L-FABP directly interacts with PPARα in cultured primary hepatocytes

Abstract

Although studies with liver type fatty acid binding protein (L-FABP) gene ablated mice demonstrate a physiological role for L-FABP in hepatic fatty acid metabolism, little is known about the mechanisms whereby L-FABP elicits these effects. Studies indicate that L-FABP may function to shuttle lipids to the nucleus, thereby increasing the availability of ligands of nuclear receptors, such as peroxisome proliferator-activated receptor-α (PPARα). The data herein suggest that such mechanisms involve direct interaction of L-FABP with PPARα. L-FABP was shown to directly interact with PPARα in vitro through co-immunoprecipitation (co-IP) of pure proteins, altered circular dichroic (CD) spectra, and altered fluorescence spectra. In vitro fluorescence resonance energy transfer (FRET) between Cy3-labeled PPARα and Cy5-labeled L-FABP proteins showed that these proteins bound with high affinity (Kd approximately 156 nM) and in close proximity (intermolecular distance of 52Å). This interaction was further substantiated by co-IP of both proteins from liver homogenates of wild-type mice. Moreover, double immunogold electron microscopy and FRET confocal microscopy of cultured primary hepatocytes showed that L-FABP was in close proximity to PPARα (intermolecular distance 40–49Å) in vivo. Taken together, these studies were consistent with L-FABP regulating PPARα transcriptional activity in hepatocytes through direct interaction with PPARα. Our in vitro and imaging experiments demonstrate high affinity, structural molecular interaction of L-FABP with PPARα and suggest a functional role for L-FABP interaction with PPARα in long chain fatty acid (LCFA) metabolism.

Keywords

• cytoplasmic lipid binding protein
• fluorescence
• FRET
• liver fatty acid binding protein
• nuclei
• peroxisome proliferator activated receptor