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在所有哺乳动物中都有表达，但受物种特有的调控方式。因此，研究它在人类中的调节和功能是理解人类细胞生物学所必需的，并可能对理解哺乳动物的进化有意义。Discovery Grant应用程序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发现了两种控制诱导人iNOS表达的新机制。第一种是NO对iNOS蛋白表达的正反馈放大，这是通过激活哺乳动物靶标雷帕霉素(MTOR)从而阻止蛋白酶体介导的iNOS蛋白降解而发生的。MTOR是一种丝氨酸/苏氨酸激酶，通过增加mRNA的翻译来调节细胞的生长和代谢。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。我们确定的第二个调控机制是在常氧条件下，转录因子缺氧诱导因子1(HIF-1)在细胞因子介导的iNOS基因表达诱导中的需要。HIF-1通常控制细胞对低氧的反应，但越来越多地与非低氧反应有关。我们不知道细胞因子是如何诱导HIF-1激活以增加iNOS表达的。鉴于上述悬而未决的问题，我们将研究mTOR和HIF-1诱导iNOS表达的鲜为人知的机制。我们还将研究诱导型一氧化氮合酶衍生的一氧化氮对细胞生物学反应的影响。这一发现有可能扩大我们对iNOS和NO生物学的相关知识，并发现mTOR和HIF-1的新的细胞生物学功能。 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.探讨细胞因子诱导HIF-1活化上调iNOS表达的机制。我们将研究细胞因子诱导iNOS基因表达的HIF-1反式激活的信号机制，以及HIF-1a中这一效应所需的结构基序。 3.检测iNOS来源的NO的细胞生物学效应。我们将使用RNA-seq来确定iNOS衍生的NO如何调节全球基因表达签名。这将提供由iNOS衍生的NO控制的细胞过程的信息。然后将通过实验检验NO在候选细胞过程中的作用。

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.