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. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46 is the most 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 adjacent resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. Mutants (C46A and C53A), not forming Cys46-Cys53 disulfide crosslinking, increased oxidation of Cys106 to sulfinic and sulfonic acids. Furthermore, we found that DJ-1 C46A mutant has distorted unstable structure identified by biochemical assay and 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 all three Cys residues including Cys46-Cys53 disulfide crosslinking are required for maintaining the structural integrity, the regulation process and cellular function as an antioxidant protein. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

Effects of DJ‑1 on apoptosis and mitophagy of glomerular podocytes

“…The crystal structure of human DJ-1 contains three cysteine residues at amino acids 46, 53 and 106 (Cys46, Cys53 and Cys106), which is its main functional domain. Notably, DJ-1 is a sensitive oxidative stress sensor in vivo and can determine the intracellular redox level according to its different oxidation states [non-oxidation, SO2 - (free radical), SO3 - (free radical)] ( 20 ). DJ-1 has powerful anti-oxidative, immunoregulatory, anti-apoptotic and molecular chaperone functions ( 21 ).…”

Persulfidation of DJ-1 : Mechanism and Consequences

“…DJ-1 has been known to be sulfinylated for years, with both C106[20,21] and C46[24,72] 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.…”

“…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[53,72]. Indeed, the determination of the Tm of various forms of DJ-1 is highly sensitive to the experimental conditions.…”

The Tale of DJ-1 (PARK7): A Swiss Army Knife in Biomedical and Psychological Research

“…The stepwise oxidation of these Cys thiols into S-sulfenic, S-sulfinic, and S-sulfonic acids enables the antioxidant effects of DJ-1. Excessive oxidation of Cys thiol decreases its nucleophilicity, thereby affecting the enzymatic activities of DJ-1 [ 58 ]. As an essential redox sensor, DJ-1 play significant roles in antioxidative cytoprotection under oxidative insult.…”