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

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.

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

人可溶性鸟苷酸环化酶（sGC）作为 NO 的受体，是 NO信号转导通路中一个关键的核心金属酶。NO 与 sGC 结合后激活 sGC，从而催化其底物 GTP 转化为 cGMP。NO 信号通路发生紊乱则将导致多种疾病的发生，如心血管疾病、心力衰竭、肺动脉高压和神经退行性疾病等。因此，揭示 sGC 蛋白的结构基础，阐明 sGC 介导 NO 信号转导的分子机制将有助于理解 NO 信号紊乱所致疾病的病理机制，为新型心脑血管等疾病药物的设计和开发提供新思路。在本项目中，我们在分子和细胞水平上，采用荧光探针标记和融合荧光蛋白等方法，运用寿命成像激光共聚焦显微镜系统地研究了sGC 被 NO 激活过程中结构域之间、亚基之间的相互作用，及 sGC 构象变化过程，揭示了 sGC 介导 NO 信号转导的分子机制。基于FlAsH-EDT2荧光标记探针与 heme 之间的能量转移方法，通过检测sGC heme氧化丢失过程中FlAsH-EDT2的荧光变化，揭示了sGC heme氧化丢失的分子机制。该结果对于设计和开发靶向无 heme 和 heme 氧化的sGC 的心血管疾病药物具有重要的指导意义。首次发现同源二聚体的sGC 1亚基具有亚硝酸盐还原酶的功能，研究了 sGC β1亚基同源二聚体的亚硝酸盐还原酶的分子机制。该结果对于理解体内 sGC β1同源二聚体的功能具有重要的意义，并推测体内sGC可能存在自激活的过程。