Autophagy ( IF 13.3 ) Pub Date : 2020-05-26 , DOI: 10.1080/15548627.2020.1761652 Shuo Tian 1 , Shouheng Jin 1 , Yaoxing Wu 1 , Tao Liu 1 , Man Luo 1 , Jiayu Ou 1, 2 , Weihong Xie 1 , Jun Cui 1, 2
ABSTRACT
Macroautophagy/autophagy, a eukaryotic homeostatic process that sequesters cytoplasmic constituents for lysosomal degradation, is orchestrated by a number of autophagy-related (ATG) proteins tightly controlled by post-translational modifications. However, the involvement of reversible ubiquitination in the regulation of autophagy remains largely unclear. Here, we performed a single-guide RNA-based screening assay to investigate the functions of deubiquitinating enzymes (DUBs) in regulating autophagy. We identified previously unrecognized roles of several DUBs in modulating autophagy at multiple levels by targeting various ATG proteins. Mechanistically, we demonstrated that STAMBP/AMSH (STAM-binding protein) promotes the stabilization of ULK1 by removing its lysine 48 (K48)-linked ubiquitination, whereas OTUD7B mediates the degradation of PIK3 C3 by enhancing its K48-linked ubiquitination, thus positively or negatively affects autophagy flux, respectively. Together, our study elaborated on the broad involvement of DUBs in regulating autophagy and uncovered the critical roles of the reversible ubiquitination in the modification of ATG proteins.
Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; DUB: deubiquitinating enzyme; EBSS: Earle’s balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OTUD7B: OTU domain-containing protein 7B; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; sgRNA: single-guide RNA; SQSTM1/p62: sequestosome 1; STAMBP/AMSH: STAM-binding protein; ULK1: unc-51 like autophagy activating kinase 1; USP: ubiquitin specific peptidase.
中文翻译:
自噬中功能性去泛素化酶的高通量筛选
摘要
巨自噬/自噬是一种真核生物稳态过程,可隔离细胞质成分以进行溶酶体降解,由许多受翻译后修饰严格控制的自噬相关 (ATG) 蛋白协调。然而,可逆泛素化在自噬调节中的参与在很大程度上仍不清楚。在这里,我们进行了基于单向导 RNA 的筛选试验,以研究去泛素化酶 (DUB) 在调节自噬中的功能。我们通过靶向各种 ATG 蛋白,确定了几个 DUB 在多个水平上调节自噬中以前未被认识到的作用。从机制上讲,我们证明了 STAMBP/AMSH(STAM 结合蛋白)通过去除其赖氨酸 48(K48)连接的泛素化来促进 ULK1 的稳定,而 OTUD7B 通过增强其 K48 连接的泛素化来介导 PIK3 C3 的降解,从而分别对自噬通量产生正面或负面影响。总之,我们的研究详细阐述了 DUB 广泛参与调节自噬,并揭示了可逆泛素化在 ATG 蛋白修饰中的关键作用。
缩写:ATG:自噬相关;Baf A 1:巴弗洛霉素A 1;DUB:去泛素化酶;EBSS:厄尔平衡盐溶液;KO:淘汰赛;MAP1LC3/LC3:微管相关蛋白1轻链3;OTUD7B:含OTU结构域的蛋白7B;PIK3C3:磷脂酰肌醇 3-激酶催化亚基 3 型;sgRNA:单向导RNA;SQSTM1/p62:sequestosome 1;STAMBP/AMSH:STAM 结合蛋白;ULK1:unc-51 类似自噬激活激酶 1;USP:泛素特异性肽酶。