Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Biochemistry

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Maurine Linder

Abstract

Protein S-acylation is the post-translational modification of proteins with long-chain fatty acids at cysteine residues via a thioester linkage. The most commonly attached lipid is 16-carbon palmitate, thus the process is often called palmitoylation. Unlike other lipid modifications, protein S-acylation is reversible. Consequently, cells use acylation/deacylation cycles to regulate protein localization, stability, and activity. A family of integral membrane enzymes called DHHC proteins because of a conserved Asp-His-His-Cys motif, catalyze protein S-acylation within cells. DHHC proteins have been associated with human diseases including cancers, Huntington's disease, and mental retardation. However little is known about their function or regulation. This work focuses on developing a better mechanistic understanding of mammalian DHHC proteins. To facilitate biochemical characterization, a protocol was developed to express and purify recombinant DHHC proteins from insect cells using recombinant baculovirus. Protein S-acyltransferase: PAT) activity was measured by in vitro assays using radiolabeled palmitoyl-coenzyme A and purified protein substrates. Assay conditions were optimized to maximize PAT activity. In vitro, both protein substrate and DHHC protein incorporated palmitate. This latter process is termed enzyme autoacylation and lead to the hypothesis that DHHC proteins use a two-step ping-pong mechanism with an acyl-enzyme transfer intermediate. A fluorescent peptide, high performance liquid chromatography-based PAT assay was developed for classic steady-state kinetic experiments. Single turnover assays supported the hypothesis with radiolabeled fatty acid transferring from acyl-DHHC to protein substrate. Unexpectedly, these investigations also revealed that DHHC proteins display different acyl-CoA chain length preferences. A mechanism for this difference is proposed and tested. Inhibitors of DHHC proteins will be useful tools for studying protein S-acylation within cells and as potential pharmaceutical agents. Others in the field have identified classes of compounds that reduce cellular S-acylation. Four representative compounds and the known palmitoylation inhibitor 2-bromopalmitate: 2BP) were tested for inhibition of DHHC-mediated S-acylation with four DHHC proteins and their cognate substrates in vitro. Two compounds, 2BP and 2-(2-Hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one: compound V), inhibited autoacylation and acyl-transfer of all DHHCs tested. These compounds were further characterized for reversibility and time-dependence. Given their modest potency and lack of specificity, new screens for inhibitors of DHHC proteins are needed.

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Permanent URL: http://dx.doi.org/10.7936/K7ZP444S

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