NO signaling by a Soluble Guanylyl Cyclase-Thioredoxin transnitrosation complex

 说明书(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能刺激可溶性鸟苷酸环化酶(SGC)产生cGMP，并能刺激其下游信号通路，因而广为人知。然而，NO也可以通过S亚硝化(在蛋白质的半胱氨酸上添加NO部分，SNO)来共价修饰半胱氨酸。尽管这种可逆的翻译后修饰被越来越多地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却知之甚少。我们的合作团队已经做出了令人兴奋的观察，即关键的NO受体sGC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)来增加亚硝化。此外，这种增加的亚硝化作用是由于sGC与硫氧还蛋白1(Trx1)的结合，硫氧还蛋白1(Trx1)是一种具有心脏保护作用的硫醇氧化还原蛋白，具有反硝化和反硝化活性。最初的质谱分析和生化分析表明，sGC反式亚硝化Trx1，而Trx1反过来亚硝化特定的靶子集，这一发现得到了心肌细胞shTrx1基因敲除实验的支持。这些新颖的观察结果导致了一种挑衅性的想法，即sGC通过包括S亚硝化的Trx1中间体的跨亚硝化级联反应来调节S亚硝化特异性。基于这一假设，本研究旨在回答三个关键问题。目的：Trx1的sGC转硝化机制是什么？我们将通过突变和生化分析确定负责sGC将SNO转移到Trx1并参与相互作用的关键半胱氨酸(Cys)。AIM2：sGC/Trx1转亚硝化级联反应的特定靶点是什么，靶点特异性的机制是什么？利用新的和高度特异的蛋白质组学方法，我们将定量在亚硝化和氧化条件下受sGC/Trx1转亚硝化级联反应调控的SNO-蛋白质组，并确定目标蛋白质之间的共同序列基序。目的：sGC介导的转亚硝化反应是一种抗细胞凋亡机制吗？NO信号和Trx1是应激反应中抗细胞凋亡的重要组成部分。在已确定的sGC/Trx1转亚硝化靶点中，有一个细胞内氯离子通道4(CLIC4)，它是一种细胞凋亡的调节因子，其 核转位是通过特定的亚硝化作用来调节的。我们将确定CLIC4的sGC/Trx1转硝化是否是血管紧张素II诱导心肌细胞凋亡的重要抑制机制，从而揭示这一新发现的转硝化级联反应在心力衰竭后心脏重塑中的潜在作用。这个多PI项目可以 从而发现了由特异性S亚硝化作用驱动的新的心脏保护通路。

 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.