O-GlcNAcylation of ATG4B positively regulates autophagy by increasing its hydroxylase activity

Abstract

Autophagy is a catabolic degradation process and maintains cellular homeostasis. And autophagy is activated in response to various stress conditions. Although O-GlcNAcylation functions a sensor for nutrient and stress, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we identified that ATG4B is novel target for O-GlcNAcylation under metabolic stress condition. Treatment with PugNAc, an O-GlcNAcase inhibitor increased activation of autophagy in SH-SY5Y cells. Both bimolecular fluorescence complementation and immunoprecipitation assay indicated that OGT directly interacts with ATG4B in SH-SY5Y cells. We also found that the O-GlcNAcylated ATG4B was increased in autophagy activation conditions, and down-regulation of OGT reduces O-GlcNAcylation of ATG4B under low glucose condition. Furthermore, the proteolytic activity of ATG4B for LC3 cleavage was enhanced in PugNAc-treated cells. Taken together, these results imply that O-GlcNAcylation of ATG4B regulates autophagy activation by increasing its proteolytic activity under metabolic stress condition.

Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells

“…This assay has supported significant discoveries that helped to understand the post-translational regulation of ATG4B. These include the identification of the ubiquitin ligase RNF5 as a key regulator of ATG4B stability ( Kuang et al, 2012 ), the O-GlcNAc modification of ATG4B to increase its proteolytic activity ( Jo et al, 2016 ), and the regulation of ATG4B activity by phosphorylation ( Yang et al, 2015 ; Pengo et al, 2017 ). Here, we present a small molecule and siRNA screen to identify regulators of ATG4B activity in cell-based assays.…”

“…There are four members of the ATG4 family in mammalian cells that are partially redundant in substrate processing, but have also distinct specificities. ATG4B, the main isoform of the ATG4 family of proteins is regulated by different types of post-translational modifications, including ubiquitination ( Kuang et al, 2012 ), O-GlcNAcylation ( Jo et al, 2016 ), S -nitrosylation ( Li et al, 2017 ), capase mediated proteolysis ( Betin and Lane, 2009 ; Betin et al, 2012 ), redox mechanisms ( Scherz-Shouval et al, 2007 ; Qiao et al, 2015 ; Heintze et al, 2016 ) and phosphorylation ( Yang et al, 2015 ; Huang et al, 2017 ; Pengo et al, 2017 ; Sanchez-Wandelmer et al, 2017 ; Ni et al, 2018 ). It is not well understood how ATG4B hydrolase activity toward its two substrates pro-LC3 and LC3-II could be differentially regulated, but recently it has been pointed out that post-translational modifications may control the ATG4B proteolytic and de-lipidation activity.…”

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

“…However, overall expression of ATG4 proteins in healthy cells is quite stable and ubiquitous. ATG4B undergoes several types of post-translational modifications under multiple conditions [60], including ubiquitination [59], O-GlcNAcylation [61], S-nitrosylation [62], caspase-mediated proteolysis [42,63], redox mechanisms [64][65][66], and phosphorylation [12][13][14][15]67].…”

Autophagy-regulating protease Atg4: structure, function, regulation and inhibition

“… 42 Atg4B is also modified with O-linked β- N -acetylglucosamine under metabolic stress condition, which was reported to increase Atg4B activity and activates autophagy although modification sites were not determined. 59 Ubiquitination also regulates Atg4 activity. Atg4B is recognized by a membrane-associated E3 ligase RNF5 and ubiquitinated, and finally degraded by the proteasome.…”