Regulation and function of human inducible nitric oxide synthase

人诱导型一氧化氮合酶的调节和功能

• 批准号: RGPIN-2019-05192
• 负责人: Choy, Jonathan
• 金额: $ 2.33万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761192
• 关键词: Regulation; function; human; inducible; nitric

Inducible nitric oxide synthase (iNOS) produces the bioactive gas NO that has many cell and physiological functions.  iNOS is expressed in all mammals but is regulated in a species specific manner.  As such, studying its regulation and function in humans is needed to understand human cell biology and may have implications for understanding mammalian evolution. The short-term objectives of this Discovery Grant application are to examine new post-translational and transcriptional processes that regulate human iNOS expression and to identify new cell endogenous effects of iNOS-derived NO. The proposed studies are based on our identification of two novel mechanisms that control the induction of human iNOS expression. The first is a positive feedback amplification of iNOS protein expression by NO, which occurs through the activation of mammalian target of rapamycin (mTOR) that subsequently prevents the proteasome-mediated degradation of iNOS protein. mTOR is a serine/threonine kinase that regulates cell growth and metabolism through increasing mRNA translation. The mechanism by which mTOR increases iNOS protein levels is interesting because little is known about how this kinase affects post-translational processes, such as protein degradation. The second regulatory mechanism that we identified is the need for the transcription factor hypoxia inducible factor-1 (HIF-1) in the cytokine-mediated induction of iNOS gene expression in normoxic conditions. HIF-1 normally controls the response of cells to hypoxia but is increasingly being implicated in non-hypoxia responses. We do not know how cytokines induce the activation of HIF-1 to increase iNOS expression. Given the outstanding issues described, we will examine the poorly understood mechanisms by which mTOR and HIF-1 act to induce iNOS expression.  We will also examine the effects of iNOS-derived NO on cell biological responses. The findings have the potential to expand our knowledge related to the biology of iNOS and NO as well as to identify new cell biological functions of mTOR and HIF-1. The specific aims will be: 1.Determine how mTOR amplifies iNOS protein levels. We will identify post-translational modifications of iNOS that are controlled by NO and mTOR. The effect of these modifications on iNOS protein stability will be determined by mutation studies. 2.Examine the mechanism by which cytokines induce HIF-1 activation to up-regulate iNOS expression. We will examine the signaling mechanisms by which cytokines induce HIF-1 trans-activation of iNOS gene expression as well as the structural motifs in HIF-1a that are needed for this effect. 3.Examine the cell biological effects of iNOS-derived NO. We will use RNA-seq to determine how global gene expression signatures are regulated by iNOS-derived NO. This will provide information on cellular processes that are controlled by iNOS-derived NO. The role of NO in the candidate cellular processes will then be examined experimentally.

诱导型一氧化氮合酶 (iNOS) 产生具有多种细胞和生理功能的生物活性气体 NO，iNOS 在所有哺乳动物中表达，但以物种特异性方式进行调节，因此，需要研究其在人类中的调节和功能。这项发现资助申请的短期目标是研究调节人类 iNOS 表达的新翻译后和转录过程，并确定新的细胞内源性效应。 iNOS 衍生的 NO 所提出的研究基于我们对控制人类 iNOS 表达诱导的两种新机制的鉴定，第一个是 NO 对 iNOS 蛋白表达的正反馈放大，这是通过激活哺乳动物雷帕霉素靶点而发生的。 (mTOR) 随后阻止蛋白酶体介导的 iNOS 蛋白降解 mTOR 是一种丝氨酸/苏氨酸激酶，通过增加 mRNA 翻译来调节细胞生长和代谢 mTOR 增加 iNOS 的机制。蛋白质水平很有趣，因为人们对这种激酶如何影响翻译后过程（例如蛋白质降解）知之甚少。我们确定的第二个调节机制是细胞因子中转录因子缺氧诱导因子-1 (HIF-1) 的需要。在常氧条件下，HIF-1 介导的 iNOS 基因表达诱导通常控制细胞对缺氧的反应，但我们不知道细胞因子如何诱导 HIF-1 的激活。鉴于所描述的突出问题，我们将研究尚不清楚的 mTOR 和 HIF-1 诱导 iNOS 表达的机制。我们还将研究 iNOS 衍生的 NO 对细胞生物反应的影响。扩大我们与 iNOS 和 NO 生物学相关的知识以及确定 mTOR 和 HIF-1 的新细胞生物学功能的潜力。具体目标是： 1.确定 mTOR 如何放大 iNOS 蛋白水平。确定由 NO 和 mTOR 控制的 iNOS 翻译后修饰。这些修饰对 iNOS 蛋白稳定性的影响将通过突变研究确定。 2.检查细胞因子诱导 HIF-1 激活以上调 iNOS 表达的机制。我们将研究细胞因子诱导 HIF-1 反式激活 iNOS 基因表达的信号传导机制以及该作用所需的 HIF-1a 结构基序 3.检查细胞生物学。我们将使用 RNA-seq 来确定 iNOS 衍生的 NO 如何调节全局基因表达特征，这将提供有关 iNOS 衍生的 NO 控制的细胞过程的信息。然后将通过实验检查候选细胞过程。