Regulation of Soluble Guanylyl Cyclase, the NO-Receptor

描述(申请人提供)：自从发现内皮衍生松弛因子(EDRF)是内源性有毒气体一氧化氮(NO)以来，大量的生理功能被归因于NO。尽管NO的重要性得到了广泛的认可，但人们对NO受体--可溶性鸟苷酸环化酶(SGC)的调节机制知之甚少。SGC是一种含血红素的杂二聚体，催化底物GTP形成cGMP。在NO结合后，sGC被激活数百倍。SGC是一种含有NO受体-血红素结构域、二聚化结构域和效应催化结构域的多结构域信号酶。目前尚不清楚NO信号是如何传播到sGC的催化域的。我们最近发现，SGc是通过S亚硝化反应脱敏的，即在特定半胱氨酸(Cys)的游离硫醇上加一个NO部分。S现在有大量证据表明，硫醇氧化还原反应是sGC功能的关键和动态调节因素，sGC的硫醇修饰具有临床意义，因为它们与高血压患者血管反应性降低有关。然而，活性半胱氨酸修饰调控sGC激活、NO信号转导到催化区、sGC基础活性、结构域相互作用和非血红素亲和力的机制尚不清楚。建议的研究试图了解sGC调节机制的结构和分子基础，以及这些机制的破坏如何有助于心血管病理的发展。我们将利用纯化的酶、细胞系统和动物模型进行以下目的的结构-功能研究、分子动力学模拟、生化和动力学分析以及应用生理学：1)确定硫醇半胱氨酸在sGC激活的分子机制中的作用；2)研究硫醇还原蛋白硫氧还蛋白对sGC的调节作用；3)确定sGC的硫醇依赖功能障碍如何参与高血压和心肌肥厚。了解sGC的调节机制将是揭示某些类型的高血压、动脉粥样硬化和勃起功能障碍的分子基础的关键，这些疾病影响着6000多万美国人。

DESCRIPTION (provided by applicant): Since the discovery that the endothelium derived relaxing factor (EDRF) was the endogenous toxic gas nitric oxide (NO), an astonishing number of physiological functions have been attributed to NO. Despite the widely recognized importance of NO, little is known about the mechanism of regulation of the NO receptor, the soluble guanylyl cyclase (sGC). sGC is a heme containing heterodimer that catalyzes the formation of cGMP from the substrate GTP. Upon binding of NO, the sGC is activated several hundred fold. The sGC is a multi-domain signaling enzyme that contains the NO receptor-heme domain, a dimerization domain and the effector-catalytic domain. It is not known how the NO signal is propagated to the catalytic domain of sGC. We recently discovered that sGC is desensitized by S-nitrosylation, the addition of a NO moiety to the free thiol of a specific cysteine (Cys). There s now substantial evidence that thiol redox reactions are critical and dynamic regulators of sGC function and that thiol modifications of sGC have clinical relevance as they are associated with decreased vascular reactivity in hypertension. However, the mechanisms by which modification of reactive Cys modulate sGC activation, NO signal transduction to the catalytic domain, sGC basal activity, domain interactions and NO-heme affinity have yet to be explored. The proposed studies seek to understand the structural and molecular basis of mechanisms of regulation of the sGC and how disruption of these mechanisms contributes to development of cardiovascular pathologies. We will use purified enzymes, cellular system and animals models to conduct structure-function studies, molecular dynamics simulation, biochemical and kinetics analysis and applied physiology for the following aims: 1) define the role of thiol Cys in the molecular mechanism of activation of sGC; 2) investigate the modulation of sGC by the thiol-reducing protein thioredoxin and 3) establish how thiol-dependent dysfunction of sGC contributes to hypertension and cardiac hypertrophy. Understanding the mechanisms of regulation of sGC will be key to uncovering the molecular basis of some types of hypertension, atherosclerosis and erectile dysfunction, which affect more than 60 millions Americans.