Regulation of Soluble guanylyl cyclase, the NO-receptor

描述(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中心，特别是在心血管系统和中枢和外周神经系统。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。当NO与血红素结合后，sGC被激活数百倍，人们认为NO-cGMP信号级联在调节血压、突触可塑性和抑制血小板聚集方面起着关键作用。建议的研究试图了解sGC调控机制的结构基础，并确定其活性的调节因子：1)我们最近的工作表明sGC含有变构调节位点。我们试图确定决定所提议的变构位点的结构和性质的残基。在腺酰环化酶同源性的指导下，我们进行了突变分析，确定了似乎介导变构激活的两个亚基之间相互作用的特定区域。我们将通过生化研究和分子模拟相结合的方法来表征这些突变体。2)我们实验室和其他实验室最近的工作表明，sGC存在内源性调节因子。我们已经发现了一种新的内源性激活剂，但尚未鉴定。我们将确定此激活器并为其他人筛选。3)野生型和突变型sGC将用于测试来自合成激活剂YC-1的新生成的化学库。活性化合物和sGC将被用作分子建模的模板。这个模型结合构效关系研究将被用来产生关于NO-cGMP信号通路的生理调节的详细和可检验的假说。了解sGC的调节机制和识别调节分子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。

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.