Palmitoylation is the reversible addition of a 16 carbon fatty acid group to cysteine residues on proteins. The forward reaction is catalysed by DHHC palmitoyl acyl transferase (PAT) enzymes and there are 23 human isoforms. DHHC5 is a cell surface localised PAT proposed to contribute to cardiac reperfusion injury. A peptide array based on the DHHC5 extended C tail was used to screen rat cardiac lysates for DHHC5 interactors, which were subsequently identified by mass spectrometry. Biotinylated peptides covering the C-tail of DHHC5 were incubated with cardiac lysates and captured on streptavidin sepharose beads. Following the removal of contaminants, targets were identified as potential substrates of DHHC5 if they appear in the cardiac palmitoyl proteome or the SwissPalm database. Included are several kinases, phosphatases, ion transporters, G protein alpha subunits (Galphai2) as well as GLUT4 and FAT/CD36. DHHC5 interactors identified in the screen were expressed with an N terminal GFP tag for detection in both HEK293 and Crispr-engineered FT293 DHHC5 KO cells. Acyl resin assisted capture (AcylRAC) was used to assess their palmitoylation. Both FAT/CD36 and Galphai2 were less palmitoylated in DHHC5 KO cells, and this was restored on over expression of DHHC5 (but not DHHC17), indicating these are DHHC5 substrates. Further work will investigate the functional consequences of DHHC5 palmitoylation of CD36 and Galphai2, as well as validating other putative DHHC5 candidate substrates.
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