Mitochondrial thioredoxin-2/peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress

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Abstract

A dominant-negative, active-site mutant (C93S-Trx2) of mitochondrial thioredoxin-2 (Trx2) was expressed in cells to study the function of the thioredoxin system in protection against mitochondrial oxidative stress. C93S-Trx2 was detected as a disulfide with mitochondrial peroxiredoxin-3 (Prx3) but not peroxiredoxin-5 (Prx5). C93S-Trx2 enhanced sensitivity to cell death induced by tert-butylhydroperoxide or by tumor necrosis factor-α (TNF-α). In cells treated with buthionine sulfoximine (BSO) to deplete glutathione (GSH), endogenous Trx2 was oxidized, C93S-Trx2 potentiated toxicity, and overexpression of Trx2 protected against toxicity. Thus, the results show that Trx2 interacts with Prx3 in vivo and that the Trx2/Prx3 system functions in parallel with the GSH system to protect mitochondria from oxidative stress. The additive protection by Trx2 and GSH shows that Trx2 and GSH systems are both functionally important at low oxidative stress conditions.

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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.

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