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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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L-FABP was identified as a 4-HNE modified protein in cytosolic fractions of mice chronically treated with ethanol utilizing 2D SDS-PAGE and LC-MS.(A) 48 proteins in 39 spots were identified from 2D gels by matching up immunoblots probed with an anti-4-HNE antibody created in our lab. Immunoblots from both control and ethanol-fed mice reveal an increase in the total pool of 4-HNE modified proteins. A second set of gels were probed for L-FABP, while a third set was stained with imperial and used for protein excision, tryptic digest, and LC-MS identification. (B) In vivo stability of L-FABP was assessed via measurement of ubiquitin-tagged protein by co-immunoprecipitation with ubiquitin antibody. (C) Relative quantification of poly-ubiquitinated L-FABP was normalized to total L-FABP in the liver (P<0.001, n = 6). (D) FABP1 mRNA was measured by qPCR (P<0.05, n = 3).
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pone-0038459-g001: L-FABP was identified as a 4-HNE modified protein in cytosolic fractions of mice chronically treated with ethanol utilizing 2D SDS-PAGE and LC-MS.(A) 48 proteins in 39 spots were identified from 2D gels by matching up immunoblots probed with an anti-4-HNE antibody created in our lab. Immunoblots from both control and ethanol-fed mice reveal an increase in the total pool of 4-HNE modified proteins. A second set of gels were probed for L-FABP, while a third set was stained with imperial and used for protein excision, tryptic digest, and LC-MS identification. (B) In vivo stability of L-FABP was assessed via measurement of ubiquitin-tagged protein by co-immunoprecipitation with ubiquitin antibody. (C) Relative quantification of poly-ubiquitinated L-FABP was normalized to total L-FABP in the liver (P<0.001, n = 6). (D) FABP1 mRNA was measured by qPCR (P<0.05, n = 3).

Mentions: LPO is a known consequence to sustained ethanol consumption, and identifying protein targets for modification by reactive aldehydes may further elucidate mechanistic information for the progression of specific diseases. Mice fed an ethanol-containing diet for 6 weeks has been shown to result in significant hepatic alterations consistent with early liver injury, including increased plasma ALT activity, increased liver triglyceride accumulation, and hepatomegaly [19], [20]. As shown in Figure 1A, the 2D gel immunostained for 4-HNE protein adducts of the ethanol-fed mice displayed a marked increase in 4-HNE-modified proteins. We have identified 48 unique proteins from 39 4-HNE immunopositive spots from hepatic cytoplasmic fractions of mice fed chronically with ethanol for 6 weeks. Of the 39 spots, 34 received significant protein identifications (Table S1). Interestingly, 4-HNE immunopositive spots are present in the control gels, but to a lesser degree as compared with the ethanol group. The presence of 4-HNE in the control is expected as submicromolar to micromolar concentrations of 4-HNE have been shown to induce homeostatic, cell-specific effects such as stimulation of proliferation and regulating signaling pathways [37]. Of the proteins identified, our focus remained on L-FABP due to its pivotal role in hepatic FA uptake and trafficking. Following tryptic digest, 4 peptides of L-FABP were identified from the 2D gels, with 34% sequence coverage and a significant MASCOT score of 303. To verify spot identification, paired gels were probed with an antibody directed against L-FABP. Compared with the control gel, the ethanol-fed sample showed a reduction in the total expression of L-FABP; correlating with previous data observed in rats [18]. In Figure 1B, we confirm the ∼80% reduction in L-FABP by 1D Western Blot analysis in whole cell lysates of control and ethanol-fed mice (P<0.001, n = 6). In Figure 1B and 1C the stability of L-FABP was assessed in vivo. Co-immunoprecipitation of ubiquitinated L-FABP revealed a significant increase in poly-ubiquitinated L-FABP in response to ethanol treatment when normalized to the total L-FABP protein pool (P<0.001, n = 6). Figure 1D shows RT-PCR of FABP1 mRNA, which revealed a 1.6 fold decrease in message following ethanol treatment. Relative expression of FABP1 in control (1.00±0.084) and ethanol (0.617±0.069) livers (P<0.05, n = 3).


