Nitric Oxide Signaling And Soluble Guanylate Cyclase

一氧化氮信号传导对几种生理功能是至关重要的，并且在细胞中的功能障碍是重要的。 在这种信号级联中涉及多种疾病，例如勃起功能障碍， 心脏病、神经变性、中风、高血压和胃肠道疾病。 可溶性鸟苷酸环化酶（sGC）的激活和失活是至关重要的 一氧化氮（NO）信号传导。NO在两个水平上调节sGC，这与 NO信号传导的许多药理学观察描述了紧张性和急性 NO在神经元信号传导中的这些作用的幅度和持续时间， 心脏功能、血管张力和血管舒张对于这些器官的正常功能至关重要。 系统，但两个NO效应的机制还没有得到彻底的研究。一 已经出现了通过sGC进行NO信号传导的新范例。了解sGC如何 从低激活状态到高激活状态的切换是这种新范例的核心。我们的具体 目的包括：（i）表征sGC的NO活化，重点是研究 低和高活性状态的生理相关性，（ii） 变构核苷酸和激活剂结合位点，以及核苷酸在 调节酶的NO活化，和（iii）确定氧化的 对sGC的损害以及它在人类疾病中的作用。实验方法将 包括物理生物化学方法，如质谱和快速反应 野生型和定点突变的动力学、克隆、表达、纯化和表征 sGC的突变体，并在各种细胞系统中进行实验，以延长sGC的表达。 将发现转化为体内环境。本提案的核心目标是制定一个全面的 对NO和sGC之间复杂关系的新认识。我们寻求 开发sGC激活和NO失活的完整分子水平视图， 核苷酸（ATP和GTP）。这项工作的扩展到生理功能将 为认识和治疗一氧化氮信号转导障碍提供了理论依据， 人类疾病

Nitric oxide signaling is critical to several physiological functions, and dysfunction in the in this signaling cascade is implicated in multiple diseases such as erectile dysfunction, heart disease, neurodegeneration, stroke, hypertension, and gastrointestinal disease. Activation and deactivation of soluble guanylate cyclase (sGC) is of central importance in nitric oxide (NO) signaling. NO regulates sGC at two levels and this is consistent with numerous pharmacological observations of NO signaling that describe tonic and acute roles for NO. The amplitude and duration of these effects of NO in neuronal signaling, cardiac function, vascular tone and vasodilation are vital to the proper function of these systems, but the mechanism for two NO effects has not been thoroughly investigated. A new paradigm for NO signaling through sGC has emerged. Understanding how sGC switches from a low to high activation state is central to this new paradigm. Our specific aims include: (i) Characterization of NO activation of sGC, with emphasis on studies of the physiological relevance of low and high activity states, (ii) Characterization of the allosteric nucleotide and activator binding site(s), and the role of nucleotide in modulating NO activation of the enzyme, and (iii) determining the effect of oxidative damage to sGC and the role of this in human disease. Experimental approaches will include physical biochemical methods such as mass spectrometry and rapid-reaction kinetics, cloning, expression, purification and characterization of wild type and sitedirected mutants of sGC, and experiments in various cellular systems to extend the findings into an in vivo setting. It is a central goal of this proposal to develop an entirely new understanding of the complex relationship between NO and sGC. We seek to develop a complete molecular level view of sGC activation and deactivation by NO and nucleotides (ATP and GTP). The extension of this work into physiological function will provide a rational basis for the understanding and treatment of NO signaling disorders in human disease.