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.

Persulfidation of DJ-1 : Mechanism and Consequences

“…The persulfidation of DJ-1 slightly stabilized the protein thermally compared to the wt form, as indicated by the 5°C variation of their respective Tm. Unfortunately, we were unable to obtain a clean melting curve with the sulfinylated form of DJ-1 and to directly compare the inherent stabilization afforded by each PTM, most probably because we had to work in the absence of DTT which reduces intra-disulfide bridges formed upon oxidation of DJ-1 [56,74]. Indeed, the determination of the Tm of various forms of DJ-1 is highly sensitive to the experimental conditions.…”

“…DJ-1 has been known to be sulfinylated for years, with both C106 [22,23] and C46 [26,74] being target cysteines. The sulfinylation of C106 acts as a redox switch for DJ-1 activity, while the role of the modification of C46 is still obscure but likely of physiological significance since C46-SO 2 H has been described to be a substrate for sulfiredoxin Srx.…”