Limits...
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.

Show MeSH

Related in: MedlinePlus

Adduction affects protein stability only when in the apo form.Thermal denaturation of apo and holo native and 4-HNE treated protein revealed differences in stability profiles. (A) The stability of apo rL-FABP decreases in a 4-HNE concentration dependent manner. (B) Holo rL-FABP thermal stabillity is only moderately affected by 4-HNE adducts. Blue - 0X 4-HNE (0 µM); green - 0.1X 4-HNE (4.93 µM); yellow - 1X 4-HNE (49.26 µM); orange - 5X 4-HNE (246.31 µM).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368874&req=5

pone-0038459-g005: Adduction affects protein stability only when in the apo form.Thermal denaturation of apo and holo native and 4-HNE treated protein revealed differences in stability profiles. (A) The stability of apo rL-FABP decreases in a 4-HNE concentration dependent manner. (B) Holo rL-FABP thermal stabillity is only moderately affected by 4-HNE adducts. Blue - 0X 4-HNE (0 µM); green - 0.1X 4-HNE (4.93 µM); yellow - 1X 4-HNE (49.26 µM); orange - 5X 4-HNE (246.31 µM).

Mentions: Consequences of 4-HNE modification commonly include protein inactivation and destabilization [40]. To further examine the effects of aldehyde adduction and destabilization of L-FABP, a thermal denaturation assay was employed. Figure 5A shows that apo rL-FABP (Tm = 71.82±0.09°C) is sensitive to degradation following 4-HNE modification in a concentration dependent manner (0.1X, Tm = 70.09±0.004°C; 1X, Tm = 68.56±0.05°C; 5X, Tm = 66.38±0.15°C; p<0.01). Contrary to apo rL-FABP, Figure 5B demonstrates that the thermal stability of holo rL-FABP (Tm = 86.09±0.57°C) is only moderately affected following 4-HNE adduction (0.1X, Tm = 83.84±0.39°C; 1X, Tm = 84.89±0.48°C; 5X, Tm = 83.698±0.53°C). Apo and holo stability may also be related by comparing the slope of the thermal melts, with the apo being far steeper than the holo indicating a faster transition between the folded and unfolded state. Consistent with previous reports, it is evident that lipid binding by FABP results in a significantly more stable product [41]. The Tm from the holo rL-FABP remains semi-quantitative as maximal temperature (98°C) does not result in a fully aggregated state. These data suggest that 4-HNE adduction only minimally affects holo rL-FABP protein stability.


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)

Adduction affects protein stability only when in the apo form.Thermal denaturation of apo and holo native and 4-HNE treated protein revealed differences in stability profiles. (A) The stability of apo rL-FABP decreases in a 4-HNE concentration dependent manner. (B) Holo rL-FABP thermal stabillity is only moderately affected by 4-HNE adducts. Blue - 0X 4-HNE (0 µM); green - 0.1X 4-HNE (4.93 µM); yellow - 1X 4-HNE (49.26 µM); orange - 5X 4-HNE (246.31 µM).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038459-g005: Adduction affects protein stability only when in the apo form.Thermal denaturation of apo and holo native and 4-HNE treated protein revealed differences in stability profiles. (A) The stability of apo rL-FABP decreases in a 4-HNE concentration dependent manner. (B) Holo rL-FABP thermal stabillity is only moderately affected by 4-HNE adducts. Blue - 0X 4-HNE (0 µM); green - 0.1X 4-HNE (4.93 µM); yellow - 1X 4-HNE (49.26 µM); orange - 5X 4-HNE (246.31 µM).
Mentions: Consequences of 4-HNE modification commonly include protein inactivation and destabilization [40]. To further examine the effects of aldehyde adduction and destabilization of L-FABP, a thermal denaturation assay was employed. Figure 5A shows that apo rL-FABP (Tm = 71.82±0.09°C) is sensitive to degradation following 4-HNE modification in a concentration dependent manner (0.1X, Tm = 70.09±0.004°C; 1X, Tm = 68.56±0.05°C; 5X, Tm = 66.38±0.15°C; p<0.01). Contrary to apo rL-FABP, Figure 5B demonstrates that the thermal stability of holo rL-FABP (Tm = 86.09±0.57°C) is only moderately affected following 4-HNE adduction (0.1X, Tm = 83.84±0.39°C; 1X, Tm = 84.89±0.48°C; 5X, Tm = 83.698±0.53°C). Apo and holo stability may also be related by comparing the slope of the thermal melts, with the apo being far steeper than the holo indicating a faster transition between the folded and unfolded state. Consistent with previous reports, it is evident that lipid binding by FABP results in a significantly more stable product [41]. The Tm from the holo rL-FABP remains semi-quantitative as maximal temperature (98°C) does not result in a fully aggregated state. These data suggest that 4-HNE adduction only minimally affects holo rL-FABP protein stability.

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