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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 binding profile of apo rL-FABP is adversely affected following 4-HNE adduction.Utilizing the fluorescent probe, ANS, equilibrium binding studies of native and 4-HNE modified r-LFABP revealed alterations in both affinity for ANS and maximal binding capacity for ligands when the protein is modified. PG, an arginine blocker, is used as a control for 50% of maximal binding. Blue - 0X 4-HNE; green-0.1X 4-HNE (5.93 µM); yellow-1X 4-HNE (53.95 µM); orange-5X 4-HNE (245.09 µM); red-PG (10 mM); purple-5X 4-HNE + PG.
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pone-0038459-g003: The binding profile of apo rL-FABP is adversely affected following 4-HNE adduction.Utilizing the fluorescent probe, ANS, equilibrium binding studies of native and 4-HNE modified r-LFABP revealed alterations in both affinity for ANS and maximal binding capacity for ligands when the protein is modified. PG, an arginine blocker, is used as a control for 50% of maximal binding. Blue - 0X 4-HNE; green-0.1X 4-HNE (5.93 µM); yellow-1X 4-HNE (53.95 µM); orange-5X 4-HNE (245.09 µM); red-PG (10 mM); purple-5X 4-HNE + PG.

Mentions: To further assess the functional effects of 4-HNE adduction on L-FABP, a well-established binding assay utilizing ANS was performed. As shown in Figure 3, a significant, concentration-dependent reduction in the capacity and affinity for ANS was observed following adduction with pathologically relevant concentrations of 4-HNE. Table 2 contains the regression analysis comparing the native and 4-HNE adducted rL-FABP. The dissociation constants in the primary binding site show increased affinity for ANS at lower concentrations of 4-HNE (0.1X, kd1 = 0.132±0.074 µM and 1X, 0.135±0.074 µM compared to native (0X, 0.347±0.153 µM ) but relatively minor changes at the highest concentration assessed (5X, kd1 = 0.395±0.101 µM ). The secondary binding site, however, displayed an approximate 6-fold decrease in the affinity for ANS following 4-HNE modification at the highest 4-HNE treatment (0x, kd2 = 6.17±1.44 µM compared to 5X, kd2 = 34.20±13.85 µM). The Bmax1 values of 4-HNE modified rL-FABP compared to the native protein demonstrate reduced capacity in the primary site following 4-HNE modification in a concentration dependent manner. The Bmax1 is reduced the most by PG (83, 200±23,200 AFU) compared to native (540,200±142,500 AFU), consistent to previously published reports [31]. We also observed that Bmax2 values remain relatively unchanged following 4-HNE adduction.


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 binding profile of apo rL-FABP is adversely affected following 4-HNE adduction.Utilizing the fluorescent probe, ANS, equilibrium binding studies of native and 4-HNE modified r-LFABP revealed alterations in both affinity for ANS and maximal binding capacity for ligands when the protein is modified. PG, an arginine blocker, is used as a control for 50% of maximal binding. Blue - 0X 4-HNE; green-0.1X 4-HNE (5.93 µM); yellow-1X 4-HNE (53.95 µM); orange-5X 4-HNE (245.09 µM); red-PG (10 mM); purple-5X 4-HNE + PG.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038459-g003: The binding profile of apo rL-FABP is adversely affected following 4-HNE adduction.Utilizing the fluorescent probe, ANS, equilibrium binding studies of native and 4-HNE modified r-LFABP revealed alterations in both affinity for ANS and maximal binding capacity for ligands when the protein is modified. PG, an arginine blocker, is used as a control for 50% of maximal binding. Blue - 0X 4-HNE; green-0.1X 4-HNE (5.93 µM); yellow-1X 4-HNE (53.95 µM); orange-5X 4-HNE (245.09 µM); red-PG (10 mM); purple-5X 4-HNE + PG.
Mentions: To further assess the functional effects of 4-HNE adduction on L-FABP, a well-established binding assay utilizing ANS was performed. As shown in Figure 3, a significant, concentration-dependent reduction in the capacity and affinity for ANS was observed following adduction with pathologically relevant concentrations of 4-HNE. Table 2 contains the regression analysis comparing the native and 4-HNE adducted rL-FABP. The dissociation constants in the primary binding site show increased affinity for ANS at lower concentrations of 4-HNE (0.1X, kd1 = 0.132±0.074 µM and 1X, 0.135±0.074 µM compared to native (0X, 0.347±0.153 µM ) but relatively minor changes at the highest concentration assessed (5X, kd1 = 0.395±0.101 µM ). The secondary binding site, however, displayed an approximate 6-fold decrease in the affinity for ANS following 4-HNE modification at the highest 4-HNE treatment (0x, kd2 = 6.17±1.44 µM compared to 5X, kd2 = 34.20±13.85 µM). The Bmax1 values of 4-HNE modified rL-FABP compared to the native protein demonstrate reduced capacity in the primary site following 4-HNE modification in a concentration dependent manner. The Bmax1 is reduced the most by PG (83, 200±23,200 AFU) compared to native (540,200±142,500 AFU), consistent to previously published reports [31]. We also observed that Bmax2 values remain relatively unchanged following 4-HNE adduction.

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