Abstract

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.

Highlights

  • Thioredoxin reductase 1 (TrxR1) is the only known reductant of thioredoxin

  • The mechanism by which the thioredoxin 1 (Trx1) redox state is maintained in TrxR1 activity-depleted cells is unclear so far

  • To perform the enzyme assay, 20 nM TrxR1, 0.2 mM NADPH, the indicated amounts of ATG (Sigma), the indicated amounts of ebselen (Daiichi Pharmaceutical Co., Tokyo, Japan) and 3 ␮M Trx1 were added into the reaction solution, and reactions were initiated by the addition of 0.16 mM insulin

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Summary

Background

Results: The glutaredoxin system showed the capacity of reducing thioredoxin 1 Depletion of both TrxR1 activity and glutathione in cells induced Trx oxidation and cell death. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx oxidation, reactive oxygen species accumulation, and cell death. The glutaredoxin system and glutathione have a backup role to keep Trx reduced in cells with loss of TrxR1 activity. The mechanism by which the Trx redox state is maintained in TrxR1 activity-depleted cells is unclear so far. Excessive oxidation of Trx was the key factor responsible for cell death, rather than the loss of TrxR1 activity On this basis, we show that ATG and ebselen combined displayed synergistic cytotoxicity on cells through Trx oxidation

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