Wo-tailed t-test). doi:10.1371/journal.pone.0065725.gFigure 4. Increase in hydrophobic domain of protein exposure in dbdb and Sod12/2 mice. Total BisANS labeling of sciatic nerve homogenates for (A) dbdb and (B) Sod12/2 mice are presented. Results are expressed as mean 6 SEM (n = 6; *p,0.05 by two-tailed t-test). doi:10.1371/journal.pone.0065725.gProtein Oxidation, Misfolding and DemyelinationFigure 5. Determination of PMP22 protein carbonylation, Oltipraz changes in hydrophobicity, and aggregation in Dbdb mice. (A) A KyteDoolittle Hydropathy plot was determined for PMP22 hydrophobicity. FTC labeled (B) cytosolic and (C) detergent-soluble protein fractions were immunoprecipitated with anti-PMP22 polyclonal antibody, loaded onto SDS-PAGE gels and analyzed for protein carbonylation. (D) The detergent soluble fraction was analyzed by Western blot against PMP22. The higher order PMP22 aggregates are indicated by *. Results are expressed as mean 6 SEM (n = 6; *p,0.05 by two-tailed t-test). doi:10.1371/journal.pone.0065725.gFruquintinib thickness and was in agreement with previous studies in dbdb mice [2] and human type II diabetes [3]. Sod12/2 mice exhibited all of the physiological changes observed in dbdb mice (with the exception of myelin thickness) at 6 months of age and biochemical changes (elevation of protein carbonylation and alteration in 16985061 protein surface hydrophobicity), however, the changes at this age were not as robust as that observed in 5-mo-old dbdb mice. It is interesting to note that Sod12/2 mice also exhibited a reduction in sciatic NCV and a modest increase in tdml, which was also lessrobust than that observed in dbdb mice. There was clearly an agerelated effect in Sod12/2 mice as myelin thickness was significantly reduced by 20 months of age which affirms that oxidative stress is indeed one of the critical determinants for demyelination in peripheral neuropathies. We postulate that the subtle effects on myelin thickness observed in Sod12/2 mice compared to dbdb mice at 6 months of age may be due to a lesser degree of oxidative damage and misfolding in these mice. As both sugar aldehydes (arabinose, glyoxal, methyl glyoxal, glycoaldehyde, and 3-deox-Protein Oxidation, Misfolding and Demyelinationhydropathy plot analysis performed in this study revealed that the primary structure of PMP22 has four distinct regions in the primary sequence that are more hydrophobic than the rest of the sequence which could explain the propensity of PMP22 to aggregate in dbdb mice. It is also important to note that the mutation of leucine19 on PMP22 to proline (first trans-membrane domain) favors b-sheet conformation while the wild-type PMP22 adopts a stable a-helical conformation. This transition from ahelix to b-sheet is likely to favor the aggregation of PMP22 [49]. We predict that carbonylation at this region on PMP22 may shift its conformation from a-helix to b-sheet and promote its aggregation. This can plausibly explain the increase in PMP22 aggregation as a consequence of elevated oxidative stress in both our in vivo and in vitro studies. In conclusion, our data indicates that reduction in NCV associated with altered myelin morphology in dbdb and Sod12/2 mice may be a consequence of elevated global protein carbonylation and exposure of surface hydrophobic domain on proteins. Our future studies will determine if the protection of sciatic nerve/ myelin proteins from oxidative damage will attenuate the loss in NCV and myelin thickness observed in peripheral ne.Wo-tailed t-test). doi:10.1371/journal.pone.0065725.gFigure 4. Increase in hydrophobic domain of protein exposure in dbdb and Sod12/2 mice. Total BisANS labeling of sciatic nerve homogenates for (A) dbdb and (B) Sod12/2 mice are presented. Results are expressed as mean 6 SEM (n = 6; *p,0.05 by two-tailed t-test). doi:10.1371/journal.pone.0065725.gProtein Oxidation, Misfolding and DemyelinationFigure 5. Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in Dbdb mice. (A) A KyteDoolittle Hydropathy plot was determined for PMP22 hydrophobicity. FTC labeled (B) cytosolic and (C) detergent-soluble protein fractions were immunoprecipitated with anti-PMP22 polyclonal antibody, loaded onto SDS-PAGE gels and analyzed for protein carbonylation. (D) The detergent soluble fraction was analyzed by Western blot against PMP22. The higher order PMP22 aggregates are indicated by *. Results are expressed as mean 6 SEM (n = 6; *p,0.05 by two-tailed t-test). doi:10.1371/journal.pone.0065725.gthickness and was in agreement with previous studies in dbdb mice [2] and human type II diabetes [3]. Sod12/2 mice exhibited all of the physiological changes observed in dbdb mice (with the exception of myelin thickness) at 6 months of age and biochemical changes (elevation of protein carbonylation and alteration in 16985061 protein surface hydrophobicity), however, the changes at this age were not as robust as that observed in 5-mo-old dbdb mice. It is interesting to note that Sod12/2 mice also exhibited a reduction in sciatic NCV and a modest increase in tdml, which was also lessrobust than that observed in dbdb mice. There was clearly an agerelated effect in Sod12/2 mice as myelin thickness was significantly reduced by 20 months of age which affirms that oxidative stress is indeed one of the critical determinants for demyelination in peripheral neuropathies. We postulate that the subtle effects on myelin thickness observed in Sod12/2 mice compared to dbdb mice at 6 months of age may be due to a lesser degree of oxidative damage and misfolding in these mice. As both sugar aldehydes (arabinose, glyoxal, methyl glyoxal, glycoaldehyde, and 3-deox-Protein Oxidation, Misfolding and Demyelinationhydropathy plot analysis performed in this study revealed that the primary structure of PMP22 has four distinct regions in the primary sequence that are more hydrophobic than the rest of the sequence which could explain the propensity of PMP22 to aggregate in dbdb mice. It is also important to note that the mutation of leucine19 on PMP22 to proline (first trans-membrane domain) favors b-sheet conformation while the wild-type PMP22 adopts a stable a-helical conformation. This transition from ahelix to b-sheet is likely to favor the aggregation of PMP22 [49]. We predict that carbonylation at this region on PMP22 may shift its conformation from a-helix to b-sheet and promote its aggregation. This can plausibly explain the increase in PMP22 aggregation as a consequence of elevated oxidative stress in both our in vivo and in vitro studies. In conclusion, our data indicates that reduction in NCV associated with altered myelin morphology in dbdb and Sod12/2 mice may be a consequence of elevated global protein carbonylation and exposure of surface hydrophobic domain on proteins. Our future studies will determine if the protection of sciatic nerve/ myelin proteins from oxidative damage will attenuate the loss in NCV and myelin thickness observed in peripheral ne.
