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

BOTTASSO, N; FALOMIR LOCKHART LJ; CÓRSICO B; RODRIGUEZ SAWICKI, L; SCAGLIA, N; FRANCHINI GR; STORCH J

Río de Janeiro

Congreso; V Latin American Protein Society Meeting; 2016

Latin American Protein Society

Two isoforms of Fatty Acid Binding Proteins (FABPs) are abundantly expressed within intestinal epithelial cells: liver FABP (LFABP) and intestinal FABP (IFABP). Together they represent 1-2% of total proteins present within the enterocytes. Both proteins are associated with intracellular dietary lipid transport and trafficking towards diverse cell fates, although their specific functions are not well understood and are thought to differ. Using the Caco-2 intestinal cell model, we undertook protein:protein interaction studies, to determine the protein partners for each of these FABPs.Firstly, to evaluate LFABP interactions with other proteins present in Caco-2 cell lysate we applied the Far Western blot technique followed by MS analysis. We identified two candidate proteins: HSP60 and calreticulin. Secondly, IFABP-protein interactions were analyzed by Co-immunoprecipitation (CoIP) . Based on results reported in the literature for other FABPs we tested and confirmed the interaction between IFABP and a member of the PPAR (Peroxisome Proliferator Activated Receptors) transcription factors family, PPARɣ. Unexpectedly, we found an apparent decrease in PPARɣ molecular weight in the CoIP eluate compared to the band present in the Caco-2 lysate. We are now studying the possible causes for this observation.Thirdly, we examined PPARɣ expression levels in an LFABP ablated Caco-2 cell line, generated in our lab applying an antisense technique. In modified cells LFABP expression represents 47% of control cells. The results showed a concomitant decrease in PPARɣ levels in LFABP silenced cells relative to controls. In summary, our work shows for the first time the interaction of FABPs with different proteins expressed in Caco-2 enterocytes. Currently, we are focusing on recombinant PPARɣ purification to analyze the interaction with FABPs in vitro, as well as on studying changes in the expression of PPARɣ downstream genes in cultured cells, using inhibitors and activators of FABPs and PPARɣ.