Regulation and function of human inducible nitric oxide synthase

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在所有哺乳动物中均有表达，但受物种特定方式的调控。因此，研究其在人类中的调节和功能是了解人类细胞生物学的必要条件，并可能对理解哺乳动物的进化具有指导意义。这项发现基金申请的短期目标是研究调节人类iNOS表达的新的翻译后和转录过程，并确定iNOS衍生的NO的新的细胞内源性效应。提出的研究是基于我们确定的两种控制诱导人类iNOS表达的新机制。第一种是NO对iNOS蛋白表达的正反馈扩增，这是通过激活哺乳动物雷帕霉素靶蛋白（mTOR）来实现的，该靶蛋白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翻译调节细胞生长和代谢。mTOR增加iNOS蛋白水平的机制很有趣，因为人们对这种激酶如何影响翻译后过程（如蛋白质降解）知之甚少。我们确定的第二种调节机制是在常氧条件下，细胞因子介导的iNOS基因表达诱导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，但越来越多地涉及非缺氧反应。我们不知道细胞因子是如何诱导HIF-1激活从而增加iNOS表达的。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生物学的认识，并确定mTOR和HIF-1的新的细胞生物学功能。

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.