Lipid Metabolic Labeling to Study Site- and Lipid-Specific Long-Chain S-Acylation Dynamics

Long-chain S-acylation is a post-translational modification that regulates key cellular processes, including signal transduction and metabolic regulation. However, the dynamic nature and site- and lipid-specific patterns of long-chain protein acylation remain poorly understood. Site- and lipid-specific metabolic labeling with various ω-alkynyl fatty acids uncovered the site-specific heterogeneity of long-chain protein S-acylation. Cells use various fatty acids for long-chain S-acylation, including C16:0, C18:0, and C18:1 on cysteines, while N-myristoylation preferentially incorporates C14:0 on N-terminal glycine residues. Our results demonstrate that long-chain S-acylation sites can exhibit both lipid heterogeneity and specificity and reveal that both enzymatic specificity and metabolic context can influence fatty acid incorporation. Exploration of dynamic protein long-chain S-acylation uncovered that acyl-protein thioesterases targeted by Palmostatin B regulate long-chain S-acylation involving various lipids, including C16:0, C18:0, and C18:1. Moreover, the site- and lipid-specific strategy uncovered dynamic long-chain S-acylation in a hydrophobic loop of ABHD17B that requires insertion into the lipid bilayer for efficient catalytic activity, suggesting tunability of ABHD17B enzyme activity through long-chain S-acylation. Our approach represents a significant advancement in lipid metabolic labeling methodologies, offering enhanced efficiency and sensitivity for studying S-acylation dynamics with lipid and site specificity.