Regulation and function of human inducible nitric oxide synthase

诱导型一氧化氮合酶（Inducible nitric oxide synthase，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调节。 因此，需要研究其在人类中的调节和功能，以了解人类细胞生物学，并可能对理解哺乳动物进化产生影响。 这个发现补助金申请的短期目标是研究新的翻译后和转录过程，调节人iNOS的表达，并确定新的细胞内源性的iNOS衍生NO的影响。拟议的研究是基于我们确定的两个新的机制，控制诱导人iNOS的表达。第一种是NO对iNOS蛋白表达的正反馈放大，其通过激活哺乳动物雷帕霉素靶蛋白（mTOR）而发生，所述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蛋白水平的机制是有趣的，因为对这种激酶如何影响翻译后过程（如蛋白质降解）知之甚少。 我们确定的第二个调节机制是在常氧条件下，在精氨酸介导的诱导iNOS基因表达中需要转录因子缺氧诱导因子1（HIF-1）。 HIF-1通常控制细胞对缺氧的反应，但越来越多地参与非缺氧反应。 我们不知道细胞因子如何诱导HIF-1的活化以增加iNOS的表达。 考虑到所描述的突出问题，我们将研究mTOR和HIF-1诱导iNOS表达的机制。 我们还将研究iNOS衍生的NO对细胞生物学反应的影响。 这些发现有可能扩大我们对iNOS和NO生物学的了解，并确定mTOR和HIF-1的新细胞生物学功能。 具体目标是： 1.确定mTOR如何放大iNOS蛋白水平。 我们将确定由NO和mTOR控制的iNOS的翻译后修饰。 这些修饰对iNOS蛋白稳定性的影响将通过突变研究来确定。 2.检查细胞因子诱导HIF-1活化以上调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HIF-1a中这种效应所需的结构基序。 3.我们将使用RNA-seq来确定全球基因表达的签名是如何由iNOS衍生的NO调节。这将提供有关由iNOS衍生的NO控制的细胞过程的信息。NO在候选细胞过程中的作用将通过实验进行研究。

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 factor1 (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.