The functional capacity of genetically-encoded proteins is powerfully expanded by reversible posttranslational acetylation. Our long term goal is to understand the catalytic mechanism and substrate specificity of protein deacetylases, the enzymes which hydrolyze acetyl groups from the epsilon-NH2 side chain of lysyl residues within histone N-termini as well as proteins such as p53 and HIV-Tat. Protein deacetylases play a critical role in transcriptional repression, chromatin remodeling, and modulation of activities of oncogenic proteins, transcription factors, and mitotic proteins. Inhibitors of these enzymes are powerful anticancer agents that bring about morphological changes in cancer cells, reverting transformed cells to a normal-like phenotype by stimulating cellular differentiation and cell cycle machinery. Understanding the mechanisms, specificity, and global function of these important proteins will have pronounced impact on our ability to eradicate human cancer and better our understanding of the complex biology of transcription. In this proposal we seek to gain a complete view of histone deacetylase mechanism, specificity, and inhibition.
In Aim 1 we will rigorously characterize the catalytic amidohydrolase mechanism of Hos3 from S. cerevisiae, a prototypical Class I/II zinc-dependent histone deacetylase.
In Aim 2, analysis of the zinc-dependent enzymes will be complemented by a full characterization of S. cerevisiae Hst2, a prototypical member of the newly discovered NAD+-dependent (Sir2- like) Class III histone deacetylases. Finally, in Aim 3 we will examine the substrate specificity of histone deacetylases using well defined site-specifically modified peptides, full-length histones, and compositionally defined synthetic nucleosome assemblies. The effects of microsequence context, posttranslational modification state/position, partner protein and DNA directive groups, isoform selectivity, and histone/nucleosome composition and conformation on deacetylase specificity will be determined. Together, these studies will provide a clear picture of protein deacetylase mechanism, inhibition, and substrate specificity, and will direct the development of mechanism-based, isoform-selective inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065539-03
Application #
6708088
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Jones, Warren
Project Start
2002-03-01
Project End
2007-02-28
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
3
Fiscal Year
2004
Total Cost
$259,943
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Burg, Jonathan M; Link, Jennifer E; Morgan, Brittany S et al. (2015) KDM1 class flavin-dependent protein lysine demethylases. Biopolymers 104:213-46
Bellucci, Joseph J; Amiram, Miriam; Bhattacharyya, Jayanta et al. (2013) Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides. Angew Chem Int Ed Engl 52:3703-8
Folk, Drew S; Torosian, Justin C; Hwang, Sunhee et al. (2012) Monitoring ýý-secretase activity in living cells with a membrane-anchored FRET probe. Angew Chem Int Ed Engl 51:10795-9
Pollock, Julie A; Larrea, Michelle D; Jasper, Jeff S et al. (2012) Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERýý-dependent and -independent manners. ACS Chem Biol 7:1221-31
Cropp, T Ashton; McCafferty, Dewey G (2011) Found in translation: applications of protein and peptide molecular diversity. Curr Opin Chem Biol 15:347-9
Gaweska, Helena; Henderson Pozzi, Michelle; Schmidt, Dawn M Z et al. (2009) Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1. Biochemistry 48:5440-5
Gooden, David M; Schmidt, Dawn M Z; Pollock, Julie A et al. (2008) Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B. Bioorg Med Chem Lett 18:3047-51
Schmidt, Dawn M Z; McCafferty, Dewey G (2007) trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1. Biochemistry 46:4408-16
Lee, Min Gyu; Wynder, Christopher; Schmidt, Dawn M et al. (2006) Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol 13:563-7
Frankel, Brenda A; Bentley, Matthew; Kruger, Ryan G et al. (2004) Vinyl sulfones: inhibitors of SrtA, a transpeptidase required for cell wall protein anchoring and virulence in Staphylococcus aureus. J Am Chem Soc 126:3404-5

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