Structure, Function, and Inhibition of Protein Depalmitoylases

Abstract

Proteins are often regulated through the addition of chemical modifications that modulate localization, activity, and interactions. Protein S-palmitoylation describes the attachment of long-chain fatty acids to cysteine residues in proteins to promote membrane association, which contributes to the regulation of a number of cellular processes. Altered S-palmitoylation contributes to the pathogenesis of neurological disorders, cancer, and many other diseases. Importantly, dynamic S-palmitoylation is required to modulate the activity of proteins in response to various extracellular signaling events. This includes various membrane-associated or membrane proteins such as G proteins, small GTPases, receptors, scaffold proteins, cytoskeletal proteins, and kinases. These observations suggest that enzymes catalyzing the removal of palmitate must also play a critical role in modulating the activity of hundreds of essential proteins. Therefore, it is important to characterize how S-palmitoylation is regulated, which will provide a more complete molecular picture of how cells adjust to various signaling events. Over the last decade, potent inhibitors of depalmitoylases have been developed, yet the basic mechanisms and the cellular functions of depalmitoylases remain poorly characterized. Since depalmitoylation can modulate select cell signaling events, I hypothesized that depalmitoylase enzymes have dedicated substrates and differentially contribute to the global dynamic S-palmitoylation regulation. My dissertation addresses this hypothesis by focusing on the structure, function, and inhibition of the depalmitoylating enzymes APT1 and APT2. The first chapter of this thesis provides an overview of depalmitoylating enzymes. Second, I used isoform-selective inhibitors to understand the structure, substrate engagement and selectivity of APT1 and APT2. Third, I validated the selectivity of the APT2 inhibitor ML349 by affinity enrichment and proteomics. Finally, I explore the dynamic rate of palmitoylation regulated by depalmitoylases in cells. Overall, my research has clarified the mechanism and function of protein de-palmitoylation in cell regulation.