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

项目摘要 本项目的目的是提供有关生物体的功能和调节的机制信息， 心脏棕榈酰蛋白质组（棕榈酰组）在健康，以及如何改变棕榈酰化状态的 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修饰 修饰，其中脂肪酰基链，最常见的是棕榈酰基链，共价连接到硫醇侧 半胱氨酸链这种疏水性的增加驱动蛋白质运输并改变它们的相互作用 与相邻分子的结合棕榈酰化很重要，原因有两个：（1）患病率-约20%的人类 蛋白质是棕榈酰化的，（2）可逆的-提供蛋白质分布的动态控制， 功能最近，我们从人、狗和大鼠心脏中全面纯化了棕榈酰化蛋白， 使用蛋白质组学方法鉴定了454种形成核心“心脏棕榈酰组”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，并将其概括为“亚细胞” 微域组织者。我们进一步鉴定了11种棕榈酰化（DHHC）酶， 心目前的建议是建立在这些最近的进展，并解决两个主要问题。 首先，考虑到已知在心脏（11）和底物（454）中表达的DHHC酶的数量， 在心肌细胞中DHHC酶/底物关系是如何确定的？目标1将测试 DHHC酶作为跨膜蛋白在胞质溶胶中不能自由移动的， 心肌细胞中划分的“区域”和其区域内的蛋白质是潜在的底物。 第二，当心脏棕榈酰组失调时，心脏棕榈酰组在疾病发展中的作用是什么？ 我们将使用junctophilin-2（JPH 2）作为案例研究。JPH 2是主要的jSR/PM系链。它需要 棕榈酰化其半胱氨酸侧链以加强jSR/PM连接。JPH 2中的遗传变异 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JPH 2在人肝细胞中的分布和功能中的作用。 肌细胞目的3将探索一些JPH 2遗传变异体损害 棕榈酰化，并且JPH 2棕榈酰化的这种缺陷有助于它们在大肠杆菌中的致病性。 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）CICR（钙诱导的钙释放）的调节， 心肌细胞，和（6）分析/定量翻译后修饰。这种组合 使我们能够从单个分子到整体蛋白质组探测心脏棕榈酰组。我们还将 提供有关JPH 2结构和功能的基本信息，这对于 了解为什么JPH 2中与疾病相关的遗传变异是致病性的。 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