Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations

Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition. <b>Abbreviations:</b> ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4’,6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell

氯喹（Chloroquine, CQ）作为一种溶酶体亲和试剂，常被用于抑制溶酶体降解与巨自噬（macroautophagy，又称自噬（autophagy））。本研究探讨了CQ对细胞分泌过程的非细胞自主效应。实验证实，CQ介导的溶酶体与自噬抑制可改变细胞分泌组（secretome）的组成，并诱导Atg8同源蛋白及自噬受体的释放。具体而言，Atg8家族蛋白以依赖脂酰化修饰的方式被包裹于小型细胞外囊泡（small extracellular vesicles, sEVs）中并完成分泌。CQ处理可显著增强sEV内Atg8家族蛋白的释放水平。通过使用全长ATG16L1以及一种可使Atg8家族蛋白仅在双层膜（而非单层膜）上发生脂酰化修饰的ATG16L1突变体，我们证明LC3B（microtubule associated protein 1 light chain 3 beta）存在于两种不同的sEV群体中：一类富集SDCBP/Syntenin-1、CD63以及内体膜脂酰化LC3B；另一类虽含有LC3B但未富集SDCBP/Syntenin-1或CD63，我们的实验数据支持该类囊泡来源于双层膜结构。本研究结果凸显了sEV异质性与组分组成的环境依赖性，并阐明了溶酶体抑制条件下自噬通路与sEV组分的整合调控机制。<b>缩略语列表：</b> ACTB：肌动蛋白β（actin beta）；ANOVA：方差分析（analysis of variance）；ATG4B：自噬相关4B半胱氨酸肽酶（autophagy related 4B cysteine peptidase）；Atg8：自噬相关8（autophagy related 8）；ATG16L1：自噬相关16样蛋白1（autophagy related 16 like 1）；ATP5F1A/ATP5a：ATP合酶F1亚基α（ATP synthase F1 subunit alpha）；CALCOCO2：钙结合卷曲螺旋结构域蛋白2（calcium binding and coiled-coil domain 2）；CASP3：半胱氨酸天冬氨酸蛋白酶3（caspase 3）；CASP7：半胱氨酸天冬氨酸蛋白酶7（caspase 7）；CQ：氯喹（chloroquine）；CD9：CD9分子（CD9 molecule）；CD63：CD63分子（CD63 molecule）；DAPI：4',6-二脒基-2-苯基吲哚（4’,6-diamidino-2-phenylindole）；DQ-BSA：染料淬灭牛血清白蛋白（dye quenched-bovine serum albumin）；ER：内质网（endoplasmic reticulum）；ERN1/IRE1a：内质网至细胞核信号转导1（endoplasmic reticulum to nucleus signaling 1）；EV：细胞外囊泡（extracellular vesicles）；FBS：胎牛血清（fetal bovine serum）；FDR：错误发现率（false discovery rate）；GABARAP：GABA A型受体相关蛋白（GABA type A receptor-associated protein）；GABARAPL2：GABA A型受体相关蛋白样2（GABA type A receptor associated protein like 2）；GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；GFP：绿色荧光蛋白（green fluorescent protein）；GO：基因本体论（gene ontology）；HCQ：羟氯喹（hydroxychloroquine）；HSP90AA1：热休克蛋白90α家族A类成员1（heat shock protein 90 alpha family class A member 1）；IP：免疫沉淀（immunoprecipitation）；KO：基因敲除（knockout）；LAMP2：溶酶体相关膜蛋白2（lysosomal associated membrane protein 2）；LIR：LC3互作区域（LC3-interacting region）；LMNA：核纤层蛋白A/C（lamin A/C）；MAP1LC3B/LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；MS：质谱分析（mass spectrometry）；NBR1：NBR1自噬受体（NBR1 autophagy cargo receptor）；NCOA4：核受体辅激活因子4（nuclear receptor coactivator 4）；NTA：纳米颗粒追踪分析（nanoparticle tracking analysis）；PE：磷脂酰乙醇胺（phosphatidylethanolamine）；PECA：探针水平表达变化平均法（probe-level expression change averaging）；SDCBP/syntenin-1：Syndecan结合蛋白（syndecan binding protein）；SD：标准差（standard deviation）；SE：分泌型（secreted）；sEV：小型细胞外囊泡（small extracellular vesicles）；SQSTM1/p62：隔离体1（sequestosome 1，又称p62）；TAX1BP1：Tax1结合蛋白1（Tax1 binding protein 1）；TEM：透射电子显微镜（transmission electron microscopy）；TMT：串联质量标签（tandem-mass tag）；TSG101：肿瘤易感基因101（tumor susceptibility 101）；ULK1：UNC-51样自噬激活激酶1（unc-51 like autophagy activating kinase 1）；WC：全细胞（whole cell）