NITRIC OXIDE SIGNALING IN ALLERGIC AIRWAY DISEASE

描述（由申请人提供）：哮喘的特征是气道高反应性和重塑，以及传导气道的慢性炎症。气道炎症反应中的一个关键事件是上皮细胞核因子的激活？B（NF-？B），一种调节参与炎症过程的许多基因表达的转录因子。一氧化氮（NO）合酶的诱导是气道炎症的共同特征，各种细胞研究表明NO对NF？B介导的基因表达，推测是通过关键蛋白质的S-亚硝化。由于炎性病症涉及粒细胞的募集和氧化剂产生酶如嗜酸性粒细胞过氧化物酶（EPO）的活化，NO-的代谢可能由于氧化转化为潜在的促炎活性氮物质（RNS）而改变，并且NO-的生物活性（通过S-亚硝化）可能降低。事实上，在严重哮喘患者的气道分泌物中发现了低于正常的S-亚硝基硫醇水平，并且通过增加酪氨酸硝化证实了RNS的形成。因此，我们推测，增加氧化NO”代谢的结果在减少S-亚硝化的NF-？B途径，从而促进其慢性激活，并因此增加气道炎症。由于缺乏足够的工具来检测完整细胞或组织中的S-亚硝基蛋白，因此解决这种假设是困难的。我们已经适应了最近开发的程序，基于化学衍生选择性生物素化和/或纯化S-亚硝化蛋白质，使我们能够检测完整组织切片中的S-亚硝化蛋白质，并收集S-亚硝化蛋白质，用于通过更全面的蛋白质组学分析识别细胞靶点。我们计划使用这些方法，以确定在S-亚硝化的变化与NO代谢的小鼠模型过敏性气道炎症（目的1），并确定在S-亚硝化的变化与NF-？B激活和基因表达，在培养的气道上皮细胞（目的2）和过敏性气道炎症小鼠气道（目的3）。最后，与使用各种（敲除）模型的缺陷，在EPO或髓过氧化物酶（MPO）和过氧化氢酶的过度表达，我们计划探讨的作用，氧化剂产生酶的改变在NO”代谢，并在S-亚硝化的蛋白质参与NF-？B激活和基因表达。总的来说，我们预计这些研究将提供因果关系之间的NO代谢，NF-？B活化和慢性气道炎症。

DESCRIPTION (provided by applicant): Asthma is characterized by airway hyperreactivity and remodeling, and chronic inflammation of the conducting airways. A critical event in the inflammatory response with the airways is the epithelial activation of nuclear factor ?B (NF-?B), a transcription factor that regulates the expression of many genes involved in the inflammatory process. Induction of nitric oxide (NO) synthase is a common feature of airway inflammation, and various cell studies have illustrated inhibitory effects of NO" on NF ?B mediated gene expression, presumably by S-nitrosation of critical proteins. Since inflammatory conditions involve recruitment of granulocytes and activation of oxidant-producing enzymes, such as eosinophil peroxidase (EPO), metabolism of NO" is likely to be altered because of oxidative conversion to potentially proinflammatory reactive nitrogen species (RNS), and bioactivity of NO" (through S-nitrosation) may be reduced. Indeed, subnormal S-nitrosothiol levels have been found in airway secretions of severe asthmatics, and formation of RNS has been demonstrated by increased tyrosine nitration. We therefore hypothesize that increased oxidative NO" metabolism results in reduced S-nitrosation of components of the NF-?B pathway, thereby promoting its chronic activation and consequently augmenting airway inflammation. Addressing such a hypothesis has been difficult because of a lack of adequate tools to detect S-nitrosoproteins in intact cells or tissues. We have adapted a recently developed procedure, based on chemical derivatization to selectively biotinylate and/or purify S-nitrosated proteins, allowing us to detect S-nitrosated proteins in intact tissue sections and to collect S-nitrosated proteins for identification of cellular targets by more global proteomic analysis. We plan to use these approaches to determine changes in S-nitrosation in relation to NO metabolism in a mouse model of allergic airway inflammation (Aim 1), and to identify changes in S-nitrosation in specific proteins in relation to alterations in NF-?B activation and gene expression, in cultured airway epithelial cells (Aim 2) and in airway of mice with allergic airway inflammation (Aim 3). Finally, with the use of various (knock-out) models of deficiency in EPO or myeloperoxidase (MPO) and of overexpression of catalase, we plan to explore a role of oxidant-producing enzymes in alterations in NO" metabolism, and in changes in S-nitrosation of proteins involved in NF-?B activation and gene expression. Collectively, we anticipate that these studies will provide causal links between NO" metabolism, NF-?B activation, and chronic airway inflammation.