S-nitrosothiol breakdown by airway epithelial cells

描述(申请人提供)：用一氧化氮修饰半胱氨酸残基会改变一系列蛋白质的功能。这种蛋白质修饰代表了一种生理信号机制，称为S-亚硝化，已被认为类似于磷酸化。它调节炎症细胞和其他与肺部生物学有关的细胞的许多生物活性。然而，与磷酸化不同，人们对S-亚硝化信号的调控知之甚少。在本课题组前期工作中，我们研究了内源性支气管扩张剂S-亚硝基-谷胱甘肽和S-亚硝胺在呼吸道中的代谢调节。我们现在已经完成了这个项目的大部分目标。事实上，GSNO现在正在作为一种吸入疗法进入临床试验。然而，关于人体呼吸道S-亚硝硫醇代谢的关键问题仍然存在，因为它与肺中的特定分子靶点有关。例如，我们和其他人已经证明，在哮喘动物模型中，GSNO还原酶在呼吸道上皮细胞的表达是呼吸道高反应性的重要决定因素。此外，我们还发现了证据表明，GSNO的分解产物S-亚硝基-L-半胱氨酸通过肌球蛋白的S亚硝化引起立体选择性的非周期GMP依赖性的平滑肌松弛。此外，一些研究人员已经报道，GSNO可以调节与肺部疾病相关的蛋白质的功能和细胞内运输，包括囊性纤维化跨膜调节蛋白。我们开发了测量细胞S的新技术-亚硝硫醇。在本项目中，我们将应用这些新技术在人类呼吸道组织中研究1)GsNO还原酶表达的调节(目标1)；2)GsNO还原酶和其他S亚硝硫醇调节酶对平滑肌功能的影响(目标2)；以及3)S亚硝硫醇调节酶对上皮细胞中S亚硝硫醇修饰的蛋白质代谢和运输的影响，特别是在GSNO治疗的背景下(目标3)。我们相信，我们的出版记录和初步数据表明，我们可以在拟议的时间框架内实现这些目标。此外，我们的数据还表明，这些目标是创新的、科学合理的，并对哮喘和囊性纤维化的治疗具有潜在的相关性。项目简介：我们的项目专注于信号分子S-亚硝硫醇的肺部新陈代谢。我们发现有证据表明，呼吸道中S-亚硝硫醇代谢异常可能有助于解释许多哮喘患者的气道狭窄。此外，事实证明，了解这种新陈代谢可能对开发某些治疗哮喘和囊性纤维化的新疗法很重要。

DESCRIPTION (provided by applicant): Modification of cysteine residues by nitric oxide changes the function of a broad spectrum of proteins. This protein modification represents a physiological signaling mechanism, termed S- nitrosylation, that has been proposed to be analogous to phosphorylation. It regulates many of the bioactivities of inflammatory cells and other cells relevant to pulmonary biology. However, unlike phosphorylation, the regulation of S-nitrosylation signaling is poorly understood. In our previous project, we studied the metabolic regulation in the airway of S-nitrosoglutathione (GSNO), an endogenous bronchodilator and S-nitrosylating agent. We have now accomplished most of the aims of this project. Indeed, GSNO is now entering clinical trials as an inhalational therapy. However, critical questions remain regarding human airway S-nitrosothiol metabolism as it relates to specific molecular targets in the lung. For example, we and others have shown that the airway epithelial expression of the enzyme, GSNO reductase, is an important determinant of airway hyper-responsiveness in animal models of asthma. Moreover, we have found evidence that the GSNO breakdown product, S-nitroso-L-cysteine, causes stereoselective, cyclic GMP-independent smooth muscle relaxation through S-nitrosylation of myosin. Further, several investigators have reported that GSNO can regulate the function and intracellular trafficking of proteins relevant to lung disease, including the cystic fibrosis transmembrane regulatory protein. We have developed novel techniques for measuring cellular S-nitrosothiols. In the current project, we will apply these novel techniques to study, in human airway tissue, 1) the regulation of GSNO reductase expression (Aim 1); 2) the effect of GSNO reductase and other S-nitrosothiol regulatory enzymes on smooth muscle function (Aim 2); and 3) the effect of S-nitrosothiol regulatory enzymes on the metabolism and trafficking of S-nitrosothiol-modified proteins in epithelial cells, particularly in the context of GSNO therapy (Aim 3). We believe that our publication record and preliminary data suggest that we can accomplish these aims in the proposed time frame. Further, our data also suggest that these aims are innovative, scientifically sound and of potential relevance to the treatment of asthma and cystic fibrosis. Project Narrative: Our project is focused on the pulmonary metabolism of signaling molecules known as S-nitrosothiols. We have found evidence that abnormal S-nitrosothiol metabolism in the airway may help to explain airway narrowing in many patients with asthma. Further, it turns out that understanding this metabolism is likely to be important for the development of certain new therapies for asthma and cystic fibrosis.