatases that down-regulate mitogenic signal tranduction through dephosphorylation of the MAP kinases. Mutations in several of these enzyme families can cause miss-regulation of gene transcription and lead to such diseases as cancer. Through these studies will come an understanding of biological function and enzyme mechanism, which will undoubtedly lead to the design of rational therapeutics that target these novel enzymes or the pathways they regulate.
Publications of Note
Kim, Y., Tanner, K.G. and Denu, J.M., A continuous, non-radioactive assay for histone acetyltransferases, Analytical Biochemistry, 280, 308-314 (2000).
Fjeld, C.C., Rice, A.E., Kim, Y., Gee, K.R. and Denu, J.M. Mechanistic basis for catalytic activation of mitogen-activated protein kinase phosphatase 3 by extracellular signal-regulated kinase, J. Biol. Chem., 275, 6749-6757 (2000).
Cheung, P., Tanner, K.G., Cheung, W.L., Sassone-Corsi, P., Denu, J.M. and Allis, C.D., Synergistic coupling of histone H3 phosphorylation and acetylation in response to mitogen stimulation, Molecular Cell, 5, 905-915 (2000).
Tanner, K.G., Langer, M. L., Kim, Y. and Denu, J.M., Kinetic mechanism of the histone acetyltransferase GCN5 from yeast, J. Biol. Chem., 275, 22048- 22055 (2000).
Tanner, K.G., Langer, M.R. and Denu, J.M. Kinetic mechanism of human histone acetyltransferase P/CAF, Biochemistry, 39, 11961-11969 (2000).
Tanner, K.G., Landry, J., Sternglanz, R. and Denu, J.M. Silent information regulator 2 Family of NAD- dependent histone/protein deacetylases generates a unique product 1-O-acetyl ADP ribose, Proc. Natl. Acad. Sci. U S A, 97, 14178-14182 (2000).
Roth, S.Y., Denu, J.M. and Allis, C. D. Histone acetyltransferases. Annu. Rev. Biochem. 70, 81-120 (2001).
Rigas, J.D., Hoff, R. H., Rice, A.E., Hengge, A.C. and Denu, J.M. Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity hosphatase MKP3 by extracellular regulated kinase, Biochemistry, 40, 4398-4406 (2001).
Jackson, M. and Denu, J.M. Molecular reactions of protein phosphatases--Insights from structure and chemistry, Chemical Reviews, 101, 2313-2340 (2001).
Denu, J.M. and Tanner, K.T. Redox regulation of protein tyrosine phosphatases by hydrogen peroxide: Detecting sulfenic acid intermediates and examining reversible inactivation, Methods In Enzymology 348, 297-305 (2002).
Langer, M. R., Tanner, K.G. and Denu, J.M. Mutational analysis of conserved residues in the GCN5 family of histone acetyltransferases, J. Biol. Chem., 276, 31321-31331; (2001).
Kumano, M., Carroll, D.J., Denu, J.M. and Foltz, K. R. Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization, Developmental Biology 236, 244-257 (2001).
Todd, J.L., Rigas, J.D. and Denu, J.M. Dual specificity Protein Tyrosine phosphates VHR down-regulates c-Jun N-terminal Kinase (JNK). Oncogene 21, 2573-2583 (2002).
Schumacher, M.A., Todd, J.L., Rice, A.E., Tanner, K.G., and Denu, J.M. Structural basis for the recognition of biphosphorylated MAP kinase peptide by human VHR protein phosphatase, Biochemistry, 41, 3009-3017 (2002).
Bashor, C., Denu, J.M., Brennan, R.G., and Ullman, B. Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani. Biochemistry 41, 4020-31(2002).
Borra, M., OíNeill, F.J., Jackson, M.D., Marshall, B., Verdin, E. Foltz, K., and Denu, J.M. Conserved enzymatic production and biological effect of O-Acetyl ADP ribose by Sir2-like NAD+-dependent deacteylases. J. Biol. Chem. 277, 12632-41 (2002).
Ludlam, W.H., Kneeland, T., Taylor, M.H., Tanner, K.G., Denu, J.M., Goodman, R.H. and Smolik, S.M. The acetyltransferase acitivity of CBP is required for wingless activation and H4 acetylation in Drosophila melanogaster. Mol. and Cell Biol. 22, 3832-41 (2002).
Jackson, M.D. and Denu, J. M. Structural identification of 2í- and 3í-O-Acetyl-ADP Ribose as novel metabolites derived from the Sir2 family of
Langer, M.R., C.J. Fry, C.L. Peterson and J. M. Denu Modulating acetyl-CoA binding in the GCN5 family of histone acetyltransferases. J. Biol. Chem. 277, 27337-27344 (2002).
Boyer, L. A., Langer, M. R, Crowley, K. A., Tan S., Denu, J. M., Peterson, C. L., Essential role of the SANT domain for the function of multiple chromatin remodeling enzymes. Molecular Cell, 10, 935-942 (2002).
Rafty, L. A., Schmidt, M. T., Perraud, A. L., Scharenberg, A. M., Denu, J. M., Analysis of O-Acetyl- ADP-ribose as a Target for Nudix ADP-ribose Hydrolases. J Biol Chem. Dec 6;277(49):47114-47122 (2002).
Dutnall R. N. and Denu, J. M., Methyl magic and HAT tricks. Nat Struct Biol. Dec;9(12):888-91 (2002).
Denu, J. M. Linking chromatin function with metabolic networks: Sir2 (silent information regulator 2) family of NAD+-dependent Deacetylases. Trends Biochem. Sci. 28, 41-48, Jan 2003
North, B. J., Marshall, B. L., Borra, M. T. Denu, J. M. and Verdin, E. The human Sir2 ortholog, hSIRT2 is an NAD+-dependent Tubulin deacetylase. Mol Cell 11, 437-444 (2003).
Jackson, M. D., Fjeld, C. C. and Denu, J. M. Probing the Function of Conserved Residues in the Serine/Threonine Phosphatase PP2C(. Biochemistry 42(28):8513-8521 (2003).
Jackson, M. D., Schmidt, M. T., Oppenheimer, N. J. and Denu, J. M. Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases. J. Biol. Chem. 278(51): 50985- 98 (2003).
Borra, M. T. and Denu, J. M. Quantitative assays for characterization of the Sir2 family of NAD+- dependent deacetylases, Methods in Enzymology, in press 2003.
Kim Y, Rice A. E., Denu J. M. Intramolecular Dephosphorylation of ERK by MKP3. Biochemistry. 42(51): 15197-207 (2003).
Borra, M. T. and Denu, J. M. Quantitative assays for characterization of the Sir2 family of NAD+-dependent deacetylases, Methods in Enzymology, 376:171-87 (2004).
Grzyska, P. K., Kim, Y., Jackson, M. D., Hengge, A. C. and Denu, J. M. Probing the transition state structure of dual-specificity protein phosphatases using a physiological substrate mimic. Biochemistry, 43(27): 8807-14. (2004).
Borra, M. T., Langer, M. R., Slama, J. T. and Denu, J. M. Substrate Specificity and Kinetic Mechanism of the Sir2 Family of NAD+-dependent Histone/Protein Deacetylases. Biochemistry 43(30):9877-9887 (2004).
Schmidt, M. T., Smith, B. C., Jackson, M. D. and Denu, J. M. Co-enzyme specificity ofSir2 protein deacetylases: Implications for physiological regulation. J. Biol. Chem. Jul 21 [Epub ahead of print] (2004).
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University
of Wisconsin - Department
of Biomolecular Chemistry
First published: 01/01/05 Last updated:
1/18/05 Email Biomolecular Chemistry
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