NO signaling by a Soluble Guanylyl Cyclase-Thioredoxin transnitrosation complex

可溶性鸟苷酸环化酶-硫氧还蛋白转亚硝基复合物的 NO 信号转导

• 批准号: 8894270
• 负责人: ANNIE V BEUVE
• 金额: $ 41.16万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8894270
• 关键词: Angiotensin II; Apoptosis; Apoptosis Regulator; Apoptotic; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; Cardiac; Cardiac Myocytes; Cardiovascular Physiology; Cardiovascular system; Cell Line; Cell Nucleus; Cell Survival; Cells; Cessation of life; Complex; Consensus Sequence; Cyclic GMP; Cysteine; Data; Environment; Functional disorder; Guanosine Triphosphate; Heart; Heart Hypertrophy; Heart failure; In Vitro; Investigation; Lead; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Modeling; Modification; Mus; Mutagenesis; Myocardium; Nitric Oxide; Nitrosation; Nuclear Translocation; Oxidation-Reduction; Pathway interactions; Play; Post-Translational Protein Processing; Process; Property; Proteins; Proteome; Proteomics; Regulation; Role; SKIL gene; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Specificity; Stress; Sulfhydryl Compounds; Thioredoxin; Transgenic Mice; angiogenesis; base; cGMP production; heme a; human CLIC4 protein; mutant; nitrosative stress; novel; oxidation; prevent; protein function; protein protein interaction; public health relevance; research study; response; small hairpin RNA

 DESCRIPTION (provided by applicant): Nitric oxide (NO) is an important signaling molecule that regulates diverse functions relevant to cardiovascular function, apoptosis and angiogenesis. NO is best known for its ability to stimulate soluble guanylyl cyclase (sGC) to produce cGMP and stimulate its downstream signaling pathways. However, NO can also covalently modify cysteines via S-nitrosation (addition of a NO moiety to the cysteine of a protein, SNO). Although this reversible post-translational modification is increasingly recognized as an important regulatory mechanism of protein function, and to play a role in cardiac protection, dynamic regulation of protein nitrosation specificity is poorly understood. Our collaborative team has made the exciting observation that sGC, the key NO receptor, modulates the level of nitrosation of specific proteins in cardiomyocytes and smooth muscle cells. Preliminary data showed that sGC increases nitrosation by a protein-protein interaction-driven SNO transfer (transnitrosation). Moreover, this increased nitrosation is due, for a specific subset of proteins, to the association of sGC with thioredoxin 1 (Trx1), a cardiac protective thiol-redox protein with both transnitrosation and denitrosation activities. Initial mass spectrometry and biochemical analyses showed that sGC transnitrosates Trx1, which in turn nitrosates a specific subset of targets, a finding supported by shTrx1 knockdown experiments in cardiomyocytes. These novel observations lead to the provocative idea that sGC modulates S-nitrosation specificity via a transnitrosation cascade that includes an S-nitrosated Trx1 intermediate. This study aims to answer three critical questions based on this hypothesis. Aim1: What is the mechanism of sGC transnitrosation of Trx1? We will identify key cysteines (Cys) responsible for sGC transfer of SNO to Trx1 and for interaction via mutagenesis and biochemical analyses. Aim2: What are the specific targets of the sGC/Trx1 transnitrosation cascade and the mechanisms underlying target specificity? Using novel and highly specific proteomics approaches, we will quantify the SNO-proteomes modulated by the sGC/Trx1 transnitrosation cascade under nitrosative and oxidative conditions and determine consensus sequence motifs among the target proteins. Aim3: Is sGC-mediated transnitrosation an anti-apoptotic mechanism? NO signaling and Trx1 are crucial components of the anti-apoptotic response to stress. Among the sGC/Trx1 transnitrosation targets identified is the chloride intracellular channel 4 (CLIC4), a regulator of apoptosis, whose nuclear translocation is modulated via specific nitrosation. We will determine whether sGC/Trx1 transnitrosation of CLIC4 is an important inhibitory mechanism of angiotensin II-induced apoptosis of cardiomyocytes, underlying the potential role of this newly discovered transnitrosation cascade in cardiac remodeling following heart failure. This multi-PI project could lead to the discovery of novel cardioprotective pathway driven by specific S-nitrosation.

 描述（由申请人提供）：一氧化氮（NO）是一种重要的信号分子，调节与心血管功能、细胞凋亡和血管生成相关的多种功能。NO最为人所知的是其刺激可溶性鸟苷酸环化酶（sGC）产生cGMP并刺激其下游信号通路的能力。然而，NO也可以通过S-亚硝化（将NO部分添加到蛋白质的半胱氨酸，SNO）共价修饰半胱氨酸。虽然这种可逆的翻译后修饰越来越被认为是蛋白质功能的重要调节机制，并在心脏保护中发挥作用，但对蛋白质亚硝化特异性的动态调节知之甚少。我们的合作团队已经做出了令人兴奋的观察，即sGC，关键的NO受体，调节心肌细胞和平滑肌细胞中特定蛋白质的亚硝化水平。初步数据显示，sGC通过蛋白质-蛋白质相互作用驱动的SNO转移（转亚硝化）增加亚硝化。此外，这种增加的亚硝化作用是由于，对于特定的蛋白质子集，sGC与硫氧还蛋白1（Trx 1），心脏保护性巯基氧化还原蛋白与转亚硝化和脱亚硝化活性的协会。最初的质谱和生化分析表明，sGC transnitrosates Trx 1，这反过来又亚硝化了一个特定的目标子集，这一发现得到了心肌细胞中shTrx 1敲低实验的支持。这些新的观察结果导致了一个挑衅性的想法，即sGC通过包括S-亚硝化Trx 1中间体的转亚硝化级联来调节S-亚硝化特异性。本研究旨在回答基于这一假设的三个关键问题。目的1：Trx 1的sGC转亚硝化机制是什么？我们将通过诱变和生化分析确定负责sGC将SNO转移到Trx 1以及相互作用的关键半胱氨酸（Cys）。目的2：sGC/Trx 1转亚硝化级联反应的特异性靶点和靶点特异性机制是什么？使用新的和高度特异性的蛋白质组学方法，我们将量化的sGC/Trx 1 transnitrosation级联亚硝化和氧化条件下调制的SNO-蛋白质组，并确定目标蛋白质之间的共识序列基序。目的3：sGC介导的转亚硝化是一种抗凋亡机制吗？NO信号和Trx 1是抗凋亡应激反应的重要组成部分。在sGC/Trx 1转亚硝化作用靶点中，确定了细胞内氯离子通道4（CLIC 4），这是一种凋亡调节因子， 核转位通过特异性亚硝化作用调节。我们将确定CLIC 4的sGC/Trx 1转亚硝化是否是血管紧张素II诱导的心肌细胞凋亡的重要抑制机制，这是新发现的转亚硝化级联在心力衰竭后心脏重塑中的潜在作用的基础。这个多PI项目可以 导致发现由特异性S-亚硝化驱动的新的心脏保护途径。