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.