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Characterization of 4-HNE modified L-FABP reveals alterations in structural and functional dynamics.

Smathers RL, Fritz KS, Galligan JJ, Shearn CT, Reigan P, Marks MJ, Petersen DR - PLoS ONE (2012)

Bottom Line: 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.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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.

ABSTRACT
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.

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Affinity and total binding capacity for fatty acids is diminished following 4-HNE modification.The displacement of ANS by natural ligands of L-FABP is shown in both native and 1X 4-HNE (53.95 µM) treated rL-FABP. Displacement profiles of rL-FABP reveal loss of both the affinity and the total capacity for SA (A), OA (B), and LA (C).
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pone-0038459-g004: Affinity and total binding capacity for fatty acids is diminished following 4-HNE modification.The displacement of ANS by natural ligands of L-FABP is shown in both native and 1X 4-HNE (53.95 µM) treated rL-FABP. Displacement profiles of rL-FABP reveal loss of both the affinity and the total capacity for SA (A), OA (B), and LA (C).

Mentions: The effects of 4-HNE modification on the affinity and capacity of L-FABP towards natural ligands, including FA, were analyzed using a well-documented ANS displacement assay. As demonstrated in Figure 4 and Table 3, 4-HNE reduces the maximal binding to the FA tested by 41% for SA, 32.7% for OA, and 49% for LA, respectively. The inhibition constants of adducted rL-FABP increased from the native protein for SA and LA, but decreased for OA. These data indicate that 4-HNE modification may affect the specificity towards ligands despite showing a decrease in the overall capacity of the system. The Hill constants indicate positive cooperativity for WT and 4-HNE adducted rL-FABP; with negative cooperativity for 4-HNE adducted rL-FABP displaced with OA.


Characterization of 4-HNE modified L-FABP reveals alterations in structural and functional dynamics.

Smathers RL, Fritz KS, Galligan JJ, Shearn CT, Reigan P, Marks MJ, Petersen DR - PLoS ONE (2012)

Affinity and total binding capacity for fatty acids is diminished following 4-HNE modification.The displacement of ANS by natural ligands of L-FABP is shown in both native and 1X 4-HNE (53.95 µM) treated rL-FABP. Displacement profiles of rL-FABP reveal loss of both the affinity and the total capacity for SA (A), OA (B), and LA (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368874&req=5

pone-0038459-g004: Affinity and total binding capacity for fatty acids is diminished following 4-HNE modification.The displacement of ANS by natural ligands of L-FABP is shown in both native and 1X 4-HNE (53.95 µM) treated rL-FABP. Displacement profiles of rL-FABP reveal loss of both the affinity and the total capacity for SA (A), OA (B), and LA (C).
Mentions: The effects of 4-HNE modification on the affinity and capacity of L-FABP towards natural ligands, including FA, were analyzed using a well-documented ANS displacement assay. As demonstrated in Figure 4 and Table 3, 4-HNE reduces the maximal binding to the FA tested by 41% for SA, 32.7% for OA, and 49% for LA, respectively. The inhibition constants of adducted rL-FABP increased from the native protein for SA and LA, but decreased for OA. These data indicate that 4-HNE modification may affect the specificity towards ligands despite showing a decrease in the overall capacity of the system. The Hill constants indicate positive cooperativity for WT and 4-HNE adducted rL-FABP; with negative cooperativity for 4-HNE adducted rL-FABP displaced with OA.

Bottom Line: 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.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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus