LUBAC and OTULIN regulate autophagy initiation and maturation by mediating the linear ubiquitination and the stabilization of ATG13
LUBAC and OTULIN regulate autophagy initiation and maturation by mediating the linear ubiquitination and the stabilization of ATG13
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LUBAC 和 OTULIN 通过介导线性泛素化和 ATG13 的稳定来调节自噬的启动和成熟
DOI：
10.1080/15548627.2020.1781393
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发表时间：
2020-06-28
期刊：
影响因子：
13.3
通讯作者：
Liu, Yanfen
中科院分区：
文献类型：
--
作者：
Chu, Yuanyuan;Kang, Yingjin;Liu, Yanfen
摘要巨自噬/自噬是一种膜介导的细胞内降解途径，大量的胞浆内容物通过该途径被溶酶体消化。自噬的启动和成熟步骤是如何调控的，目前尚不清楚。在这项研究中，我们发现E3泛素连接酶复合体、线性泛素链组装复合体(LUBAC)和脱泛素化酶(DUB)OTULIN位于吞噬体区，控制自噬的启动和成熟。自噬诱导时，LUBAC关键成分RNF31/HOIP易位到Lc3点区域。RNF31基因敲除抑制了自噬的启动，细胞对细菌感染更敏感。然而，OTULIN基因敲除促进了自噬的启动，但阻止了自噬的成熟。在OTULIN基因敲除细胞中，过量泛素化的ATG13蛋白被招募到吞噬体中进行长期扩张，因此抑制了自噬小体的成熟。总之，我们的研究提供了证据，证明LUBAC和OTULIN通过介导ATG13的线性泛素化和稳定化来协同调节自噬的启动和自噬小体的成熟。缩写：ATG：自噬相关；CALCOCO2/NDP52：钙结合和螺旋卷曲结构域2；CQ：氯喹；CUL1-FBXL20：CULL1-F盒和富含亮氨酸的重复蛋白20；CUL3-KLHL20：CULL3-海带样家族成员20；CUL4-AMBRA1：CULL4-自噬和BECLIN 1调节因子1；CyLD：CyLD赖氨酸63脱泛素酶；DAPI：4‘，6-二氨基-2-苯基吲哚；DUB：脱泛素酶；EBSS：Earle平衡盐水液；GFP：绿色荧光蛋白；GST：谷胱甘肽转移酶S酶；IKBKG/Nemo：核因子kappaB亚单位抑制物；伽马；DUB：脱泛素酶；EBSS：耳氏平衡盐水液；GFP：绿色荧光蛋白；GST：谷胱甘肽S-酶；IKBKG/Nemo：核因子kappaB亚单位抑制物；LUBAC：线性泛素链组装复合体；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：MIT相互作用基序；MRFP：单体红色荧光蛋白；NEDD4：NEDD4 E3泛素蛋白连接酶；NFKB：核因子-kappaB复合体；OPTN：Optineurin；OTULIN：具有线性连接特异性的Otu脱泛素酶；PIK3C3/Vps34：磷脂酰肌醇3-催化亚单位3；PtdIns：磷脂酰肌醇；PtdIns3K：III类磷脂酰肌醇3-复合体；PtdIns3P：磷脂酰肌醇3-磷酸；RBC1/HOK1：含锌的脱泛素酶；PIK3C3/Vps34：磷脂酰肌醇3-催化亚基3；PtdIns：磷脂酰肌醇；PtdIns3K：III类磷脂酰肌醇3-复合体；PtdIns3P：磷脂酰肌醇3-磷酸；RBK1/HOK1：含锌的脱泛素酶；PIK3C3/Vps34：磷脂酰肌醇3-催化亚单位3；PtdIns：磷脂酰肌醇；PtdIns3K：III类磷脂酰肌醇3-复合体激酶；PtdIns3P：磷脂酰肌醇3-磷酸；RBC1/ILK1：含手指的脱泛素酶；PIK3C3/Vps34：磷脂酰肌醇3-催化亚单位3；PtdIns：磷脂酰肌醇；PtdIns3K：III类磷脂酰肌醇3-复合体；PtdIns3P：磷脂酰肌醇3-磷酸；RBC1/HOK1：含锌的脱泛素酶；PtdIns：磷脂酰肌醇；PtdInsRB1CC1/FIP200：Rb1诱导的卷曲1；RIPK1：受体相互作用的丝氨酸/苏氨酸激酶1；RNF216：环指蛋白216；RNF31/HOIP：环指蛋白31；RT-PCR：逆转录聚合酶链式反应；鼠伤寒沙门氏菌：沙门氏菌；沙门氏菌；Sharpin：小腿相关的RH结构域相互作用因子；SMURF1：Smad特异的E3泛素蛋白连接酶1；SQSTM1：隔离小体1；STING：干扰素反应刺激因子cGAMP；STUB1/CHIP：STIP1同源和含U盒蛋白1；肿瘤坏死因子；TNFAIP3/A20：肿瘤坏死因子诱导蛋白；TRAF6：肿瘤坏死相关受体相关因子；6TRIM32：含32个三体基序；UBAN：TNIP/ABIN和IKBKG/NEMO蛋白中的泛素结合；ULK1/2：UNC-51样自噬激活蛋白1/2；USP：泛素特异性多肽酶；UVRAG：与紫外线辐射相关；VCPIP1：含有Valosin的蛋白相互作用蛋白1；WIPI2：WD重复结构域，磷酸肌醇相互作用蛋白2；ZBTB16-CUL3-RBX1：含锌指和BTB结构域的蛋白16-cullin 3-Ringbox 1；ZRANB1：含锌指RANBP2的蛋白1。
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.