The goals of this research are to better understand the mechanism, specificity and biological function of several medically important and unique classes of zinc and/or iron metalloenzymes. (1) Protein farnesyltransferase and protein geranylgeranyltransferaseI catalyze the prenylation of many proteins in important signal transduction pathways. We propose to investigate the structure of the catalytic transition state by measuring kinetic isotope effects and the determinants of substrate specificity and product dissociation by mutagenesis. (2) Proteins modified by the prenylation pathwayswill be identified by first assaying the affinity and catalytic activity of libraries of peptides derived from the human genome and then verified as prenylation targets in vivo using modified prenyldiphosphate substrates and antibody detection. Furthermore, immunoprecipitation and proteomic analysis will be used to interrogate native expression of prenylated proteins. Identification of substrates of this pathway is an important step toward understanding the biological functions of these modifications and the downstreamtargets of the chemotherapeutic inhibitors of this pathwaythat are currently in clinical trials. (3) The enzyme UDP-3-0-(R-3-hydroxymyristoyl)-N- acetylglucosamine deacetylase (LpxC) catalyzesthe committed step in the synthesis of Lipid A in gram negative bacteria, making LpxC an antibacterial target. We propose to characterize the catalytic mechanism and metal specificity (Fe or Zn) using structure variation together with kinetic and thermodynamicstudies. These data will facilitate the design of potent LpxC inhibitors as novel antibiotics especially especially against those organisms associated with cystic fibrosis and some of the potential bioterror agents listed as MlAID category A and B priority pathogens. (4) Histone deacetylases catalyze the deacetylation of acetylated lysine residues involved in the regulation of gene expression and cell differentiation; inhibitors of HDACs are currently in clinical trials as anticancer drugs. We proposeto investigate the identity of the catalytic metal in vivo; the catalytic mechanism; and the substrate specificity, using both known substrates and proteomic approaches. Altering the active site metal may be an important regulatory mechanism of metal-dependent deacetylases. The information gained from these experiments will aid our understanding of the biological function of these important post-translational modifications and enhance the development of novel inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM040602-20S1
Application #
7659889
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Ikeda, Richard A
Project Start
1988-07-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2010-03-31
Support Year
20
Fiscal Year
2008
Total Cost
$42,543
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Castañeda, Carol Ann; Wolfson, Noah A; Leng, Katherine R et al. (2017) HDAC8 substrate selectivity is determined by long- and short-range interactions leading to enhanced reactivity for full-length histone substrates compared with peptides. J Biol Chem 292:21568-21577
Niu, Shuai; Kim, Byung Chul; Fierke, Carol A et al. (2017) Ion Mobility-Mass Spectrometry Reveals Evidence of Specific Complex Formation between Human Histone Deacetylase 8 and Poly-r(C)-binding Protein 1. Int J Mass Spectrom 420:9-15
Lopez, Jeffrey E; Haynes, Sarah E; Majmudar, Jaimeen D et al. (2017) HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking. J Am Chem Soc 139:16222-16227
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Temple, Kayla J; Wright, Elia N; Fierke, Carol A et al. (2016) Synthesis of Non-natural, Frame-Shifted Isoprenoid Diphosphate Analogues. Org Lett 18:6038-6041
Temple, Kayla J; Wright, Elia N; Fierke, Carol A et al. (2016) Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs. Bioorg Med Chem Lett 26:3503-7
Bergom, Carmen; Hauser, Andrew D; Rymaszewski, Amy et al. (2016) The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases. J Biol Chem 291:6534-45
Gantt, Sister M Lucy; Decroos, Christophe; Lee, Matthew S et al. (2016) General Base-General Acid Catalysis in Human Histone Deacetylase 8. Biochemistry 55:820-32
López, Jeffrey E; Sullivan, Eric D; Fierke, Carol A (2016) Metal-dependent Deacetylases: Cancer and Epigenetic Regulators. ACS Chem Biol 11:706-16
Jennings, Benjamin C; Danowitz, Amy M; Wang, Yen-Chih et al. (2016) Analogs of farnesyl diphosphate alter CaaX substrate specificity and reactions rates of protein farnesyltransferase. Bioorg Med Chem Lett 26:1333-6

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