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 信号传导

• 批准号: 10475129
• 负责人: ANNIE V BEUVE
• 金额: $ 43.71万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475129
• 关键词: Actins; Adenoviruses; Affect; Angiotensin II; Apoptosis; Binding; Biochemical; Bioinformatics; Biological Assay; Blood Vessels; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Line; Cell physiology; Cells; Co-Immunoprecipitations; Complex; Consensus; Cyclic GMP; Cysteine; Environment; Equilibrium; Functional disorder; Funding; Guanosine Triphosphate; Heart Hypertrophy; Heart failure; Homeostasis; Hypertension; Impairment; Investigation; Knock-in; Knock-in Mouse; Lead; Ligation; Mass Spectrum Analysis; Measures; Mediator of activation protein; Metabolic Pathway; Modeling; Modification; Mus; Mutation Analysis; Nitric Oxide; Nitrosation; OLFM4 gene; Oxidation-Reduction; Oxidative Stress; Oxides; Pathologic; Pathway interactions; Peptides; Physiological; Post-Translational Protein Processing; Property; Proteins; Proteomics; Regulation; Role; SKIL gene; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Skeleton; Smooth Muscle; Soluble Guanylate Cyclase; Specificity; Stress; Sulfhydryl Compounds; System; TXN gene; Vasodilation; Wild Type Mouse; angiogenesis; blood pressure regulation; cGMP production; cardioprotection; desensitization; heme a; in vivo; mouse model; novel; oxidation; polymerization; protective effect; protein function; protein protein interaction; response

PROJECT SUMMARY Nitric oxide (NO) is an important signaling molecule that regulates diverse functions relevant to vascular function, apoptosis and angiogenesis. NO is best known for its ability to stimulate soluble guanylyl cyclase (now called GC1) to produce cGMP and stimulate its downstream signaling pathways. However, NO can also covalently modify cysteines (Cys) via S-nitrosation or S-nitrosylation (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, dynamic regulation of protein nitrosation specificity is poorly understood. Our most recent investigations reveal that GC1 has a transnitrosylase activity, i.e. GC1 has the ability to directly transfer SNO to specific targets by protein-protein interaction (transnitrosation). This transnitrosation activity does not require the cGMP forming activity of GC1 and can be accomplished by a single subunit of GC1 (formation of cGMP requires 2 subunits). Furthermore, we showed that one transnitrosation target of GC1 is oxidized thioredoxin 1 (oTrx1), a thiol-redox protein that modulates cellular S-nitrosation. In fact, oxidative/nitrosative conditions appear to favor the GC1-Trx1 complex. Using advanced proteomics approaches, we recently identified the Cys in GC1 and Trx1 that are involved in the SNO transfer in a purified system, and the Cys of proteins targeted by the GC1/Trx1 transnitrosation cascade in smooth muscle and cardiac cells. Our hypothesis is that the function of GC1 transnitrosation activity is an adaptive response to oxidative stress and potentially compensates for the dysfunction of the canonical NO-GC1-cGMP pathway that occurs in oxidative conditions. To explore this provocative hypothesis, we propose to conduct mutational analysis of the Cys we have identified to characterize the mechanism of transnitrosation in smooth muscle and cardiac cells. By comparing the targets of GC1, Trx1 and both we will determine the mechanisms underlying target specificity. We will determine how GC1/Trx1 transnitrosation of specific targets affects their cellular function. For this, we will use cell lines and primary cells isolated from a novel mouse knock-in (KI) of a Cys of GC1 involved in transnitrosation. To determine the physiological relevance of GC1- and GC1/Trx1-transnitrosation in the cardiovascular system and the adaptive response to stress, we will use the Cys KI mouse model and inhibitory peptides that disrupt the GC1/Trx1 transnitrosating complex under Angiotensin II-induced oxidative stress. This project could lead to the discovery of novel cardiovascular protective pathways driven by specific S- nitrosation.

项目摘要 一氧化氮（NO）是一种重要的信号分子，调节血管内皮细胞的多种功能， 功能、凋亡和血管生成。NO最为人所知的是它能够刺激可溶性鸟苷酰 环化酶（现在称为GC 1）产生cGMP并刺激其下游信号通路。 然而，NO也可以通过S-亚硝基化或S-亚硝基化（加成）共价修饰半胱氨酸（Cys NO部分与蛋白质的半胱氨酸，SNO）。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， 对蛋白质亚硝化特异性的动态调节知之甚少。我们最近的调查 表明GC 1具有转亚硝基酶活性，即GC 1具有将SNO直接转移到 通过蛋白质-蛋白质相互作用（transnitrosation）特异性靶点。这种转亚硝化活性不 需要GC 1的cGMP形成活性，并且可以通过GC 1的单个亚基来完成 （cGMP的形成需要2个亚基）。此外，我们还发现， GC 1是氧化硫氧还蛋白1（oTrx 1），一种调节细胞S-亚硝化的巯基氧化还原蛋白。事实上， 氧化/亚硝化条件似乎有利于GC 1-Trx 1复合物。利用先进的蛋白质组学 最近，我们在GC 1和Trx 1中发现了Cys，它们参与了SNO的转移， 纯化的系统，和蛋白质的半胱氨酸靶向的GC 1/Trx 1转亚硝化级联在平滑的 肌肉和心脏细胞。我们的假设是，GC 1的转亚硝化活性的功能是一个 适应性反应氧化应激和潜在的补偿功能障碍的典型 NO-GC 1-cGMP途径发生在氧化条件下。为了探索这个挑衅性的假设， 我们建议对我们已经确定的半胱氨酸进行突变分析，以描述其机制， 平滑肌和心脏细胞中的转亚硝作用。通过比较GC 1、Trx 1和两者的靶点， 我们将确定靶特异性的潜在机制。我们将确定GC 1/Trx 1 特定靶点的亚硝化会影响其细胞功能。为此，我们将使用细胞系， 原代细胞分离自一种新的小鼠基因敲入（KI）GC 1的Cys参与转亚硝化。 确定GC 1-和GC 1/Trx 1-转亚硝化在心血管疾病中的生理相关性。 系统和对应激的适应性反应，我们将使用Cys KI小鼠模型和抑制肽 在血管紧张素II诱导的氧化应激下破坏GC 1/Trx 1转亚硝化复合物。这 该项目可能导致发现由特定S- 亚硝化