Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione

Abstract

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The K(m) and k(cat) values for mitochondrial and cytosolic thioredoxins (9.5 microm and 131 s(-1), 34 microm and 197 s(-1), respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 microm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths.

FIGURE 1.

Subcellular localization of GFP-fused mitochondrial TGR and mtTrx. NIH3T3 cells were transiently transfected either with the non-recombinant pEGFP vector (A) or with the pEGFP-derived constructs carrying mitochondrial TGR (B) and mtTrx (C) N-terminally fused to GFP. Images were obtained at 8 h post-transfection using an I-confocal microscope. A set of three panels is shown for each construct. Left panels show green fluorescence corresponding to transiently expressed GFP fusion proteins. Center panels show the red fluorescence of the mitochondrial dye (MitoTracker). The right panels show merged images from left and center panels.

FIGURE 2.

Analysis of recombinant proteins. A, SDS-PAGE analysis of purified recombinant proteins. After purification on a nickel-nitrilotriacetic acid column and desalting, recombinant proteins were run on a 12.5% polyacrylamide gel. 1 μg of each recombinant protein was loaded on the gel. Lanes 1–5, TGR_GCUG, TR_GCUG, TGR_GUCG, TGR_GCCG, TGR_GC*, respectively; lane 6, molecular weight markers; lane 7, mtTrx; and lane 8, cTrx. The positions of molecular weight marker are indicated on the right. B and C. ⁷⁵Se incorporation into recombinant TGRs. BL21(DE3) cells expressing TGR_GCUG, TGR_GUCG, TGR_GCCG, and TGR_GC* were induced with 100 μm isopropyl 1-thio-β-d-galactopyranoside for 3 h at 37 °C. 50 μCi of ⁷⁵Se were added to 10-ml cultures 30 min before induction. Total cell protein samples were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. B, Coomassie Blue staining of the polyvinylidene difluoride membrane. C, ⁷⁵Se detection by phosphorimaging device analysis. Lanes 1–4, 10 μl of total cell protein samples from cells expressing recombinant TGR_GCUG, TGR_GUCG, TGR_GCCG, and TGR_GC*, respectively; lane 5, molecular weight marker. FDH-O, formate dehydrogenase O (110 kDa), the single selenoprotein expressed by E. coli under aerobic conditions. The bands around 66 kDa are indicated by an asterisk on lanes 1 and 2 on the right panel and correspond to ⁷⁵Se-labeled Sec-containing recombinant TGRs. Lower molecular mass bands on these lanes probably correspond to secondary initiation or degradation products of these Sec-containing recombinants. The absence of bands on lanes 3 and 4 indicates no unspecific selenium incorporation was detected. The positions of molecular weight markers are indicated on the right.

FIGURE 3.

Kinetic parameters of TGR_GCUG with mtTrx and cTrx. Apparent K_m and k_cat of TGR_GCUG with mtTrx and cTrx were obtained using the Trx-coupled assay. The initial reaction velocities (v₀) at different Trx concentrations were measured at a constant and saturating NADPH concentration (200 μm) and a constant enzyme concentration (0.5 nm). Plots of v₀ versus substrate concentration for mtTrx and cTrx are shown. The data were fitted to the Michaelis-Menten equation using Origin 7.5 software. Apparent K_m and v_max were obtained from these fittings and apparent k_cat was calculated from apparent v_max. Apparent K_m, k_cat, and k_cat/K_m values are indicated. The enzyme (TGR_GCUG) concentration used for k_cat calculation was corrected according to its selenium content.

FIGURE 4.

TR activity of TGR mutants. The TR activities of wild-type and mutant TGRs were compared using the DTNB assay. The assay was carried out at constant and saturating concentrations of DTNB and NADPH (5 mm and 200 μm, respectively) and different concentrations of each enzyme. The plots of initial velocities (v₀) versus enzyme concentration are shown. The selenoenzyme (TGR_GCUG, TR_GCUG, and TGR_GUCG) concentrations used for k_cat calculations were corrected according to their selenium contents.

FIGURE 5.

