Identification of Novel Denitrosylases

描述(申请人提供)：翻译后的蛋白质修饰S-亚硝化，一氧化氮(NO)基团与半胱氨酸的共价加成形成S-亚硝基蛋白质(SNO-蛋白质)，在哺乳动物系统中介导了NO对细胞功能的普遍影响，并且调节失调的S-亚硝化已与广泛的人类疾病有关。越来越多的证据表明，酶介导的脱氮作用，即从SNO-蛋白质中去除NO基团，在调节蛋白质S-亚硝化的水平和动态中起着至关重要的作用，但还没有系统地鉴定脱硝基酶或对其底物进行描述。在此之前，我们使用 S-亚硝基谷胱甘肽还原酶(GSNOR)不直接作用于SNO-蛋白，而是通过至少部分SNO-蛋白和S-亚硝基谷胱甘肽之间的细胞平衡来调节蛋白质S-亚硝化。最近，我们在大肠杆菌中的分析发现了一种新的SNO-蛋白质脱氮酶(首次在微生物中描述)。在这里提出的研究中，我们将使用大肠杆菌作为一个模型系统来系统地鉴定脱氮酶，部分是基于我们的发现，特定的转录因子(TF)是S亚硝化的，并在亚硝化胁迫下被激活。因此，上调的独特调控子至少部分通过诱导脱氮活性来调控细胞SNO蛋白水平。在目标1中，我们将重点介绍新发现的脱氮酶和二硫醇还原酶硫氧还蛋白，该酶以前被我们鉴定为哺乳动物细胞中的SNO-蛋白质脱氮酶，我们将使用我们介绍的固相蛋白质组学方法来确定作为这些酶的底物的SNO-蛋白质(在亚硝酸盐胁迫下诱导)。我们已经发现，在缺乏所有已知的脱氮酶的细胞中，包括Tf本身在内的多种蛋白质可以快速脱氮，在目标2中，我们将：a)询问SNO-Tf相互作用组，b)建立脱氮酶活性的生化筛选，以确定负责的脱氮酶(S)。在目标3中，我们将筛选在S-Tf亚硝化过程中诱导的调节子的成分，以实现新的脱氮活性。因此，这些目标集中在新的脱氮酶及其底物的鉴定上。拟议的研究与人类的病理生理学有直接的相关性，因为我们先前已经证实，在微生物中发现的反硝化活性在系统发育中可能是高度保守的，因此我们的分析很可能揭示新的酶活性，在分析人类疾病中调节失调的S-亚硝化方面具有广泛的视野。此外，由于脱氮酶保护细菌免受亚硝化应激的影响，亚硝化应激是哺乳动物天然免疫的主要组成部分，我们的研究可能指出在治疗细菌发病机制方面的潜在治疗靶点。

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.