S-nitrosothiol breakdown by airway epithelial cells

描述（由申请人提供）：一氧化氮对半胱氨酸残基的修饰改变了广谱蛋白质的功能。这种蛋白质修饰代表了一种称为S-亚硝基化的生理信号传导机制，其已被提出类似于磷酸化。它调节炎性细胞和与肺生物学相关的其他细胞的许多生物活性。然而，与磷酸化不同，S-亚硝基化信号的调节知之甚少。在我们以前的项目中，我们研究了内源性支气管扩张剂和S-亚硝基化剂S-亚硝基谷胱甘肽（GSNO）在气道中的代谢调节。我们已经实现了这个项目的大部分目标。事实上，GSNO现在正作为吸入疗法进入临床试验。然而，关键问题仍然是关于人气道S-亚硝基硫醇代谢，因为它涉及到特定的分子靶点在肺。例如，我们和其他人已经表明，气道上皮表达的酶，GSNO还原酶，是一个重要的决定因素，气道高反应性的哮喘动物模型。此外，我们已经发现证据表明，GSNO分解产物，S-亚硝基-L-半胱氨酸，通过肌球蛋白的S-亚硝基化引起立体选择性，周期GMP-独立的平滑肌松弛。此外，一些研究者已经报道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。我们已经开发了新的技术来测量细胞S-亚硝基硫醇。在本项目中，我们将应用这些新技术研究人气道组织中GSNO还原酶表达的调节（目的1），GSNO还原酶和其他S-亚硝基硫醇调节酶对平滑肌功能的影响（目的2），和3）S-亚硝基硫醇调节酶对上皮细胞中S-亚硝基硫醇修饰的蛋白质的代谢和运输的影响，特别是在GSNO治疗的情况下（目的3）。我们认为，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，与哮喘和囊性纤维化的治疗有潜在的相关性。项目叙述：我们的项目主要集中在被称为S-亚硝基硫醇的信号分子的肺部代谢。我们发现的证据表明，S-亚硝基硫醇代谢异常的气道可能有助于解释许多哮喘患者的气道狭窄。此外，事实证明，了解这种代谢可能对开发哮喘和囊性纤维化的某些新疗法很重要。

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.