Mitochondrial thioredoxin-2/peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress
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Cell culture and treatments
HeLa, HEK-293 and COS7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin and streptomycin, and grown in a humidified atmosphere of 5% CO2 at 37°C. Transfections with either pCDNA3.1-v5, pCDNA3.1-v5-Trx2, or pCDNA3.1-V5-C93S-Trx2, were performed following the procedure for Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with t-BH (100 μM) or TNF-α (10 ng/ml) for 24 h for viability experiments. For
Construction and expression of Trx2 and Trx2 mutants
Trx2 contains the conserved redox-active site, which is numbered C90,C93 in translated precursor form prior to import into mitochondria (identical to C33,C36 in processed mitochondrial form and corresponding to C32,C35 in E. coli Trx and human Trx1). Previous research with E. coli Trx and human Trx1 showed that the cysteine corresponding to C90 of Trx2 is involved in the initial step of the catalytic cycle, forming an intermolecular disulfide with target proteins [11], [12]. By analogy, C90
Discussion
Mitochondria contain multiple systems that protect against oxidative damage, including both GSH- and Trx2-dependent systems for elimination of peroxides. However, detailed knowledge of the functions of these systems in mitochondria is complicated by overlapping activities and interactions with components of the larger cytoplasmic compartment. The present data show that C93S-Trx2 forms a relatively abundant disulfide with Prx3. In contrast, no interaction with Prx5 was detected. This latter
Acknowledgment
This research was supported by NIH Grant R01 ES09047.
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Present address: Department of Cell Biology, Capital University of Medical Sciences, Beijing 100069, PR China.