UBE3C tunes autophagy via ATG4B ubiquitination

ABSTRACT

ATG4B is a core protein and essential for cleaving precursor MAP1LC3/LC3 or deconjugating lipidated LC3-II to drive the formation of autophagosomes. The protein stability and activity of ATG4B regulated by post-translational modification (ubiquitination) will directly affect macroautophagy/autophagy. However, the mechanism involved in ATG4B ubiquitination is largely unclear. In this study, a new E3 ligase of ATG4B, UBE3C, was identified by mass spectra. UBE3C mainly assembles K33-branched ubiquitin chains on ATG4B at Lys119 without causing ATG4B degradation. In addition, the increased ubiquitination of ATG4B caused by UBE3C overexpression inhibits autophagy flux in both normal and starvation conditions, which might be due to the reduced activity of ATG4B and ATG4B-LC3 interaction. This reduction could be reversed once the lysine 119 of ATG4B was mutated to arginine. More important, under starvation conditions the interaction between ATG4B and UBE3C apparently decreased followed by the removal of the K33-branched ubiquitin chain of ATG4B. Thus, starvation-induced autophagy could be partially suppressed by an increased ubiquitination level of ATG4B. In conclusion, our research reveals a novel modification mode of ATG4B in which UBE3C can fine tune ATG4B activity by specific ubiquitination regulating autophagy without causing ATG4B degradation.

Abbreviation: ATG: autophagy-related; Baf: bafilomycin A₁; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; GFP: green fluorescent protein; HA-Ub: HA-tagged ubiquitin; IF: immunofluorescence; IP: immunoprecipitation; K: lysine; KO: knockout; K0: all K-to-R mutant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NC: negative control; R: arginine; WCL: whole cell lysate; WT: wild-type.

KEYWORDS:

• ATG4B
• autophagy
• UBE3C
• ubiquitination
• protein interaction

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31970699, 32100610), the Guangdong Basic and Applied Basic Research Foundation (2021A1515010766, 2019A1515011030), the Guangdong Provincial Key Laboratory of Construction Foundation (2019B030301005), and the Key-Area Research and Development Program of Guangdong Province (2020B1111110003).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2299514

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.