Cyclophilin a binds to peroxiredoxins and activates its peroxidase activity

Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes ha ve been identified in mammalian tissues. Prxs are members of a group of per oxidases that have conserved reactive cysteine residue(s) in the active sit e(s). An immediate physiological electron donor for the peroxidase catalysi s for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (I-Cys Prx) is still unclear. To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI pro tein overlay assay. A 20-kDa binding protein was identified by the Prx VI p rotein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx Vl-binding protein as a cyclophilin A (CyP-A). The binding of re combinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies. hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes. hCyP-A supported antioxidant activi ty of Prx II and Prx VI both against thiol (dithiothreitol)-containing meta l-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems. P rx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent. These results strongly suggest tha t CyP-A not only binds to Prx proteins but also supports its peroxidase act ivity as an immediate electron donor. In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Pr x.