L-FABP directly interacts with PPARalpha in cultured primary hepatocytes.
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-alpha (PPARalpha). The data herein suggest that such mechanisms involve direct interaction of L-FABP with PPARalpha. L-FABP was shown to directly interact with PPARalpha 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 PPARalpha 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 52A). 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 PPARalpha (intermolecular distance 40-49A) in vivo. Taken together, these studies were consistent with L-FABP regulating PPARalpha transcriptional activity in hepatocytes through direct interaction with PPARalpha. Our in vitro and imaging experiments demonstrate high affinity, structural molecular interaction of L-FABP with PPARalpha and suggest a functional role for L-FABP interaction with PPARalpha in long chain fatty acid (LCFA) metabolism.
Hostetler
Heather A
HA
Department of Physiology and Pharmacology, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843, USA.
McIntosh
Avery L
AL
Atshaves
Barbara P
BP
Storey
Stephen M
SM
Payne
H Ross
HR
Kier
Ann B
AB
Schroeder
Friedhelm
F
D000818:Animals; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D008024:Ligands; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D047493:PPAR alpha / Q000737:chemistry / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1 / Q000378:metabolism
C493320:Fabp1 protein, mouse; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008024:Ligands; D047493:PPAR alpha; D011994:Recombinant Proteins; D051780:Sterol Regulatory Element Binding Protein 1; D019308:Palmitic Acid