Abstract
Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson’s disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.
Highlights
Mutations in parkinsonism associated deglycase (PARK7) gene, which encodes for the human DJ-1/PARK7 protein, are associated with autosomal recessive early-onset forms of Parkinson’s disease (PD) [1]. hDJ-1 has been described as a cytoprotective multifunctional enzyme with antioxidant, protease, glyoxalase and deglycase activities [2]
To test for esterase activity of hDJ-1 in vitro, human DJ-1 His-tagged, as well as mutants C106D and M26I were expressed in Escherichia coli BL21 (DE3) and purified in accordance to well-established protocols [8]
The supernatants of crude homogenates from all aforementioned clones were subjected to downstream purification by using a Ni2+-affinity column which enriched hDJ-1 and its mutant forms (C106D and M26I) to high purity as determined by Western blotting by employing a rabbit polyclonal anti-DJ-1 antibody raised against a synthetic peptide corresponding to residues near the N-terminus of DJ-1
Summary
Mutations in parkinsonism associated deglycase (PARK7) gene, which encodes for the human DJ-1 (hDJ-1)/PARK7 protein, are associated with autosomal recessive early-onset forms of Parkinson’s disease (PD) [1]. hDJ-1 has been described as a cytoprotective multifunctional enzyme with antioxidant, protease, glyoxalase and deglycase activities [2]. The oxidative-sensing and protease activities of DJ-1 have been suggested to require the redox-sensing catalytic cysteine residue (C106) and other hydrophilic amino acid residues (H126, E18) [5,7]. The acute exposure of cells transiently expressing heterologous hDJ-1 to oxidative stress enhances the proteolytic cleavage of the zymogen of DJ-1, its proteolytic activity decreases in vitro while the cytoprotective activities of DJ-1 are increased [8], suggesting that prosurvival effects and proteolytic activity of DJ-1 are inversely related. The fact that the spatial arrangement of the amino acids of the catalytic triad does not favor protease activity [7], it is conceivable that hDJ-1 favors catalysis of multiple substrates including esters in a similar manner to papain. As postulated elsewhere [7], it is not known whether the catalytic site of DJ-1 is capable of performing other enzymatic activities besides its known protease, deglycase and glyoxalase activities
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