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L. A. Brennan, W. Lee, M. Markosian, M. Demos, F. Giblin, M. Kantorow; Restoration of Cytochrome C and Crystallin Function by Msra Repair Suggests an Important Role for Methionine Oxidation in Lens Epithelium Function and Cataract. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2540.
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Protein methionine sulfoxide (PMSO) formation is a major feature of the aging lens and cataract. PMSO has been detected in lens crystallins indicating a role for PMSO in lens fibers that could include aggregation and cataract. We have shown that the PMSO repair enzyme MsrA is essential for lens cell mitochondrial function and its deletion causes cataract in mice. Here we explored the effects of PMSO oxidation and MsrA repair on crystallin and cyt c functions in the lens in vitro and in vivo with an emphasis on lens epithelial cell function.
Immunohistochemical staining, western analysis and immunoprecipitation, were used to localize cyt c, MsrA and crystallin in the whole lens and lens cells. crystallin chaperone activity was assessed using both thermal and chemical denaturation assays. Cyt c redox status was assessed using cytochrome c oxidase and peroxidase assays. Sites of methionine oxidation were determined by cyanogen bromide cleavage and mass spec. Effects of methionine oxidation on these proteins were correlated in the lenses of wt and MsrA-knockout mice.
Cyt c is mainly localized to the lens epithelium while crystallin and MsrA are found throughout the lens. All three proteins were found in the mitochondrial of lens epithelial cells. Oxidation of methionines in cyt c resulted in loss of cytochrome c oxidase activity and increased peroxidase activity, while oxidation of methionines in crystallin results in loss of chaperone activity. MsrA is capable of restoring the functions of both oxidized cyt c and crystallin. Analysis of cyt c and crystallin from the lenses of MsrA knockout mice showed aggregation of both the proteins compared to wt-lenses. crystallin showed specific methionine oxidation in B crystallin protein.
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