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Elevated protein carbonylation, and misfolding in sciatic nerve from db/db and Sod1(-/-) mice: plausible link between oxidative stress and demyelination.

Hamilton RT, Bhattacharya A, Walsh ME, Shi Y, Wei R, Zhang Y, Rodriguez KA, Buffenstein R, Chaudhuri AR, Van Remmen H - PLoS ONE (2013)

Bottom Line: Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice.Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice.Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, USA.

ABSTRACT
Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1(-/-) ) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.

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Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in Dbdb mice.(A) A Kyte-Doolittle 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 ± SEM (n = 6; *p<0.05 by two-tailed t-test).
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pone-0065725-g005: Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in Dbdb mice.(A) A Kyte-Doolittle 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 ± SEM (n = 6; *p<0.05 by two-tailed t-test).

Mentions: Because we found a global increase in cytosolic and detergent-soluble sciatic nerve protein carbonyls in dbdb mice and increase in global exposure of hydrophobic pockets, we next investigated if peripheral myelin proteins undergo carbonylation and aggregation in diabetic neuropathy. We chose PMP22 to test our hypothesis as it is one of the abundant proteins (2–5%) in peripheral myelin [11]. Moreover, PMP22 has been found as a key player in multiple Charcot Marie Tooth Disease 1a neuropathies and has been reported to be mutated and aggregated in several neuropathies [32], [33], [34], [35]. We first asked whether the primary sequence of PMP22 can predict and identify any domain that have motif(s) of hydrophobicity as hydrophobic domains in general initiate the aggregation process. We utilized the primary sequence of PMP22 obtained from protein search and online software Kyte-Doolittle Hydropathy plots for this theoretical study. We found that certain regions in PMP22 have motifs of hydrophobicity which can predict its region-specific hydrophobicity (Figure 5A). We next determined if PMP22 is preferentially carbonylated in dbdb mice and found that immunoprecipitated cytosolic PMP22 is heavily carbonylated (1.7±0.3 fold increase over dbm, p<0.05, Figure 5B).


Elevated protein carbonylation, and misfolding in sciatic nerve from db/db and Sod1(-/-) mice: plausible link between oxidative stress and demyelination.

Hamilton RT, Bhattacharya A, Walsh ME, Shi Y, Wei R, Zhang Y, Rodriguez KA, Buffenstein R, Chaudhuri AR, Van Remmen H - PLoS ONE (2013)

Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in Dbdb mice.(A) A Kyte-Doolittle 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 ± SEM (n = 6; *p<0.05 by two-tailed t-test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672154&req=5

pone-0065725-g005: Determination of PMP22 protein carbonylation, changes in hydrophobicity, and aggregation in Dbdb mice.(A) A Kyte-Doolittle 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 ± SEM (n = 6; *p<0.05 by two-tailed t-test).
Mentions: Because we found a global increase in cytosolic and detergent-soluble sciatic nerve protein carbonyls in dbdb mice and increase in global exposure of hydrophobic pockets, we next investigated if peripheral myelin proteins undergo carbonylation and aggregation in diabetic neuropathy. We chose PMP22 to test our hypothesis as it is one of the abundant proteins (2–5%) in peripheral myelin [11]. Moreover, PMP22 has been found as a key player in multiple Charcot Marie Tooth Disease 1a neuropathies and has been reported to be mutated and aggregated in several neuropathies [32], [33], [34], [35]. We first asked whether the primary sequence of PMP22 can predict and identify any domain that have motif(s) of hydrophobicity as hydrophobic domains in general initiate the aggregation process. We utilized the primary sequence of PMP22 obtained from protein search and online software Kyte-Doolittle Hydropathy plots for this theoretical study. We found that certain regions in PMP22 have motifs of hydrophobicity which can predict its region-specific hydrophobicity (Figure 5A). We next determined if PMP22 is preferentially carbonylated in dbdb mice and found that immunoprecipitated cytosolic PMP22 is heavily carbonylated (1.7±0.3 fold increase over dbm, p<0.05, Figure 5B).

Bottom Line: Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice.Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice.Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, USA.

ABSTRACT
Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1(-/-) ) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.

Show MeSH
Related in: MedlinePlus