K48/K63-linked polyubiquitination of ATG9A at K581/K838 by TRAF6 E3 ligase

Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. However, the molecular mechanisms underlying the activation of oxidative stress-mediated autophagic response remain elusive. Here we show that ubiquitin E3 ligase TNF receptor-associated factor 6 (TRAF6) and the deubiquitinase A20 coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The reactive oxygen species (ROS)-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of class III phosphatidylinositol-3-kinase (PI3KC3/VPS34) UVRAG complex, thereby stimulating autophagy. Notably, depletion of ATG9A or expression of the ATG9A ubiquitination mutants markedly decreases ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination and enhances the assembly of the UVRAG-containing VPS34 complex in an ATG9A-dependent manner. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreased phosphorylation of IRF-3 in LPS-stimulated macrophages. Our findings provide important insight into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.

高反应性氧化分子的过度生成与积累会引发氧化应激，并对细胞组分造成氧化损伤。越来越多的研究证据表明，细胞自噬（autophagy）可通过降解并回收细胞内受损组分，减轻细胞氧化损伤并维持氧化还原稳态（redox homeostasis）。然而，氧化应激介导的自噬反应激活背后的分子机制仍有待阐明。本研究发现，泛素E3连接酶（ubiquitin E3 ligase）肿瘤坏死因子受体相关因子6（TRAF6）与去泛素化酶（deubiquitinase）A20协同调控细胞响应氧化应激时ATG9A的泛素化（ubiquitination）与自噬激活。活性氧（reactive oxygen species, ROS）依赖的TRAF6介导的ATG9A非蛋白水解型K48/63位连接泛素化，可增强其与Beclin 1的结合以及III型磷脂酰肌醇-3-激酶（PI3KC3/VPS34）UVRAG复合物的组装，进而刺激自噬。值得注意的是，敲除ATG9A或表达ATG9A泛素化突变体，可显著降低活性氧诱导的VPS34激活与自噬水平。本研究进一步发现，脂多糖（lipopolysaccharide, LPS）诱导的活性氧生成，同样可通过ATG9A依赖的方式，促进TRAF6介导的ATG9A泛素化并增强含UVRAG的VPS34复合物的组装。敲除ATG9A会导致脂多糖刺激的巨噬细胞中Toll样受体4（TLR4）内体运输异常以及干扰素调节因子3（IRF-3）磷酸化水平降低。本研究结果为K48/K63位连接的ATG9A泛素化如何调控氧化应激诱导的自噬提供了重要的机制见解。