Nitric Oxide Signaling and Soluble Guanylate Cyclase

描述（由申请人提供）：本提案的广泛、长期目标是：1）开发一氧化氮（NO）激活和失活可溶性鸟苷酸环化酶（sGC）的完整分子水平视图，以及2）通过操纵NO为理解和治疗人类疾病提供合理基础。具体目标是：1）sGC的NO活化和失活的表征，包括第二NO结合位点的性质，2）核苷酸变构位点的表征，包括该位点在调节酶的NO活化中的作用，3）对sGC功能重要的（a）分子内和（B）分子间蛋白质-蛋白质相互作用的作图，4）开发对铁-血红素结合位点特异的新型烷基-NO配体，用作光谱探针、潜在的激动剂和/或拮抗剂，和工具，以帮助确定非血红素NO结合位点，和5）获得sGC的晶体结构。实现这些目标的研究设计和方法将是利用基础分子生物学获得纯化蛋白。然后，用生物化学方法研究sGC的功能，包括活性实验和电子吸收、共振拉曼和电子顺磁光谱。这些研究旨在开发sGC功能的生理模型。了解这一功能对于设计治疗剂来治疗涉及NO信号通路的疾病至关重要。一氧化氮和一氧化氮合酶抑制剂在临床上用于治疗疾病，如败血性休克、肺动脉高压、男性阳痿和心绞痛。基于操纵NO在人类生物学中的功能的新疗法将继续对人类健康和疾病治疗/预防产生重大影响。拟议的研究检查了NO激活sGC的细节，这是这种操作的核心。

DESCRIPTION (provided by applicant): The broad, long-term objectives of this proposal are: 1) To develop a complete molecular level view of soluble guanylate cyclase (sGC) activation and deactivation by nitric oxide (NO), and 2) To provide a rational basis for the understanding and treatment of human disease through the manipulation of NO. The specific aims are: 1) The characterization of NO activation and deactivation of sGC, including the nature of the second NO binding site, 2) The characterization of the nucleotide allosteric site, including the role of this site in modulating NO activation of the enzyme, 3) The mapping of the (a) intramolecular and (b) intermolecular protein-protein interactions that are important for sGC function, 4) To develop novel alkyl-NO ligands that are specific for the iron-heme binding site, to be used as spectroscopic probes, potential agonists and/or antagonists, and tools to help identify non-heme NO binding sites, and 5) obtain a crystal structure of sGC. The research design and methods for achieving these goals will be the use of basic molecular biology to acquire purified protein. Then, sGC function will be investigated with biochemical methods, including activity experiments and electronic absorption, resonance Raman, and electron paramagnetic spectroscopy. These studies are directed towards the development of a physiological model of sGC function. Understanding this function is critical for the design of therapeutic agents to treat diseases that involve the NO signaling pathway. Nitric oxide and nitric oxide synthase inhibitors are in clinical use in the treatment of diseases such as septic shock, pulmonary hypertension, male impotence, and angina pectortis. New therapies based on the manipulation of NO function in human biology will continue to have significant impact on human health and disease treatment/prevention. The proposed research examines the details of NO activation of sGC, the heart of this manipulation.