DEVELOPMENT OF RECOMBINANT PROTEIN MODELS TO EVALUATE CHEMICAL PROBES FOR CYSTEINE OXIDATION

Oxidative stress is caused by excess reactive oxygen species (ROS) within the cell and risks the onset of diseases in humans. Antioxidant enzymes prevent oxidative stress through biochemically sensitive thiol (-SH) groups in cysteines that form oxidative post-translational modifications (OxiPTMs) to chemically neutralize ROS. Chemical probes detect specific OxiPTMs at cysteine regulatory nodes, but there is a lack of well-validated model proteins available for probe testing. PARK7 (DJ-1), PRDX6, and GPx3 are three antioxidant enzymes containing chemically active cysteines that we aim to develop as model proteins. Here, we confirmed differential reactivities of the two cysteines within human peroxiredoxin 6 (HsPRDX6) by substituting each cysteine with alanine or serine and comparing the OxiPTMs formed in response to oxidation using Western blotting. Future investigation of differential reactivities of cysteines within PARK7 and GPx3 will contribute to the better understanding of cysteine’s biological roles and verify their suitability as model proteins.