MGRN1/TSG101/RAB7信号通路对小鼠睾丸支持细胞自噬的调控

我们及前人早期研究发现MGRN1参与生精过程，然机制不明。近期研究发现MGRN1下调后自噬体与溶酶体的膜融合受阻以致自噬体降解下降而数量增多，MGRN1通过泛素化TSG101调控该过程，而TSG101不直接参与膜融合，那么其下游介导膜融合的蛋白是什么？我们近期研究发现小鼠睾丸支持细胞中MGRN1下调后自噬体增多，且自噬体上膜融合关键蛋白Rab7募集下降。故首次提出小鼠睾丸支持细胞中MGRN1调节自噬的新机制：MGRN1通过泛素化TSG101调控Rab7在自噬体上的募集调节自噬体与溶酶体的膜融合而调控自噬。本课题拟构建相关表达载体或干扰载体，分别正反向调节MGRN1、TSG101、RAB7后，检测三者的变化及相互影响，并利用电镜，Western blot等技术检测自噬的相关指标，最终确定MGRN1/TSG101/Rab7信号通路对小鼠睾丸支持细胞自噬的调控及机制，为男性不育的诊治提供新靶点。

It has been reported that MGRN1 is involved in the process of spermatogenesis, while the detailed molecular mechanism is still unknown. A recent study found that Mahogunin (MGRN1) regulated membrane fusion between autophagosomes and lysosomes via ubiquitination of TSG101 and more autophagosomes was observed if functional depletion of MGRN1. While TSG101 acts as a member of ESCRT proteins that are involved in MVB biogenesis but have not been demonstrated to mediate membrane fusion, it remains an open question how TSG101 mediate autophagosome fusion. Our previous work had found that in mice sertoli cells knockdown of MGRN1 led to the decrease of the recruitment of Rab7 to autophagosomes and increase of the number of autophagosomes. Therefore, we speculate that MGRN1 regulates fusion between autophagosomes and lysosomes by the recruitment of Rab7 to autophagosomes via ubiquitination of TSG101. In this project, we plan to overexpress and/or knowdown the expression of MGRN1、TSG101、RAB7 in mice sertoli cells via their respective overexpression and/or interference vectors,and then study the impact betwen MGRN1、TSG101 and Rab7,as well as the autophagy in mice sertoli cells by western blot and transmission electron microscope. Finally, we will evaluate whether MGRN1/TSG101/RAB7 signaling pathway regulates the autophagy of mice sertoli cells. By doing so we could provide a new target for the diagnosis and therapy of male infertility.