S-Nitrosylation and Cardioprotection

描述（由申请人提供）：缺血性心脏病是美国最常见的死亡原因之一，造成巨大的医疗负担。神经保护性干预措施对减轻这种负担有很大的希望，但很少有实验发现成功地转化为有效的治疗方法。这可能是由于缺乏关于心脏保护机制细节的知识。一氧化氮，无论是内源性产生或外源性管理，已被证明是心脏保护的重要组成部分。我们最近的研究已经证明了一氧化氮衍生蛋白S-亚硝基化水平的增加与心脏保护之间的关联。S-亚硝基化是一种可逆的基于巯基的修饰，其由一氧化氮部分与半胱氨酸残基的游离巯基的共价连接产生。一氧化氮 合成酶同工型代表心肌细胞中内源性一氧化氮产生的主要来源。S-亚硝基化被认为通过调节靶蛋白的活性和/或功能以及通过阻断不可逆半胱氨酸氧化的破坏作用来提供保护作用。我们最近的数据与S-亚硝基化的总体保护作用一致，但分子机制，特定蛋白质靶点的相对重要性， 并且S-亚硝基化的病理生理学意义尚不清楚。因此，本提案的目标是通过研究S-亚硝基化的生理和病理方面来确定心肌中蛋白质S-亚硝基化的特异性和机制后果。为了建立S-亚硝基化的机制细节，一种新的方法来确定被S-亚硝基化修饰的给定蛋白质的百分比（即，S-亚硝基化占用）正在开发中，这将用于检查区室化的S-亚硝基化信号传导。半胱氨酸诱变也将用于检查S-亚硝基化对蛋白质功能的影响。我们以前确定了许多S-亚硝基化位点的心脏保护，从而使我们能够专注于特定的半胱氨酸残基，在原位改变。该提议的具体目的如下：1）确定S-亚硝基化占据是否在心肌细胞的不同细胞区室之间变化，并评估这如何影响信号传导，2）确定S-亚硝基化是否改变蛋白质-蛋白质相互作用，改变蛋白质定位，以及 促进蛋白质反式-S-亚硝基化，和3）确定S-亚硝基化在心肌离子通道活性调节中的特定作用。该提案的结果将定义S-亚硝基化在心脏中的作用，并通过建立S-亚硝基化的机制作用来推进该领域，从而为潜在的治疗靶点提供关键的见解。

DESCRIPTION (provided by applicant): 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 rol 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.