NITRIC OXIDE SIGNALING IN ALLERGIC AIRWAY DISEASE

描述(由申请人提供)：哮喘的特征是呼吸道高反应性和重塑，以及传导气道的慢性炎症。呼吸道炎症反应中的一个关键事件是核因子B(NF-B)的上皮激活，核因子-B是一种转录因子，调节参与炎症过程的许多基因的表达。一氧化氮合酶(NO)的诱导是呼吸道炎症的一个共同特征，各种细胞研究表明，NO“对NF？B介导的基因表达具有抑制作用，这可能是通过S亚硝化关键蛋白来实现的。由于炎症条件涉及粒细胞的募集和产生氧化剂的酶的激活，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性反应氮物种(RNS)而改变，而NO的生物活性(通过S亚硝化)可能会降低。事实上，在重症哮喘患者的呼吸道分泌物中发现了低于正常水平的S-亚硝硫醇，并通过增加酪氨酸硝化证明了RNS的形成。因此，我们假设，氧化NO“代谢增加导致核因子-βB途径各部分的S亚硝化反应减少，从而促进其慢性激活，从而增强呼吸道炎症。解决这样一个假设一直很困难，因为缺乏足够的工具来检测完整细胞或组织中的S亚硝酸蛋白。我们采用了最近开发的一种基于化学衍生化的方法来选择性地生物素化和/或纯化S亚硝化的蛋白质，使我们能够在完整的组织切片中检测到S亚硝化的蛋白质，并收集S亚硝化的蛋白质，以便通过更全面的蛋白质组分析来识别细胞靶标。我们计划使用这些方法来确定过敏性气道炎小鼠模型(目标1)中与NO代谢有关的S亚硝化的变化，并确定S亚硝化在特定蛋白质中的变化与核因子-？B激活和基因表达的变化，以及在培养的气道上皮细胞(目标2)和过敏性气道炎小鼠的气道(目标3)中的变化。最后，通过使用EPO或髓过氧化物酶缺乏和过氧化氢酶过度表达的各种(基因敲除)模型，我们计划探索氧化剂产生酶在NO代谢改变中的作用，以及参与核因子-β活化和基因表达的蛋白质的S亚硝化改变中的作用。总之，我们预计这些研究将提供一氧化氮代谢、核因子-β激活和慢性呼吸道炎症之间的因果联系。

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.