Negative Regulation of Autophagy by UBA6-BIRC6–Mediated Ubiquitination of LC3

Abstract

Although the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. The ability to manipulate autophagy is important not only for addressing fundamental biological questions, but also for its possible application to the treatment of various human diseases. To identify novel regulators of autophagy, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroblastoma cells gene-edited to express the endogenous autophagy effector LC3B fused to a tandem of GFP and mCherry.Using this methodology, we identified the ubiquitin-activating (E1) enzyme UBA6 and the hybrid ubiquitin-conjugating (E2)/ubiquitin-ligase (E3) enzyme BIRC6 as important autophagy regulators. We found that these two enzymes cooperate to monoubiquitinate LC3B on lysine-51, targeting it for degradation by the proteasome. Knockout of UBA6 or BIRC6 increased the levels of LC3B as well as autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, depletion of UBA6 or BIRC6 KO decreased the formation of aggresomelike induced structures in H4 cells, and aggregates of an a-synuclein mutant in the axon of rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B, possibly to prevent the deleterious effects of excessive autophagy. Inhibition of UBA6 or BIRC6, on the other hand, could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders.

The conformational and mutational landscape of the ubiquitin-like marker for autophagosome formation in cancer

“…Strikingly, the mutant P32Q showed a strong expression decrease in respect to the wildtype form, validating our predictions on the effect of P32Q mutation on LC3B stability and the neutral effects of I35V, M60V, K65E and K49N. Our results on the variants Y113C and K49N were also in line with recent papers, indicating that these mutations do not have a strong effect on protein stability [34,60,93]. Our scans predict Y113C as destabilizing the protein architecture, thus we wonder if this discrepancy could be related to the fact that the high-throughput mutational scans do not provide enough conformational sampling to model this mutation.…”

Novel and conventional inhibitors of canonical autophagy differently affect LC3‐associated phagocytosis

“…Thus, our data suggest that EACC seems to accelerate the turnover of LC3 by proteasomal degradation. Indeed, it has previously been shown that LC3 is degraded by the proteasome following ubiquitination [52].…”

Atg8-Family Proteins—Structural Features and Molecular Interactions in Autophagy and Beyond

“…Ubiquitination is another type of PTM modulating autophagy in an LC3- or GABARAP-selective manner. Monoubiquitination of LC3 (and not GABARAPs) at K51 driven by the coordinated action of ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3 for the proteasomal degradation and negatively regulates autophagy [ 89 ]. Mib1-driven mono- and polyubiquitination of the GABARAP (not LC3) on K13 and K23 within N -terminal α-helical subdomain occurs through K48-chains and targets GABARAPs to proteasomal degradation [ 72 ].…”