An immunoprecipitation-based assay to assess cysteine-106-dependent catalytic activity of human-protein DJ-1 in cell line lysates.

DJ-1 is a protein with a wide range of protective cellular functions and implicated in several pathologies, from neurodegenerative Parkinson's disease to cancer. Its physiological functions rely on its ability to form protein complexes and on the highly conserved, redox-sensitive, cysteine residue C106 located in the enzyme's active site. The later plays a key role in the protection against the modification of biomolecules by glycolytic metabolites. However, to date, only an assay based on the highly efficient enzymatic hydrolysis of the reactive intermediate cyclic 3-phosphoglyceric anhydride (cPGA) by DJ-1 can quantify its activity in biological fluids. In this work, we propose a new immunoprecipitation assay using fluorescence to assess DJ-1 catalytic activity in crude cell lysates. This assay efficiently differentiates a wild-type cell line from its DJ-1 knock-out version, and the activity recorded in five human cell line lysates were validated by the good correlation obtained with the activities observed using the cPGA assay. To conclude, this assay is a complementary expansion to the toolbox for studying DJ-1 activity and the associated C106 redox state in cell lysates, as it makes for some of the shortcomings of the previous assay.