Ubiquitination is a novel post-translational modification of VMP1 in autophagy of human tumor cells

FELIPE JAVIER RENNA; JULIANA HAYDEE ENRIQUÉ STEINBERG ; MARIA MANIFAVA; CLAUDIO GOBZALEZ; MARIANA SOFIA TADIC; TAMARA ORQUERA; CAROLINA VECINO ; ALEJANDRO JAVIER ROPOLO; MARIO ROSSI ; NICHOLAS KTISTAKIS ; MARIA I. VACCARO

BioRxiv

BioRxiv

Lugar: Nueva York; Año: 2023

2692-8205

Autophagy is a tightly regulated catabolicprocess involved in the degradation and recycling of proteins and organelles.Ubiquitination plays an important role in the regulation of autophagy. VacuoleMembrane Protein 1 (VMP1) is an essential autophagy protein whose expression inpancreatic cancer stem cells, carrying activated Kirsten rat sarcoma viraloncogene homolog (KRAS), triggers autophagy and enables therapy resistance.Using biochemical and cellular approaches we identified ubiquitination as apost-translational modification of VMP1 from the initial steps in autophagosomebiogenesis, remaining ubiquitinated as part of the autophagosome membranethroughout autophagic flux until autolysosome formation. However, VMP1 is notdegraded by autophagy nor by the ubiquitin-proteasomal system. Massspectrometry and immunoprecipitation showed that Cell division cycle proteincdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complexassociated with cancer Cullin?RING ubiquitin ligase complex 4 (CRL4), is anovel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1ubiquitination decreases under the CRL inhibitor MLN4924 and increases with theCdt2 overexpression. Our results indicate that ubiquitination is a novelpost-translational modification of VMP1 during autophagy in human tumor cells.VMP1 ubiquitination may be of clinical relevance in tumor cell therapyresistance.@font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-469750017 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin-top:0cm;margin-right:0cm;margin-bottom:8.0pt;margin-left:0cm;line-height:107%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-font-kerning:1.0pt;mso-ligatures:standardcontextual;mso-ansi-language:EN-US;mso-fareast-language:EN-US;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:11.0pt;mso-ansi-font-size:11.0pt;mso-bidi-font-size:11.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-font-kerning:1.0pt;mso-ligatures:standardcontextual;mso-ansi-language:EN-US;mso-fareast-language:EN-US;}.MsoPapDefault{mso-style-type:export-only;margin-bottom:8.0pt;line-height:107%;}div.WordSection1{page:WordSection1;}