ABSTRACT

Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a deubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. RNF31 knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. OTULIN knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In OTULIN knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.

Abbreviations: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; CUL1-FBXL20: cullin 1-F-box and leucine rich repeat protein 20; CUL3-KLHL20: cullin 3-kelch like family member 20; CUL4-AMBRA1: cullin 4-autophagy and beclin 1 regulator 1; CYLD: CYLD lysine 63 deubiquitinase; DAPI: 4′,6-diamidino-2-phenylindole; DUB: deubiquitinating enzyme; EBSS: Earle’s Balanced Salt Solution; GFP: green fluorescent protein; GST: glutathione S-transferase; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; LUBAC: linear ubiquitin chain assembly complex; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3B; MIM: MIT-interacting motif; mRFP: monomeric red fluorescent protein; NEDD4: NEDD4 E3 ubiquitin protein ligase; NFKB: NF-kappaB complex; OPTN: optineurin; OTULIN: OTU deubiquitinase with linear linkage specificity; PIK3C3/Vps34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns: phosphatidylinositol; PtdIns3K: class III phosphatidylinositol 3-kinase complex; PtdIns3P: phosphatidylinositol 3-phosphate; RBCK1/HOIL1: RANBP2-type and C3HC4-type zinc finger containing 1; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RIPK1: receptor interacting serine/threonine kinase 1; RNF216: ring finger protein 216; RNF31/HOIP: ring finger protein 31; RT-PCR: reverse transcriptase polymerase chain reaction; S. Typhimurium: Salmonella enterica serovar Typhimurium; SHARPIN: SHANK associated RH domain interactor; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING: stimulator of interferon response cGAMP interactor 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TNF/TNF-alpha: tumor necrosis factor; TNFAIP3/A20: TNF alpha induced protein 3; TRAF6: TNF receptor associated factor 6; TRIM32: tripartite motif containing 32; UBAN: ubiquitin binding in TNIP/ABIN and IKBKG/NEMO proteins; ULK1/2: unc-51 like autophagy activating kinase 1/2; USP: ubiquitin specific peptidase; UVRAG: UV radiation resistance associated; VCPIP1: valosin containing protein interacting protein 1; WIPI2: WD repeat domain, phosphoinositide interacting protein 2; ZBTB16-CUL3-RBX1: zinc finger and BTB domain containing protein 16-cullin 3-ring-box 1; ZRANB1: zinc finger RANBP2-type containing 1.

摘要

巨自噬/自噬是一种膜介导的细胞内降解途径，通过该途径，大量的细胞质内容物在溶酶体中被消化。自噬起始和成熟步骤是如何调节的尚不清楚。在这项研究中，我们发现 E3 泛素连接酶复合物、线性泛素链组装复合物 (LUBAC) 和去泛素化酶 (DUB) OTULIN 定位于吞噬细胞区域以控制自噬的启动和成熟。当自噬被诱导时，LUBAC 关键成分 RNF31/HOIP 易位到 LC3 泪点区域。RNF31敲低抑制自噬启动，细胞对细菌感染更敏感。然而，OTULIN敲低促进自噬启动但阻止自噬成熟。在欧图林敲低细胞后，过量的泛素化 ATG13 蛋白被募集到吞噬细胞中以延长扩增时间，从而抑制自噬体成熟。总之，我们的研究提供了证据，证明 LUBAC 和 OTULIN 通过介导线性泛素化和 ATG13 的稳定来协同调节自噬起始和自噬体成熟。

缩写：ATG：自噬相关；CALCOCO2/NDP52：钙结合和卷曲螺旋结构域 2；CQ：氯喹；CUL1-FBXL20：cullin 1-F-box 和富含亮氨酸的重复蛋白 20；CUL3-KLHL20：cullin 3-kelch 像家庭成员 20；CUL4-AMBRA1：cullin 4-自噬和 beclin 1 调节剂 1；CYLD：CYLD 赖氨酸 63 去泛素酶；DAPI：4',6-二脒基-2-苯基吲哚；DUB：去泛素化酶；EBSS：厄尔平衡盐溶液；GFP：绿色荧光蛋白；GST：谷胱甘肽 S-转移酶；IKBKG/NEMO：核因子κB激酶调节亚基γ抑制剂；LUBAC：线性泛素链组装复合物；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；MAP1LC3B/LC3B：微管相关蛋白1轻链3B；MIM：麻省理工学院交互主题；mRFP：单体红色荧光蛋白；NEDD4：NEDD4 E3泛素蛋白连接酶；NFKB：NF-κB复合物；OPTN：optineurin；OTULIN：具有线性连锁特异性的 OTU 去泛素酶；PIK3C3/Vps34：磷脂酰肌醇 3-激酶催化亚基 3 型；PtdIns：磷脂酰肌醇；PtdIns3K：III类磷脂酰肌醇3-激酶复合物；PtdIns3P：3-磷酸磷脂酰肌醇；RBCK1/HOIL1：RANBP2型和C3HC4型锌指含1个；RB1CC1/FIP200：RB1-诱导型线圈1；RIPK1：受体相互作用的丝氨酸/苏氨酸激酶 1；RNF216：无名指蛋白 216；RNF31/HOIP：无名指蛋白 31；RT-PCR：逆转录聚合酶链式反应；鼠伤寒沙门氏菌：PtdIns3P：3-磷酸磷脂酰肌醇；RBCK1/HOIL1：RANBP2型和C3HC4型锌指含1个；RB1CC1/FIP200：RB1-诱导型线圈1；RIPK1：受体相互作用的丝氨酸/苏氨酸激酶 1；RNF216：无名指蛋白 216；RNF31/HOIP：无名指蛋白 31；RT-PCR：逆转录聚合酶链式反应；鼠伤寒沙门氏菌：PtdIns3P：3-磷酸磷脂酰肌醇；RBCK1/HOIL1：RANBP2型和C3HC4型锌指含1个；RB1CC1/FIP200：RB1-诱导型线圈1；RIPK1：受体相互作用的丝氨酸/苏氨酸激酶 1；RNF216：无名指蛋白 216；RNF31/HOIP：无名指蛋白 31；RT-PCR：逆转录聚合酶链式反应；鼠伤寒沙门氏菌：肠沙门氏菌鼠伤寒血清型; SHARPIN：SHANK 相关 RH 域交互器；SMURF1：SMAD 特异性 E3 泛素蛋白连接酶 1；SQSTM1：隔离体 1；STING：干扰素反应 cGAMP 相互作用物 1 的刺激物；STUB1/CHIP：STIP1同源性和含有蛋白质1的U-box；TNF/TNF-α：肿瘤坏死因子；TNFAIP3/A20：TNFα诱导蛋白3；TRAF6：TNF受体相关因子6；TRIM32：包含 32 个的三方基序；UBAN：TNIP/ABIN 和 IKBKG/NEMO 蛋白中的泛素结合；ULK1/2：unc-51 样自噬激活激酶 1/2；USP：泛素特异性肽酶；UVRAG：相关联的抗紫外线辐射；VCPIP1：含有蛋白质相互作用蛋白1的valosin；WIPI2：WD重复结构域，磷酸肌醇相互作用蛋白2；ZBTB16-CUL3-RBX1：锌指和 BTB 结构域，含有蛋白质 16-cullin 3-ring-box 1；ZRANB1：