NO signaling by a Soluble Guanylyl Cyclase -Thioredoxin transnitrosation complex

项目总结 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最为人所知的是它能刺激可溶性的鸟苷酸 环化酶(现在称为Gc1)产生cGMP并刺激其下游的信号通路。 然而，NO也可以通过S亚硝化或S亚硝化(加成)来共价修饰半胱氨酸(Cys 与蛋白质的半胱氨酸不同的部分，SNO)。尽管这种可逆的翻译后翻译 修饰是蛋白质功能的一种重要调节机制， 蛋白质亚硝化特异性的动态调节还知之甚少。我们最新的调查 揭示Gc1具有转硝酸酶活性，即Gc1具有将SNO直接转移到 通过蛋白质-蛋白质相互作用(转亚硝化)的特定靶标。这种转硝化作用不会 需要Gc1的cGMP形成活性，可由Gc1的单个亚基完成 (cGMP的形成需要2个亚基)。此外，我们还证明了一个转亚硝化靶标 Gc1是氧化硫氧还蛋白1(OTrx1)，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。使用高级蛋白质组学 方法，我们最近在Gc1和Trx1中发现了参与SNO转移的Cys 纯化系统，以及Gc1/Trx1转亚硝化级联反应中靶向蛋白的Cys 肌肉和心肌细胞。我们的假设是Gc1转亚硝化活性的函数是 对氧化应激的适应性反应和潜在的对规范的功能障碍的补偿 发生在氧化条件下的NO-Gc1-cGMP途径。为了探索这一具有挑衅性的假设， 我们建议对我们已经确定的半胱氨酸进行突变分析，以表征其机制 在平滑肌和心肌细胞中的反硝化作用。通过比较Gc1、Trx1和两者的目标 我们将确定靶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作用会影响其细胞功能。为此，我们将使用细胞系和 从参与反硝化作用的Gc1半胱氨酸的一种新的小鼠敲入(KI)中分离出的原代细胞。 确定Gc1-和Gc1/Trx1-转硝化在心血管中的生理学相关性 系统对应激的适应性反应，我们将采用Cys Ki小鼠模型和抑制性多肽 在血管紧张素II诱导的氧化应激下，这会破坏Gc1/Trx1转亚硝化复合体。这 该项目可能导致发现由特定S驱动的新的心血管保护通路- 亚硝化作用。

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.