Regulation of Soluble Guanylyl Cyclase, the NO-Receptor

可溶性鸟苷酸环化酶（NO 受体）的调节

• 批准号: 7596175
• 负责人: ANNIE V BEUVE
• 金额: $ 33.52万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7596175
• 关键词: Address; Affect; American; Atherosclerosis; Binding; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Catalytic Domain; Cells; Collaborations; Cyclic GMP; Cysteine; Cytoplasmic Protein; Data; Dimerization; Endothelium-Dependent Relaxing Factors; Enzymes; Erectile dysfunction; Exposure to; Functional disorder; Gases; Guanosine Triphosphate; Heme; Hypertension; In Vitro; Kinetics; Laboratories; Link; Modeling; Molecular; Mutate; Mutation; Neurons; Nitric Oxide; Oxides; Oxidoreductase; Pathway interactions; Phenotype; Physiological; Physiology; Platelet aggregation; Play; Post-Translational Protein Processing; Process; Production; Property; Protein Disulfide Isomerase; Regulation; Resolution; Role; Signal Transduction; Soluble Guanylate Cyclase; Structural Models; Structure; Sulfhydryl Compounds; Synaptic plasticity; System; Tertiary Protein Structure; Vasodilation; Work; YC-1; base; desensitization; heme a; heme receptor; in vitro activity; in vivo; inhibitor/antagonist; mutant; response; small molecule

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.

说明（由申请人提供）：自从发现内皮源性舒张因子（EDRF）是内源性气体一氧化氮（NO）以来，大量的生理功能已归因于NO。尽管NO的重要性被广泛认识，但对NO受体（可溶性鸟苷酸环化酶（sGC））的调节机制知之甚少。sGC是一种含血红素的异二聚体，可催化底物GTP形成cGMP。当NO与血红素结合时，sGC被激活数百倍。sGC是包含受体-血红素结构域、二聚化结构域和效应子-催化结构域的多结构域信号传导酶。目前还不知道NO信号如何传播到催化域。提出的研究试图了解sGC调节机制的结构和分子基础：1）NO信号如何从受体-血红素结构域传递到催化效应结构域，2）为什么sGC在长时间暴露于NO后对NO无反应，3）sGC活性的内源性调节剂在这种抑制中发挥作用。1)我们最初对sGC血红素结构域和二聚化结构域进行的基于结构的突变分析（与我们的合作者货车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)在我们寻求理解sGC的脱敏机制时，我们发现sGC在体外和体内被S-亚硝基化，并且S-亚硝基化与对NO刺激的响应性丧失相关，而基础活性保持不变。S-亚硝基化是一种翻译后修饰，其中NO部分被添加到特定半胱氨酸的游离巯基。我们将通过鉴定和突变S-亚硝基化半胱氨酸来研究sGC的脱敏机制，并分析所得的表型。3)我们实验室和其他实验室最近的工作表明，sGC存在内源性调节剂。我们已经发现蛋白质二硫键异构酶（PDI）抑制NO刺激的sGC活性。我们将描述抑制的机制，并解决其生理相关性。 了解sGC的调节机制并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并开发其代偿疗法的关键，这些疾病影响超过6000万美国人。一氧化氮（NO）在血管中诱导产生小分子信使cGMP，其松弛血管。NO-cGMP通路的功能障碍是许多心血管疾病的原因，包括高血压、勃起功能障碍和动脉粥样硬化，这些疾病影响了6000多万美国人。我们试图了解这些分子的产生是如何由身体控制的。