Abstract
Peroxiredoxins are important regulators of cellular peroxide metabolism. As antioxidants, they restrict oxidation of other cell proteins, but as signaling molecules they can act as sensors and promote thiol protein oxidation via a redox relay mechanism. The presence of peroxiredoxins could therefore influence other thiol proteins, even in cells experiencing endogenous redox activity. To investigate this for the two cytoplasmic peroxiredoxins, Prdx1 and Prdx2, we have compared the thiol proteome of wildtype Jurkat cells with cells in which either one was knocked out. Using mass spectrometry and isotope tagging, approximately 10,000 common CysSH-containing peptides were detected for each WT/KO comparison. Knockout of Prdx1 or Prdx2 resulted in a change in redox state of a small selection of Cys residues, with less than 100 giving more than a 2-fold difference. Strikingly, a large proportion of these, including those that showed the greatest change, were common to both KOs. Some Cys residues showed more oxidation in the knockouts, whereas others showed less. The candidate proteins have diverse functions and have not been known to be oxidant sensitive. No differences were seen in redox state of Cys residues of other Prdxs and oxidant sensitive proteins. A change in expression in Prdx2 knockout cells was indicated for seven cytoskeletal or regulatory thiol proteins, three of which were tested and validated by western blotting. Little firm evidence was found for thiol redox changes dependent on either Prdx that could be attributed to oxidation via a relay mechanism.
• Redox proteomics was used to compare levels of reduced Cys residues in wildtype and Prdx1 or Prdx knockout Jurkat cells.
• Less than 1 % of the 10,000 Cys-containing peptides detected showed changes.
• A large proportion of changes were common to both knockouts.
• No strong evidence for knockout of one of the Prdxs affecting the redox state of other Cys proteins.