Characterization of 4-HNE modified L-FABP reveals alterations in structural and functional dynamics.
4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd(1) = 0.395 µM and Kd(2) = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD.
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
Rebecca L
RL
Smathers
Kristofer S
KS
Fritz
James J
JJ
Galligan
Colin T
CT
Shearn
Philip
P
Reigan
Michael J
MJ
Marks
Dennis R
DR
Petersen
D000447:Aldehydes / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D000431:Ethanol / Q000009:adverse effects; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D047468:Immunoprecipitation; D008108:Liver Diseases, Alcoholic / Q000378:metabolism / Q000503:physiopathology*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054875:Ubiquitination
D000447:Aldehydes; D000817:Anilino Naphthalenesulfonates; D017931:DNA Primers; C493320:Fabp1 protein, mouse; D050556:Fatty Acid-Binding Proteins; D000431:Ethanol; C027132:1-anilino-8-naphthalenesulfonate; C027576:4-hydroxy-2-nonenal