⾼基⽒體蛋⽩降解經由細胞⾃噬調控

Abstract

細胞通過自噬去除受損的胞器。然而，通過高基氏體自噬引發的蛋白質降解仍是有爭議的議題。在本篇研究，我們建立了兩種不同的方法可獲得高基氏體自噬證據。一是使用光主動誘導高基氏體損傷，另一種是無損傷方式應用高基氏體蛋白轉染並長時間觀察。 在主動損傷高基氏體的策略中，我們用化學和遺傳學方式。將具高基氏體專一性的光敏劑結合光照以觸發活性氧的產生，可實現高基氏體損傷。此外，雷射可達成破壞細胞中的小部分之高基氏體，發光二極管照射則可破壞全部的高基氏體。 我們偵測到受損高基氏體可吸引自噬轉接蛋白p62，泛素和自噬標記蛋白LC3聚集。還證實了LC3顆粒中含有成熟的自噬小體圍繞著高基氏體碎片。ATG4B、LC3點突變和ATG5的基因抑制消除了自噬小體的形成。長時間顯微鏡觀察下，高基氏體被自噬小體吞噬並進入溶酶體，並通過SNARE蛋白STX17調控降解，而氨基酸剔除可加速降解。我們還發現E3連接酶HACE1可能調控受損高基氏體泛素化。結論是，我們發現損壞之高基氏體降解可通過細胞自噬機制調控。 在被動非破壞方法中，我們用基因轉染方法在細胞表現高基氏體蛋白，並長期培養後觀察到大量高基氏體顆粒在細胞質積累。出乎意料的是，我們發現大多數高基氏體與LC3顆粒共定位，但此現象並未在LC3點突變或是ATG16L1敲除細胞中被觀察到。此外，高爾基顆粒也與LAMP1共定位，並具溶酶體特性。敲弱自噬基因導致高基氏體蛋白含量上升，並形成破碎小型的高基氏體。而ATG5基因敲除之小鼠胚胎成纖維細胞也顯示異常高基氏體形態。敲弱STX17導致高基氏體膨脹堆積和尺寸擴大。以上結果可證明，我們可能發現了一個新穎的運輸過程，高基氏體到溶酶體的蛋白質的運輸途徑是由細胞自噬機制所調節。

Cells remove damaged organelles through autophagy. However, Golgi apparatus (GA) degradation via autophagy (Golgiphagy) is controversy. Here we have established two different schemes to get the evidences for Golgiphagy. One used light actively induced GA damage and another applied transfection of GA proteins without damage to observe passively. In active damage GA therapy, we used chemical and genetically Golgi-localized photosensitizers respectively light illumination to trigger reactive oxygen species production and it can be achieved specific GA impairment. Besides, laser bleached small fractions of GA in single cell and LED illuminated global organelles on whole cells. After GA impairment, we detected autophagy adaptors p62、ubiquitin and autophagosome marker LC3B on injured GA. It was also confirmed LC3 punta contained mature autophagosome which were surrounding debris of GA. Overexpression of ATG4B、LC3 mutant and knockdown of ATG5 abolished autophagosome formation. Long-term time lapse imaging showed that the autophagosome-engulfed GA entered into lysosome for degradation by SNARE protein STX17 mediation and EBSS starvation accelerated degradation process. We also found E3 ligase HACE1 could mediate damaged GA ubiquitination. According to the results, damage GA degradation is regulated by autophagy. In passive non-damage method, we also used genetically Golgi-localized proteins and observed a large number Golgi punta accumulated in cytosol in long-term incubation. Surprisingly, we found most Golgi punta co-localized with wild-type LC3 but not in mutant and the phenomenon was inhibited in ATG16L1 knockout cell line. Furthermore, most Golgi punta also co-localized with LAMP1 and was characterized as lysosome. Knockdown of autophagic genes caused GA protein levels were increased and it was fragmented to form mini-Golgi stacks in cytosol and ATG5-/- MEFs also showed abnormal GA morphology. Knockdown of STX17 led to more Golgi punta accumulation and size expansion. These results can be demonstrated a novel Golgi to lysosome trafficking pathway for protein turnover is regulated by autophagy.