Regulation of soluble guanylyl cyclase, the NO-receptor

描述（由申请人提供）：由一氧化氮（NO）调节的细胞过程是生物学和疾病的许多方面的核心，特别是在心血管系统和中枢和外周神经系统中。尽管NO的重要性已被广泛认识，但对NO受体（可溶性鸟苷环化酶）的调控机制知之甚少。sGC是一种含血红素的异源二聚体，可催化底物GTP生成cGMP。当NO与血红素结合时，sGC被激活数百倍，而NO- cgmp信号级联被认为是调节血压、突触可塑性和抑制血小板聚集的关键参与者。我们的研究旨在了解sGC调控机制的结构基础，并确定其活性的调节剂：1)我们最近的工作表明sGC包含一个变构调节位点。我们试图确定残基决定的结构和提议的变构位点的性质。在与腺苷酸环化酶同源性的指导下，我们进行了突变分析，确定了两个亚基之间相互作用的特定区域，这些区域似乎介导了变构活化。我们将通过结合分子模型的生化研究来描述这些突变体。2)我们实验室和其他人最近的研究表明，sGC存在一种内源性调节因子。我们已经发现但尚未确定一种新的内源性激活剂。我们将识别此激活器并筛选其他激活器。3)野生型和突变型sGC将用于测试由合成活化剂YC-1衍生的新生成的化学文库。活性化合物和sGC将被用作分子模型的模板。该模型与结构-活性关系研究相结合，将用于产生关于NO-cGMP信号通路生理调节的详细和可测试的假设。了解sGC的调控机制并确定其调控分子将是揭示某些类型高血压、动脉粥样硬化和勃起功能障碍分子基础的关键。

DESCRIPTION (provided by applicant): The cellular processes that are regulated by nitric oxide (NO) are central to many aspects of biology and disease, particularly in the cardiovascular system and the central and peripheral nervous systems. 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 to the heme, the sGC is activated several hundred-fold and it is thought that the NO-cGMP signaling cascade is a key player in regulation of blood pressure, synaptic plasticity and inhibition of platelet aggregation. The proposed studies seek to understand the structural basis of mechanisms of regulation of the sGC and to identify modulators of its activity: 1) Our recent work suggests that sGC contains an allosteric regulatory site. We seek to identify residues that dictate the structure and nature of the proposed allosteric site. Guided by homology with adenylyl cyclases, we conducted a mutational analysis that identifies specific regions of interactions between the two subunits that seem to mediate allosteric activation. We shall characterize these mutants by biochemical studies integrated with molecular modeling. 2) Recent work from our laboratory and others, suggests that there is an endogenous regulator for the sGC. We have discovered but not yet identified a novel endogenous activator. We will identify this activator and screen for others. 3) The wild-type and mutant sGC will be used to test a newly generated chemical library derived from a synthetic activator, YC-1. Active compounds and sGC will be used as templates for molecular modeling. This modeling combined with structure-activity relationship studies will be used to generate detailed and testable hypotheses regarding the physiological modulation of the NO-cGMP signaling pathway. Understanding the mechanisms of regulation of sGC and identifying regulatory molecules will be key to uncovering the molecular basis of some types of hypertension, atherosclerosis and erectile dysfunction.