Proximity proteomics on RPE1 stable cell lines with miniTurbo-p62 and ATG5 Knock-down to investigate p62 filaments and calcium-rich p62-enwrapped lipid droplets

The selective autophagy receptor p62/SQSTM1 (from hereon p62) is known to form higher-order filaments in vitro and to undergo liquid-liquid phase separation when mixed with poly-ubiquitin. We determined the full-length cryo-EM structure of p62 and elucidated a structured double helical filament scaffold composed of the PB1-domain associated with the flexible C-terminal part residing in the lumen and the solvent-accessible major groove. At different pH values and upon binding to soluble LC3, LC3-conjugated membranes and poly-ubiquitin, we observed p62 filament re-arrangements in the form of structural unwinding, disassembly, lateral association and bundling, respectively. In the cellular environment, under conditions of ATG5 knockdown leading to stalled autophagy, we imaged high-contrast layers consisting of p62 oligomers enwrapping lipid droplets by cryogenic electron tomography in cellulo, which we identified as calcium as well as phosphorus by compositional spectroscopy analysis. Together, we visualized the cellular ultrastructure of p62 oligomers with high calcium content as a potential early stage of autophagy. Therefore, we performed proximity biotinylation and proteomics to identify calcium transporters and binding proteins in proximity to the p62 encapsulated lipid droplets.

选择性自噬受体p62/SQSTM1（selective autophagy receptor p62/SQSTM1，以下简称p62）已知可在体外形成高阶多聚丝状体，并在与多泛素链（poly-ubiquitin）混合时发生液-液相分离（liquid-liquid phase separation）。我们解析了p62的全长冷冻电镜（cryo-electron microscopy, cryo-EM）结构，并阐明了一种结构化双螺旋丝状体支架：该支架由PB1结构域（PB1-domain）构成，其柔性C末端部分位于管腔内，并形成溶剂可及的主沟槽。在不同pH条件下、结合可溶性LC3时、结合LC3偶联膜时以及结合多泛素链时，我们分别观察到p62丝状体发生结构解旋、解聚、侧向缔合与聚集重排。在细胞环境中，当ATG5基因敲低（ATG5 knockdown）导致自噬停滞（stalled autophagy）时，我们通过细胞内（in cellulo）冷冻电子断层成像（cryogenic electron tomography）技术，成像到了由p62寡聚体构成的高对比度层状结构，该结构包裹着脂滴（lipid droplets）；通过成分光谱分析（compositional spectroscopy analysis），我们确定该层状结构中含有钙与磷元素。综上，我们可视化了富含钙的p62寡聚体细胞超微结构（cellular ultrastructure），其可能代表自噬的早期阶段。据此，我们开展了邻近生物素标记（proximity biotinylation）与蛋白质组学（proteomics）实验，以鉴定p62包被脂滴附近的钙转运蛋白与结合蛋白。