Macroautophagy/autophagy plays a pivotal role in cytoplasmic material recycling and metabolic turnover, in which ATG4B functions as a “scissor” for processing pro-LC3 and lipidated LC3 to drive the autophagy progress. Mounting evidence has demonstrated the tight connection between ROS and autophagy during various pathological situations. Coincidentally, several studies have shown that ATG4B is potentially regulated by redox modification, but the underlying molecular mechanism and its relationship with autophagy is ambiguous. In this study, we verified that ATG4B activity was definitely regulated in a reversible redox manner. We also determined that Cys292 and Cys361 are essential sites of ATG4B to form reversible intramolecular disulfide bonds that respond to oxidative stress. Interestingly, we unraveled a new phenomenon that ATG4B concurrently formed disulfide-linked oligomers at Cys292 and Cys361, and that both sites underwent redox modifications thereby modulating ATG4B activity. Finally, increased autophagic flux and decreased oxidation sensitivity were observed in Cys292 and Cys361 double site-mutated cells under normal growth conditions. In conclusion, our research reveals a novel molecular mechanism that oxidative modification at Cys292 and Cys361 sites regulates ATG4B function, which modulates autophagy.Abbreviations: Air-ox: air-oxidation; ATG4B: autophagy related 4B cysteine peptidase; BCNU: 1,3-bis(2-chloroethyl)-1-nitrosourea; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; DTT: dithiothreitol; GSH: reduced glutathione; GSNO: S-nitrosoglutathione; GSSG: oxidized glutathione; HMW: high molecular weight; H₂O₂: hydrogen peroxide; NAC: N-acetyl-L-cysteine; NEM: N-ethylmaleimide; PE: phosphatidylethanolamine; PTM: post-translational modification; ROS, reactive oxygen species; WT: wild type

巨自噬/自噬在细胞质物质循环和代谢周转中起着关键作用，其中 ATG4B 作为“剪刀”来处理 pro-LC3 和脂化 LC3 以推动自噬进程。越来越多的证据表明，在各种病理情况下，ROS 与自噬之间存在紧密联系。巧合的是，一些研究表明 ATG4B 可能受到氧化还原修饰的调节，但潜在的分子机制及其与自噬的关系尚不清楚。在这项研究中，我们证实 ATG4B 活性确实以可逆的氧化还原方式受到调节。我们还确定 Cys292 和 Cys361 是 ATG4B 形成响应氧化应激的可逆分子内二硫键的重要位点。有趣的是，我们揭示了一个新现象，即 ATG4B 同时在 Cys292 和 Cys361 处形成二硫键连接的寡聚物，并且两个位点都经过氧化还原修饰从而调节 ATG4B 活性。最后，在正常生长条件下，在 Cys292 和 Cys361 双位点突变细胞中观察到自噬通量增加和氧化敏感性降低。总之，我们的研究揭示了一种新的分子机制，即 Cys292 和 Cys361 位点的氧化修饰调节 ATG4B 功能，从而调节自噬。缩写：Air-ox：空气氧化；ATG4B：自噬相关4B半胱氨酸肽酶；BCNU：1,3-双(2-氯乙基)-1-亚硝基脲；CBB：考马斯亮蓝；CM：完全培养基；CQ：氯喹；DTT：二硫苏糖醇；GSH：还原型谷胱甘肽；GSNO：S-亚硝基谷胱甘肽；GSSG：氧化型谷胱甘肽；HMW：高分子量；H ₂ O ₂：过氧化氢；NAC：N-乙酰基-L-半胱氨酸；NEM：N-乙基马来酰亚胺；PE：磷脂酰乙醇胺；PTM：翻译后修饰；ROS，活性氧；WT：野生型