Cellular regulation of nitric oxide synthase expression and S-nitrosylation

一氧化氮合酶表达和 S-亚硝基化的细胞调节

• 批准号: RGPIN-2014-06583
• 负责人: Choy, Jonathan
• 金额: $ 2.99万
• 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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=612313
• 关键词: Cellular; regulation; nitric; oxide; synthase

Nitric oxide (NO) is a bioactive gas that controls a wide array of cell biological processes. It exerts its biological effects mainly by causing targeted protein S-nitrosylation, which is the covalent addition of a nitroso moiety onto target cysteines and is an enzymatically-controlled process in cells. S-nitrosylation controls protein function and has been proposed to be of similar importance to cell biology as protein phosphorylation. However, its regulation and biological effects are relatively understudied. Because of the biological importance of NO, the long-term goal of my research program is to understand the biological effects and cellular regulation of this gas. In order to fully understand the biology of NO, it is important to also study the mechanisms by which its production is regulated. NO is synthesized by enzymes called nitric oxide synthases (NOSs), of which there are three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Cytokines modulate the expression of all NOSs, and we have studied the cytokine regulation of iNOS and eNOS expression. With regard to iNOS, we have shown that NO initiates a positive feedback loop that amplifies iNOS expression by inducing Ras S-nitrosylation. We also know that NO-mediated amplification of iNOS expression occurs through increased mRNA translation. However, we do not know how Ras S-nitrosylation is regulated in cells and how iNOS mRNA translation is controlled by the NO-mediated feedback pathway that we have identified. These questions need to be investigated. In addition to iNOS, we have also determined that the cytokine IL-17 up-regulates eNOS expression through an unknown post-translational mechanism. Based on our findings, the short-term objectives of this Discovery Grant are to define the cellular regulation of Ras S-nitrosylation and post-transcriptional regulation of iNOS and eNOS expression. The work proposed will advance our understanding of the cell biological effects of NO and of NOS regulation. The three aims are: Aim 1: Determine the mechanism controlling Ras S-nitrosylation and its effect on Ras function. We have determined that iNOS-derived NO leads to Ras S-nitrosylation but do not know the mechanism by which this occurs. Much of the S-nitrosylation that occurs in cells is enzymatically-regulated by nitrosylases, which catalyze the reaction, and by de-nitrosylases, which reverse it. We will identify the nitrosylases and de-nitrosylases that target Ras. The effect of S-nitrosylation on Ras function will also be determined by examining the effect of this protein modification on the association of Ras with signaling molecules and on Ras localization. Aim 2: Determine the mechanism by which NO amplifies iNOS mRNA translation . We have characterized a new NO-mediated signaling pathway that amplifies iNOS mRNA translation but do not know the specific translational mechanisms involved. We will determine this by identifying iNOS mRNA binding proteins that are affected by NO. The function of candidate proteins will then be determined by inhibiting their expression with siRNA and by mutating their RNA binding motifs in iNOS mRNA sequences. Aim 3: Determine the post-translational mechanism by which IL-17 increases eNOS protein levels. We have shown that IL-17 increases eNOS protein levels through a post-translational process but do not know the mechanism. The effect of IL-17 on eNOS post-translational modifications that are known to affect protein stability, such as phosphorylation and ubiqutinylation, will be determined by proteomic analysis. The role of candidate protein modifications will then be determined by mutating the modification site(s).

一氧化氮 (NO) 是一种生物活性气体，可控制多种细胞生物过程。它主要通过引起目标蛋白S-亚硝基化来发挥其生物学作用，这是亚硝基部分共价添加到目标半胱氨酸上，是细胞中酶促控制的过程。 S-亚硝基化控制蛋白质功能，并且已被认为对细胞生物学与蛋白质磷酸化具有相似的重要性。然而，其调节和生物效应的研究相对较少。由于一氧化氮的生物学重要性，我的研究计划的长期目标是了解这种气体的生物效应和细胞调节。 为了充分了解 NO 的生物学特性，研究其产生的调节机制也很重要。 NO 由称为一氧化氮合酶 (NOS) 的酶合成，其中有三种亚型：神经型 (nNOS)、诱导型 (iNOS) 和内皮型 (eNOS)。细胞因子调节所有NOS的表达，我们研究了细胞因子对iNOS和eNOS表达的调节。关于 iNOS，我们已经证明 NO 启动正反馈循环，通过诱导 Ras S-亚硝基化来放大 iNOS 表达。我们还知道，NO 介导的 iNOS 表达放大是通过增加 mRNA 翻译而发生的。然而，我们不知道Ras S-亚硝基化在细胞中是如何调节的，也不知道我们已经确定的NO介导的反馈途径如何控制iNOS mRNA翻译。这些问题都需要调查。除了 iNOS 之外，我们还确定细胞因子 IL-17 通过未知的翻译后机制上调 eNOS 表达。 根据我们的研究结果，本次发现资助的短期目标是确定 Ras S-亚硝基化的细胞调节以及 iNOS 和 eNOS 表达的转录后调节。这项工作将增进我们对 NO 和 NOS 调节的细胞生物学效应的理解。这三个目标是： 目标 1：确定 Ras S-亚硝基化的控制机制及其对 Ras 功能的影响。 我们已经确定 iNOS 衍生的 NO 会导致 Ras S-亚硝基化，但不知道其发生的机制。细胞中发生的大部分 S-亚硝基化反应均受到催化反应的亚硝基化酶和逆转反应的去亚硝基化酶的酶促调节。我们将鉴定针对 Ras 的亚硝基酶和去亚硝基酶。 S-亚硝基化对 Ras 功能的影响也将通过检查这种蛋白质修饰对 Ras 与信号分子的关联以及 Ras 定位的影响来确定。 目标 2：确定 NO 放大 iNOS mRNA 翻译的机制。 我们已经表征了一种新的 NO 介导的信号通路，该通路可放大 iNOS mRNA 翻译，但不知道所涉及的具体翻译机制。我们将通过鉴定受 NO 影响的 iNOS mRNA 结合蛋白来确定这一点。然后通过用 siRNA 抑制候选蛋白的表达并突变 iNOS mRNA 序列中的 RNA 结合基序来确定候选蛋白的功能。 目标 3：确定 IL-17 增加 eNOS 蛋白水平的翻译后机制。 我们已经证明 IL-17 通过翻译后过程增加 eNOS 蛋白水平，但不知道其机制。 IL-17 对 eNOS 翻译后修饰（已知会影响蛋白质稳定性，例如磷酸化和泛素化）的影响将通过蛋白质组学分析来确定。然后，通过修饰位点的突变来确定候选蛋白质修饰的作用。