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核因子的上皮活化？B (NF - ?B)，一种调节许多参与炎症过程的基因表达的转录因子。诱导一氧化氮（NO）合成酶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，可能是通过关键蛋白的s -亚硝化作用。由于炎症条件涉及粒细胞的募集和氧化剂产生酶的激活，如嗜酸性粒细胞过氧化物酶（EPO）， NO的代谢可能会因为氧化转化为潜在的促炎活性氮（RNS）而改变，NO的生物活性（通过s -亚硝化）可能会降低。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，并且通过酪氨酸硝化增加证明了RNS的形成。因此，我们假设氧化NO"代谢的增加导致NF-？B途径，从而促进其慢性激活，从而增加气道炎症。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，因此解决这样的假设是困难的。我们采用了最近开发的一种基于化学衍生化的程序来选择性地生物素化和/或纯化s -亚硝化蛋白，使我们能够在完整的组织切片中检测s -亚硝化蛋白，并通过更全面的蛋白质组学分析收集s -亚硝化蛋白以鉴定细胞靶标。我们计划使用这些方法来确定过敏性气道炎症小鼠模型中s -亚硝化与NO代谢的变化（Aim 1），并确定特定蛋白质中s -亚硝化的变化与NF-？B在培养的气道上皮细胞（Aim 2）和变应性气道炎症小鼠气道中的激活和基因表达（Aim 3）。最后，通过使用EPO或髓过氧化物酶（MPO）缺乏和过氧化氢酶过表达的各种（敲除）模型，我们计划探索产生氧化剂的酶在NO代谢改变中的作用，以及在NF-？B活化及基因表达。总的来说，我们预计这些研究将提供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.