RACK1在IRE1相关肝脏内质网应激保护中的关键作用及分子机制研究

Inositol-requiring enzyme1(IRE1) plays as the key sensor of endoplasmic reticulum(ER) stress. Our previous studies showed that activating IRE1-related signal pathway of ER stress was involved in the protection of ischemic preconditioning against ischemia/reperfusion (I/R) injury. Furthermore, we found that RACK1 bound to IRE1 and induced the recruitment and activation of AMPK in ER stress. Active AMPK kinase further phosphorylated IRE1 in a spatially regulated pattern and activated IRE1 phosphorylated Bcl2, which contributed to the cytoprotective effect in hepatocytes. These results indicates that RACK1 is essential in the cytoprotective effect of IRE1-related ER stress. However, the underlying mechanism of recruitment and spatiotemporal dynamics of IRE1 signal pathway induced by RACK1 remains unclear. In this study, we propose to further investigate the role of RACK1 in cytoprotective effect of IRE1-related ER stress in vivo by RACK1 liver targeting knockout mice model. We will explore the spatiotemporal dynamics and regulatory mechanism of RACK1 to IRE1 signal pathway by molecular and functional proteomics methods. This study will provide insight of molecular mechanism of ER stress preconditioning and novel molecular targets for clinical pharmacological interventions to I/R injury in liver.

IRE1是内质网应激的核心感受器，我们前期研究发现IRE1及相关内质网应激通路的激活是缺血预处理保护肝脏缺血/再灌注损伤的重要机制。进一步研究提示肝细胞内质网应激条件下RACK1与IRE1的结合可引起细胞内IRE1相关蛋白AMPK的募集和激活，活化的AMPK与IRE1结合并促进IRE1磷酸化，导致Bcl2的激活，发挥抗凋亡和肝细胞保护作用。因此，RACK1是IRE1相关内质网应激肝细胞保护通路中的必要元件。但是，RACK1与IRE1及相关蛋白的募集及其时空效应的分子机制尚不明了。本研究拟利用Cre-loxp重组酶系统构建条件性RACK1肝脏定向敲除小鼠，从器官层面进一步证实RACK1在IRE1相关内质网应激肝细胞保护中的作用，在此基础上运用分子生物学方法和功能蛋白组学技术，研究RACK1对IRE1及相关通路的时空效应和关键调控机制，为临床肝脏药物靶向预处理保护提供可能的分子靶点和理论依据。

内质网应激与肝脏缺血再灌注损伤的关系密切，适当的内质网应激可以导致肝脏对缺血再灌注损伤产生一定程度的耐受，但当损伤程度剧烈或内质网应激过分刺激下，肝脏的损伤反而加重。我们通过研究提示肝细胞内质网应激条件下RACK1与IRE1的结合可引起细胞内IRE1相关蛋白AMPK的募集和激活，活化的AMPK与IRE1结合并促进IRE1磷酸化，导致Bcl2的激活，发挥抗凋亡和肝细胞保护作用。因此，RACK1是IRE1相关内质网应激肝细胞保护通路中的必要元件。但是，RACK1与IRE1及相关蛋白的募集及其时空效应的分子机制尚不明了。本课题组成功利用Cre-loxp重组酶系统构建条件性RACK1肝脏定向敲除小鼠，从器官层面进一步证实RACK1通过IRE1相关内质网应激途径肝细胞保护中的作用，在此基础上运用分子生物学方法和功能蛋白组学技术，研究RACK1对IRE1及相关通路的时空效应和关键调控机制，为临床肝脏药物靶向预处理保护提供可能的分子靶点和理论依据，另外我们还观察到细胞铁死亡参与肝脏缺血再灌注的现象，RACK1可以通过减轻铁死亡进而减轻肝脏缺血再灌注损伤，其间接作用是通过增强GPX4分子的表达进而降低铁死亡程度。