A Display Thiol-Proteomics Approach to Characterize Global Redox Modification of Proteins by Selenium: Implications for the Anticancer Action of Selenium

摘要

Background: The generation of a monomethylated selenium metabolite is critical for the anticancer activity of selenium. Because of its strong nucleophilicity, the metabolite can react directly with protein thiols to cause redox modification. These chemical changes have never been examined systematically before because of the lack of a reliable methodology to study reactive protein thiols globally in cells and to quantify their redox status. Materials and Methods: PC-3 human prostate cancer cells were treated with methylseleninic acid (MSA) for 0.5, 1, 2, 3, 6, 12 or 24 h. A reactive thiol specific reagent, BIAM, was used to detect the extent of global redox changes on a 2D gel electrophoresis display. The data were analyzed by the Self Organizing Maps clustering algorithm. Protein identification was done by MALDI-TOF and ESI-tandem mass spectrometry. Results: Out of a total jof 194 reactive thiol-containingprotein spots on the 2D gel display, 100 of them (cluster 1) were not sensitive to MSA modulation. The remaining 94 were categorized into three distinct patterns. Cluster 2 (60 proteins) showed an immediate and sustained loss of reactive thiols for at least 24 h; cluster 3 (19 proteins) showed a transient loss of reactive thiols followed by a rapid rebound; and cluster 4 (15 proteins) showed a transient gain followed by a rapid return to normal. In contrast, there were minimal protein redox changes in control cells (not treated with MSA) over the same period of time. A total of 85 proteins were identified of which 40 were in clusters 2 to 4. The proteins which are sensitive to redox modification by MSA are distributed in various subcellular compartments. Western blot analysis showed that a number of chaperones were significantly induced by MSA. Conclusion: Global redox modification of proteins can be a major driving force of cellular stress, since these changes are likely to lead to protein unfolding, misfolding or aggregation. The induction of chaperones in cells treated with MSA is consistent with this interpretation since chaperones are charged with rescuing misfolded proteins. The above scenario is discussed in relation to an adaptive response which ultimately determines how cells respond to treatment with selenium.