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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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4-HNE adducts were identified on apo and holo rL-FABP via MALDI-TOF/TOF Mass Spectrometery.Following incubation with reactive aldehyde, rL-FABP was found to be modified in both the apo and holo form. MS/MS spectra from 4-HNE treated apo rL-FABP revealed the presence of two protein adducts, at residues Lys57 and Cys69. The same experiments were conducted with holo rL-FABP and revealed an additional four 4-HNE adducts on Lys31, Lys46, Lys6, and His 43. Identified b/y ions are labeled within the spectrum and shown along the peptide backbone above the spectrum. The parent ion of the MS/MS fragment is shown as an inset in the upper right corner of the spectrum.
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pone-0038459-g002: 4-HNE adducts were identified on apo and holo rL-FABP via MALDI-TOF/TOF Mass Spectrometery.Following incubation with reactive aldehyde, rL-FABP was found to be modified in both the apo and holo form. MS/MS spectra from 4-HNE treated apo rL-FABP revealed the presence of two protein adducts, at residues Lys57 and Cys69. The same experiments were conducted with holo rL-FABP and revealed an additional four 4-HNE adducts on Lys31, Lys46, Lys6, and His 43. Identified b/y ions are labeled within the spectrum and shown along the peptide backbone above the spectrum. The parent ion of the MS/MS fragment is shown as an inset in the upper right corner of the spectrum.

Mentions: L-FABP exhibits a high affinity for a vast range of hydrophobic ligands, including LCFA [38]. Structurally, L-FABP changes conformation depending on the apo or holo state [39]; it was therefore hypothesized that 4-HNE adduction would differ based on the binding state of the protein. Based on the complications of identifying in vivo modifications, adduction of rL-FABP by 4-HNE was performed in vitro to identify specific sites of modification in apo and linoleate-bound protein. As demonstrated in Table 1, MALDI-TOF/TOF analysis of digested rL-FABP revealed two MA 4-HNE adducts on apo rL-FABP, at Lys57 and Cys69 with sequence coverage of 89% and 87%, respectfully. The holo form revealed six total adducts with 90% sequence coverage. These were identified at Lys31, His43, Lys57 and Cys69 MA and Lys6 and Lys46 SB adducts. Figure 2A and 2B display the MS/MS spectrum of parent ions containing Cys69 of apo rL-FABP and Lys46 of holo rL-FABP, where relative y and b ions confirm all 4-HNE adducts on rL-FABP. The remaining MS/MS spectra of the 4-HNE adducted peptides also confirmed modifications on Lys57, His43, Lys6, and Lys31 of rL-FABP (Figures S1 and S2).


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)

4-HNE adducts were identified on apo and holo rL-FABP via MALDI-TOF/TOF Mass Spectrometery.Following incubation with reactive aldehyde, rL-FABP was found to be modified in both the apo and holo form. MS/MS spectra from 4-HNE treated apo rL-FABP revealed the presence of two protein adducts, at residues Lys57 and Cys69. The same experiments were conducted with holo rL-FABP and revealed an additional four 4-HNE adducts on Lys31, Lys46, Lys6, and His 43. Identified b/y ions are labeled within the spectrum and shown along the peptide backbone above the spectrum. The parent ion of the MS/MS fragment is shown as an inset in the upper right corner of the spectrum.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038459-g002: 4-HNE adducts were identified on apo and holo rL-FABP via MALDI-TOF/TOF Mass Spectrometery.Following incubation with reactive aldehyde, rL-FABP was found to be modified in both the apo and holo form. MS/MS spectra from 4-HNE treated apo rL-FABP revealed the presence of two protein adducts, at residues Lys57 and Cys69. The same experiments were conducted with holo rL-FABP and revealed an additional four 4-HNE adducts on Lys31, Lys46, Lys6, and His 43. Identified b/y ions are labeled within the spectrum and shown along the peptide backbone above the spectrum. The parent ion of the MS/MS fragment is shown as an inset in the upper right corner of the spectrum.
Mentions: L-FABP exhibits a high affinity for a vast range of hydrophobic ligands, including LCFA [38]. Structurally, L-FABP changes conformation depending on the apo or holo state [39]; it was therefore hypothesized that 4-HNE adduction would differ based on the binding state of the protein. Based on the complications of identifying in vivo modifications, adduction of rL-FABP by 4-HNE was performed in vitro to identify specific sites of modification in apo and linoleate-bound protein. As demonstrated in Table 1, MALDI-TOF/TOF analysis of digested rL-FABP revealed two MA 4-HNE adducts on apo rL-FABP, at Lys57 and Cys69 with sequence coverage of 89% and 87%, respectfully. The holo form revealed six total adducts with 90% sequence coverage. These were identified at Lys31, His43, Lys57 and Cys69 MA and Lys6 and Lys46 SB adducts. Figure 2A and 2B display the MS/MS spectrum of parent ions containing Cys69 of apo rL-FABP and Lys46 of holo rL-FABP, where relative y and b ions confirm all 4-HNE adducts on rL-FABP. The remaining MS/MS spectra of the 4-HNE adducted peptides also confirmed modifications on Lys57, His43, Lys6, and Lys31 of rL-FABP (Figures S1 and S2).

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