Protein S-Palmitoylation in the Heart: Function and Regulation in Health and Disease

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PROJECT SUMMARY The objective of this project is to provide mechanistic information on the function and regulation of cardiac palmitoyl-proteome (palmitoylome) in health, and how changes in the palmitoylation status of key proteins in the heart contribute to disease development. Palmitoylation is a post-translational modification, where a fatty acyl chain, most often palmitoyl chain, is covalently linked to the thiol side chain of cysteine. This increase in hydrophobicity drives protein trafficking and changes their interaction with neighboring molecules. Palmitoylation is important for 2 reasons: (1) prevalence - ~20% of human proteins are palmitoylatable, (2) reversible - providing dynamic control of protein distribution and function. Recently, we globally purified palmitoylated proteins from human, dog and rat hearts, and used proteomic approach to identify 454 proteins forming a core 'cardiac palmitoylome'. Our study defined the scope of protein palmitoylation in the heart, and broadly characterized them as 'subcellular microdomain organizers'. We further identified 11 palmitoylating (DHHC) enzymes expressed in the heart. The current proposal is built upon these recent progresses, and addresses 2 main questions. First, given the numbers of known DHHC enzymes expressed in the heart (11) and substrates (454), how is the DHHC enzyme/substrate relationship determined in cardiac myocytes? Aim 1 will test the hypothesis that DHHC enzymes, as transmembrane proteins not freely mobile in cytosol, have their 'territories' demarcated in cardiac myocytes, and proteins within their territories are potential substrates. Second, what is the role of cardiac palmitoylome in disease development when dysregulated? We will use junctophilin-2 (JPH2) as a case study. JPH2 is the major jSR/PM tether. It requires palmitoylation of its cysteine side chains to strengthen the jSR/PM junctions. Genetic variants in JPH2 have been linked to cardiomyopathies, but the mechanisms underlying their pathogenicity are not clear. Aim 2 will investigate the role of palmitoylation in determining JPH2's distribution and functions in myocytes. Aim 3 will explore the possibility that some JPH2 genetic variants compromise palmitoylation, and this deficiency in JPH2 palmitoylation contributes to their pathogenicity in cardiomyopathies. Our research team combines 6 areas of expertise: (1) multiscale detection/quantification of protein palmitoylation, (2) quantitative proteomics, (3) high-resolution imaging/analysis, (4) bioinformatics/biostatistics, (5) modulation of CICR (Ca-induced Ca release) in cardiomyocytes, and (6) profiling/quantification of post-translational modifications. This combination allows us to probe the cardiac palmitoylome from single molecules to global proteome. We will also provide fundamental information on the structure and function of JPH2, which is critical for understanding why disease-related genetic variants in JPH2 are pathogenic. 1