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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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The structural integrity of the binding pocket is significantly affected following 4-HNE adduction.Lipophilic surface maps were utilized to visualize ligand portals and the internal binding cavity of native and 4-HNE adducted L-FABP. Areas colored brown indicate more lipophilic sites of the protein, while the blue indicates more hydrophilic portions. Both apo (A) and holo (B) L-FABP have a well-defined binding area within the protein cavity. Upon 4-HNE adduction, it is clear that both the integrity and size of the binding pocket diminishes in the “open chain” apo (C), holo (D), and the HA structures (E,F).
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pone-0038459-g007: The structural integrity of the binding pocket is significantly affected following 4-HNE adduction.Lipophilic surface maps were utilized to visualize ligand portals and the internal binding cavity of native and 4-HNE adducted L-FABP. Areas colored brown indicate more lipophilic sites of the protein, while the blue indicates more hydrophilic portions. Both apo (A) and holo (B) L-FABP have a well-defined binding area within the protein cavity. Upon 4-HNE adduction, it is clear that both the integrity and size of the binding pocket diminishes in the “open chain” apo (C), holo (D), and the HA structures (E,F).

Mentions: To further examine the effects of peripheral 4-HNE adduction, Figure 7 shows the lipophilic surface maps that were applied to visualize ligand portals and the internal binding cavity of native and 4-HNE adducted L-FABP. Figure 7A shows that in the apo state, a well-defined ligand entrance site at portal 1 and interior binding cavity exist throughout the β-barrel with an internal binding area of 1,116.38 Å3. Figures 7C and E reveal that upon 4-HNE adduction, the binding pocket exhibits diminished solvent accessibility at portal 1 and reduced areas of 634.125 Å3 and 597.125 Å3, respectively. In the holo state, more dramatic effects of aldehyde adduction are evident, where the binding pocket loses almost all structural integrity. Native holo L-FABP retains an internal binding area of 705 Å3 in the presence of two LA ligands as shown by Figure 7B; Figure 7D and F reveal that the 4-HNE modified L-FABP possess areas of 143.375 Å3 and 404 Å3 with reduced ligand access at portal 1 compared to native holo L-FABP.


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)

The structural integrity of the binding pocket is significantly affected following 4-HNE adduction.Lipophilic surface maps were utilized to visualize ligand portals and the internal binding cavity of native and 4-HNE adducted L-FABP. Areas colored brown indicate more lipophilic sites of the protein, while the blue indicates more hydrophilic portions. Both apo (A) and holo (B) L-FABP have a well-defined binding area within the protein cavity. Upon 4-HNE adduction, it is clear that both the integrity and size of the binding pocket diminishes in the “open chain” apo (C), holo (D), and the HA structures (E,F).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038459-g007: The structural integrity of the binding pocket is significantly affected following 4-HNE adduction.Lipophilic surface maps were utilized to visualize ligand portals and the internal binding cavity of native and 4-HNE adducted L-FABP. Areas colored brown indicate more lipophilic sites of the protein, while the blue indicates more hydrophilic portions. Both apo (A) and holo (B) L-FABP have a well-defined binding area within the protein cavity. Upon 4-HNE adduction, it is clear that both the integrity and size of the binding pocket diminishes in the “open chain” apo (C), holo (D), and the HA structures (E,F).
Mentions: To further examine the effects of peripheral 4-HNE adduction, Figure 7 shows the lipophilic surface maps that were applied to visualize ligand portals and the internal binding cavity of native and 4-HNE adducted L-FABP. Figure 7A shows that in the apo state, a well-defined ligand entrance site at portal 1 and interior binding cavity exist throughout the β-barrel with an internal binding area of 1,116.38 Å3. Figures 7C and E reveal that upon 4-HNE adduction, the binding pocket exhibits diminished solvent accessibility at portal 1 and reduced areas of 634.125 Å3 and 597.125 Å3, respectively. In the holo state, more dramatic effects of aldehyde adduction are evident, where the binding pocket loses almost all structural integrity. Native holo L-FABP retains an internal binding area of 705 Å3 in the presence of two LA ligands as shown by Figure 7B; Figure 7D and F reveal that the 4-HNE modified L-FABP possess areas of 143.375 Å3 and 404 Å3 with reduced ligand access at portal 1 compared to native holo L-FABP.

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