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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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Molecular modeling simulations reveal minimal conformational changes in both apo and holo L-FABP as a result of peripheral 4-HNE adducts.Native apo (A) and holo (B) L-FABP is shown as the grey ribbon structure, “open chain” 4-HNE adducts and HA 4-HNE structures are shown as overlays over native protein, in blue (C and D) and purple (E and F), respectively. All structures are shown as a “front view” and 90° turned “side view”. Reference residues surrounding the three ligand portals are shown in black of the apo native structure and have been highlighted previously [62]. Root-mean-square deviation (RMSD) values were obtained comparing 4-HNE modified protein to the native. Residues found to be modified with 4-HNE are labeled in red, and are referenced in all protein structures. Docked LA is colored according to the 4-HNE adduction state: native  =  orange, 4-HNE  =  yellow, 4-HNE HA  =  green.
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pone-0038459-g006: Molecular modeling simulations reveal minimal conformational changes in both apo and holo L-FABP as a result of peripheral 4-HNE adducts.Native apo (A) and holo (B) L-FABP is shown as the grey ribbon structure, “open chain” 4-HNE adducts and HA 4-HNE structures are shown as overlays over native protein, in blue (C and D) and purple (E and F), respectively. All structures are shown as a “front view” and 90° turned “side view”. Reference residues surrounding the three ligand portals are shown in black of the apo native structure and have been highlighted previously [62]. Root-mean-square deviation (RMSD) values were obtained comparing 4-HNE modified protein to the native. Residues found to be modified with 4-HNE are labeled in red, and are referenced in all protein structures. Docked LA is colored according to the 4-HNE adduction state: native  =  orange, 4-HNE  =  yellow, 4-HNE HA  =  green.

Mentions: We have identified peripheral 4-HNE adducts on both the apo and holo rL-FABP utilizing MALDI-TOF/TOF studies. The structural consequence of these modifications was further investigated in the present study utilizing in silico simulations. Figure 6 demonstrates that the minimizations of these models resulted in minor structural differences between native, 4-HNE adducted, and the cyclized 4-HNE HA L-FABP in apo and holo states, respectively. Table 4 reveals the degree of structural shift between the alpha carbons (Cα) of portal 1 (Lys28, Lys31, Pro56), portal 2 (Ile22, Glu77, Lys96), and portal 3 (Ile48, Asp88, Gly105) in the 4-HNE adducted structures and the native protein. These indicate subtle structural changes occurring throughout the entire protein as a consequence of peripheral 4-HNE adduction, with the greatest shifts being under 3 Å.


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)

Molecular modeling simulations reveal minimal conformational changes in both apo and holo L-FABP as a result of peripheral 4-HNE adducts.Native apo (A) and holo (B) L-FABP is shown as the grey ribbon structure, “open chain” 4-HNE adducts and HA 4-HNE structures are shown as overlays over native protein, in blue (C and D) and purple (E and F), respectively. All structures are shown as a “front view” and 90° turned “side view”. Reference residues surrounding the three ligand portals are shown in black of the apo native structure and have been highlighted previously [62]. Root-mean-square deviation (RMSD) values were obtained comparing 4-HNE modified protein to the native. Residues found to be modified with 4-HNE are labeled in red, and are referenced in all protein structures. Docked LA is colored according to the 4-HNE adduction state: native  =  orange, 4-HNE  =  yellow, 4-HNE HA  =  green.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038459-g006: Molecular modeling simulations reveal minimal conformational changes in both apo and holo L-FABP as a result of peripheral 4-HNE adducts.Native apo (A) and holo (B) L-FABP is shown as the grey ribbon structure, “open chain” 4-HNE adducts and HA 4-HNE structures are shown as overlays over native protein, in blue (C and D) and purple (E and F), respectively. All structures are shown as a “front view” and 90° turned “side view”. Reference residues surrounding the three ligand portals are shown in black of the apo native structure and have been highlighted previously [62]. Root-mean-square deviation (RMSD) values were obtained comparing 4-HNE modified protein to the native. Residues found to be modified with 4-HNE are labeled in red, and are referenced in all protein structures. Docked LA is colored according to the 4-HNE adduction state: native  =  orange, 4-HNE  =  yellow, 4-HNE HA  =  green.
Mentions: We have identified peripheral 4-HNE adducts on both the apo and holo rL-FABP utilizing MALDI-TOF/TOF studies. The structural consequence of these modifications was further investigated in the present study utilizing in silico simulations. Figure 6 demonstrates that the minimizations of these models resulted in minor structural differences between native, 4-HNE adducted, and the cyclized 4-HNE HA L-FABP in apo and holo states, respectively. Table 4 reveals the degree of structural shift between the alpha carbons (Cα) of portal 1 (Lys28, Lys31, Pro56), portal 2 (Ile22, Glu77, Lys96), and portal 3 (Ile48, Asp88, Gly105) in the 4-HNE adducted structures and the native protein. These indicate subtle structural changes occurring throughout the entire protein as a consequence of peripheral 4-HNE adduction, with the greatest shifts being under 3 Å.

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