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.

描述（由申请人提供）：由于发现衍生的松弛因子（EDRF）的发现是内源性气一氧化氮（NO），因此已经将大量生理功能归因于NO。尽管NO具有广泛认可的重要性，但对NO受体调节的机理，可溶性鸟叶基环酶（SGC）知之甚少。 SGC是一种含血红素的异二聚体，可催化底物GTP的CGMP形成。 NO与血红素结合后，将SGC激活几百倍。 SGC是一种多域信号传导酶，其中包含受体血红素结构域，二聚域和效应催化域。尚不清楚如何将NO信号传播到催化域。拟议的研究试图了解SGC调控机制的结构和分子基础：1）如何将NO信号从受体血红素结构域传输到催化剂效应领域，2）为什么SGC长期暴露于NO，无需启用，不响应于NO，3）在SGC中发挥作用的内在模拟器中，该角色扮演的角色扮演了这一攻击的角色。 1）我们对SGC血红素结构域和二聚体域的初始基于结构的突变分析（与我们的合作者van den Akker博士解决）揭示了这些域中的特定区域，这些区域对于无信号传导至关重要。我们将研究这些突变体如何影响SGC的激活。在我们与古代保守领域的结构建模和同源性的指导下，我们还寻求探究与SGC变构激活有关的SGC域之间的相互作用。 2）为了了解SGC脱敏的机制，我们发现SGC在体外和体内是硝基基化的，并且S-硝基化与对无刺激的反应性的丧失相关，而基础活性则保持不变。 S-亚硝基化是一种翻译后修饰，其中未在特定半胱氨酸的自由硫醇中添加一个部分。我们将通过鉴定和突变S-亚硝基化的半胱氨酸并分析所得的表型来研究SGC脱敏的机制。 3）我们实验室和其他人的最新工作表明，SGC有内源性调节剂。我们发现蛋白质二硫化物异构酶（PDI）抑制了未刺激的SGC活性。我们将表征抑制的机制，并解决其生理相关性。 了解SGC调节和确定调节分子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和开发补偿性疗法的关键，这会影响超过6000万美国人的氧化氧化物（NO）。氧化物（NO）在血管中诱导了小型cgmelecules cgmp cgmp cgmp syperserment cgmp的生产。 NO-CGMP途径的功能障碍导致许多心血管疾病，包括高血压，勃起功能障碍和动脉粥样硬化，影响超过6000万美国人。我们试图了解这些分子的产生是如何由人体控制的。