S-Nitrosylation and Cardioprotection

项目摘要/摘要 缺血性心脏病是美国最常见的死亡原因之一，是导致 巨大的医疗负担。心脏保护干预为减轻这一负担带来了巨大的希望， 但很少有实验发现成功地转化为有效的疗法。这很可能是由于 缺乏关于心脏保护机制细节的知识。一氧化氮，要么产生 内源性或外源性给药，已被证明是 心脏保护。我们最近的研究表明，一氧化氮水平的升高与 氧化物衍生蛋白S-亚硝化与心脏保护。S-亚硝化是一种可逆的、以硫醇为基础的 通过将一氧化氮部分共价连接到自由硫醇基团上而产生的修饰 半胱氨酸残留物。一氧化氮合酶亚型是内源性一氧化氮的主要来源。 在心肌细胞中产生。S-亚硝酰化被认为是通过调节 靶蛋白的活性和/或功能，以及通过阻断半胱氨酸的损伤效应 氧化。我们最近的数据与S的整体保护作用是一致的-亚硝化，但分子 机制(S)，特定蛋白质靶点的相对重要性，以及S的病理生理意义。 亚硝化作用尚不清楚。因此，这项建议的目标是界定其特殊性和机械性。 蛋白S亚硝化对心肌的生理和病理影响 S谈--亚硝化。一种新的测定S的方法--亚硝化法建立其机理细节 被S-亚硝化修饰的给定蛋白质的百分比(即S-亚硝化占有率)是 开发的，这将用于检查划分的S-亚硝化信号。半胱氨酸诱变 还将用于检测S亚硝化对蛋白质功能的影响。我们之前认出了很多S-- 在心脏保护中的亚硝化部位，从而使我们能够专注于特定的半胱氨酸残基在 SITE。本提案的具体目的如下：1)确定S-亚硝化的占有率是否在 心肌细胞的不同细胞间隔并评估这如何影响信号，2)确定S- 亚硝化改变蛋白质相互作用，改变蛋白质定位，促进蛋白质反式-S-- 3)确定S-亚硝化在心肌离子通道调节中的具体作用。 活动。这项提议的结果将明确S-亚硝化在心脏中的作用，并通过以下方式推进该领域 为S建立一个机械作用--亚硝化，从而为潜在的治疗靶点提供关键的洞察力。

PROJECT SUMMARY/ABSTRACT Ischemic heart disease is one of the most common causes of death in the United States and is responsible for a tremendous healthcare burden. Cardioprotective interventions hold great promise for lessening this burden, but few experimental discoveries have translated successfully into effective therapeutics. This is likely due to a lack of knowledge with regard to the mechanistic details of cardioprotection. Nitric oxide, either produced endogenously or administered exogenously, has been shown to be an important component of cardioprotection. Our recent studies have demonstrated an association between increased levels of nitric oxide-derived protein S-nitrosylation and cardioprotection. S-nitrosylation is a reversible, thiol-based modification that is produced from the covalent attachment of a nitric oxide moiety to the free thiol group of a cysteine residue. The nitric oxide synthase isoforms represent the major source of endogenous nitric oxide production in the cardiac myocyte. S-nitrosylation is thought to provide protective effects by modulating the activity and/or function of target proteins, and by blocking the damaging effects of irreversible cysteine oxidation. Our recent data are consistent with an overall protective role for S-nitrosylation, but the molecular mechanism(s), the relative importance of specific protein targets, and the pathophysiological significance of S- nitrosylation is unknown. Thus, the goal of this proposal is to define the specificity and mechanistic consequences of protein S-nitrosylation in the myocardium by investigating the physiologic and pathologic aspects of S-nitrosylation. To establish the mechanistic details of S-nitrosylation, a novel method to determine the percentage of a given protein that is modified by S-nitrosylation (i.e., S-nitrosylation occupancy) is being developed and this will be used to examine compartmentalized S-nitrosylation signaling. Cysteine mutagenesis will also be used to examine the effects of S-nitrosylation on protein function. We previously identified many S- nitrosylation sites in cardioprotection, thus allowing us to focus on specific cysteine residues that are altered in situ. The specific aims of this proposal are as follows: 1) determine if S-nitrosylation occupancy varies between different cellular compartments of the cardiomyocyte and evaluate how this affects signaling, 2) determine if S- nitrosylation changes protein-protein interaction, alters protein localization, and promotes protein trans-S- nitrosylation, and 3) determine the specific role of S-nitrosylation in the regulation of myocardial ion channel activity. The results of this proposal will define the role of S-nitrosylation in the heart and advance the field by establishing a mechanistic role for S-nitrosylation, thus lending critical insight into potential therapeutic targets.