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-硝基化修饰后翻译后蛋白质，将一氧化氧化物（NO）组的共价添加到Cys硫醇中形成S-硝基蛋白质（SNO蛋白）（SNO蛋白），介导了NO在无处不在的乳细胞系统中的无处不在的一部分，并且均与乳腺菌功能相关联，并介导了Mammalian Systems and nosemect s-N no he nosect and nosecret s-n no he no he nosecred so noy s.-N no he no he no he no he nos rysrect s-N人类疾病。越来越多的证据表明，酶介导的脱酰基化的基本作用，即从SNO蛋白中去除NO组，在调节蛋白S-硝基化的水平和动力学中，但没有系统地识别否硝基糖基酶或其底物的分配。以前，我们使用过 大肠杆菌作为模型系统，以识别一种在进化保守的酶机制，该酶机制调节了硝基苯基化，S-硝基谷硫酸硫代还原酶（GSNOR），该机制并非直接作用于SNO蛋白质，而是通过在某些Sno n-Nnnetose和S-Nnnetrose之间进行细胞平衡来调节蛋白S-硝基化。最近，我们在大肠杆菌中的分析确定了一种新型的SNO蛋白硝化酶（一种在微生物中描述的）。在此处提出的研究中，我们将采用大肠杆菌作为模型系统来识别系统性降解酶，部分基于我们的发现，即在硝化应力下特定的转录因子（TF）是硝基化并激活的。因此，在上调的独特调节子控制细胞SNO蛋白质水平，至少部分通过诱导硝基化活性。在AIM 1中，我们将重点关注新鉴定的硝酸酶和二硫醇还原酶硫蛋白蛋白，以前被我们鉴定为哺乳动物细胞中的SNO蛋白替代葡萄糖蛋白硝化酶，我们将采用我们引入的固体相蛋白质组学方法来确定SNO蛋白质（由Nitrosations诱导的二烯型），这些方法由Nitrosations Servantates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的脱硝基酶的细胞中迅速脱硝基化，并且在AIM 2中，我们将：a）询问SNO-TF相互作用组并b）建立脱硝基酶活性的生化筛选，以识别负责的硝酸化酶（S）。在AIM 3中，我们将筛选在TF的S-亚硝基化以用于新硝基化活性后诱导的调节子的成分。因此，这些目的融合了新型非硝基糖基酶及其底物的鉴定。拟议的研究与人类病理生理学具有直接相关性，因为我们以前已经确定，在微生物中发现的非硝基化激活可能通过系统发育高度保守，因此我们的分析很可能揭示了在人类病中不良调节的S-硝基化分析中广泛perview的新型酶促活性。此外，作为脱硝基糖基酶保护细菌免受哺乳动物先天免疫的主要成分的硝化应激，我们的研究可能指出了细菌发病机理的潜在治疗靶标。