Cellular Regulation of Nitric Oxide in Airway Inflammation

描述(申请人提供)：诱导型一氧化氮合酶(INOS)过度产生一氧化氮(NO)与哮喘的气道炎发病机制有关。这项研究的长期目标是了解iNOS活性的调节，并设计新的方法来调节它。虽然人们对影响iNOS合成和催化活性的因素知之甚少，但对其细胞调控却知之甚少。我们最近发现iNOS是通过泛素-蛋白酶体途径降解的。泛素连接系统的特异性主要由识别和结合靶蛋白的特定泛素连接酶(E3)提供。因此，鉴定和鉴定iNOS的E3泛素连接酶是理解iNOS细胞调控机制的关键步骤之一。我们的初步数据确定了一个含有F-box的蛋白；我们将其命名为FBSinos，它很可能是iNOS的E3泛素连接酶。更多的初步数据表明，细胞通过对iNOS的时间和空间调节来调节NO的合成。这些机制包括相对较快的iNOS周转速度和iNOS在核周的隔离，我们称之为“生理性侵袭性”。有趣的是，我们的初步数据表明，FBXINOS不仅对iNOS的周转很重要，而且对iNOS的亚细胞靶向也很重要。我们建议检验以下假设：包含F-box的蛋白FBCinos是iNOS的E3连接酶，它在iNOS细胞调控中发挥核心作用。这种FBCinos的细胞调控进一步调节iNOS和NO的细胞水平。为了验证这一假设，我们提出了以下具体目标的研究：1)FBXINOS对iNOS的调节特性。2)确定FBXINOS泛素连接酶靶向侵袭者iNOS的机制。(3)iNOS和FBCinos在气道炎症中的调控特征。研究将在表达诱导型一氧化氮合酶的培养细胞和在气液界面培养的原代支气管上皮细胞中进行。这项拟议研究的基本原理是，一旦了解了这些机制，它们将极大地增加我们对iNOS细胞处理方式的理解。未来的治疗策略可以被设计成在疾病状态下调节这些细胞反应。

DESCRIPTION (provided by applicant): Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of airway inflammation of asthma. The long-term goal of this research is to understand the regulation of iNOS activity and to devise novel methods to regulate it. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its cellular regulation. We have recently shown that iNOS is degraded through the ubiquitin-proteasome pathway. The specificity of the ubiqutination system, is mainly provided by the specific ubiquitin ligase enzyme (E3) that recognizes and binds to the target protein. Therefore, identification and characterization of the E3 ubiquitin ligase for iNOS should be regarded as one of the most critical steps for understanding the mechanisms of iNOS cellular regulation. Our preliminary data identified an F-box-containing protein; we termed FBXinos that is a likely candidate to be the E3 ubiquitin ligase for iNOS. Additional preliminary data suggest that cells regulate NO synthesis by temporal and spatial regulation of iNOS. These mechanisms include a relatively rapid rate of iNOS turnover and sequestration of iNOS to a perinuclear location we termed the "physiologic aggresome". Interestingly, our preliminary data suggest that FBXinos is important not only for iNOS turnover but also for subcellular targeting of iNOS. We propose to test the following hypothesis: An F-box-containing protein, FBXinos, is the E3 ligase for iNOS and it plays a central role in iNOS cellular regulation. The cellular regulation of this FBXinos further modulates iNOS and NO cellular levels. To test this hypothesis we propose studies with the following specific aims: 1) Characterization of the regulation of iNOS by FBXinos. 2) Determine the mechanisms by which FBXinos ubiquitin ligase targets iNOS to the aggresome. 3) Characterization of the regulation of iNOS and FBXinos in airway inflammation. Studies will be conducted in cultured cells expressing iNOS and in primary bronchial epithelial cells cultured at the air/liquid interphase. The rationale for the proposed studies is that once these mechanisms are understood, they would greatly increase our understanding of cellular handling of iNOS. Future therapeutic strategies can be designed to regulate these cellular responses in disease states.