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. 2012 Nov 2;287(45):38210-9.
doi: 10.1074/jbc.M112.392225. Epub 2012 Sep 13.

Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose

Yatao Du  1 , Huihui Zhang, Jun Lu, Arne Holmgren
Affiliations

Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose

Yatao Du et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Effects of ATG and AF on cell viability and TrxR activity in HeLa cells. A, HeLa cells were treated with different concentrations of ATG or AF for 24, 48, and 72 h. Cell viability was determined with the MTT assay. Error bars show mean ± S.D. B and C, HeLa cells were treated with different concentrations of ATG (B) or AF (C) for 24 h. After treatment, TrxR activities were determined with the fluorescent insulin assay. Error bars show mean ± S.D. n = 2; *, p < 0.05, Student's t test, ATG or AF treated cells versus control.
FIGURE 2.
FIGURE 2.
Redox state of Trx1/2 in HeLa cells exposed to ATG and AF. A, principle of redox Western blot analysis. To prepare mobility standards, cell lysates are denatured with urea and fully reduced with DTT. Then, varying molar ratios of IAA to IAM are incubated with the reduced thioredoxin containing five cysteines, producing six protein isoforms with introduced number of acidic carboxymethyl thiol adducts (-SA−) and neutral amidomethyl thiol adducts (-SM). During urea-PAGE, the ionized -SA− group resulted in faster protein migration toward the anode. Therefore, the six isoforms are separated and used as a mobility standard for representing the number of -SA−. To determine the redox state of thioredoxin in compound-treated cells, HeLa cells were lysed in urea lysis buffer containing IAA. After the free thiols of thioredoxin were alkylated by IAA, cell lysates were precipitated by ice-cold acetone-HCl. The precipitate was washed with ice-cold acetone-HCl two more times to remove excess IAA. Then the precipitate was resuspended in urea lysis buffer containing DTT to reduce thioredoxin containing an inter- or intramolecular disulfide bridge. The free thiols of thioredoxin were then alkylated by IAM. The alkylated thioredoxins in cell lysates were separated according to the charge amount. B, HeLa cells were treated by indicated concentrations of ATG (lanes 3-5) or AF (lanes 6-8) for 24 h, and then the redox states of Trx1 and Trx2 in HeLa cells were detected with redox state Western blot analysis under steady-state conditions. Lane 1, mobility standards. Lane 2, HeLa cells without treatment.
FIGURE 3.
FIGURE 3.
Viabilities of human cancer cells exposed to ATG and ebselen. HeLa cells, SH-SY5Y cells, HCT116 cells, U2020 cells, Du145 cells, and fibroblast cells were treated with the indicated concentrations of ATG for 6 h, and then ebselen was added into the medium. After 24 h treatment, the cell viabilities were measured with an MTT assay. Error bars show mean ± S.D. n ≥ 3; *, p < 0.05; **, p < 0.01; Student's t test, treated cells versus control.
FIGURE 4.
FIGURE 4.
ATG and ebselen synergically affected the Trx system activity in vitro. Trx system activity was determined using an insulin assay in the presence or absence of ATG and/or ebselen. A, wild-type Trx1. B, C62S/C73S Trx1. C, C69S Trx1. To perform the enzyme assay, 20 nm TrxR1, 0.2 mm NADPH, the indicated amounts of ATG, the indicated amounts of ebselen, and 3 μm Trx1 were added into the reaction solution, and then reactions were initiated by the addition of 0.16 mm insulin. D, 4.5 μm pre-reduced TrxR1 was incubated with 10 μm ATG and/or 50 μm ebselen for 20 min. Then, 48 μm Trx1 was incubated with the mixture for 30 min. The reaction mixtures were then subjected to SDS-PAGE gel in the absence (lanes 1-4) or presence (lanes 5-8) of DTT. M, protein marker. E, the reaction mixtures were subjected to Western blot analysis using Trx1 antibody (lanes 1 and 2) and TrxR1 antibody (lanes 3 and 4).
FIGURE 5.
FIGURE 5.
The Redox state of Trx1/2 in HeLa cells exposed to ATG and ebselen. A, HeLa cells were treated with the indicated concentration of ebselen (lanes 2-4) for 24 h, and then the redox states of Trx1 and Trx2 in HeLa cells were detected with a redox state Western blot analysis. Lane 1, mobility standards. B, HeLa cells were treated by the indicated concentrations of ATG for 6 h, and then ebselen was added into the medium (lanes 2-9). After 24 h, the redox state of Trx1 and Trx2 in HeLa cells were detected with a redox state Western blot analysis. Lane 1, mobility standards. C, HeLa cells were treated with 20 μm ATG for 6 h (lane 3-5), and then ebselen was added into the medium (lane 3-5). At the indicated time point, the redox states of Trx1 and Trx2 in HeLa cells were detected with a redox state Western blot analysis. Lane 1, mobility standards. Lane 2, HeLa cells without treatment.
FIGURE 6.
FIGURE 6.
Measurement of ROS generation and TrxR activity in HeLa cells exposed to ATG and ebselen. A, HeLa cells were treated with the indicated concentrations of ATG or ebselen or ATG plus ebselen for 30 min. After washing with PBS and treatment with 10 μm carboxy-H2DCFDA for 30 min, cells were washed again to remove excess H2DCFDA. Fluorescence was analyzed as described under “Experimental Procedures.” Error bars show mean ± S.D. n ≥ 3. **, p < 0.01, Student's t test, treated cells versus control. B, HeLa cells were treated with different concentrations of ATG or ebselen or ATG plus ebselen for 24 h. After treatment, TrxR activities were determined with a fluorescent insulin assay. Error bars show mean ± S.D. n = 2. **, p < 0.01, Student's t test, treated cells versus control.
FIGURE 7.
FIGURE 7.
The glutathione system was a backup of TrxR1for reduction of Trx1. A, Trx1-S2 was prepared as described under “Experimental Procedures” and confirmed by redox urea-PAGE. Lane 1, mobility standard. Lane 2, Trx1-S2. B, 60 nm glutathione reductase, 0.2 mm NADPH, and 50 μm Trx1-S2 were added to cuvettes for the GSH reduction assay in the presence of 1, 3, 6, and 10 mm GSH (four solid lines from bottom to top). 0.2 mm NADPH and 50 μm Trx1-S2 were added to cuvettes for the TrxR1 reduction assay in the presence of 5 and 10 nm TrxR1 (two dotted lines from bottom to top). The absorbance at 340 nm was followed against an identical blank without Trx1-S2. A second-order polynomial regression line was fit in the inset. C, 60 nm glutathione reductase, 0.2 mm NADPH, 1 mm GSH, indicated amounts of Grx1 and 50 μm Trx1-S2 was added to cuvettes. The absorbance at 340 nm was followed against an identical blank without Trx1-S2. D, HeLa cells were treated with the indicated concentrations of ATG and/or BSO. After 48-h treatment, the redox state of Trx1 was detected using a redox Western blot analysis. The cell viability was measured with an MTT assay. Error bars show mean ± S.D. n = 4. **, p < 0.01, Student's t test, treated cells versus control.
SCHEME 1.
SCHEME 1.
Thioredoxin oxidation caused by ATG and ebselen induced cell death.
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