Stepwise Oxidations Play Key Roles in the Structural and Functional Regulations of DJ-1

Abstract

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. We found that Cys46 is also reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe that selectively reacts with redox sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. We also found that Cys46-Cys53 disulfide crosslinking affects the oxidative state of the third Cys106, which shows the crosstalk among three cysteine residues of DJ-1. Furthermore, we demonstrated that DJ-1 C46A mutant, not forming Cys46-Cys53 intra-disulfide bond, lost structural stability of DJ-1 employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike wild type DJ-1. These findings suggest that DJ-1 regulates its structure and activities by concerted oxidative modifications of three cysteine residues. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.