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

基本信息

批准号：
10260574
负责人：
ANNIE V BEUVE
金额：
$ 44.61万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
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/10260574
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

项目摘要

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）是一个重要的信号分子，可调节与血管相关的多种功能功能，凋亡和血管生成。 NO以其刺激可溶性Guanylyl的能力而闻名循环酶（现在称为GC1）产生CGMP并刺激其下游信号通路。但是，也不能通过S-硝化或S-硝基化来共价修改半胱氨酸（Cys）（添加蛋白质的半胱氨酸的无部分，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，蛋白质亚硝化特异性的动态调节知识不足。我们最近的调查揭示GC1具有跨硝基酶活性，即GC1具有将SNO直接传递到的能力蛋白质 - 蛋白质相互作用（跨硝化）的特定靶标。这种转让活动没有需要GC1的CGMP形成活性，可以通过GC1的单个亚基来完成（CGMP的形成需要2个亚基）。此外，我们证明了一个经过的跨硝化目标 GC1被氧化硫氧还蛋白1（OTRX1），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。使用高级蛋白质组学方法是，我们最近确定了与SnO转移有关的GC1和TRX1中的CY 纯化的系统，以及由GC1/TRX1转硝化级联对靶向的蛋白质的CY 肌肉和心脏细胞。我们的假设是GC1转硝化活性的功能是对氧化应激的自适应反应，并有可能补偿规范的功能障碍在氧化条件下发生的NO-GC1-CGMP途径。探索这个挑衅的假设，我们建议对我们确定的CYS进行突变分析以表征机制平滑肌和心脏细胞中的跨硝化作用。通过比较gc1，trx1的目标我们将确定目标特异性的基础机制。我们将确定GC1/TRX1如何特定靶标的跨硝化影响其细胞功能。为此，我们将使用单元线和从涉及转硝化的GC1的新型小鼠敲入（Ki）中分离出来的原代细胞。确定心血管中GC1-和GC1/TRX1-TRANSNINTROSATION的生理相关性系统和对压力的自适应反应，我们将使用Cys Ki小鼠模型和抑制性肽这破坏了血管紧张素II诱导的氧化应激下的GC1/TRX1转硝化复合物。这项目可能导致发现由特定S-驱动的新型心血管保护途径硝化。