FIGURE 2.

Substitutions at position 18 of DJ-1 impact Cys¹⁰⁶- formation. A, oxidation of Cys¹⁰⁶ in vitro to Cys¹⁰⁶-. Mass spectrometry was used to monitor the oxidation of DJ-1 as a function of hydrogen peroxide concentration in solution. The fraction of protein oxidized was calculated as a ratio of the integrated area of the oxidized protein peak to the total area of both the oxidized and reduced peaks. A comparison of the oxidation curves of these proteins shows that every substitution at position 18 results in diminished oxidation compared with the wild-type protein, although the extent of this diminution varies among the three substitutions. E18D abolishes the ability of Cys¹⁰⁶ to be oxidized to cysteine-sulfinic acid, and E18N oxidizes very easily at low H₂O₂ levels. B, oxidation of DJ-1 in vivo. Human M17 neuroblastoma cells were transfected with V5-tagged versions of the indicated DJ-1 constructs (wild type (WT), E18N, E18Q, E18D, and C106A, from top to bottom) and exposed to 300 μm paraquat for 24 h. Protein extracts were separated on two-dimensional gels and blotted for DJ-1. Estimated pI values for each isoform are indicated above the blots. Images are representative of duplicate experiments for each construct.