VMP1 ubiquitination by the E3 ligase complexVMP1 ubiquitination is a novel regulatory mechanism of autophagy in human tumor cells

RENNA F; VACCARO MI

Berna

Congreso; Swiss Apoptosis & Autophagy Meeting (SA2M) 2021; 2021

Swiss Apoptosis & Autophagy Society

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteinsand organelles. This process has been linked to the resistance of pancreatic cancer stem cells toanticancer drugs. Ubiquitination plays an important role in the regulation of autophagy. VMP1 is anessential autophagy protein whose expression in pancreatic cancer stem cells, carrying activated KRAS,enables therapeutic resistance.Using biochemical and cellular approaches we investigated the role of VMP1 ubiquitination in theregulation of autophagy in the cell lines HEK 293T, HeLa and PANC1. In silico analysis of VMP1 structurerevealed two high-confi dence ubiquitination sites. Co-immunoprecipitation of VMP1-Ub from cellsconcomitantly transfected with VMP1-EGFP and Ub-Flag expression plasmids, as well as co-distributionof VMP1 and ubiquitin in LC3-labeled autophagosomes, confi rmed VMP1 ubiquitination duringautophagy. In order to know whether ubiquitination marks VMP1 for degradation, we inhibited theproteasome using MG132 and the lysosome with Chloroquine. We found that VMP1 levels were notaffected after proteasome inhibition nor after lysosome inactivation, suggesting that VMP1 is neitherdegraded by the ubiquitin-proteasome system nor is it a cargo of autophagy. To investigate whetherVMP1 ubiquitination regulates its role in autophagy, we performed distribution analyses for VMP1 andmarkers of autophagic structures. We found highly signifi cant colocalization between VMP1 andubiquitin in ATG13-labeled omegasomes and in LC3-labeled autophagosomes, as well as in LAMP1-marked autolysosomes. In Atg5 -/- MEF cells we found VMP1 distributed in dots which colocalizes withubiquitin but not with LC3, suggesting that VMP1 ubiquitination occurs upstream to LC3 conjugation.Moreover, in some Atg5 wt cells we found big membrane vesicles with VMP1 signal that do notcolocalize with ubiquitin nor LC3 indicating VMP1 is ubiquitinated when it is part of the autophagystructures. Finally, by proteomic analysis and co-immunoprecipitation assay we found the subunit Cdt2of the E3 ligase CRL4/Cdt2 as an interactor of VMP1, suggesting VMP1 ubiquitination reaction iscatalyzed by this enzymatic complex.Our results suggest that VMP1 ubiquitination is required for the regulation of the autophagic pathway inpancreatic cancer cells. We conclude that ubiquitination is a regulatory mechanism in VMP1-inducedautophagy and suggest a possible clinical relevance of modulating VMP1 ubiquitination amongtherapeutic strategies in pancreatic cancer.