The Pancreatitis-Induced Membrane Protein VMP1 that Triggers Autophagy Interacts with S100A10.

PARDO, ROMINA; LO RÉ, ANDREA; GRASSO, DANIEL; ROPOLO, ALEJANDRO; BOGGIO, VERONICA; IOVANNA, JUAN L.; VACCARO, MARIA I.; LO RE A; GRASSO D; ROPOLO A; BOGGIO V; IOVANNA JL; VACCARO MI

SanDiego, USA

Congreso; Digestive Disease Week 2008; 2008

American Gastroenterological Association Institute (AGA)

TITLE: The Pancreatitis-Induced Membrane Protein VMP1 that Triggers Autophagy Interacts with S100A10.AUTHORS (LAST NAME, FIRST NAME): Pardo, Romina; Lo Ré, Andrea; Grasso, Daniel; Ropolo, Alejandro; Boggio, Veronica; Iovanna, Juan L.; Vaccaro, Maria I.INSTITUTIONS: School of Medicine, University of Buenos Aires, Buenos Aires, Argentina. ABSTRACT BODY: The VMP1 gene was characterized by its early and strong expression during the acute phase of pancreatitis. VMP1 is necessary in autophagy and its expression induces the formation of autophagosomes. In order to analyze the function of VMP1 at a molecular level, the purpose of this work was to search for interacting proteins by the Two-Hybrid strategy. This in vivo system is based on putting in contact the protein of interest with each of the proteins that are expressed by a cell line. We amplified by PCR 4 fragments from the VMP1 hydrophilic domains and then subcloned them in the pSOS vector. Three independent Two-Hybrid experiments were performed with each of the VMP1 fragments using a HeLa cells library. The checked positive clones were amplified by PCR, purified and finally sequenced. Of the 94 positive clones that were obtained, 11 of them corresponded to interacting proteins: S100A10, ElF, EEF1G, FADD, HSPA5, AlphaL-1 Fucosidase, Ribosomal protein S10, Kinesin 2, TARBP2, LARP1 y USP9X. A bibliographic search was done for each of the interactors that were found and those related with the role of VMP1 were selected. We started studying the S100A10 protein because it’s over expressed in pancreatic tumors and it’s related with vesicular transport. By performing pull-down essays we could confirmed its interaction with VMP1. Besides this, it was analyzed by confocal microscopy the effect of the interaction between VMP1 and S100A10 on the formation of the autophagosome; the recruitment of the fusion fluorescent protein pRFP-LC3 was used as marker. We found that over expression of S100A10 reduced significantly the recruitment of LC3 induced by VMP1 over expression. In conclusion we were able to find out a group of genes that potentially interact with VMP1 in vivo and we demonstrated that the interaction VMP1-S100A10 reduces the formation of autophagosomes.