Identification of Novel Denitrosylases

新型脱硝基酶的鉴定

• 批准号: 8686880
• 负责人: JONATHAN S. STAMLER
• 金额: $ 36.11万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8686880
• 关键词: Bacteria; Biochemical; Biological Models; Cell physiology; Cells; Co-Immunoprecipitations; Coupled; Enzymes; Equilibrium; Escherichia coli; Eukaryotic Cell; Evaluation; Excision; Functional disorder; Genes; Goals; Human; Individual; Infection; Mammalian Cell; Mediating; Methods; Microarray Analysis; Modeling; Natural Immunity; Nitric Oxide; Nitric Oxide Donors; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Pathogenesis; Phase; Phylogeny; Plants; Play; Post-Translational Protein Processing; Protein Isoforms; Protein S; Proteins; Proteomics; Reactive Nitrogen Species; Reactive Oxygen Species; Regulon; Reporting; Role; S-Nitrosoglutathione; S-Nitrosothiols; Side; Signal Transduction; Solid; Source; Substrate Specificity; Sulfhydryl Compounds; System; Thioredoxin; antimicrobial; antimicrobial drug; base; candidate identification; dithiol; enzyme substrate; glutaredoxin; human disease; macrophage; microbial; microorganism; nitrosative stress; novel; protective effect; public health relevance; response; therapeutic target; thioredoxin reductase; transcription factor

DESCRIPTION (provided by applicant): Post-translational protein modification by S-nitrosylation, the covalent addition of a nitric oxide (NO) group to a Cys thiol to form an S-nitroso-protein (SNO-protein), mediates a large part of the ubiquitous influence of NO on cellular function in mammalian systems, and dysregulated S-nitrosylation has been associated with a broad spectrum of human diseases. Increasing evidence points to essential roles for enzymatically mediated denitrosylation, that is, the removal of the NO group from SNO-proteins, in regulating the levels and dynamics of protein S-nitrosylation, but there has been no systematic identification of denitrosylases or delineation of their substrates. Previously, we used E. coli as a model system to identify an evolutionarily conserved enzymatic mechanism that regulates denitrosylation, S-nitrosoglutathione reductase (GSNOR), which does not act directly on SNO-proteins but regulates protein S-nitrosylation by virtue of the cellular equilibrium between at least some SNO-proteins and S-nitrosoglutathione. More recently, our analysis in E. coli has identified a novel SNO-protein denitrosylase (the first described in microorganisms). In the studies proposed here, we will employ E. coli as a model system to identify systematically denitrosylases, based partly on our finding that a specific transcription factor (TF) is S-nitrosylated and activated under nitrosative stress. The unique regulon that is consequently up-regulated governs cellular SNO-protein levels, at least in part through the induction of denitrosylating activity. In Aim 1, we will focus on the newly identified denitrosylase and on the dithiol reductase thioredoxin, previously identified by us as a SNO-protein denitrosylase in mammalian cells, and we will employ solid-phase proteomic methods introduced by us to determine the SNO-proteins (induced by nitrosative stress) that serve as substrates of these enzymes. We have found that multiple proteins, including TF itself, are rapidly denitrosylated in cells deficient in all known denitrosylases, and in Aim 2 we will: a) interrogate the SNO-TF interactome and b) establish a biochemical screen for denitrosylase activity, to identify the responsible denitrosylase(s). In Aim 3, we will screen the components of the regulon that is induced upon S-nitrosylation of TF for novel denitrosylating activities. Thus, these Aims converge on the identification of novel denitrosylases and their substrates. The proposed studies have direct relevance for human pathophysiology, because we have established previously that denitrosylating activates discovered in microorganisms are likely to be highly conserved through phylogeny, and our analysis is thus likely to reveal novel enzymatic activities of broad purview in the analysis of dysregulated S-nitrosylation in human disease. In addition, inasmuch as denitrosylases protect bacteria against the nitrosative stress that is a principal component of mammalian innate immunity, our studies may point to potential therapeutic targets in the treatment of bacterial pathogenesis.

描述（由申请人提供）：通过 S-亚硝基化进行翻译后蛋白质修饰，即一氧化氮 (NO) 基团与 Cys 硫醇共价添加以形成 S-亚硝基蛋白（SNO-蛋白），介导 NO 对哺乳动物系统中细胞功能的大部分普遍影响，并且 S-亚硝基化失调与广谱相关 的人类疾病。越来越多的证据表明，酶介导的脱亚硝基化（即从 SNO 蛋白中去除 NO 基团）在调节蛋白质 S-亚硝基化的水平和动态方面发挥着重要作用，但目前还没有对脱亚硝基酶的系统鉴定或对其底物的描述。之前我们用过 大肠杆菌作为模型系统，用于识别调节去亚硝基化的进化上保守的酶机制，即 S-亚硝基谷胱甘肽还原酶 (GSNOR)，它不直接作用于 SNO-蛋白质，而是通过至少一些 SNO-蛋白质和 S-亚硝基谷胱甘肽之间的细胞平衡来调节蛋白质 S-亚硝基化。最近，我们对大肠杆菌的分析发现了一种新型 SNO 蛋白脱亚硝基酶（首次在微生物中描述）。在此提出的研究中，我们将采用大肠杆菌作为模型系统来系统地识别脱亚硝基酶，部分基于我们的发现，即特定转录因子 (TF) 在亚硝化应激下被 S-亚硝基化并被激活。因此上调的独特调节子至少部分通过诱导去亚硝基化活性来控制细胞 SNO 蛋白水平。在目标 1 中，我们将重点关注新鉴定的脱亚硝基酶和二硫醇还原酶硫氧还蛋白（之前已被我们鉴定为哺乳动物细胞中的 SNO 蛋白脱亚硝基酶），并且我们将采用我们引入的固相蛋白质组学方法来确定作为这些酶底物的 SNO 蛋白（由亚硝化应激诱导）。我们发现，包括 TF 本身在内的多种蛋白质在缺乏所有已知脱亚硝基酶的细胞中快速去亚硝基化，在目标 2 中，我们将：a) 询问 SNO-TF 相互作用组，b) 建立脱亚硝基酶活性的生化筛选，以鉴定负责的脱亚硝基酶。在目标 3 中，我们将筛选 TF S-亚硝基化诱导的调节子成分，以发现新的脱亚硝基化活性。因此，这些目标集中在新型脱硝基酶及其底物的鉴定上。所提出的研究与人类病理生理学具有直接相关性，因为我们之前已经确定，在微生物中发现的去亚硝基化激活可能在系统发育中高度保守，因此我们的分析可能会揭示在人类疾病中 S-亚硝基化失调分析中具有广泛范围的新酶活性。此外，由于脱亚硝基酶可以保护细菌免受亚硝化应激的影响，而亚硝化应激是哺乳动物先天免疫的主要组成部分，因此我们的研究可能会指出治疗细菌发病机制的潜在治疗靶点。