人可溶性鸟苷酸环化酶介导一氧化氮信号转导的结构基础和调控分子机制研究

项目名称： 人可溶性鸟苷酸环化酶介导一氧化氮信号转导的结构基础和调控分子机制研究

项目编号： No.21472027

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 环境科学、安全科学

项目作者： 谭相石

作者单位： 复旦大学

项目金额： 90万元

中文摘要： 内源性化学小分子NO作为信使分子在血液循环和神经系统中有非常重要的生理功能。基于化学小分子探针的信号转导分子机制和调控机制研究是化学生物学的前沿热点。本项目以人源性可溶性鸟苷酸环化酶（sGC）介导NO信号转导的分子机制和调控机制为研究对象，基于sGC莹光标记（FLAsH）和融合荧光蛋白，利用化学小分子探针（NO/CO和sGC的激活剂/激动剂等），通过荧光抗淬灭（FlAsH-Heme之间的FRET） 的方法研究sGC中血红素辅基的氧化丢失以及在NO信号转导过程中血红素辅基的变化，阐明血红素中心键合NO/CO信号分子,介导和调控NO信号转导的结构基础和调控分子机制；利用基因重组、基因突变、蛋白质修饰等技术手段，揭示sGC的两个亚基之间及其每个亚基的结构域之间的协调分子机制；阐明sGC的结构改变/基因突变及血红素氧化丢失等导致的NO信号转导异常的结构基础,探索NO信号转导障碍导致疾病的分子机制和

中文关键词： 可溶性鸟苷酸环化酶；信号转导；荧光探针；sGC；一氧化氮

英文摘要： Nitric oxide, a signaling molecule in the cardiovascular system, has been receiving increasing attention. Soluble guanylate cyclase (sGC), as a NO sensor/receptor, is activated by NO to catalyze the conversion of guanosine 5'-triphosphate (GTP) to cyclic guanylate monophosphate (cGMP) in mediating NO-signaling transduction. The dysfunction of the NO signaling results in many pathological disorders, including several cardiovascular diseases such as arterial hypertension, pulmonary hypertension, heart failure and so on. The molecular mechanism for sGC to mediate and regulate the NO signaling is unclear up to now. This project aims to investigate the structural basis and molecular mechanism for human sGC to mediate and regulate NO signal transduction in both the molecular and cellular level. To this end, the full length and truncated recombinant sGC proteins and mutants attached biarsenical fluorophor FlAsH or fused fluorescent proteins will be prepared and characterized by UV/Vis, CD, EPR, fluorescent spectroscopy. The FRET between heme and the fluorophor FlAsH will be monitored when the oxidized heme gets out of pocket. The roles of key residues and structural domains in sGC will be revealed based on the FRET approach. The structure-property-reactivity-function relationship of sGC to mediate NO signaling will be illustrated. The NO signaling regulation mechanism will be studied by the usage of chemical probes (NO/CO, activator or stimulator of sGC). Fluorescence dequenching, based on the interaction of the optical active prosthetic heme group and the attached biarsenical fluorophor FlAsH can be used to detect changes in living cellular sGC. The association rate constant measurements of two subunit of sGC in living cells will be performed by microinjections using Eppendorf FemtoJet attached to Eppendorf Inject Man NI2 micromanipulator. Structural basis for sGC to mediate and regulate NO signaling will be uncovered by structural determination of sGC containing various structural domains. The molecular mechanism of NO signaling disorder related to cardiovascular diseases will be explored and new insights into developing Heme-dependent and heme-independent drugs will be provided.

英文关键词： NO;sGC;Soluble guanylate cyclase;Signaling transduction;Fluorescent probe