Molecular Regulation of Human iNOS by Phosphorylation

描述(申请人提供)：诱导型一氧化氮合酶(INOS)导致一氧化氮(NO)的过量产生与包括呼吸道肺部炎症在内的几种疾病的发病机制有关。这一建议的总体目标是了解iNOS细胞周转的翻译后机制，从而为未来旨在控制NO合成的研究奠定基础。我们的初步数据表明，iNOS在泛素化之前受到磷酸化的调节。 我们建议检验以下假设：a)连接泛素和iNOS的特定泛素连接酶优先与磷酸化的iNOS结合。因此，特异性诱导型一氧化氮合酶残基的磷酸化(S)对诱导型一氧化氮合酶的泛素化和降解至关重要。B)细胞通过维持受细胞因素调节的iNOS的强劲周转率来严格控制NO的合成。 为了验证这些假设，我们提出了以下特定目标的研究：目标1：阐明磷酸化在iNOS周转中的作用并确定特定的磷酸化残基；目标2：表征iNOS周转速率和调节它的细胞因素。建议研究的理由是，一旦了解了这些机制，就可以设计治疗策略来改变这些途径并调节iNOS的周转。

DESCRIPTION (provided by applicant): Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway lung inflammation. The overall objective of this proposal is to understand the posttranslational mechanisms of cellular turnover of iNOS and thus lay the groundwork for future studies aiming at controlling NO synthesis. Our preliminary data suggest that iNOS is subject to regulation by phosphorylation precedes its ubiquitination. We propose to test the following hypotheses: A) The specific ubiquitin ligase that links ubiquitin to iNOS binds preferentially to phosphorylated iNOS. Thus phosphorylation of specific iNOS residue(s) is critical for iNOS ubiquitination and degraqdation. B) Cells maintain a tight control over NO synthesis by maintaining a robust rate of iNOS turnover that is modulated by cellular factors. To test these hypotheses we propose studies with the following Specific Aims; Aim 1: Elucidation of the role of phosphorylation in iNOS turnover and determining specific phosporylated residues; Aim 2: Characterization of the rate of iNOS turnover and cellular factors that modulate it. The rational for the proposed studies is that once these mechanisms are understood, therapeutic strategies can be designed to alter these pathways and modulate iNOS turnover.