Proteotoxic stress disrupts epithelial integrity by inducing MTOR sequestration and autophagy overactivation

Macroautophagy/autophagy, an evolutionarily conserved degradation system, serves to clear intracellular components through the lysosomal pathway. Mounting evidence has revealed cytoprotective roles of autophagy; however, the intracellular causes of overactivated autophagy, which has cytotoxic effects, remain elusive. Here we show that sustained proteotoxic stress induced by loss of the RING and Kelch repeat-containing protein C53A5.6/RIKE-1 induces sequestration of LET-363/MTOR complex and overactivation of autophagy, and consequently impairs epithelial integrity in <i>C. elegans</i>. In C53A5.6/RIKE-1-deficient animals, blocking autophagosome formation effectively prevents excessive endosomal degradation, mitigates mislocalization of intestinal membrane components and restores intestinal lumen morphology. However, autophagy inhibition does not affect LET-363/MTOR aggregation in animals with compromised C53A5.6/RIKE-1 function. Improving proteostasis capacity by reducing DAF-2 insulin/IGF1 signaling markedly relieves the aggregation of LET-363/MTOR and alleviates autophagy overactivation, which in turn reverses derailed endosomal trafficking and rescues epithelial morphogenesis defects in C53A5.6/RIKE-1-deficient animals. Hence, our studies reveal that C53A5.6/RIKE-1-mediated proteostasis is critical for maintaining the basal level of autophagy and epithelial integrity. <b>Abbreviations:</b> ACT-5: actin 5; ACTB: actin beta; ALs: autolysosomes; APs: autophagosomes; AJM-1: apical junction molecule; ATG: autophagy related; C. elegans: Caenorhabditis elegans; CPL-1: cathepsin L family; DAF: abnormal dauer formation; DLG-1: Drosophila discs large homolog; ERM-1: ezrin/radixin/moesin; EPG: ectopic P granule; GFP: freen fluorescent protein; HLH-30: helix loop helix; HSP: heat shock protein; LAAT-1: lysosome associated amino acid transporter; LET: lethal; LGG-1: LC3, GABARAP and GATE-16 family; LMP-1: LAMP (lysosome-associated membrane protein) homolog; MTOR: mechanistic target of rapamycin kinase; NUC-1: abnormal nuclease; PEPT-1/OPT-2: Peptide transporter family; PGP-1: P-glycoprotein related; RAB: RAB family; RIKE-1: RING and Kelch repeat-containing protein; SLCF-1: solute carrier family; SQST-1: sequestosome related; SPTL-1: serine palmitoyl transferase family.

巨自噬（Macroautophagy/autophagy）是一种进化上保守的降解系统，通过溶酶体途径清除细胞内组分。越来越多的研究证据揭示了自噬的细胞保护作用；然而，具有细胞毒性效应的过度活化自噬的细胞内诱因仍尚不明确。
本研究发现，缺失含RING和Kelch重复结构域的蛋白C53A5.6/RIKE-1（RING and Kelch repeat-containing protein）所诱导的持续性蛋白毒性应激，会导致LET-363/MTOR（mechanistic target of rapamycin kinase）复合物发生聚集，并引发自噬过度活化，最终损害秀丽隐杆线虫（Caenorhabditis elegans，以下简称C. elegans）的上皮完整性。
在C53A5.6/RIKE-1缺陷型线虫中，阻断自噬体形成可有效抑制过度的内体降解，缓解肠道膜组分的错定位，并恢复肠道管腔形态。但抑制自噬并不会影响C53A5.6/RIKE-1功能受损动物体内的LET-363/MTOR聚集。通过降低DAF-2胰岛素/胰岛素样生长因子1（insulin/IGF1）信号通路来增强蛋白稳态能力，可显著缓解LET-363/MTOR的聚集，减轻自噬过度活化，进而逆转紊乱的内体运输，并挽救C53A5.6/RIKE-1缺陷型动物的上皮形态发生缺陷。因此，本研究揭示了C53A5.6/RIKE-1介导的蛋白稳态对于维持基础水平自噬与上皮完整性至关重要。
**缩写说明：** ACT-5：肌动蛋白5（actin 5）；ACTB：β-肌动蛋白（actin beta）；ALs：自噬溶酶体（autolysosomes）；APs：自噬体（autophagosomes）；AJM-1：顶连接分子（apical junction molecule）；ATG：自噬相关（autophagy related）；C. elegans：秀丽隐杆线虫（Caenorhabditis elegans）；CPL-1：组织蛋白酶L家族（cathepsin L family）；DAF：异常滞育形成（abnormal dauer formation）；DLG-1：果蝇盘大同源物（Drosophila discs large homolog）；ERM-1：埃兹蛋白/根蛋白/膜突蛋白家族（ezrin/radixin/moesin）；EPG：异位P颗粒（ectopic P granule）；GFP：绿色荧光蛋白（green fluorescent protein）；HLH-30：螺旋-环-螺旋蛋白（helix loop helix）；HSP：热休克蛋白（heat shock protein）；LAAT-1：溶酶体相关氨基酸转运蛋白（lysosome associated amino acid transporter）；LET：致死（lethal）；LGG-1：LC3、GABARAP与GATE-16家族（LC3, GABARAP and GATE-16 family）；LMP-1：溶酶体相关膜蛋白（LAMP, lysosome-associated membrane protein）同源物；MTOR：雷帕霉素机制性靶标激酶（mechanistic target of rapamycin kinase）；NUC-1：异常核酸酶（abnormal nuclease）；PEPT-1/OPT-2：肽转运蛋白家族（Peptide transporter family）；PGP-1：P-糖蛋白相关蛋白（P-glycoprotein related）；RAB：RAB家族；RIKE-1：含RING和Kelch重复结构域的蛋白（RING and Kelch repeat-containing protein）；SLCF-1：溶质转运蛋白家族（solute carrier family）；SQST-1：Sequestosome相关蛋白（sequestosome related）；SPTL-1：丝氨酸棕榈酰转移酶家族（serine palmitoyl transferase family）。