Canada's Michael Smith Genome Sciences Centre, BC Cancer Research Institute, Vancouver, BC, Canada.
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
Autophagy. 2022 Nov;18(11):2547-2560. doi: 10.1080/15548627.2022.2039535. Epub 2022 Feb 28.
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. 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（CQ）是一种溶酶体趋向性药物，常用于抑制溶酶体降解和巨自噬/自噬。在这里，我们研究了 CQ 对细胞外分泌的影响。我们发现溶酶体和自噬抑制作用改变了分泌组，并诱导了 Atg8 同源物和自噬受体的释放。Atg8 家族蛋白，特别是以脂化依赖的方式分泌到小细胞外囊泡（sEVs）中。CQ 处理增强了 Atg8 家族蛋白在 sEVs 中的释放。使用全长 ATG16L1 和一种能够在双但不在单膜上进行 Atg8 家族蛋白脂化的 ATG16L1 突变体，我们证明 LC3B 以两种不同的 sEV 群体释放：一种富含 SDCBP/Syntenin-1、CD63 和富含脂质的 LC3B 的内体，另一种含有 LC3B 但不富含 SDCBP/Syntenin-1 或 CD63 的 sEV，我们的数据支持其源自双膜来源。我们的研究结果强调了 sEV 异质性和组成的上下文依赖性，并说明了自噬和 sEV 组成在应对溶酶体抑制时的整合。ACTB：肌动蛋白 beta；ANOVA：方差分析；ATG4B：自噬相关 4B 半胱氨酸肽酶；Atg8：自噬相关 8；ATG16L1：自噬相关 16 样 1；ATP5F1A/ATP5a：ATP 合酶 F1 亚基 alpha；CALCOCO2：钙结合和卷曲螺旋结构域 2；CASP3：半胱天冬酶 3；CASP7：半胱天冬酶 7；CQ：氯喹；CD9：CD9 分子；CD63：CD63 分子；DAPI：4'，6-二脒基-2-苯基吲哚；DQ-BSA：染料猝灭-牛血清白蛋白；ER：内质网；ERN1/IRE1a：内质网到核信号 1；EV：细胞外囊泡；FBS：胎牛血清；FDR：错误发现率；GABARAP：GABA 型 A 受体相关蛋白；GABARAPL2：GABA 型 A 受体相关蛋白样 2；GAPDH：甘油醛-3-磷酸脱氢酶；GFP：绿色荧光蛋白；GO：基因本体论；HCQ：羟氯喹；HSP90AA1：热休克蛋白 90 alpha 家族成员 1；IP：免疫沉淀；KO：敲除；LAMP2：溶酶体相关膜蛋白 2；LIR：LC3 相互作用区；LMNA：核纤层蛋白 A/C；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3 beta；MS：质谱；NBR1：NBR1 自噬货物受体；NCOA4：核受体共激活因子 4；NTA：纳米颗粒跟踪分析；PE：磷脂酰乙醇胺；PECA：探针级表达变化平均；SDCBP/syntenin-1： syndecan 结合蛋白；SD：标准偏差；SE：分泌；sEV：小细胞外囊泡；SQSTM1/p62：sequestosome 1；TAX1BP1：Tax1 结合蛋白 1；TEM：透射电子显微镜；TMT：串联质量标签；TSG101：肿瘤易感性 101；ULK1：unc-51 样自噬激活激酶 1；WC：全细胞。
