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

Abstract

Background: Mitochondrial thymidine kinase 2 phosphorylates thymidine and deoxycytidine and is essential for mitochondrial function. Results: S-Glutathionylation of TK2 reduced activity in vitro and in vivo and affected its stability in H 2 O 2 -treated mitochondria and human cells. Conclusion: Oxidative stress induces mitochondrial TK2 S-glutathionylation and down-regulation. Significance: S-Glutathionylation is a new TK2 regulatory mechanism possibly contributing to mitochondrial diseases and aging.

Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs

“…We previously showed that mitochondrial TK2 was selectively degraded in U2OS cells during oxidative stress, via a mechanism involving oxidative modifications (17). In mitochondria, dGK is a complementing enzyme for TK2 and is responsible for the phosphorylation of deoxyguanosine and deoxyadenosine.…”

“…Mitochondria were prepared by differential centrifugation, and the mitochondrial proteins were extracted as described previously (17 , and the cellular proteins were extracted by freezing and thawing three times, followed by sonication in an ice-water bath and centrifugation at 16,000 ϫ g for 20 min at 4°C. The protein concentration was determined by the Bradford method (Bio-Rad protein assay), using bovine serum albumin (BSA) as a standard.…”

“…Mouse monoclonal antibodies against ␣-and ␤-tubulin, cytochrome c oxidase subunit II (COX II), and cytochrome c oxidase subunit IV (COX IV) were purchased from Abcam. Polyclonal rabbit anti-human TK2 and anti-human dGK antibodies were produced using synthetic peptides of C-terminal sequences (TK2, NRDRILTPENRKHC; dGK, EEVTKQEDLMRVC) as the antigens (GenScript) and were purified from rabbit antisera by affinity chromatography on Sepharose resin (CNBr-activated Sepharose; GE Healthcare) coupled with the peptide antigen (17). A mouse monoclonal anti-human TK1 antibody (19) obtained from AroCell AB (Uppsala, Sweden) and a polyclonal rabbit anti-human dCK antibody (20) were used to detect the TK1 and dCK proteins, respectively.…”

“…Approximately 6 ϫ 10 6 cells were seeded in 175-cm 2 tissue culture flasks and incubated in the presence or absence of 20 M AZT alone or in combination with 20 M uridine in the medium for 3 days. The mitochondria were then isolated, and the levels of TK2 and dGK in the mitochondrial extracts were determined by Western blot analysis using polyclonal rabbit anti-human TK2 and dGK antibodies (17). To assess the effects of AZT in the absence or presence of Urd on cytosolic salvage enzymes (TK1 and dCK), total cell lysates (ϳ100 g protein) were resolved by 12% SDS-PAGE, and the levels of TK1 and dGK were determined by Western blotting using antibodies against human TK1 (19) and dCK (20).…”

“…Some of these analogs are also known to induce oxidative stress via increased reactive oxygen species (ROS) production (13)(14)(15)(16). Recently, we showed that TK2 activity and protein levels were downregulated during oxidative stress (17) and that incubation with ddI led to reduced TK2 and dGK protein levels in U2OS cells (18). In the present study, we explored the effects of AZT on mitochondrial TK2 and dGK levels in cultured U2OS cells.…”