Amino acid and protein specificity of protein fatty acylation in C. elegans .

Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins of S -acyl moieties differ from N - and O -fatty acylation. Here, we show that fatty acylation patterns in Caenorhabditis elegans differ markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteine S -acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry-capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013 S -acylated proteins and 510 hydroxylamine-resistant N - or O -acylated proteins. Subsets of S -acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including the S -acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.
Competing Interests: Competing interests statement:H.L. is a founder of, consultant, and a stockholder for Sedec Therapeutics. F.C.S. is a founder of, consultant, and a stockholder for Ascribe Bioscience and Holoclara Inc.