cGMP与Tropomyosin相互作用在肺动脉高压发生发展中的作用机制研究

Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate.(NO-sGC-cGMP) signal pathway plays an important role in the occurrence and development of pulmonary arterial hypertension(PAH). However, the molecular mechanism by which NO-sGC-cGMP causes the process of the PAH development remains to be further defined. In our previous studies, we have utilized the Biotin-cGMP pull down assay to search for the proteins regulated by cGMP, and we found that cGMP interacted with tropomyosin, a factor that responsible for regulating the interaction between actin and myosin and is therefore a regulator of muscle contraction. Meanwhile, we identified isoform 4 of TPM1 was expressed in the human pulmonary artery smooth muscle cells, and analyzed the specificity of the interaction between cGMP and tropomyosin in vitro. Based on the result of pull down assay, we would further confirm the interaction between cGMP and tropomyosin and identificate the binding sites of the interaction. Moreover, we would further identificate the interaction might bridge the affinity effect of tropomyosin and actin, which would lead to the alteration of the structure and function of the Actin-Tropomyosin-Myosin complex. In summary, we would elucidate the downstream signal pathway of NO-sGC-cGMP. This work will contribute to the discovery of novel targeted agents, and provide new insights into the development of new approaches for PAH therapy.

一氧化氮—可溶性鸟苷酸环化酶—环磷酸鸟苷（NO-sGC-cGMP）细胞信号转导通路与肺动脉高压的发生密切相关。我们前期工作利用Biotin-cGMP pull-down实验找到cGMP可能相互作用蛋白原肌球蛋白（tropomyosin）。我们确定人肺动脉平滑肌细胞中特异表达的原肌球蛋白异构体类型，运用体外研究方法验证了cGMP和原肌球蛋白结合的特异性。由此提出cGMP与原肌球蛋白相互作用后是否通过Actin-Tropomyosin-Myosin收缩蛋白复合物调控肺血管的收缩和舒张。本项目将进一步证明cGMP与原肌球蛋白结合的特异性，找到二者结合具体位点，以及阐明cGMP与原肌球蛋白结合后调控Actin-Tropomyosin-Myosin收缩蛋白复合物的亲和力从而介导血管收缩或舒张。总之，本课题对NO-sGC-cGMP下游信号通路的阐明，将为肺动脉高压发生发展的机制研究提供更多理论支持。

一氧化氮—可溶性鸟苷酸环化酶—环磷酸鸟苷（NO-sGC-cGMP）细胞信号转导通路与肺动脉高压的发生密切相关，但NO-sGC-cGMP下游通过何种作用机制参与调控肺动脉高压目前并不清楚。我们推测，cGMP作为体内第二信使，可能与某些重要蛋白质发生相互作用。我们前期工作利用Biotin-cGMP pull-down实验找到cGMP可能相互作用蛋白原肌球蛋白（tropomyosin）,确定人肺动脉平滑肌细胞中特异表达的原肌球蛋白异构体类型为isoform 4，并利用抗体检测pull-down的方法证实了cGMP与原肌球蛋白的相互作用。本课题在前期工作基础之上运用浓度梯度实验和竞争性抑制实验进一步验证cGMP和原肌球蛋白结合的特异性，利用tropomyosin的片段缺失突变体证实tropomyosin蛋白的68-208aa片段是tropomyosin与cGMP的相互作用位点。RT-PCR、Western blot和免疫荧光实验证明cGMP与tropomyosin的结合不影响tropomyosin的表达水平和亚细胞定位。本课题也进一步利用等温滴定量热法证明cGMP竞争性结合tropomyosin后减少Actin-Tropomyosin-Myosin的相互作用，进而降低Actin-Tropomyosin-Myosin收缩蛋白复合物功能，从而使血管舒张。总之，本课题对NO-sGC-cGMP下游信号通路的阐明，为肺动脉高压血管收缩舒张的调控提出新的理论基础，为临床肺动脉高压的干预提供全新的靶标，并为增强其临床实际应用潜能提供强有力的理论支持。