Identification of Novel Denitrosylases

描述（由申请人提供）：通过S-亚硝基化（将一氧化氮（NO）基团共价加成至Cys巯基以形成S-亚硝基蛋白（SNO-蛋白））进行的翻译后蛋白质修饰介导了NO对哺乳动物系统中细胞功能的大部分普遍影响，并且失调的S-亚硝基化与广谱人类疾病相关。越来越多的证据表明，酶介导的脱亚硝基化，即从SNO-蛋白质中去除NO基团，在调节蛋白质S-亚硝基化的水平和动力学中起着重要作用，但还没有系统地鉴定脱亚硝基化酶或描绘其底物。在此之前，我们使用 E.大肠杆菌作为一个模型系统，以确定一个进化上保守的酶机制，调节脱亚硝基化，S-亚硝基谷胱甘肽还原酶（GSNOR），它不直接作用于SNO-蛋白质，但调节蛋白质S-亚硝基化凭借至少一些SNO-蛋白质和S-亚硝基谷胱甘肽之间的细胞平衡。最近，我们在E.大肠杆菌中发现了一种新的SNO蛋白脱硝酶（首次在微生物中发现）。在本研究中，我们将采用E.大肠杆菌作为模式系统，以确定系统的脱亚硝基酶，部分基于我们的发现，一个特定的转录因子（TF）是S-亚硝基化和亚硝化胁迫下激活。因此上调的独特调节子至少部分通过诱导脱亚硝基化活性来控制细胞SNO蛋白水平。在目标1中，我们将专注于新发现的脱硝酶和二巯基还原酶硫氧还蛋白，我们以前确定为SNO-蛋白脱硝酶在哺乳动物细胞中，我们将采用固相蛋白质组学方法，我们介绍，以确定SNO-蛋白（诱导亚硝化应激），作为这些酶的底物。我们已经发现多种蛋白质，包括TF本身，在缺乏所有已知的脱亚硝基酶的细胞中快速脱亚硝基化，并且在目标2中，我们将：a）询问SNO-TF相互作用组和B）建立脱亚硝基化酶活性的生物化学筛选，以鉴定负责的脱亚硝基化酶。在目标3中，我们将筛选在TF的S-亚硝基化后诱导的调节子的组分以用于新的脱亚硝基化活性。因此，这些目标集中在鉴定新的脱亚硝基酶及其底物上。所提出的研究与人类病理生理学直接相关，因为我们先前已经确定，在微生物中发现的脱亚硝基化激活可能是高度保守的，因此我们的分析可能揭示新的酶活性，在人类疾病中S-亚硝基化失调的分析中具有广泛的范围。此外，由于脱亚硝基酶保护细菌免受亚硝化应激，这是哺乳动物先天免疫的主要组成部分，我们的研究可能指向潜在的治疗目标，在治疗细菌的发病机制。

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.