Oxidative Stress Induced S-glutathionylation and Proteolytic Degradation of Mitochondrial Thymidine Kinase 2

Abstract Protein glutathionylation in response to oxidative stress can affect both the stability and activity of target proteins. Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in mitochondrial DNA precursor synthesis. Using an antibody specific for glutathione (GSH), S-glutathionylated TK2 was detected after the addition of glutathione disulfide (GSSG) but not GSH. This was reversed by the addition of dithiothreitol, suggesting that S-glutathionylation of TK2 is reversible. Site-directed mutagenesis of the cysteine residues and subsequent analysis of mutant enzymes demonstrated that Cys-189 and Cys-264 were specifically glutathionylated by GSSG. These cysteine residues do not appear to be part of the active site, as demonstrated by kinetic studies of the mutant enzymes. Treatment of isolated rat mitochondria with hydrogen peroxide resulted in S-glutathionylation of added recombinant TK2. Treatment of intact cells with hydrogen peroxide led to reduction of mitochondrial TK2 activity and protein levels, as well as S-glutathionylation of TK2. Furthermore, the addition of S-glutathionylated recombinant TK2 to mitochondria isolated from hydrogen peroxide-treated cells led to degradation of the S-glutathionylated TK2, which was not observed with unmodified TK2. S-Glutathionylation on Cys-189 was responsible for the observed selective degradation of TK2 in mitochondria. These results strongly suggest that oxidative damage-induced S-glutathionylation and degradation of TK2 have significant impact on mitochondrial DNA precursor synthesis.