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: 10580267
• 负责人: ANNIE V BEUVE
• 金额: $ 8.66万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10580267
• 关键词: Actins; Adenoviruses; Administrative Supplement; Affect; Angiotensin II; Apoptosis; Binding; Biochemical; Bioinformatics; Biological Assay; Blood Vessels; CRISPR/Cas technology; Calcium; Cardiac; Cardiovascular system; Cell Line; Cell physiology; Cells; Co-Immunoprecipitations; Complex; Consensus; Cyclic GMP; Cysteine; Environment; Equilibrium; Equipment; 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; Technology; Vasodilation; Wild Type Mouse; angiogenesis; blood pressure regulation; cGMP production; cardioprotection; desensitization; heme a; improved; in vivo; intravital microscopy; mouse model; novel; oxidation; parent grant; polymerization; pressure; protective effect; protein function; protein protein interaction; response

PROJECT SUMMARY (PARENT GRANT) This is an application for an administrative supplemental equipment under our parent grant GM112415. The equipment we are requesting is at the center of our experimental strategy. This equipment is a Pressure Myograph P114 system that will replace and upgrade an intravital microscopy system that is not anymore available to us. We will not be able to complete Aim3 without this type of equipment and because of the upgraded technology we will improve and expand the investigations of Aim1 and Aim2 of the project of our parent grant summarized below: 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（父母赠款） 这是根据我们的父母赠款GM112415的行政补充设备的申请。这 我们要求的设备是我们实验策略的中心。该设备是压力 Myograph P114系统将替换并升级不再存在的插入式显微镜系统 向我们使用。没有这种设备，我们将无法完成AIM3，并且由于升级 技术我们将改善和扩大对我们父母赠款项目的AIM1和AIM2的调查 摘要下面： 一氧化氮（NO）是一个重要的信号分子，可调节与血管功能相关的多种功能， 凋亡和血管生成。 NO以其刺激可溶性鸟叶环酶的能力而闻名（现在称为 GC1）产生CGMP并刺激其下游信号通路。但是，也不能共价 通过S-硝化或S-硝基化修饰半胱氨酸（CYS）（在蛋白质的半胱氨酸中添加无部分， SNO）。尽管这种可逆的翻译后修饰越来越被认为是重要的 蛋白质功能的调节机制，蛋白质硝化特异性的动态调节很差 理解。我们最近的研究表明，GC1具有跨硝基酶活性，即GC1具有 能够通过蛋白质蛋白质相互作用（转硝化）将SNO直接传递到特定靶标。这 转硝化活性不需要GC1的CGMP形成活性，可以通过一个单一完成 GC1的亚基（CGMP的形成需要2个亚基）。此外，我们证明了一种经势化 GC1的靶标是氧化硫氧还蛋白1（OTRX1），这是一种调节细胞S-硝化化的硫代氧蛋白。实际上， 氧化/亚硝化条件似乎有利于GC1-TRX1复合物。使用先进的蛋白质组学方法， 最近，我们确定了与纯化系统中SNO转移有关的GC1和TRX1中的CYS，以及 由平滑肌和心脏细胞中GC1/TRX1跨硝化级联靶向的蛋白质的Cys。我们的 假设是GC1转硝化活性的功能是对氧化应激和 有可能补偿发生在氧化中的规范NO-GC1-CGMP途径的功能障碍 状况。为了探讨这一挑衅性假设，我们建议对CYS进行突变分析 已经确定表征平滑肌和心脏细胞中跨硝化机制。经过 比较GC1，TRX1的靶标，以及我们将确定目标特异性的基础机制。 我们将确定特定靶标的GC1/TRX1转硝化如何影响其细胞功能。为此，我们 将使用从涉及的GC1的Cys的新型小鼠敲入（Ki）中分离出的细胞系和主细胞 跨硝化。确定gc1-和gc1/trx1- transn硝化的生理相关性 心血管系统和对压力的自适应反应，我们将使用CYS KI鼠标模型和抑制作用 在血管紧张素II诱导的氧化应激下破坏GC1/TRX1转硝化复合物的肽。这 项目可能导致发现由特定S-硝化驱动的新型心血管保护途径。