Regulation of Soluble Guanylyl Cyclase, the NO-Receptor

描述（由申请人提供）：自从发现内皮衍生放松因子（EDRF）是内源性气体一氧化氮（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。sGC是一种多结构域信号转导酶，包含受体-血红素结构域、二聚化结构域和效应-催化结构域。目前尚不清楚NO信号是如何传播到催化域的。本研究旨在了解sGC调控机制的结构和分子基础：1)NO信号如何从受体-血红素域传递到催化-效应域；2)sGC在长时间暴露于NO后对NO无反应的原因；3)sGC活性的内源性调节剂是否在这种抑制中发挥作用。1)我们对sGC血红素结构域和二聚化结构域的初步基于结构的突变分析（与我们的合作者van den Akker博士一起解决）揭示了这些结构域中对NO信号传导至关重要的特定区域。我们将研究这些突变体如何影响sGC的NO激活。在我们的结构建模和与古老保守结构域的同源性的指导下，我们还试图探索参与sGC变构激活的sGC结构域之间的相互作用。2)为了了解sGC脱敏的机制，我们发现sGC在体外和体内都是S-亚硝基化的，并且S-亚硝基化与对NO刺激的反应性丧失有关，而基础活性保持不变。s -亚硝基化是一种翻译后修饰，将NO片段添加到特定半胱氨酸的游离硫醇中。我们将通过鉴定和突变s -亚硝基化半胱氨酸来研究sGC脱敏的机制，并分析由此产生的表型。3)我们实验室和其他人最近的工作表明，sGC存在内源性调节剂。我们发现蛋白质二硫异构酶（PDI）抑制no刺激的sGC活性。我们将描述抑制的机制和解决其生理相关性。

DESCRIPTION (provided by applicant): Since the discovery that the endothelium derived relaxing factor (EDRF) was the endogenous 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 to the heme, the sGC is activated several hundred fold. The sGC is a multi-domain signaling enzyme that contains the 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. The proposed studies seek to understand the structural and molecular basis of mechanisms of regulation of the sGC: 1) how the NO signal is transmitted from the receptor-heme domain to the catalytic-effector domain, 2) why sGC, following prolonged exposure to NO, becomes unresponsive to NO, 3) do endogenous modulators of sGC activity play a role in this inhibition. 1) Our initial structure-based mutational analysis of the sGC heme domain and dimerization domain (solved with our collaborator Dr. van den Akker) revealed specific regions in these domains that are crucial for NO signaling. We shall investigate how these mutants affect NO activation of sGC. Guided by our structural modeling and homology with ancient conserved domains, we also seek to probe the interactions between the sGC domains that are involved in sGC allosteric activation. 2) In our quest to understand the mechanism of desensitization of sGC, we discovered that sGC is S- nitrosylated in vitro and in vivo and that S-nitrosylation correlates with the loss of responsiveness to NO- stimulation, while the basal activity remains unaltered. S-nitrosylation is a post-translational modification in which a NO moiety is added to the free-thiol of specific cysteines. We will study the mechanism of desensitization of sGC by identifying and mutating the S-nitrosylated cysteines and analyze the resulting phenotypes. 3) Recent work from our laboratory and others suggests that there are endogenous modulators for the sGC. We have discovered that protein disulfide isomerase (PDI) inhibits NO-stimulated sGC activity. We will characterize the mechanism of inhibition and address its physiological relevance. Understanding the mechanisms of regulation of sGC and identifying regulatory molecules will be key to uncovering the molecular basis of and developing compensatory therapies for some types of hypertension, atherosclerosis and erectile dysfunction, which affect more than 60 million Americans.Nitric oxide (NO) induces in the blood vessels the production of a small molecule messenger cGMP which relaxes the vasculature. Dysfunction in the NO-cGMP pathway is responsible for many cardiovascular diseases including hypertension, erectile dysfunction and atherosclerosis which affect more than 60 million Americans. We seek to understand how the production of these molecules is controlled by the body.