Abstract
Peroxiredoxin 1 (Prdx1) is an abundant cytoplasmic human thiol peroxidase. It functions to detoxify reactive oxygen species, in particular, hydrogen peroxide (H2O2). The canonical mechanism of Prdx1 activity involves the reaction of the Prdx1 homodimer with H2O2 to form an intermolecular disulphide bond between two active site cysteines, C52 and C173. Prdx1 is then regenerated to the reduced form by the thioredoxin/thioredoxin reductase system. Recent evidence suggests that in eukaryotes including humans, Prdx1 and its homologues participate in redox signalling relays in which upon oxidation, Prdx1 is able to selectively oxidise the cysteine thiols of interaction partners, modulating their activity. One such partner is apoptosis signalling kinase 1 (ASK1), a mitogen activated protein kinase involved in the transduction of cellular stress stimuli such as oxidative stress into the activation of proapoptotic downstream effectors. To investigate this interaction, recombinant human Prdx1 and ASK1 were studied in vitro under different conditions and in the presence or absence of components of the thioredoxin/thioredoxin reductase system, with the aim of isolating and characterising mixed disulfide species containing Prdx1 and ASK1. Additionally, the equilibrium between the decameric and dimeric forms of Prdx1 was investigated biophysically using microcalorimetry and dynamic light scattering.
Prdx1-ASK1 mixed disulfides were generated and characterised by SDS-PAGE, western blotting, and mass spectrometry, but required the addition of the thiol conjugating agent dithionitrobenzoic acid to form. In the context of previously known kinetic data, the results suggest that Prdx1 requires the presence of one or more adaptor proteins to interact with and oxidise ASK1 in vivo, or that this interaction depends on the quaternary topology of Prdx1 to occur.