Sun Chaonan, Chen Yuxin, Gu Qianqian, Fu Yuanyuan, Wang Yao, Liu Cui, Xie Huazhong, Liao Yong, Zheng Zhihua, Liu Peiqing, Li Min
School of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery, Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-Sen University, Guangzhou, Guangdong, China.
Chongqing Medical and Pharmaceutical College, Chongqing, China.
Autophagy. 2024 Mar;20(3):645-658. doi: 10.1080/15548627.2023.2299514. Epub 2024 Jan 3.
ATG4B is a core protein and essential for cleaving precursor MAP1LC3/LC3 or deconjugating lipidated LC3-II to drive the formation of autophagosomes. The protein stability and activity of ATG4B regulated by post-translational modification (ubiquitination) will directly affect macroautophagy/autophagy. However, the mechanism involved in ATG4B ubiquitination is largely unclear. In this study, a new E3 ligase of ATG4B, UBE3C, was identified by mass spectra. UBE3C mainly assembles K33-branched ubiquitin chains on ATG4B at Lys119 without causing ATG4B degradation. In addition, the increased ubiquitination of ATG4B caused by UBE3C overexpression inhibits autophagy flux in both normal and starvation conditions, which might be due to the reduced activity of ATG4B and ATG4B-LC3 interaction. This reduction could be reversed once the lysine 119 of ATG4B was mutated to arginine. More important, under starvation conditions the interaction between ATG4B and UBE3C apparently decreased followed by the removal of the K33-branched ubiquitin chain of ATG4B. Thus, starvation-induced autophagy could be partially suppressed by an increased ubiquitination level of ATG4B. In conclusion, our research reveals a novel modification mode of ATG4B in which UBE3C can fine tune ATG4B activity by specific ubiquitination regulating autophagy without causing ATG4B degradation. ATG: autophagy-related; Baf: bafilomycin A; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; GFP: green fluorescent protein; HA-Ub: HA-tagged ubiquitin; IF: immunofluorescence; IP: immunoprecipitation; K: lysine; KO: knockout; K0: all K-to-R mutant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NC: negative control; R: arginine; WCL: whole cell lysate; WT: wild-type.
ATG4B是一种核心蛋白，对于切割前体MAP1LC3/LC3或将脂化的LC3-II去共轭以驱动自噬体的形成至关重要。由翻译后修饰（泛素化）调节的ATG4B的蛋白质稳定性和活性将直接影响巨自噬/自噬。然而，ATG4B泛素化所涉及的机制在很大程度上尚不清楚。在本研究中，通过质谱鉴定了一种新的ATG4B的E3连接酶UBE3C。UBE3C主要在赖氨酸119处的ATG4B上组装K33分支的泛素链，而不会导致ATG4B降解。此外，由UBE3C过表达引起的ATG4B泛素化增加在正常和饥饿条件下均抑制自噬通量，这可能是由于ATG4B活性降低以及ATG4B与LC3的相互作用减少所致。一旦ATG4B的赖氨酸119突变为精氨酸，这种减少就可以逆转。更重要的是，在饥饿条件下，ATG4B与UBE3C之间的相互作用明显减少，随后ATG4B的K33分支泛素链被去除。因此，ATG4B泛素化水平的增加可部分抑制饥饿诱导的自噬。总之，我们的研究揭示了一种新的ATG4B修饰模式，其中UBE3C可以通过特异性泛素化调节自噬来微调ATG4B活性，而不会导致ATG4B降解。ATG：自噬相关；Baf：巴弗洛霉素A；CBB：考马斯亮蓝；CM：完全培养基；CQ：氯喹；GFP：绿色荧光蛋白；HA-Ub：HA标记的泛素；IF：免疫荧光；IP：免疫沉淀；K：赖氨酸；KO：敲除；K0：所有K突变为R的突变体；MAP1LC3/LC3：微管相关蛋白1轻链3；MS：质谱；NC：阴性对照；R：精氨酸；WCL：全细胞裂解物；WT：野生型。
