Enzyme-centric chemoproteomics reveals isomer-specific S-acylation modification networks

Protein lipidation is a critical post-translational modification, but the relationship between the fine structure of fatty acids (FAs) and the specificity of lipidation remains largely unexplored. Here, we develop an enzyme-centric chemoproteomic strategy to elucidate the intricate relationships between the key players involved in the protein lipidation process. By synthesizing alkyne-tagged FA isomer probes in combination with zDHHC enzyme overexpression and quantitative proteomics, we reveal at the proteomic level how minor isomeric differences in FAs affect their modification. Protein lipidation demonstrates a marked specificity for the C = C bond isomerism, and the comprehensive network between FA isomers, zDHHC enzymes, and S-acylated proteins is mapped. Furthermore, the isomeric selectivity and the spatial binding characteristics of autoacylation intermediates are elucidated, suggesting the molecular determinants governing this specificity. This study provides deep insights into the molecular basis of protein lipidation. Protein lipidation is a crucial biological process, yet the role of fatty acid fine structure remains largely unexplored. Here, the authors develop a new strategy to demonstrate that C = C bond position can reshape the topological structure and functional output of the entire S-acylation network.