Selective targeting of the cysteine proteome by thioredoxin and glutathione redox systems

Abstract

Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system.

Fig. 1.

Determination of conditions for ARF-induced oxidation of Trx1 with minimal effect on the GSH redox system. TrxR1 activity was measured by monitoring NADPH oxidation rate at absorbance 340 nm after incubating purified TrxR1 protein with ARF as indicated (A top). To examine ARF dose-dependent oxidation in Trx1 redox state, cells treated with indicated amounts of ARF for 2 h were analyzed for E_hTrx1 (A, bottom) by using the ratio of oxidized:reduced Trx1 (quantification of two bands' intensity by densitometry) from the redox western assay (A, top), the E_o for the active site dithiol/disulfide of Trx1 and the Nernst equation (33, 34). B, To examine ARF dose-dependent effect on E_hGSSG, HT 29 cells treated with ARF as indicated amounts for 2 h were analyzed for GSH and GSSG by HPLC and E_hGSSG was calculated using the Nernst equation (E_o = −264, pH 7.4). Bar graphs are shown as mean ± S.E. (n = 3) with significance indicated at p < 0.05.

Fig. 2.

Determination of conditions for BSO-induced oxidation of GSH with minimal effect on the Trx redox system. A, To examine dose-dependent effect of BSO on GSH redox potential, cells treated with indicated amounts of BSO for 15 h were analyzed for GSH and GSSG, and E_hGSSG was calculated using the Nernst equation. B, To examine dose-dependent effect of BSO on E_hTrx1, cells treated with BSO, analyzed for reduced and oxidized Trx1 by redox Western blotting, and E_hTrx1 was calculated using the Nernst equation. Bar graphs are shown as mean ± S.E. (n = 3) with significance indicated at p < 0.05.

Fig. 3.

Oxidation of Trx1 redox state (E_hTrx1) by ARF and oxidation of GSH redox state (E_hGSSG) by BSO in HT29 cells under conditions of redox proteomics assay. To verify results from the dose-response studies under conditions of redox proteomics assay, HT29 cells were treated with 100 μm BSO or 20 μm ARF and analyzed by redox Western blotting for E_hTrx1 (A) and by HPLC analyses for E_hGSSG (B). In A, subsets of cells were treated with DTT (5 mm) and H₂O₂ (2 mm) as the reduced and oxidized controls, respectively. Quantified intensity of oxidized Trx1 (Trx1^Ox1) and reduced Trx1 (Trx^Red) bands of each treatment was used for E_hTrx1 calculation. Note that a small amount of a hyper-oxidized form of Trx1, termed Trx1^Ox2, was detected in some of the experiments with ARF. This has been previously shown to represent oxidation of a C62-C69 dithiol present in a surface α-helix (33). E_hGSSG was calculated from Nernst equation with GSH and GSSG concentrations obtained from HPLC analysis (B). Aurothioglucose (ATG) was included for comparison. Bar graphs are shown as mean ± S.E. (n = 3) with significance indicated for p < 0.05.

Fig. 4.

Redox ICAT/MS-based redox proteomics defined peptidyl Cys and proteins regulated by Trx, GSH, and Trx/GSH systems. To determine target proteins regulated by Trx and GSH system, HT29 cells treated with ARF (20 μm, 2 h) and BSO (100 μm, 15 h) were analyzed for redox ICAT-based mass spectrometry as described in the Experimental Procedures. Of the 606 total peptidyl Cys detected in samples treated with ARF, 219 were oxidized 1.3-fold or higher compared with CR (A). On the other hand, of the 594 total peptidyl Cys detected in samples treated with BSO, 60 peptidyl Cys were oxidized 1.3-fold or higher than CR (A). Distribution of ARF and BSO-induced oxidation of peptidyl Cys were visualized by pie charts (B). C, Of the 219 ARF-oxidized peptidyl Cys, 96 were oxidized by only ARF and 41 were by ARF and BSO. Of the 60 BSO-oxidized peptidyl Cys, 9 were oxidized by only BSO and 41 were by BSO and ARF.

Fig. 5.

Pathway maps regulated by ARF-oxidized peptidyl Cys. To identify functional pathways affected by disruption in Trx system, 96 peptidyl Cys oxidized by ARF were analyzed by MetaCore Bioinformatics software from Thomson Reuters (https://portal.genego.com/). Of the 39 statistically significant pathways, the top 10 pathways are shown. These include four glycolysis and gluconeogenesis, three cytoskeleton remodeling, oxidative stress by ASK1, CFTR (cystic fibrosis transmembrane conductance regulatory) protein activation, and Parkinson's disease by LRRK2 (Leucine-rich repeat serine/threonine-protein kinase 2).

Fig. 6.

Inhibition of glycolysis and gluconeogenesis pathway by ARF. Of the top 10 most significant pathways identified by MetaCore analysis (Fig. 5), glycolysis and gluconeogenesis pathway maps are shown. Pathway map details are provided in the link, http://pathwaymaps.com/maps/930/. Of the 66 enzymes and molecules involved in these pathways, seven (indicated with circle, ALDOA, ENO3, G3P2, LDHA, MDH2, PGAM1, PKM2) were significantly oxidized by ARF (see Table I also).

Fig. 7.

Inhibition of pyruvate kinase (PK) activity and decrease in lactate level by ARF. To verify ARF-inhibited glycolysis pathway identified by redox proteomics, cellular PK activity and lactate levels were measured. HT29 cells treated with ARF (20 μm, 2h) and BSO (100 μm, 15h) or none (CR) were analyzed for PK activity (mU/ml) and l-lactate level (% of control) after preparing standard curves for each PK and l-lactate using fluorometric (Ex/Em = 536/587 nm) assay kits, (Abcam). Bar graphs are shown as mean ± S.E. (n = 3). p < 0.05.

Fig. 8.

Pathway maps regulated by ARF and BSO-oxidized peptidyl Cys. To identify functional pathway maps affected by disruption in both Trx and GSH system, 41 peptidyl Cys oxidized by both ARF and BSO were analyzed by MetaCore Bioinformatics software from Thomson Reuters (https://portal.genego.com/). Six statistically significant pathways were defined including translation and cell adhesion related pathways (See Table II also).