Hysteretic behavior of GR activity of TGR. Full-time courses obtained using different assay conditions are shown. In all cases the reactions were started by the addition of TGR_GCUG at the indicated final concentrations. A, effect of enzyme concentration. Assays were performed at varying TGR_GCUG concentrations and constant NADPH and GSSG concentrations (100 μm and 1 mm, respectively). B, effect of GSSG concentration. GSSG concentration was varied at constant NADPH and enzyme concentrations (100 μm and 10 nm, respectively). Note that at 31 and 62 μm GSSG reactions come to their end at higher A₃₄₀, i.e. before depleting NADPH, because GSSG becomes the limiting reagent. C, effect of GSH concentration. GSH was included at various concentrations while maintaining constant GSSG, NADPH, and enzyme concentrations (1 mm, 125 μm, and 10 nm, respectively). D, effect of Trx concentration. cTrx was added at different concentrations to reaction mixtures containing 1 mm GSSG, 125 μm NADPH, and 10 nm TGR_GCUG. The TGR_GCUG concentration considered was corrected according to its selenium content.

FIGURE 6.

TR activity of TGR_GCUG and TR_GCUG analyzed at the hysteresis conditions for GR activity. A, the TR activities of untreated and glutathionylated TGR_GCUG were compared using the Trx-coupled assay. B, the GR activities of untreated and glutathionylated TGR_GCUG were compared at 100 μm GSSG. In both A and B the enzyme preparations were assayed at 1 nm TGR concentration and 150 μm NADPH. It should be noted that, to calculate the volume of enzyme preparation that ought to be used in the assay, glutathionylated TGR was assumed to be 2-fold diluted following desalting. This approximation could explain the slightly smaller slopes observed for this enzyme in both assays, as compared with the untreated one. C, the TR activity of TR_GCUG was evaluated using the Trx-coupled assay both in the absence and presence of high concentration (1 mm) GSSG. The enzyme was assayed at 1 nm final concentration and 150 μm NADPH. The selenoenzyme (TGR_GCUG and TR_GCUG) concentrations considered were corrected according to their selenium contents.

FIGURE 7.

GR activity of TGR mutants. Time courses obtained for the GR activity of wild-type TGR and its mutants are shown. In all cases the reaction was started by the addition of the enzymes at the indicated final concentrations. A, comparison of the GR activity of TGR_GCUG, TGR_GCCG, TGR_GC*, and TR_GCUG at 31 μm GSSG and 125 μm NADPH. B, the GR activity of TGR_GUCG at different enzyme concentrations was evaluated at 31 μm GSSG and 125 μm NADPH. C, the effect of GSH addition on the hysteretic behavior of TGR_GUCG was studied by including GSH at 1 mm in a reaction mixture containing 1 mm GSSG, 125 μm NADPH, and 25 nm TGR_GUCG. The enzyme concentrations for selenoproteins (TGR_GCUG, TR_GCUG, and TGR_GUCG) were corrected according to their selenium contents.

FIGURE 8.

Auranofin effect on E. granulosus larval worms. Protoscoleces were incubated in vitro at 37 °C, 5% CO₂ in DMEM with 10 μm auranofin, a TGR inhibitor, or its vehicle (DMSO) as a control. A, control protoscoleces after 30 h of culture. B, treated protoscoleces after 12 h of culture (all protoscoleces were dead, note the disorganization of the parenchyma and the loss of the hooks or the entire crown of hooks). C, treated protoscoleces after 30 h of culture. The scale bar on C corresponds to 100 μm.

FIGURE 9.

Hysteretic behavior and glutathionylation of TGR. At high GSSG concentrations, the GR activity of TGR exhibited hysteretic behavior (filled squares); TGR was found to be glutathionylated under hysteresis and deglutathionylated once hysteresis was abolished. At low GSSG concentrations, the GR activity did not exhibit hysteretic behavior (open squares), and the enzyme was not glutathionylated. The figure also shows that hysteretic behavior is favored by high [GSSG]/[GSH] ratios or low enzyme concentrations and is relieved at low [GSSG]/[GSH] ratios or high enzyme concentrations.