NO signaling by a Soluble Guanylyl Cyclase -Thioredoxin transnitrosation complex

项目总结(母公司赠款) 这是我们的母公司GM112415项下的行政补充设备的申请。这个 我们所要求的设备是我们实验战略的中心。这台设备是一种压力 Myoggraph p114系统将取代和升级不再存在的活体显微镜系统 对我们来说是可用的。如果没有这种类型的设备，我们将无法完成Aim3，因为升级了 我们将改进和扩大对母公司资助项目AIM1和AIM2的调查 摘要如下： 一氧化氮(NO)是一种重要的信号分子，调节与血管功能相关的多种功能， 细胞凋亡和血管生成。NO最为人所知的是它能够刺激可溶性鸟苷酸环化酶(现在称为 Gc1)产生cGMP并刺激其下游信号通路。然而，NO也可以共价 通过S亚硝化或S亚硝化修饰半胱氨酸(在蛋白质的半胱氨酸中添加NO部分， Sno)。尽管这种可逆的翻译后修饰越来越被认为是一种重要的 蛋白质功能调节机制动态调节蛋白质亚硝化专一性差 明白了。我们最新的研究表明，Gc1具有转硝酸酶活性，即Gc1具有 通过蛋白质-蛋白质相互作用(转亚硝化)将SNO直接转移到特定靶标的能力。这 反硝化活性不需要Gc1的cGMP形成活性，可以通过一个单一的 Gc1亚基(cGMP的形成需要2个亚基)。此外，我们还证明了一种转亚硝化反应 Gc1的靶点是氧化硫氧还蛋白1(OTrx1)，这是一种调节细胞S亚硝化的硫醇氧化还原蛋白。事实上, 氧化/亚硝化条件似乎有利于Gc1-Trx1复合体。使用先进的蛋白质组学方法， 我们最近在Gc1和Trx1中发现了参与纯化系统中SNO转移的Cys，并且 Gc1/Trx1转亚硝化级联反应靶向的蛋白质Cys在平滑肌和心肌细胞中的表达。我们的 假设Gc1转亚硝化活性的功能是对氧化应激的适应性反应和 潜在地补偿氧化过程中发生的典型的NO-Gc1-cGMP途径的功能障碍 条件。为了探索这一挑衅性的假设，我们建议对Cys WE进行突变分析 已经确定了在平滑肌和心肌细胞中转亚硝化的机制。通过 比较Gc1、Trx1和两者的靶点，我们将确定潜在的靶标特异性的机制。 我们将确定特定靶点的Gc1/Trx1反硝化如何影响其细胞功能。为此，我们 将使用从一种新的小鼠敲入(Ki)小鼠中分离出的细胞系和原代细胞参与Gc1的半胱氨酸 反硝化作用。为确定Gc1-和Gc1/Trx1-转亚硝化作用的生理学相关性 心血管系统与应激的适应性反应，我们将采用Cys Ki小鼠模型并加以抑制 在血管紧张素II诱导的氧化应激下破坏Gc1/Trx1转亚硝化复合体的多肽。这 该项目可能导致发现由特定的S亚硝化作用驱动的新的心血管保护通路。

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.