利用密码子扩展技术对细胞焦亡中gasdermin家族蛋白行为进行特异性荧光标记与成像研究

细胞焦亡是由炎性caspase活化诱发的程序性死亡，属于机体的重要天然免疫反应过程，在对抗外源感染和内源危险信号中发挥重要作用。近年来，细胞焦亡被发现经由以GSDMD（Gasdermin D）蛋白为代表的gasdermin家族蛋白介导。这一机制被发现时间较短，有许多细节的研究有限。一个原因是，在其荧光成像研究上，目前使用融合荧光蛋白标记GSDMD的方法无法区分caspase-1切割前后的蛋白，较大的背景干扰了对蛋白的高精度追踪。本项目通过基因密码子扩展技术向gasdermin家族蛋白中定点引入非天然氨基酸，实现对激活及膜结合的GSDMD蛋白的特异性示踪。在此之上，本项目将通过对GSDMD蛋白与膜结构的作用进行荧光成像研究，探究其行为模式，以及与细胞焦亡中其他蛋白的关联。进一步地，本项目将这一特异性荧光标记方法也推广至其他gasdermin家族蛋白，帮助探索家族成员在细胞焦亡中的生理作用。

Pyroptosis is a form of programmed cell death that induced by inflammatory-caspases. It contributes to non-specific immunity and plays an important role in fighting against infection and innate danger signals. Recently, it is reported that pyroptosis is mediated by gasdermin family proteins, especially GSDMD. The details of gasdermin family proteins’ roles in pyroptosis have not been fully elucidated since the short time after discovery of GSDMD-mediated mechanism. One of the reasons is that current method for their fluorescence imaging is not good enough. With fusing fluorescent protein to GSDMD, it is not able to distinct GSDMD proteins before and after caspase-1 cleavage. And the high background fluorescence will impair locating of GSDMD in high resolution. This object facilitates specifically fluorescent labeling of cleaved or membrane-bound GSDMD protein by genetic code expansion. Next, this object aims to investigating the interactions between GSDMD and the membrane structures in pyroptosis via specifically fluorescence imaging of membrane-bound GSDMD. Furthermore, this object will try to extend the specific labeling method to other members of gasdermin family proteins and shed light on research of their roles in pyroptosis.