biostudies-literatureUnknown301(4)Mondal TNational Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthNational Human Genome Research InstituteNational Institute of Child Health and Human DevelopmentProtein S-acylation, commonly known as protein palmitoylation, is the most prevalent form of protein lipidation with ∼6000 target proteins and in humans, is catalyzed by 23 integral membrane enzymes of the zDHHC family. Recognition of its importance in cellular physiology as well as human diseases has undergone an explosive growth in recent years. Yet, the nature of zDHHC-substrate interactions has remained poorly understood for most zDHHC enzymes. Cell-based experiments indicate a promiscuous and complex zDHHC-substrate network, whereas lack of in vitro reconstitution experiments has impeded insights into the nature of discrete zDHHC-substrate interactions. Here we report a substrate S-acylation reconstitution assay, called the Pep-PAT assay, using purified enzyme and peptide fragments ofThe Journal of biological chemistry108406https://www.ebi.ac.uk/biostudies/studies/S-EPMC12018989biostudies-literatureIn vitro reconstitution reveals substrate selectivity of protein S-acyltransferases.PMC12018989Mondal TSong JBanerjee AfalseIn vitro reconstitution reveals substrate selectivity of protein S-acyltransferases.Protein S-acylation, commonly known as protein palmitoylation, is the most prevalent form of protein lipidation with ∼6000 target proteins and in humans, is catalyzed by 23 integral membrane enzymes of the zDHHC family. Recognition of its importance in cellular physiology as well as human diseases has undergone an explosive growth in recent years. Yet, the nature of zDHHC-substrate interactions has remained poorly understood for most zDHHC enzymes. Cell-based experiments indicate a promiscuous and complex zDHHC-substrate network, whereas lack of in vitro reconstitution experiments has impeded insights into the nature of discrete zDHHC-substrate interactions. Here we report a substrate S-acylation reconstitution assay, called the Pep-PAT assay, using purified enzyme and peptide fragments of2025-01-01T00:00:00Z2025 Mar2025-07-01T03:05:44.158Z2025-07-01T03:05:44.158ZS-EPMC120189894009058210.1016/j.jbc.2025.108406