Identification of Novel Denitrosylases

新型脱硝基酶的鉴定

• 批准号: 8370000
• 负责人: JONATHAN S. STAMLER
• 金额: $ 35.22万
• 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/8370000
• 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（由申报人提供）：翻译后蛋白质的s -亚硝基化修饰，将一氧化氮（NO）基团共价加成到半胱氨酸巯基上形成s -亚硝基蛋白（SNO-protein），介导了NO在哺乳动物系统中普遍存在的对细胞功能的很大一部分影响，并且失调的s -亚硝基化与广泛的人类疾病有关。越来越多的证据表明，酶介导的脱硝基化，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力学中起重要作用，但没有系统地鉴定脱硝基化酶或描述其底物。之前，我们使用