Linked Thioredoxin-Glutathione Systems in Platyhelminth Parasites

Abstract

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated Platyhelminths (also known as flatworms) cause severe human parasitic diseases, such as schistosomiasis, cysticercosis, and hydatid disease. Parasitic platyhelminths share a unique biochemical scenario regarding thiol-based redox pathways (1-4). These organisms possess a linked thioredoxin-glutathione system that provides reducing equivalents to both the thioredoxin (Trx) 2 and the glutathione (GSH) pathways. In contrast to their mammalian hosts, platyhelminth parasites lack thioredoxin reductase (TR) and glutathione reductase (GR) and hence conventional Trx and GSH systems. The distinctive feature of the redox array of platyhelminth parasites is that reduction of Trx and GSSG is carried out by a single selenoenzyme, termed thioredoxin glutathione reductase (TGR). Thus, these organisms have an absolute reliance on TGR for redox homeostasis and DNA synthesis. Furthermore, in parasitic flatworms, mitochondrial and cytosolic TGR variants are derived from a single gene, and the encoded mature polypeptides are identical in sequence (3).TGR is a complex flavoenzyme that contains several thiolbased redox centers and is composed of an N-terminal glutaredoxin (Grx) domain fused to canonical large TR domains (5). In the platyhelminth parasite Echinococcus granulosus (class cestoda), the Grx domain has a dithiol CPYC redox center, and the TR module contains a CX 4 C redox center and the conspicuous C-terminal motif GCUG, where U is the redox active amino acid selenocysteine (Sec) (1). Detailed biochemical studies with sets of mutants of human and E. granulosus TGR have shown the following: (i) both oxidized Trx and oxidized glutathione (GSSG) reduction are dependent on the redox-active C-terminal Sec residue, and (ii) reduction of GSSG, but not of oxidized Trx, requires the Grx domain (6, 7). In the case of E. granulosus and Taenia crassiceps (class cestoda) TGRs, it has also been shown that their GR activity, but not their TR activity, is inhibited at high concentrations of oxidized glutathione (GSSG) (4, 6). This phenomenon has been proposed to be due to glutathionylation of critical Cys residues (6).

Investigations of the Catalytic Mechanism of Thioredoxin Glutathione Reductase from Schistosoma mansoni

“…Recently, a mechanism for the Grx glutathionylation activity of TGR from Echinococcus granulosus (EgTGR) has been suggested by Bonilla et al (55). They showed that the Grx glutathionylation activity of EgTGR requires the Grx domain as well as the selenocysteine residue.…”

The Redox Biology of Schistosome Parasites and Applications for Drug Development

“…These results indicate that in schistosomes Trx would only reduce GSSG under extreme oxidative stress; therefore, it is not clear whether reduction of GSSG catalyzed by Trx-1 is physiologically relevant in S mansoni . In contrast, in Echinococcus granulosus , a tapeworm parasite with a redox network similar to that found in schistosomes [165], it has been shown that that Trx is able to reduce GSSG and to catalyze deglutathionylation reactions at high concentrations of GSSG in which the activity of its TGR would be inhibited [166]. These results suggest that redox pathways may vary in different Platyhelminthes parasites.…”

“…The difference between the deglutathionylation reactions of E. granulosus TGR and S. mansoni TGR may be explained by different distances between cysteines in the Grx active sites of the two proteins. In E. granulosus TGR, the distance between Cys-31 and Cys-34 is longer than that between Cys-28 and Cys-31 in S. mansoni TGR and, therefore, the accessibility of Cys-34 to Cys-31 may be somewhat limited in E. granulosus TGR [166, 175]. …”

Thioredoxin glutathione reductase: Its role in redox biology and potential as a target for drugs against neglected diseases

“…Recently, a mechanism for the Grx deglutathionylation activity of TGR from Echinococcus granulosus (EgTGR) has been suggested [67]. It was shown that the Grx activity of EgTGR requires the Grx domain and the selenocysteine residue.…”