Enterocyte Fatty Acid Binding Proteins (FABPs): Protein interaction studies in a Caco-2 cell model

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

Mont Blanc

Congreso; 57th International Conference on the Bioscience of Lipids; 2016

Two isoforms ofFatty Acid Binding Proteins (FABPs) are abundantly expressed within intestinalepithelial cells: liver FABP (LFABP or FABP1) and intestinal FABP (IFABP orFABP2). Together they represent 1-2% of total proteins present within theenterocytes; LFABP is expressed over 40 times the concentration of IFABP. Bothproteins are associated with intracellular dietary lipid transport and traffickingtowards diverse cell fates, although their specific functions are not wellunderstood and are thought to differ. Using the Caco-2 intestinal cell model,we undertook protein:protein interaction studies, to determine the proteinpartners for each of these FABPs.Firstly, screening studies were carried out toevaluate LFABP interaction with other proteins present in Caco-2 cell lysate. Usingthe Far Western blot technique followed by MS analysis, we identified twocandidate proteins: HSP60 and calreticulin. Secondly,Co-immunoprecipitation (CoIP) was applied to study IFABP-protein interactions.Based on results reported in the literature for other FABPs we tested andconfirmed the interaction between IFABP and a member of the family of the PPAR (PeroxisomeProliferator Activated Receptors) transcription factors, PPARg. In this study weunexpectedly found an apparent decrease in PPARɣ molecular weight in the CoIPeluate compared to the band present in the Caco-2 lysate. We are now studyingthe possible causes for this observation.Thirdly,we generated an LFABP ablated Caco-2 cell line applying an antisense technique(Rodriguez Sawicki, under preparation) where we examined PPARg expressionlevels. In modified cells LFABP expression represents 47% of control cells. Theresults showed a concomitant decrease in PPARg levels in LFABP silenced cellsrelative to controls. Insummary, our work shows for the first time the interaction of FABPs with differentproteins expressed in Caco-2 enterocytes. Currently, our efforts are focused onrecombinant PPARg purification to analyze the interaction with IFABP in vitro, as well as on studying changesin the expression of PPARg downstream genes in cultured cells, using inhibitorsand activators of FABPs and PPARg.