S-Nitrosylation and Cardioprotection

S-亚硝基化和心脏保护

• 批准号: 8509246
• 负责人: Mark Jeffrey Kohr
• 金额: $ 10.75万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-23 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509246
• 关键词: Address; Affect; Binding; Cardiac; Cardiac Myocytes; Cause of Death; Cell Nucleus; Cysteine; Cytosol; Data; Fellowship; Functional disorder; Goals; Healthcare; Heart; In Situ; Intervention; Ion Channel; Knowledge; Lead; Mediating; Methods; Mitochondria; Mitochondrial Matrix; Modeling; Modification; Molecular; Morbidity - disease rate; Mutagenesis; Myocardial; Myocardial Ischemia; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pathologic; Physiological; Play; Postdoctoral Fellow; Production; Protein Isoforms; Protein S; Protein translocation; Proteins; Regulation; Relative (related person); Reperfusion Injury; Research; Role; Signal Pathway; Signal Transduction; Site; Source; Specificity; Stimulus; Sulfhydryl Compounds; Testing; Therapeutic; Translating; United States; base; extracellular; human NOS3 protein; insight; mortality; mutant; novel; oxidation; protective effect; protein function; protein protein interaction; research study; therapeutic target

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.

描述(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。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，无论是内源性的还是外源性的，都已被证明是心脏保护的重要组成部分。我们最近的研究表明，一氧化氮衍生蛋白S亚硝化水平升高与心脏保护之间存在关联。S亚硝化是一种可逆的基于硫醇的修饰，它是通过将一氧化氮部分共价连接到半胱氨酸残基的游离硫醇上而产生的。一氧化氮 合酶亚型是心肌细胞内源性一氧化氮产生的主要来源。S亚硝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的半胱氨酸氧化的损害效应来提供保护作用。我们最近的数据与S-亚硝化的整体保护作用是一致的，但分子机制(S)，特定蛋白质靶标的相对重要性， 而S-亚硝化的病理生理意义尚不清楚。因此，这项建议的目的是通过研究S亚硝化的生理学和病理学方面来确定蛋白S亚硝化在心肌中的特异性和机制后果。为了建立S-亚硝化的机理细节，正在发展一种新的方法来确定被S-亚硝化修饰的特定蛋白质的百分比(即S-亚硝化占有率)，并将用于检验分区化的S-亚硝化信号。半胱氨酸诱变也将被用来检测S亚硝化对蛋白质功能的影响。我们以前在心脏保护中发现了许多S-亚硝化位点，从而使我们能够专注于原位改变的特定半胱氨酸残基。这一建议的具体目的如下：1)确定S-亚硝酸化在心肌细胞不同细胞区段中的占有率是否不同，并评估其对信号转导的影响；2)确定S-亚硝化是否改变蛋白质-蛋白质相互作用，改变蛋白质定位，以及 促进蛋白质反式S-亚硝化；3)确定S-亚硝化在调节心肌离子通道活性中的具体作用。这项建议的结果将确定S-亚硝化在心脏中的作用，并通过建立S-亚硝化的机械作用来推动该领域的发展，从而为潜在的治疗靶点提供关键的见解。