Abstract
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.
Highlights
Autophagy is a cellular process for the lysosomal degradation of cytoplasmic materials (i.e., “cargos”) such as organelles, protein aggregates, and intracellular pathogens [1, 2]
To identify potential LC3B deubiquitinating enzyme (DUB), we conducted a CRISPRCas9 KO screen using a human H4 neuroglioma cell line that expresses LC3B endogenously tagged with tandem GFPmCherry (H4-tandem fluorescenttagged LC3B (tfLC3B) cells) [12]
The screen was based on the hypothesis that depletion of specific DUBs would increase LC3B ubiquitination and degradation, decreasing GFP and mCherry fluorescence signals
Summary
To identify potential LC3B DUBs, we conducted a CRISPRCas KO screen using a human H4 neuroglioma cell line that expresses LC3B endogenously tagged with tandem GFPmCherry (H4-tfLC3B cells) [12]. Combined treatment with bafilomycin A1 and nutrient starvation resulted in greater accumulation of LC3BII in both WT and USP10-KO cells, but, again, LC3B-II levels were lower in USP10-KO than in WT cells (Fig. 3, A–C) These findings indicated that the decreased levels of LC3B in USP10deficient cells were not due to increased lysosomal/autophagic degradation, but rather preceded the engagement of LC3B in autophagy. Similar observations were made by CHX treatment in starvation medium (Fig. S6, A–C) These experiments indicated that the lower levels of LC3B in USP10KO cells limit the availability of LC3B for recruitment of SQSTM1 and NBR1 to autophagosomes and their eventual degradation in autolysosomes under conditions of protein synthesis inhibition. To examine the effect of USP10 KO on the accumulation of ALIS, we incubated WT and USP10-KO H4 cells with 5 μg/ml puromycin for 2 and 3 h and visualized the
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