S-Nitrosation in Nitric Oxide Signaling

描述（由申请人提供）：蛋白质s -亚硝化发生在整个生物学过程中，并在所有主要疾病中发挥调节作用，包括心血管疾病、糖尿病、哮喘和癌症。然而，这些事件背后的化学和结构生物学仍未被发现。我们将通过对三个关键蛋白：硫氧还蛋白、可溶性鸟苷酸环化酶和谷胱甘肽s -亚硝基还原酶（GSNOR）的晶体学、生化和分子遗传学研究来弥补这一空白。总之，这些蛋白质执行了许多被认为发生在细胞中的基于rsno的化学反应，这将使我们能够研究这些活动在正常和疾病状态下的结构和动力学。具体目标1：涉及人硫氧还蛋白的亚硝化反应。硫氧还蛋白是蛋白质转亚硝化的关键中间体，也与包括癌症和心脏病在内的几种疾病有关。我们通过s -亚硝基谷胱甘肽（GSNO）转亚硝化获得了s -亚硝化人硫氧还蛋白的晶体结构，这是第一个描述这种结构的晶体结构。我们现在可以在体外和模型癌细胞系中探索这种修饰的结构和生物化学。特异性目的2:GSNO刺激可溶性鸟苷酸环化酶。一氧化氮信号的最佳途径是通过与可溶性鸟苷酸环化酶（sGC）中的血红素结合，启动cGMP的产生，以调节血压和记忆形成等生理活动。我们发现GSNO也是一种有效的sGC兴奋剂。我们将进行生化和分子遗传学实验来揭示GSNO在sGC功能中的作用。体内产生的大部分一氧化氮最初被谷胱甘肽捕获为GSNO，参与SNO化学并被GSNOR代谢。我们已经确定了GSNOR的高分辨率结构，并发现了该蛋白的抑制剂。我们现在提出了一个针对GSNOR的药物发现和生物化学计划。

DESCRIPTION (provided by applicant): Protein S-nitrosation occurs throughout biology and plays a regulatory role in all major diseases, including cardiovascular disease, diabetes, asthma and cancer. Yet, the chemical and structural biology underlying these events remains undiscovered. We will bridge this gap through crystallographic, biochemical and molecular genetics investigations of three key proteins: thioredoxin, soluble guanylate cyclase and glutathione S-nitroso reductase (GSNOR). Together, these proteins perform much of the RSNO-based chemistry thought to occur in the cell and will allow us to investigate the structure and dynamics of these activities in normal and disease states. Specific Aim 1: Transnitrosation reactions involving human thioredoxin. Thioredoxin has emerged as a key intermediate in protein transnitrosation and is also implicated in several diseases, including cancer and heart disease. We have achieved a crystal structure of S-nitrosated human thioredoxin through transnitrosation by S-nitrosogluatathione (GSNO), the first such structure to be described. We are now in a position to explore the structure and biochemistry of this modification in vitro and in a model cancer cell line. Specific Aim 2: GSNO stimulation of soluble guanylate cyclase. The best understood pathway for nitric oxide signaling is through binding to heme in soluble guanylate cyclase (sGC), which initiates cGMP production to regulate physiological activities such as blood pressure and memory formation. We have discovered that GSNO is also a potent stimulant of sGC. We will undertake biochemical and molecular genetic experiments to reveal the role of GSNO in sGC function Specific Aim 3: GSNO metabolism by GSNO reductase. Much of the nitric oxide produced in vivo is initially captured by glutathione as GSNO, which participates in SNO chemistry and is metabolized by GSNOR. We have determined a high resolution structure for GSNOR and discovered an inhibitor of the protein. We now propose a program in drug discovery and biochemistry targeted to GSNOR.