Protein interaction studies between enterocyte Fatty Acid Binding Proteins (FABPs) and other proteins present in Caco-2 cells

NATALIA M. BOTTASSO ARIAS; LUCIANA RODRIGUEZ SAWICKI; GISELA RAQUEL FRANCHINI; LISANDRO JORGE FALOMIR LOCKHART; NATALIA SCAGLIA; JUDITH STORCH; BETINA CÓRSICO

Congreso; V Latin American Protein Society Meeting; 2016

Two isoforms ofFatty Acid Binding Proteins (FABPs) are abundantly expressed within intestinalepithelial cells: liver FABP (LFABP) and intestinal FABP (IFABP). Together theyrepresent 1-2% of total proteins present within the enterocytes. Both proteins areassociated with intracellular dietary lipid transport and trafficking towardsdiverse cell fates, although their specific functions are not well understoodand are thought to differ. Using the Caco-2 intestinal cell model, we undertookprotein:protein interaction studies, to determine the protein partners for eachof these FABPs.Firstly, to evaluate LFABP interactions with otherproteins present in Caco-2 cell lysate we applied the Far Western blottechnique followed by MS analysis. We identified two candidate proteins: HSP60and calreticulin. Secondly,IFABP-protein interactions were analyzed by Co-immunoprecipitation (CoIP) .Based on results reported in the literature for other FABPs we tested andconfirmed the interaction between IFABP and a member of the PPAR (PeroxisomeProliferator Activated Receptors) transcription factors family, PPARg. Unexpectedly,we found an apparent decrease in PPARg molecular weight in the CoIP eluatecompared to the band present in the Caco-2 lysate. We are now studying thepossible causes for this observation.Thirdly,we examined PPARg expression levels in an LFABP ablated Caco-2 cell line,generated in our lab applying an antisense technique. In modified cells LFABPexpression represents 47% of control cells. The results showed a concomitantdecrease in PPARg levels in LFABP silenced cells relative to controls. In summary, our work showsfor the first time the interaction of FABPs with different proteins expressedin Caco-2 enterocytes. Currently, we are focusing on recombinant PPARgpurification to analyze the interaction with FABPs in vitro, as well as on studying changes in the expression of PPARgdownstream genes in cultured cells, using inhibitors and activators of FABPsand PPARg