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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=647654
• 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，可控制各种细胞生物学过程。它主要通过引起靶向蛋白S-硝基化来发挥其生物学作用，这是将氮气部分共价添加到靶半胱氨酸上，并且是细胞中酶促控制的过程。 S-亚硝基化控制蛋白质功能，并已被认为与细胞生物学相似，与蛋白质磷酸化相似。 但是，其调节和生物学作用相对研究。 由于NO的生物学重要性，我的研究计划的长期目标是了解该气体的生物学作用和细胞调节。 *为了充分了解NO的生物学，重要的是要研究其生产的机制。 NO是由称为一氧化氮合酶（NOSS）的酶合成的，其中有三种同工型：神经元（NNOS），诱导（Inos）和内皮（ENOS）。 细胞因子调节所有NOSS的表达，我们研究了iNOS和eNOS表达的细胞因子调节。 关于iNOS，我们已经表明，没有启动正反馈回路，该反馈环通过诱导ras s-硝基化来放大iNOS表达。 我们还知道，通过增加的mRNA翻译，无介导的iNOS表达扩增发生。 但是，我们不知道如何在细胞中调节Ras S-硝基化，以及如何通过我们鉴定的NO介导的反馈途径来控制INOS mRNA翻译。这些问题需要研究。除iNOS外，我们还确定了细胞因子IL-17通过未知的翻译后机制上调eNOS的表达。 *基于我们的发现，该发现赠款的短期目标是定义iNOS和eNOS表达的RAS S-硝基化和转录后调节的细胞调节。提出的工作将提高我们对NO和NOS调控的细胞生物学作用的理解。 这三个目的是：** AIM 1：确定控制RAS S-硝基化的机制及其对RAS功能的影响。 *我们已经确定，iNOS衍生的没有导致ras s-硝基化的导致，但不知道发生这种情况的机制。 在细胞中发生的许多S-硝基化是由硝基基酶酶调节的，这些硝基酶会催化反应，以及脱硝基酶，从而将其逆转。 我们将鉴定靶向RA的亚硝基酶和脱硝基酶。 S-亚硝基化对RAS功能的影响也将通过检查该蛋白质修饰对RAS与信号分子和RAS定位的缔合的影响来确定。 **目标2：确定不放大iNOS mRNA翻译的机制。 *我们表征了一种新的无介导的信号通路，该信号通路会放大iNOS mRNA翻译，但不知道涉及的特定翻译机制。 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，将通过抑制siRNA的表达并在iNOS mRNA序列中突变其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。 *我们已经表明，IL-17通过翻译后过程提高了eNOS蛋白水平，但不知道该机制。 IL-17对已知会影响蛋白质稳定性的eNOS翻译后修饰的影响，例如磷酸化和Ubiqutinylation，将通过蛋白质组学分析确定。然后，将通过突变修饰位点来确定候选蛋白质修饰的作用。