Nitric Oxide Signaling And Soluble Guanylate Cyclase

一氧化氮信号对一些生理功能至关重要，而一氧化氮信号转导系统的功能障碍 在这一信号级联中涉及多种疾病，如勃起功能障碍， 心脏病、神经退行性变、中风、高血压和胃肠道疾病。 可溶性鸟苷环化酶(SGC)的激活和失活至关重要 在一氧化氮(NO)信号中。NO在两个层面上调节sGC，这与 描述紧张性和急性的NO信号的大量药理学观察 角色不是。NO在神经元信号转导中的作用的幅度和持续时间， 心脏功能、血管紧张度和血管扩张对这些血管的正常功能至关重要。 但两种NO的作用机制还没有得到彻底的研究。一个 通过sGC传递NO信号的新范式已经出现。了解sGC如何 从低激活状态到高激活状态的切换是这一新范式的核心。我们的特定 目的包括：(1)表征sGC的NO激活，重点研究 低活性状态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(S)，以及核苷酸在 调节酶的NO激活，以及(Iii)确定氧化的效果 对sGC的损害及其在人类疾病中的作用。实验方法将 包括物理生化方法，如质谱仪和快速反应 野生型和定点基因的动力学、克隆、表达、纯化及鉴定 SGC的突变体，以及在各种细胞系统中的实验，以延长 将这些发现转化为活体环境。这项提议的核心目标是开发一种完全 对NO与sGC之间复杂关系的新认识。我们寻求 发展一个完整的分子水平的观点，sGC的激活和失活的NO和 核苷酸(ATP和GTP)。将这项工作扩展到生理功能将 为认识和治疗慢性阻塞性肺疾病患者NO信号转导障碍提供合理依据 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.