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

项目总结 本项目的目标是提供关于生物多样性的功能和调节的机械性信息。 健康人心脏棕榈酰化蛋白质组(Palmitoylome)及其棕榈酰化状态的变化 心脏中的关键蛋白质有助于疾病的发展。棕榈酰化是一种翻译后的 修饰，其中脂肪酰链，通常是棕榈酰链，共价连接到硫醇一侧 半胱氨酸链。这种疏水性的增加推动了蛋白质的运输，并改变了它们之间的相互作用 与邻近的分子相互作用。棕榈酰化之所以重要，有两个原因：(1)患病率--约20%的人类 蛋白质是可棕榈酰化的，(2)可逆-提供蛋白质分布的动态控制和 功能。最近，我们从人、狗和大鼠的心脏中全局纯化了棕榈酰化蛋白，并 用蛋白质组学方法鉴定了454种形成核心‘心脏棕榈糖体’的蛋白质。我们的研究 定义了心脏中蛋白质棕榈酰化的范围，并广泛地将其描述为亚细胞 微域组织者。我们进一步鉴定了11种棕榈酰化(DHHC)酶在 心。目前的提案建立在这些最新进展的基础上，并解决了两个主要问题。 首先，给出在心脏(11)和底物(454)中表达的已知DHHC酶的数量， 心肌细胞中DHHC酶/底物的关系是如何决定的？AIM 1将测试 DHHC酶作为跨膜蛋白不能在胞浆中自由移动的假说 在心肌细胞中划分的“区域”，以及它们区域内的蛋白质是潜在的底物。 第二，当心脏棕榈糖体失调时，它在疾病发展中的作用是什么？ 我们将使用JPH2(JPH2)作为案例研究。JPH2是主要的JSR/PM系留。它需要 其半胱氨酸侧链的棕榈酰化以加强JSR/PM连接。JPH2的遗传变异 已被认为与心肌病有关，但其致病机制尚不清楚。 目的2研究棕榈酰化在决定JPH2‘S分布和功能中的作用。 肌细胞。目标3将探索某些JPH2基因变体妥协的可能性 棕榈酰化，而JPH2棕榈酰化的这一缺陷导致它们在 心肌病。我们的研究团队结合了6个专业领域：(1)多尺度 蛋白质棕榈酰化的检测/定量，(2)定量蛋白质组学，(3)高分辨率 成像/分析，(4)生物信息学/生物统计学，(5)钙诱导钙释放的调节。 心肌细胞，以及(6)翻译后修饰的分析/量化。这种组合 使我们能够探索从单分子到整体蛋白质组的心脏棕榈醇组学。我们还将 提供有关JPH2的结构和功能的基本信息，这对 了解为什么JPH2中与疾病相关的遗传变异是致病的。 1

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