Alternative mRNAs Arising from Trans-splicing Code for Mitochondrial and Cytosolic Variants of Echinococcus granulosus Thioredoxin Glutathione Reductase

Abstract

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.

Yeast Flavohemoglobin, a Nitric Oxide Oxidoreductase, Is Located in Both the Cytosol and the Mitochondrial Matrix

“…Indeed, a growing number of mitochondrial proteins are being found in both the cytosol and the mitochondrion (48,49). These include the histidyl-tRNA synthase (50), fumarase (51,52), copper-zinc superoxide dismutase (53), and dihydroxybutanone phosphate synthase (54) from yeast, thioredoxin from Drosophila (55), the thioredoxin glutathione reductase from Echinococcus (56), and several human proteins (48) including aspartate aminotransferase (57) and the Wilson's copper transport protein (58). Close examination of several of these proteins has revealed a multiplicity of mechanisms for the localization of proteins to the mitochondrion and the cytosol (48).…”

Platyhelminth Mitochondrial and Cytosolic Redox Homeostasis Is Controlled by a Single Thioredoxin Glutathione Reductase and Dependent on Selenium and Glutathione

“…In all cases mRNA from trizoled E. granulosus protoscoleces (larval worms) was used as a template for reverse transcription and PCR, using ThermoScript reverse transcriptase (Invitrogen) and Pfu (Fermentas), respectively. Forward and reverse gene-specific primers were derived from the previously published TGR sequence (5). In the case of mutant TGRs, the reverse primers were modified appropriately.…”

“…Instead, they rely exclusively on linked thioredoxin-glutathione systems, with TGR being the key enzyme that provides reducing equivalents to both pathways. Another feature of the linked systems in platyhelminths is that cytosolic and mitochondrial TGR derive from a single gene and have identical sequence, once the leader peptide of the mitochondrial variant is removed (5). In the mammalian hosts, different thioredoxin reductase isozymes function in the cytosol and the mitochondria (11,12), TGR expression is largely restricted to testis (13), and GR exists as a distinct gene (14).…”

Selenoproteins in parasites

“…In addition, molecular characterization of the corresponding gene could also provide clues regarding the mechanism of generation of isoforms. Indeed, the analysis of TGR in E. granulosus revealed two trans-spliced cDNAs derived from a single gene [22]. These variants code for mitochondrial (mt) and cytosolic (c) TGRs, containing identical Grx and TrxR domains, but differing in their N-termini.…”

“…The biochemical characterization of E. granulosus and T. crassiceps TGR indicated that the native enzyme shuttles electrons from NADPH to oxidized Trx (TR activity), GSSG (GR activity) and glutathione-mixed disulfi des (Grx activity). The stoichiometric inhibitory effect of auranofi n on both GR and TR activities of TGR indicates that the Sec-containing C-terminal redox center participates in electron transfer to GSSG and oxidized Trx [20,22]. In addition, TR and Grx domains can function either in coupled reactions or independently.…”