Crosstalk between the ubiquitination system and the autophagic pathway

TADIC MARIANA; JULIANA HAYDEE ENRIQUÉ STEINBERG; FELIPE RENNA; ORQUERA, TAMARA

Simposio; WIA 2nd Annual Symposium (2021); 2021

Women in Autophagy

VMP1 is an essential autophagy protein whose expression in tumor cells enables therapeutic resistance. Here we investigated the role of VMP1-ubiquitination in the regulation of autophagy. In-silico analysis revealed two high-confidence ubiquitination-sites in VMP1. We found ubiquitinated-VMP1 in eluates of anti-FLAG-ImmunoPrecipitation from VMP1-GFP/Ub-FLAG co-transfected cells. We found ubiquitinated VMP1 in anti-V5-IP of VMP1-V5/Ub-FLAG co-transfection. Immunofluorescence revealed significant co-distribution of VMP1 and ubiquitin in LC3-labeled autophagosomes, confirming VMP1-ubiquitination during autophagy. To know whether ubiquitination marks VMP1 for degradation, proteasomes were inhibited with MG132 and lysosomes with Chloroquine. VMP1 levels were not affected after proteasome inhibition nor after lysosome inactivation. To investigate whether the ubiquitination of VMP1 regulates autophagy, we labelled autophagic structures and ubiquitin in cells expressing VMP1-GFP. We found highly colocalization between VMP1 and ubiquitin in ATG13-labeled omegasomes, in LC3-decorated autophagosomes, and in LAMP1-marked autolysosomes. Some non-ubiquitinated VMP1 dots are not located in autophagic structures, indicating that VMP1 is ubiquitinated when it regulates autophagy.  Next, we performed immunofluorescence of ubiquitin and LC3 in Atg5-/-MEF cells transfected with VMP1-GFP and found that VMP1-ubiquitination occurs upstream to LC3 conjugation. Mass spectrometry showed VMP1 as an interactor of the E3-ligase CRL4/Cdt2. Cdt2-Myc was found in anti-FLAG-IP immunoprecipitation of VMP1-FLAG/Cdt2-Myc co-transfected cells, and endogenous VMP1 in anti-FLAG-IP of Cdt2-FLAG transfected cells. Our results suggest that VMP1-ubiquitination by CRL4/Cdt2 is required for its role in the autophagic pathway. We conclude that ubiquitination is a regulatory mechanism in VMP1-induced autophagy and suggest a clinical relevance in tumor cell therapeutic resistance.