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.

项目概要一氧化氮（NO）是一种重要的信号分子，调节与血管相关的多种功能功能、细胞凋亡和血管生成。 NO 以其刺激可溶性鸟苷基的能力而闻名环化酶（现称为 GC1）产生 cGMP 并刺激其下游信号通路。然而，NO 也可以通过 S-亚硝化或 S-亚硝基化共价修饰半胱氨酸 (Cys)（另外 NO 部分与蛋白质半胱氨酸的连接 (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。我们最近的调查揭示GC1具有转亚硝基酶活性，即GC1具有直接将SNO转移至通过蛋白质-蛋白质相互作用（转亚硝化）确定特定目标。这种转亚硝化活性不需要 GC1 的 cGMP 形成活性，并且可以通过 GC1 的单个亚基来完成（cGMP 的形成需要 2 个亚基）。此外，我们还发现了一个转亚硝化靶标 GC1 是氧化硫氧还蛋白 1 (oTrx1)，一种调节细胞 S-亚硝化的硫醇氧化还原蛋白。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，我们最近在 GC1 和 Trx1 中鉴定了参与 SNO 转移的 Cys 纯化系统，以及 GC1/Trx1 转亚硝基级联靶向蛋白质的 Cys 肌肉和心肌细胞。我们的假设是 GC1 转亚硝基活性的功能是对氧化应激的适应性反应并可能补偿经典的功能障碍 NO-GC1-cGMP 途径发生在氧化条件下。为了探索这个具有挑战性的假设，我们建议对我们已确定的半胱氨酸进行突变分析以表征该机制平滑肌和心肌细胞中的转亚硝化作用。通过比较 GC1、Trx1 和两者的目标我们将确定目标特异性的潜在机制。我们将确定GC1/Trx1如何特定靶标的转亚硝化会影响其细胞功能。为此，我们将使用细胞系和从参与转亚硝化的 GC1 半胱氨酸的新型小鼠敲入 (KI) 中分离出的原代细胞。确定 GC1- 和 GC1/Trx1-转亚硝基在心血管中的生理相关性系统和对压力的适应性反应，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 GC1/Trx1 转亚硝基复合物。这该项目可能会发现由特定 S-驱动的新型心血管保护途径亚硝化。