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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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New portals for ligand entry/exit are observed following aldehyde adduction.(B) In the apo state, a new portal appears when 4-HNE is bound below portal 2, P4. In the holo state, a different portal appears slightly south of portal 1, P5, in 4-HNE modified L-FABP (D) and in the 4-HNE hemiacetal (F). Asterisks denote the presence of a portal: Yellow  =  Portal 2; Blue  =  Portal 1; Red  =  P4, P5. Linoleic acid bound in site 2 is also shown in holo-L-FABP: native  =  orange, 4-HNE HA  =  green. The lipophilic map is shown on the right: brown (hydrophobic) to blue scale (hydrophillic) with units given in kcal/mol.
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pone-0038459-g008: New portals for ligand entry/exit are observed following aldehyde adduction.(B) In the apo state, a new portal appears when 4-HNE is bound below portal 2, P4. In the holo state, a different portal appears slightly south of portal 1, P5, in 4-HNE modified L-FABP (D) and in the 4-HNE hemiacetal (F). Asterisks denote the presence of a portal: Yellow  =  Portal 2; Blue  =  Portal 1; Red  =  P4, P5. Linoleic acid bound in site 2 is also shown in holo-L-FABP: native  =  orange, 4-HNE HA  =  green. The lipophilic map is shown on the right: brown (hydrophobic) to blue scale (hydrophillic) with units given in kcal/mol.

Mentions: Interestingly, when adducted in the “open chain” form, a new solvent accessible portal (P4, red asterisk) appears below portal 2, between the βG-βH strand interface of apo L-FABP in Figure 8C. In the holo state, an alternative solvent accessible portal was observed at a different position on the protein. When adducted, a new portal also appears below portal 1 between βD-βE as shown in Figure 8D and F (P5, red asterisk). Reference residues around the binding portals were examined at the α carbons to detect changes in the spacial orientation following 4-HNE adduction. These amino acid maps show minimal changes in the 3-dimensional maps surrounding portals 1 and 2 (Figure S3). Therefore, we can attribute these changes in portal solvent accessibility to the changes in the amino acid side chains in the presence of 4-HNE adducts.


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)

New portals for ligand entry/exit are observed following aldehyde adduction.(B) In the apo state, a new portal appears when 4-HNE is bound below portal 2, P4. In the holo state, a different portal appears slightly south of portal 1, P5, in 4-HNE modified L-FABP (D) and in the 4-HNE hemiacetal (F). Asterisks denote the presence of a portal: Yellow  =  Portal 2; Blue  =  Portal 1; Red  =  P4, P5. Linoleic acid bound in site 2 is also shown in holo-L-FABP: native  =  orange, 4-HNE HA  =  green. The lipophilic map is shown on the right: brown (hydrophobic) to blue scale (hydrophillic) with units given in kcal/mol.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038459-g008: New portals for ligand entry/exit are observed following aldehyde adduction.(B) In the apo state, a new portal appears when 4-HNE is bound below portal 2, P4. In the holo state, a different portal appears slightly south of portal 1, P5, in 4-HNE modified L-FABP (D) and in the 4-HNE hemiacetal (F). Asterisks denote the presence of a portal: Yellow  =  Portal 2; Blue  =  Portal 1; Red  =  P4, P5. Linoleic acid bound in site 2 is also shown in holo-L-FABP: native  =  orange, 4-HNE HA  =  green. The lipophilic map is shown on the right: brown (hydrophobic) to blue scale (hydrophillic) with units given in kcal/mol.
Mentions: Interestingly, when adducted in the “open chain” form, a new solvent accessible portal (P4, red asterisk) appears below portal 2, between the βG-βH strand interface of apo L-FABP in Figure 8C. In the holo state, an alternative solvent accessible portal was observed at a different position on the protein. When adducted, a new portal also appears below portal 1 between βD-βE as shown in Figure 8D and F (P5, red asterisk). Reference residues around the binding portals were examined at the α carbons to detect changes in the spacial orientation following 4-HNE adduction. These amino acid maps show minimal changes in the 3-dimensional maps surrounding portals 1 and 2 (Figure S3). Therefore, we can attribute these changes in portal solvent accessibility to the changes in the amino acid side chains in the presence of 4-HNE adducts.

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