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)

L-FABP was identified as a 4-HNE modified protein in cytosolic fractions of mice chronically treated with ethanol utilizing 2D SDS-PAGE and LC-MS.(A) 48 proteins in 39 spots were identified from 2D gels by matching up immunoblots probed with an anti-4-HNE antibody created in our lab. Immunoblots from both control and ethanol-fed mice reveal an increase in the total pool of 4-HNE modified proteins. A second set of gels were probed for L-FABP, while a third set was stained with imperial and used for protein excision, tryptic digest, and LC-MS identification. (B) In vivo stability of L-FABP was assessed via measurement of ubiquitin-tagged protein by co-immunoprecipitation with ubiquitin antibody. (C) Relative quantification of poly-ubiquitinated L-FABP was normalized to total L-FABP in the liver (P<0.001, n = 6). (D) FABP1 mRNA was measured by qPCR (P<0.05, n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368874&req=5

pone-0038459-g001: L-FABP was identified as a 4-HNE modified protein in cytosolic fractions of mice chronically treated with ethanol utilizing 2D SDS-PAGE and LC-MS.(A) 48 proteins in 39 spots were identified from 2D gels by matching up immunoblots probed with an anti-4-HNE antibody created in our lab. Immunoblots from both control and ethanol-fed mice reveal an increase in the total pool of 4-HNE modified proteins. A second set of gels were probed for L-FABP, while a third set was stained with imperial and used for protein excision, tryptic digest, and LC-MS identification. (B) In vivo stability of L-FABP was assessed via measurement of ubiquitin-tagged protein by co-immunoprecipitation with ubiquitin antibody. (C) Relative quantification of poly-ubiquitinated L-FABP was normalized to total L-FABP in the liver (P<0.001, n = 6). (D) FABP1 mRNA was measured by qPCR (P<0.05, n = 3).
Mentions: LPO is a known consequence to sustained ethanol consumption, and identifying protein targets for modification by reactive aldehydes may further elucidate mechanistic information for the progression of specific diseases. Mice fed an ethanol-containing diet for 6 weeks has been shown to result in significant hepatic alterations consistent with early liver injury, including increased plasma ALT activity, increased liver triglyceride accumulation, and hepatomegaly [19], [20]. As shown in Figure 1A, the 2D gel immunostained for 4-HNE protein adducts of the ethanol-fed mice displayed a marked increase in 4-HNE-modified proteins. We have identified 48 unique proteins from 39 4-HNE immunopositive spots from hepatic cytoplasmic fractions of mice fed chronically with ethanol for 6 weeks. Of the 39 spots, 34 received significant protein identifications (Table S1). Interestingly, 4-HNE immunopositive spots are present in the control gels, but to a lesser degree as compared with the ethanol group. The presence of 4-HNE in the control is expected as submicromolar to micromolar concentrations of 4-HNE have been shown to induce homeostatic, cell-specific effects such as stimulation of proliferation and regulating signaling pathways [37]. Of the proteins identified, our focus remained on L-FABP due to its pivotal role in hepatic FA uptake and trafficking. Following tryptic digest, 4 peptides of L-FABP were identified from the 2D gels, with 34% sequence coverage and a significant MASCOT score of 303. To verify spot identification, paired gels were probed with an antibody directed against L-FABP. Compared with the control gel, the ethanol-fed sample showed a reduction in the total expression of L-FABP; correlating with previous data observed in rats [18]. In Figure 1B, we confirm the ∼80% reduction in L-FABP by 1D Western Blot analysis in whole cell lysates of control and ethanol-fed mice (P<0.001, n = 6). In Figure 1B and 1C the stability of L-FABP was assessed in vivo. Co-immunoprecipitation of ubiquitinated L-FABP revealed a significant increase in poly-ubiquitinated L-FABP in response to ethanol treatment when normalized to the total L-FABP protein pool (P<0.001, n = 6). Figure 1D shows RT-PCR of FABP1 mRNA, which revealed a 1.6 fold decrease in message following ethanol treatment. Relative expression of FABP1 in control (1.00±0.084) and ethanol (0.617±0.069) livers (P<0.05, n = 3).

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