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
基本信息
- 批准号:RGPIN-2014-06583
- 负责人:
- 金额:$ 2.99万
- 依托单位:
- 依托单位国家:加拿大
- 项目类别:Discovery Grants Program - Individual
- 财政年份:2017
- 资助国家:加拿大
- 起止时间:2017-01-01 至 2018-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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,控制着广泛的细胞生物学过程。它主要通过引起靶蛋白S-亚硝化来发挥其生物学效应,亚硝化是亚硝基与靶半胱氨酸的共价加成,在细胞内是一个酶控制的过程。S-亚硝化控制着蛋白质的功能,已被认为对细胞生物学具有与蛋白质磷酸化类似的重要性。然而,对其调控和生物学效应的研究相对较少。由于NO的生物学重要性,我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,还必须研究其产生的调控机制。一氧化氮是由一氧化氮合酶(NOSS)合成的,它有三种亚型:神经型(NNOS)、诱导型(INOS)和内皮型(ENOS)。细胞因子调节ALL NOS的表达,我们研究了细胞因子对iNOS和eNOS表达的调节。关于诱导型一氧化氮合酶,我们已经证明了一氧化氮通过诱导RAS-S亚硝化来启动一个正反馈环,从而放大诱导型一氧化氮合酶的表达。我们还知道,NO介导的iNOS表达的放大是通过增加mRNA的翻译来实现的。然而,我们不知道Ras-S亚硝化是如何在细胞中调节的,也不知道我们已经确定的NO介导的反馈通路如何控制iNOS mRNA的翻译。这些问题需要调查。除了iNOS,我们还确定了细胞因子IL-17通过一种未知的翻译后机制上调eNOS的表达。根据我们的发现,这项发现拨款的短期目标是确定RAS S的细胞调控-亚硝化以及iNOS和eNOS表达的转录后调控。提出的工作将促进我们对NO的细胞生物学效应和NOS调控的理解。目的1:确定RAS S亚硝化的调控机制及其对RAS功能的影响。我们已经确定诱导型一氧化氮合酶产生的NO导致RAS-S亚硝化,但不知道其发生的机制。细胞中发生的大部分S-亚硝化反应都是由催化该反应的亚硝基酶和逆转该反应的脱亚硝基酶进行酶调节的。我们将确定以RAS为靶点的亚硝酸酶和去亚硝酸酶。S亚硝化对RAS功能的影响也将通过检测这种蛋白质修饰对RAS与信号分子的结合和RAS定位的影响来确定。目的2:探讨一氧化氮(NO)促进iNOS基因翻译的机制。我们已经确定了一种新的NO介导的信号通路,该通路可以放大iNOS mRNA的翻译,但不知道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抑制候选蛋白质的表达,并通过突变iNOS mRNA序列中的RNA结合基序来确定候选蛋白质的功能。目的3:确定IL-17上调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,但机制尚不清楚。IL-17对eNOS翻译后修饰的影响将通过蛋白质组学分析来确定,这些修饰已知会影响蛋白质的稳定性,如磷酸化和泛喹化。候选蛋白质修饰的作用将通过突变修饰位点来确定(S)。
项目成果
期刊论文数量(0)
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Choy, Jonathan其他文献
High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes
- DOI:
10.1177/24725552211026245 - 发表时间:
2021-07-03 - 期刊:
- 影响因子:3.1
- 作者:
Choy, Jonathan;Kan, Yanqing;Ai, Xi - 通讯作者:
Ai, Xi
Immunobiology of Nitric Oxide and Regulation of Inducible Nitric Oxide Synthase
- DOI:
10.1007/978-3-319-54090-0_8 - 发表时间:
2017-01-01 - 期刊:
- 影响因子:0
- 作者:
Lee, Martin;Rey, Kevin;Choy, Jonathan - 通讯作者:
Choy, Jonathan
Choy, Jonathan的其他文献
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{{ truncateString('Choy, Jonathan', 18)}}的其他基金
Regulation and function of human inducible nitric oxide synthase
人诱导型一氧化氮合酶的调节和功能
- 批准号:
RGPIN-2019-05192 - 财政年份:2022
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
Regulation and function of human inducible nitric oxide synthase
人诱导型一氧化氮合酶的调节和功能
- 批准号:
RGPIN-2019-05192 - 财政年份:2021
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
Regulation and function of human inducible nitric oxide synthase
人诱导型一氧化氮合酶的调节和功能
- 批准号:
RGPIN-2019-05192 - 财政年份:2020
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
Regulation and function of human inducible nitric oxide synthase
人诱导型一氧化氮合酶的调节和功能
- 批准号:
RGPIN-2019-05192 - 财政年份:2019
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
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 - 财政年份:2018
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
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 - 财政年份:2016
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
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 - 财政年份:2015
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
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 - 财政年份:2014
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
Regulation of iNOS gene expression in human T cells
人类 T 细胞 iNOS 基因表达的调控
- 批准号:
371596-2009 - 财政年份:2013
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
Regulation of iNOS gene expression in human T cells
人类 T 细胞 iNOS 基因表达的调控
- 批准号:
371596-2009 - 财政年份:2012
- 资助金额:
$ 2.99万 - 项目类别:
Discovery Grants Program - Individual
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