Insights into Deglutathionylation Reactions

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).

Section: Discussionmentioning

confidence: 57%

“…Deglutathionylation Assay-The deglutathionylase activity was evaluated using a modification of the method described previously (20). The NADPH-dependent reduction of the glutathionylated substrate peptide SQLWC(glutathione)LSN (Peptron Inc., South Korea) was followed by the decrease in absorbance at 340 nm due to NAPDH oxidation (⑀ ϭ 6.2 mM Ϫ1 cm…”

mentioning

confidence: 99%

Linked Thioredoxin-Glutathione Systems in Platyhelminth Parasites

Bonilla

Denicola

Marino

et al. 2011

“…positive cooperative behavior of Ure2 GRX activity toward the substrate GSH is consistent with the behavior of yeast Omega GST (15) and E. coli GRX1 when assayed with substrate peptide and GSH (33), suggesting that the cooperativity provides a regulatory function in their redox roles in vivo.…”

Section: Ure2supporting

confidence: 52%

Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Zhang

Perrett

2009

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.

“…Although Trxs have been reported to catalyze deglutathionylation in some organisms (16), this reaction is generally considered to be specifically catalyzed by Grxs (17). Protein deglutathionylation can proceed via a monothiol or a dithiol pathway depending on the involvement of one or two cysteines in the catalytic mechanism (6,18,19).…”

mentioning

confidence: 99%

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

Couturier

Ströher

Albetel

et al. 2011

115

Unlike thioredoxins, glutaredoxins are involved in ironsulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys 32 ) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter-or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.