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Mechanisms that regulate the activity of the NRas depalmitoylase ABHD17 Holme, Sydney Michelle

Abstract

S-acylation is the reversible addition of a lipid to a protein, typically the 16-carbon lipid palmitate. The dynamic nature of palmitoylation enables cycling of signaling molecules, such as NRas, to maintain the plasma membrane localization that is required for oncogenic growth. The alpha/beta hydrolase domain-containing (ABHD) 17 proteins remove palmitate from protein substrates, including NRas, and thus present a promising anti-cancer therapeutic target. The ABHD17 proteins localize to the plasma membrane through a palmitoylated N-terminus, which is required for enzyme activity, but the other mechanisms that regulate activity are currently unknown. Understanding the regulation of the ABHD17 proteins will be crucial to elucidate their role in cell function and disease. Here, I used in vitro cell-based assays combined with molecular dynamics (MD) simulations performed in collaboration with Dr. S. Vanni and J. Sapia (University of Fribourg) to uncover the regulatory mechanisms of ABHD17. We have shown that the N-terminus of ABHD17A creates a predicted helix that forms the minimal region required for palmitoylation. This palmitoylated N-terminal helix is necessary and sufficient for plasma membrane localization, yet replacement with a plasma membrane anchor does not restore activity, suggesting the N-terminus has an additional function. Replacing the N-terminal palmitoylated cysteines with hydrophobic residues restores activity to ABHD17A when paired with a plasma membrane anchor, suggesting that hydrophobic interactions of this helix with the plasma membrane are critical for activity. Predictive structural analysis was used to discover a loop structure near the putative lipid-binding pocket of ABHD17. Systematic mutational analysis of this region identified three bulky hydrophobic residues that were required for activity. Importantly, mutations in either the N-terminal helix or the loop structure altered membrane association in MD simulations, and binding pocket conformation, as measured through inhibitor binding assays. This supports the model that the palmitoylated N-terminus recruits and orients ABHD17A at the plasma membrane, allowing the loop to insert in a way that favors substrate binding in the binding pocket. Collectively, these studies identify novel regulatory mechanisms of ABHD17A, which may aid in the creation of anti-cancer therapeutics and contribute to our understanding of related, uncharacterized enzymes.

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