巨自噬/自噬是一种基本的细胞分解代谢过程,将细胞质成分传递到称为自噬体的双膜囊泡中,然后与溶酶体融合,其内容物降解。自噬回收细胞质成分,包括错误折叠的蛋白质,功能失调的细胞器甚至微生物入侵者,从而在发展中发挥重要作用,免疫和细胞死亡。自噬形成是自噬的主要步骤,它由一组ATG(自噬相关)蛋白控制。ATG16L1与ATG12-ATG5缀合物相互作用以形成ATG12-ATG5-ATG16L1复合物。该复合物充当泛素样E3连接酶,催化MAP1LC3/LC3(微管相关蛋白1轻链3)的脂化,这对自噬体形成至关重要。在本研究中,我们发现ATG16L1对半胱氨酸153进行S-棕榈酰化,这是由ZDHHC7(锌指DHHC型棕榈酰转移酶7)催化的。我们观察到,重新表达ATG16L1而不是S-棕榈酰化缺陷型突变体ATG16L1C153S挽救了ATG16L1-KO(敲除)HeLa细胞中LC3脂化和自噬体形成的缺陷。此外,通过ZDHHC7表达增加ATG16L1S-棕榈酰化促进LC3-II的产生,而通过ZDHHC7缺失减少ATG16L1S-棕榈酰化抑制了LC3脂化过程和自噬体形成。机械上,在ATG16L1的Cys153残基上添加疏水性16碳棕榈酰基可增强吞噬团上ATG16L1-WIPI2B复合物和ATG16L1-RAB33B复合物的形成,从而促进LC3脂化过程和自噬体形成。总之,ATG16L1的S-棕榈酰化对于LC3的脂化过程和自噬体的形成至关重要。我们的研究揭示了ATG16L1在自噬中功能的新调控机制。
Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death.
Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12-ATG5 conjugate to form an ATG12-ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for
autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and
autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and
autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.Abbreviation: ABE: acyl-biotin exchange; ATG: autophagy related; Baf-A1: bafilomycin A1; 2-BP: 2-bromopalmitate; CCD: coiled-coil domain; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle\'s balanced salt solution; HAM: hydroxylamine; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex I; PTM: post-translational modification; RAB33B: RAB33B, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; WD: tryptophan and aspartic acid; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zinc finger DHHC-type.