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.
Highlights
The dissimilar arrangements of redox pathways as compared with their hosts, the lack of back-up systems, and the fact that parasitic organisms are subjected to the endogenous oxidative stress, and to the oxidative challenge imposed by the host’s immune system, provide a strong rationale to target platyhelminth thioredoxin-glutathione reductase (TGR)
The current model of the mechanism of reaction for TGR proposes that electrons flow from NADPH to FAD, to the C156XXXXC redox center, to the C-terminal GC595UG (U is Sec) redox center of the second subunit, and to the C31XXC redox center of the Grx domain of the first subunit
TGR has been previously shown to be present in the mitochondrial subcellular fraction of a larval worm aqueous extract [5]; the mitochondrial location of Trx was previously limited to in silico predictions in platyhelminths [7]
Summary
Selenocysteine; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5Ј-dithiobis(2-dinitrobenzoic acid); DTT, dithiothreitol; EGFP, enhanced green fluorescent protein; GR, glutathione reductase; Grx, glutaredoxin; GSSG, glutathione (oxidized form); TGR, thioredoxin glutathione reductase; TR, thioredoxin reductase; Trx, thioredoxin; SECIS, selenocysteine insertion sequence; mtTrx, mitochondrial Trx; cTrx, cytosolic Trx. The current model of the mechanism of reaction for TGR proposes that electrons flow from NADPH to FAD, to the C156XXXXC redox center (numeration according to Echinococcus granulosus TGR), to the C-terminal GC595UG (U is Sec) redox center of the second subunit, and to the C31XXC redox center of the Grx domain of the first subunit. The fully reduced enzyme can reduce either oxidized Trx using the C-terminal active site GCUG, or GSSG through the CXXC redox center of the Grx domain [13, 17]. Based on the residual GR activity of the mutant, the authors proposed an alternative view in which GSSG could be reduced directly by the CXXXXC redox center of TR domains [18]. We show that larval worms are killed by very low concentrations of auranofin, a TGR inhibitor, and discuss our results in light of the current models that have been put forward to explain the GR activity of TGR
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