Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
J Biol Chem. 2021 Jan-Jun;296:100405. doi: 10.1016/j.jbc.2021.100405. Epub 2021 Feb 10.
Components of the autophagy machinery are subject to regulation by various posttranslational modifications. Previous studies showed that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination.
自噬机制的组件受到各种翻译后修饰的调节。以前的研究表明，由泛素激活酶 UBA6 和泛素结合酶/泛素连接酶 BIRC6 催化的 LC3B 的单泛素化将 LC3B 靶向蛋白酶体降解，从而在应激条件下降低 LC3B 水平和自噬活性。然而，能够对抗这一过程的机制尚不清楚。在此，我们报告说 LC3B 的泛素化被去泛素酶 USP10 的作用所逆转。我们在一个 CRISPR-Cas9 敲除筛选中鉴定了 USP10，该筛选针对调节 LC3B 水平的泛素化相关基因。生化分析表明，沉默 USP10 通过增加泛素化和蛋白酶体降解来降低 LC3B-I 和 LC3B-II 形式的 LC3B 水平。反过来，LC3B 水平的降低导致自噬受体 SQSTM1 和 NBR1 的降解速度变慢，以及嘌呤霉素诱导的聚集体样结构的积累增加。总之，这些发现表明 LC3B 和自噬活性的水平通过 LC3B 泛素化和去泛素化的循环来控制。
