Linked Thioredoxin-Glutathione Systems in Echinococcus granulosus
Bonilla, M.1; Izmendi, D.1; Manta, B.2; Denicola, A.2; Gladyshev, V.3; Salinas, G.1
1 Cátedra de Inmunología, Universidad de la República, Montevideo, Uruguay. 2 Unidad de Fisicoquímica Biológica, Universidad de la República, Montevideo, Uruguay. 3 Department of Biochemistry, University of Nebraska-Lincoln, USA.
Thioredoxin (Trx) and glutathione (GSH) systems are the major redox dependent pathways in many organisms. They participate in redox homeostasis maintenance, supply electrons to key enzymes and regulate protein function. Platyhelminths have linked thioredoxin-glutathione systems in the mitochondria and cytosol, with the selenoenzyme thioredoxin-glutathione reductase (TGR) providing electrons to targets of both pathways. Its unusual behaviour is achieved by a Grx and TrxR domains fusion. Cytosolic (cTGR) and mitochondrial (mtTGR) variants contain identical Grx and TrxR domains and differ only at their N-termini, while cytosolic (cTrx) and putative mitochondrial (mtTrx) thioredoxin variants differ in sequence. Our aim is to characterize the thioredoxin-glutathione systems in the platyhelminth parasite Echinococcus granulosus. We are interested in (i) subcellular localization of their members and (ii) functional characterization of the enzymes. For these purposes we (i) cloned the Grx domain of TGR with or without the mitochondrial leader peptide and the mtTrx variant as N-terminal fusions to the green fluorescent protein, transfected NIH3T3 cells localised the resulting fusion proteins by confocal microscopy; and (ii) cloned the wild type form of the enzyme (TGRGCUG, U=selenocysteine) and mutants (TGRGUCG, TGRGCCG and TGRGC*), expressed the recombinant proteins in Escherichia coli and purified them to assay their activites. Recombinant mtTrx has clearly shown to translocate to mitochondria and the localisation of recombinant TGR variants has been confirmed. The UxC mutation resulted in a drastic reduction of TR activity compared to the wild type, while the truncated form of the enzyme exhibited no activity. This indicates the C-terminal domain is needed for activity and U at the penultimate residue cannot be replaced by C. Interestingly, the double mutant TGR (TGRGUCG) has lower activity than the wild type but higher than the UxC mutant, suggesting that the CxU mutation at the resolving residue can partially compensate for the activity loss caused by the penultimate UxC mutation.
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