PI(3)P结合家族蛋白WIPI1-4在自噬通路中作用的机制研究

PI(3)P binding family proteins WIPI (WD-repeat protein interacting with phosphoinositides) 1-4, which belongs to WDR (WD40-repeat domain) proteins, are regulatory beta-propeller platforms that enable the assembly of multiprotein complexes and function in many essential biological functions including cell cycle control, apoptosis, signal transduction pathways, RNA metabolism, chromatin assembly and vesicular trafficking. Previous studies have demonstrated the role of WIPIs in the pathogenesis of a variety of human diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases. Recent findings demonstrated the critical role of WIPIs in the autophagy pathway. Autophagy is a highly conserved cytoprotective mechanism for lysosomal clearance of aggregated proteins and damaged organelles. A hallmark for autophagy is the formation of double-membraned autophagosomes which is dependent of the production of PI(3)P generated by class III PI3K complex. Members of WIPI proteins are currently the only known conserved and essential PI(3)P effectors in autophagy. Our previous studies using genetic screen in C. elegans revealed a group of metazoan-specific autophagy genes, named “epg” genes. The C. elegans homologs of yeast essential autophagy gene Atg18 are atg-18 and epg-6, which function differentially in the autophagosome formation process. EPG-6 directly interacts with ATG-2 and regulates progression of omegasomes to autophagosomes, while ATG-18 may regulate association of protein aggregates, LGG-1 puncta and omegasomes. The mammalian ATG18 homologs are WIPI1-4, which could further fall into two paralogous groups, WIPI1/2 (ATG-18) and WIPI3/4 (EPG-6). How these four proteins function cooperatively in the autophagy pathway is still unclear. We plan to generate the WIPI1-4 knockout cell using CRISPR/Cas9 and knockout MEF from Wipi1-4 knockout mice. Through studying the autophagy defect in these cells, we will do hierarchal analysis of WIPIs in autophagosome formation to determine their functional difference in the autophagy pathway. We will also utilize biochemical methods to identify specific targets of WIPIs in the autophagy and other pathways. To study their function in vivo, we will further analyze the tissue-specific function of different WIPI proteins using Wipi1-4 knockout mice. This study will provide new insights into the molecular basis of autophagosome formation and potential therapeutic targets for treating related human diseases.

PI(3)P结合家族蛋白WIPI1-4是一类具有重要功能的蛋白，与肿瘤、心血管疾病、神经退行性疾病等密切相关。近年研究发现WIPI蛋白在细胞自噬过程中发挥着重要作用。本组前期利用线虫进行遗传筛选发现多个多细胞生物特有的自噬蛋白，并发现PI(3)P效应蛋白ATG18在线虫中有两个同源蛋白ATG-18和EPG-6，在自噬通路中发挥着不同作用。哺乳动物细胞中ATG18的同源蛋白进一步分化为四个WIPI1-4，它们如何共同参与和调控自噬通路目前尚不清楚。本研究组前期构建了Wipi1-4基因敲除小鼠并发现它们神经系统表型各不相同。本研究拟采用细胞生物学和生物化学等方法深入研究WIPI蛋白在自噬通路发挥功能的分子机理，并通过观察各基因敲除小鼠对不同组织和功能的影响在整体动物水平观察它们如何相互协调发挥作用。我们希望通过对WIPI蛋白功能的研究深入阐明自噬通路的分子机制并为相关疾病的治疗提供理论依据。

细胞自噬是细胞内一种重要的降解机制，在真核生物内高度保守。自噬可以清除细胞内有毒的蛋白质聚集体和受损细胞器，对维持细胞稳态具有重要作用。自噬过程需要由PI(3)P激酶复合体在自噬小体形成部位产生PI(3)P，WIPI蛋白是重要的PI(3)P效应蛋白，包含WIPI1/2和WDR45/45B，其中WIPI2和WDR45/45B为自噬过程必需。本研究深入研究了WIPI2和WDR45/45B在自噬通路中的分子机理，还通过构建Wdr45b敲除小鼠研究了WDR45B的生理功能。我们发现WIPI2可以作为衔接蛋白，介导隔离膜与内质网之间的互作。自噬小体形成的过程中，隔离膜会与内质网间不断互作，从而促进隔离膜的生长和闭合。我们发现当没有WIPI2时，DFCP1标记的欧米伽小体大量累积，但与标记了LC3的自噬小体无法共定位，说明PI3P结合蛋白WIPI2在内质网和自噬小体之间的互作中发挥了重要作用。WIPI2与ULK1/FIP200相互作用形成复合体，连接内质网和自噬小体。同时我们还发现PI3P在建立这一复合体中也起到了重要的作用。本研究首次为内质网与自噬小体之间的相互作用提供了分子机制。神经细胞是一种不分裂细胞，自噬异常对神经细胞的功能影响尤为明显。多种神经退行性疾病均与自噬异常密切相关。而自噬基因WDR45和WDR45B突变分别可以引起人类神经系统疾病BPAN和ID。我们通过构建Wdr45b敲除小鼠，发现与人类疾病特征相似，小鼠出现行为及学习记忆功能障碍，为研究智力障碍的发病的分子机理和病理过程提供了一个重要的模型。为了研究WDR45和WDR45B参与自噬通路的分子机理，我们在神经来源细胞Neuro 2a细胞敲除Wdr45/45b，并发现Wdr45/45b DKO细胞出现自噬异常。Wdr45/45b DKO细胞的自噬异常主要是由于自噬小体与溶酶体的融合效率下降导致。EPG5是介导自噬小体与溶酶体融合的栓系蛋白，WDR45和WDR45B与EPG5相互作用并帮助EPG5的晚期内吞体/溶酶体定位，进而促进膜融合复合体的形成。这些发现不但有助于我们理解WDR45/45B的功能，也对于我们理解相关人类疾病的发病机理及寻找药物靶点具有重要意义。