Posttranslational Molecular Regulation of Human iNOS

描述(申请人提供)：诱导型一氧化氮合酶(INOS)过量产生一氧化氮(NO)与许多病理疾病的发病机制有关，包括与哮喘相关的呼吸道炎症。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。对iNOS的选择性调节需要对其细胞生物学有更好的了解。虽然人们对影响iNOS合成和催化活性的因素了解很多，但对其翻译后调控却知之甚少。这项研究计划的总体目标是了解iNOS在上皮细胞中转化的翻译后机制。 我们最近发现iNOS主要是通过26S蛋白酶体降解的。此外，我们已经证明iNOS的泛素化是其降解所必需的。然而，调控这一过程的分子机制仍有待研究。我们的初步数据表明，iNOS受到磷酸化的影响，这一过程先于其泛素化。他们进一步表明，与二聚体(活性形式)相比，iNOS单体(非活性形式)可能更容易被降解。 我们假设A)：特定诱导型一氧化氮合酶残基上的磷酸化(S)调节诱导型一氧化氮合酶的泛素化，从而调节其降解。此外，参与诱导型一氧化氮合酶泛素化的特定赖氨酸残基(S)位于加氧酶结构域(二聚体界面)，从而允许诱导型一氧化氮合酶单体更快地周转，暴露的赖氨酸残基。B)细胞通过保持强大的iNOS周转率来维持对NO合成的严格控制，从而允许通过磷酸化和其他与细胞类型、底物和/或辅因子相关的细胞因子来快速调节iNOS的降解。 为了验证这些假说，我们提出了以下特定目的的研究：1)阐明磷酸化在iNOS转换中的作用。2)鉴定与iNOS泛素化有关的特定泛素连接酶活性。3)iNOS泛素化特定位点的测定。4)通过检查调节iNOS周转率的因素来表征iNOS周转率。将在表达iNOS的上皮细胞和在气/液界面培养的原代支气管上皮细胞中进行研究。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 many pathological disorders, including airway inflammation associated with asthma. The long-term goal of this research is to understand the regulation of iNOS activity and to devise novel methods to regulate it. Selective modulation of iNOS requires better understanding of its cellular biology. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its posttranslational regulation. The overall objective of this research proposal is to understand the posttranslational mechanisms of iNOS turnover in epithelial cells. We have recently shown that iNOS is primarily degraded through the 26S proteasome. In addition, we have shown that iNOS ubiquitination is required for its degradation. However, the molecular mechanisms by which this process is regulated remain to be studied. Our preliminary data suggest that iNOS is subject to phosphorylation and that this process precedes its ubiquitination. They further suggest that iNOS monomers (inactive form) may be preferentially targeted for degradation compared to dimers (active form). We hypothesize that A): phosphorylation on specific iNOS residue(s) regulates iNOS ubiquitination and hence its degradation. Furthermore, specific lysine residue(s), which are involved in iNOS ubiquitination lie within the oxygenase domain (dimer interface), thus allowing for faster turnover of iNOS monomers, with exposed lysine residues. B) Cells maintain a tight control over the regulation of NO synthesis by maintaining a robust rate of iNOS turnover, thus allowing rapid modulation of iNOS degradation by phosphorylation and by other cellular factors that relate to cell type, availability of substrate and/or cofactors. To test these hypotheses we propose studies with the following Specific Aims: 1) Elucidation of the role of phosphorylation in iNOS turnover. 2) Characterization of specific ubiquitin ligase activity responsible for iNOS ubiquitination. 3) Determination of specific sites of iNOS ubiquitination. 4) Characterization of the rate of iNOS turnover by examining factors that modulate it. Studies will be conducted in epithelial cells expressing iNOS and in primary bronchial epithelial cells cultured at the air/liquid interphase. The rational for the proposed studies is that once these mechanisms are understood, therapeutic strategies can be designed to modulate iNOS turnover.