Global identification of S-palmitoylated proteins and detection of palmitoylating (DHHC) enzymes in heart

High-throughput experiments suggest that almost 20% of human proteins may be S-palmitoylatable, a posttranslational modification (PTM) whereby fatty acyl chains, most commonly palmitoyl chain, are linked to cysteine thiol groups that impact on protein trafficking, distribution and function. In human, protein S-palmitoylation is mediated by a group of 23 palmitoylating 'Asp-His-His-Cys' domain-containing (DHHC) enzymes. There is no information on the scope of protein S-palmitoylation, or the pattern of DHHC enzyme expression, in the heart. We used resin-assisted capture to pull down S-palmitoylated proteins from human, dog, and rat hearts, followed by proteomic search to identify proteins in the pulldowns. We identified 454 proteins present in at least 2 species-specific pulldowns. These proteins are operationally called 'cardiac palmitoylome'. Enrichment analysis based on Gene Ontology terms 'cellular component' indicated that cardiac palmitoylome is involved in cellcell and cell-substrate junctions, plasma membrane microdomain organization, vesicular trafficking, and mitochondrial enzyme organization. Importantly, cardiac palmitoylome is uniquely enriched in proteins participating in the organization and function of t-tubules, costameres and intercalated discs, three microdomains critical for excitation-contraction coupling and intercellular communication of cardiomyocytes. We validated antibodies targeting DHHC enzymes, and detected eleven of them expressed in hearts across species. In conclusion, we provide resources useful for investigators interested in studying protein S-palmitoylation and its regulation by DHHC enzymes in the heart. We also discuss challenges in these efforts, and suggest methods and tools that should be developed to overcome these challenges.

Automated summary

High-throughput experiments suggest that around 20% of human proteins may undergo S-palmitoylation, with proteins in the cardiac palmitoylome mainly involved in cell-cell junctions and mitochondrial enzyme organization. The cardiac palmitoylome is uniquely enriched in proteins related to the organization of t-tubules, costameres, and intercalated discs.