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N. C. Sears, Y.-C. Chai, G. Hoppe, J. W. Crabb, J. E. Sears; Cofilin, a Key Modulator of Actin Filament Formation, Undergoes Protein S-Glutathionylation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1849.
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Thiol/disulfide exchange between protein and peptide cysteines, a process called protein S-glutathionylation, regulates protein structure and function. In order to identify proteins that are protein S-glutathionylated, we challenged cultured rat retinal pigment epithelium with various oxidants and isolated proteins that were covalently bound by glutathione.
Protein-S-glutathiolation was induced in RPE-J cells by treatment with either peroxide, t-butyl peroxide, menandione, or diamide. Protein lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to nitrocellulose for immunoblotting analysis using a monoclonal antibody to peptide disulfide adducts as a probe. Immunoblots were then used to direct retrieval of protein from Coomassie stained 2D gels for identification by quadropole mass spectrometry. A single spot of 21 kD was excised and digested in situ with trypsin (Promega, Madison WI), and analyzed by liquid chromatography electrospray tandem mass spectrometry using a CapLC system and a quadrupole time-of-flight mass spectrometer (QTOF2, Waters Corp., Milford, MA). Protein identifications from MS/MS data utilized the ProteinLynxTM Global Server (Waters Corp.) and Mascot (Matrix Science, Boston, MA) search engines and the Swiss Protein and NCBI protein sequence data bases.
A 21 kD protein was identified as cofilin using this proteomic strategy. Immunoblot analysis demonstrated that cofilin was protein S-glutathionylated to a different degree depending on the oxidative challenge (diamide > menandione > peroxide > t-butyl peroxide > control). Control samples also showed that cofilin was protein S-glutathionylated to a lesser degree under physiologic, non-oxidizing conditions.
Cofilin is reported to modulate actin filament formation, an action central to critical cellular functions. The observation that cofilin undergoes protein S-glutathionylation, even under normal non-oxidizing conditions, demonstrates that this posttranslational modification is a part of a physiological redox cycle of cofilin and suggests that actin polymerization is regulated by redox related allosteric effectors.
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