Posttranslational Molecular Regulation of Human iNOS

人类 iNOS 的翻译后分子调控

• 批准号: 6833541
• 负责人: N. Tony Eissa
• 金额: $ 37.63万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-15 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833541
• 关键词: SDS polyacrylamide gel electrophoresis; biosynthesis; cell type; enzyme activity; immunoprecipitation; ligase; mass spectrometry; nitric oxide; nitric oxide synthase; phosphorylation; posttranslational modifications; respiratory epithelium; tissue /cell culture; ubiquitin

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.

描述（由申请人提供）：诱导型一氧化氮合酶（iNOS）过度产生一氧化氮（NO）与许多病理性疾病的发病机制有关，包括与哮喘相关的气道炎症。 本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。选择性调节iNOS需要更好地了解其细胞生物学。 虽然很多人都知道的影响因素的合成和催化活性的iNOS，很少有人知道它的翻译后调节。 本研究的总体目标是了解上皮细胞中iNOS的翻译后机制。 我们最近发现，iNOS主要通过26 S蛋白酶体降解。 此外，我们已经表明，iNOS泛素化是其降解所必需的。 然而，调节这一过程的分子机制仍有待研究。 我们的初步数据表明，诱导型一氧化氮合酶是受磷酸化，这一过程之前，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）可能优先被靶向降解。 我们假设A）：特异性iNOS残基上的磷酸化调节iNOS泛素化，从而调节其降解。 此外，参与iNOS泛素化的特异性赖氨酸残基位于加氧酶结构域（二聚体界面）内，从而允许iNOS单体与暴露的赖氨酸残基更快地周转。 B）细胞通过维持稳健的iNOS更新速率来维持对NO合成调节的严格控制，从而允许通过磷酸化和通过与细胞类型、底物和/或辅因子的可用性相关的其它细胞因子来快速调节iNOS降解。 为了验证这些假设，我们提出了以下具体目的的研究：1）阐明磷酸化在iNOS周转中的作用。 2)负责iNOS泛素化的特异性泛素连接酶活性的表征。 3)确定iNOS泛素化的特异位点。 4)通过检查调节iNOS的因素来表征iNOS周转率。将在表达iNOS的上皮细胞和在空气/液体界面培养的原代支气管上皮细胞中进行研究。 提出的研究的理由是，一旦这些机制被理解，治疗策略可以被设计为调节iNOS营业额。