This proposal is a competing renewal application of R01 GM62437 that combines the use of chemical approaches, enzymologic analysis, and cellular studies to enhance our understanding of enzymes regulating protein acylation and methylation. It is now well- accepted that post-translational modifications (PTMs) involving lysine acetylation and reversible methylation on histones and other proteins are central to epigenetics. Such epigenetic modifying enzymes are viewed as attractive drug targets for cancer and other diseases. The ghrelin O-acyltransferase (GOAT) enzyme catalyzes the unusual PTM of octanoyl attachment to a Ser side chain of the peptide hormone ghrelin and inhibitors of this enzyme may be useful in the treatment of obesity and diabetes. Although there has been increasing efforts to understand the mechanisms and functions of these PTMs and the enzymes that catalyze them, there are major gaps in our understanding in these areas. Filling these knowledge gaps has the potential to provide a clearer understanding of basic biomedical processes and has the opportunity to enhance the development of novel therapeutic approaches and disease diagnostic strategies. There are four Specific Aims in this proposal. 1. Elucidate the molecular basis for protein substrate selectivity for histone acetyltransferases (HAT) using new chemical and biochemical approaches. We will develop and apply new techniques to generate histone-CoA conjugates and exploit protein microarrays to clarify structural and functional features of HAT-substrate interactions. 2. Develop and apply improved chemical tools for analyzing LSD1 histone demethylation. A combination of synthetic inhibitors and propargyl-histones will be prepared to interrogate LSD1 cellular functions and molecular interactions. 3. Clarify the effects of Lys acetylation on S-adenosyl homocysteine hydrolase and inositol monophosphate dehydrogenase-2. Expressed protein ligation will be used to install acetyl-Lys site-specifically into these metabolic enzymes to dissect potentially important nodes between protein acetylation and cellular metabolism. 4. Define major structural features of ghrelin O-acyltransferase. A combination of membrane topology mapping and bisubstrate analog crosslinking will be used to create a blueprint of this key metabolic regulatory enzyme. We believe that this research effort has the potential to greatly expand our understanding of protein post-translational modification mechanisms and functions and identify new therapeutic opportunities for treating metabolic and neoplastic diseases.

Public Health Relevance

This proposal develops and applies chemical methods for the investigation of protein regulation by post-translational modifications involving acylation and methylation. If successful, it could lead to new therapies for cancer and metabolic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37GM062437-13
Application #
8532311
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
2001-02-01
Project End
2018-02-28
Budget Start
2013-04-01
Budget End
2014-02-28
Support Year
13
Fiscal Year
2013
Total Cost
$345,339
Indirect Cost
$132,167
Name
Johns Hopkins University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Wondisford, Anne R; Xiong, Lishou; Chang, Evan et al. (2014) Control of Foxo1 gene expression by co-activator P300. J Biol Chem 289:4326-33
Maksimoska, Jasna; Segura-Peña, Dario; Cole, Philip A et al. (2014) Structure of the p300 histone acetyltransferase bound to acetyl-coenzyme A and its analogues. Biochemistry 53:3415-22
Taylor, Martin S; Ruch, Travis R; Hsiao, Po-Yuan et al. (2013) Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. J Biol Chem 288:32211-28
Schröder, Sebastian; Herker, Eva; Itzen, Friederike et al. (2013) Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells. Mol Cell 52:314-24