Platyhelminth Mitochondrial and Cytosolic Redox Homeostasis Is Controlled by a Single Thioredoxin Glutathione Reductase and Dependent on Selenium and Glutathione
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m710609200
ISSN1083-351X
AutoresMariana Bonilla, Ana Denicola, Sergey V. Novoselov, Anton A. Turanov, Anna V. Protasio, Darwin Izmendi, Vadim N. Gladyshev, Gustavo Salinas,
Tópico(s)Sulfur Compounds in Biology
ResumoPlatyhelminth 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 Km and kcat values for mitochondrial and cytosolic thioredoxins (9.5 μm and 131 s–1, 34 μm 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 μm 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. 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 Km and kcat values for mitochondrial and cytosolic thioredoxins (9.5 μm and 131 s–1, 34 μm 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 μm 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. The control of parasitic infections, which are a major cause of disability, mortality, and economic losses in many developing countries, remains as one of the most important challenges for medicine in the 21st century (1WHOPreventive Chemotherapy in Human Helminthiasis: Coordinated Use of Antihelminthic Drugs in Control Interventions: a Manual for Health Professionals and Programme Managers. WHO Press, World Health Organization, Geneva, Switzerland2006Google Scholar). In the case of the phylum platyhelminthes (flatworms), which include the causative agents of schistosomiasis (bilharzia) and hydatid disease, pharmacotherapy with praziquantel has met great success in the treatment of infection. However, drug resistance is a serious issue as it has been the case for other antiparasitic drugs (2Mansour T. Mansour T Chemotherapeutic Targets in Parasites: Contemporary Strategies. University Press, Cambridge, Cambridge2002: 58-89Google Scholar). In the case of platyhelminths, this may have severe consequences, because praziquantel is the only drug that is readily available for large scale treatment of these infections (3Doenhoff M.J. Pica-Mattoccia L. Expert Rev. Anti Infect. Ther. 2006; 4: 199-210Crossref PubMed Scopus (143) Google Scholar). Thus, the need for new drugs and/or vaccines is of great importance. In recent years, evidence has accrued that the selenocysteine (Sec) 2The abbreviations used are: Sec, selenocysteine; DMEM, Dulbecco's modified Eagle's medium; DTNB, 5,5′-dithiobis(2-dinitrobenzoic acid); DTT, dithiothreitol; EGFP, enhanced green fluorescent protein; GR, glutathione reductase; Grx, glutaredoxin; GSSG, glutathione(oxidizedform); TGR, thioredoxinglutathione reductase; TR, thioredoxin reductase; Trx, thioredoxin; SECIS, selenocysteine insertion sequence; mtTrx, mitochondrial Trx; cTrx, cytosolic Trx. -containing enzyme thioredoxin glutathione reductase (TGR) is essential for platyhelminth parasites and has emerged as a rational target for chemotherapy and/or immunotherapy (4Alger H.M. Williams D.L. Mol. Biochem. Parasitol. 2002; 121: 129-139Crossref PubMed Scopus (147) Google Scholar, 5Agorio A. Chalar C. Cardozo S. Salinas G. J. Biol. Chem. 2003; 278: 12920-12928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Rendón J.L. del Arenal I.P. Guevara-Flores A. Uribe A. Plancarte A. Mendoza-Hernández G. Mol. Biochem. Parasitol. 2004; 133: 61-69Crossref PubMed Scopus (70) Google Scholar, 7Salinas G. Selkirk M.E. Chalar C. Maizels R.M. Fernández C. Trends Parasitol. 2004; 20: 340-346Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Kuntz A.N. Davioud-Charvet E. Sayed A.A. Califf L.L. Dessolin J. Arnér E.S. Williams D.L. PLoS Med. 2007; 4: e206Crossref PubMed Scopus (295) Google Scholar). In most organisms, including the mammalian hosts of platyhelminths, cellular redox homeostasis, antioxidant defenses, and supply of reducing equivalents to several targets and essential enzymes rely on two major pathways: the glutathione (GSH) and the thioredoxin (Trx) systems, which have overlapping and differential targets and functions (9Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (539) Google Scholar, 10Winyard P.G. Moody C.J. Jacob C. Trends Biochem. Sci. 2005; 30: 453-461Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In contrast, platyhelminth parasites lack conventional thioredoxin reductase (TR) and glutathione reductase (GR), and hence conventional Trx and GSH systems (4Alger H.M. Williams D.L. Mol. Biochem. Parasitol. 2002; 121: 129-139Crossref PubMed Scopus (147) Google Scholar, 6Rendón J.L. del Arenal I.P. Guevara-Flores A. Uribe A. Plancarte A. Mendoza-Hernández G. Mol. Biochem. Parasitol. 2004; 133: 61-69Crossref PubMed Scopus (70) Google Scholar, 7Salinas G. Selkirk M.E. Chalar C. Maizels R.M. Fernández C. Trends Parasitol. 2004; 20: 340-346Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). 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 (5Agorio A. Chalar C. Cardozo S. Salinas G. J. Biol. Chem. 2003; 278: 12920-12928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In the mammalian hosts, different thioredoxin reductase isozymes function in the cytosol and the mitochondria (11Turanov A.A. Su D. Gladyshev V.N. J. Biol. Chem. 2006; 281: 22953-22963Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.-A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (149) Google Scholar), TGR expression is largely restricted to testis (13Sun Q.-A. Kirnarsky L. Sherman S. Gladyshev V.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3673-3678Crossref PubMed Scopus (242) Google Scholar), and GR exists as a distinct gene (14Kelner M.J. Montoya M.A. Biochem. Biophys. Res. Commun. 2000; 269: 366-368Crossref PubMed Scopus (44) Google Scholar). In sum, 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 not only to the endogenous oxidative stress, but also to the oxidative challenge imposed by the host's immune system, provide a strong rationale to target platyhelminth TGRs. Recent studies support this idea: inhibition of TGR expression by RNA interference caused death of the platyhelminth parasite Schistosoma mansoni, and auranofin, a potent inhibitor of TGR and Sec-containing TRs (15Gromer S. Arscott L.D. Williams Jr., C.H. Schirmer R.H. Becker K. J. Biol. Chem. 1998; 273: 20096-20101Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), caused a partial cure in experimental Schistosoma infection (8Kuntz A.N. Davioud-Charvet E. Sayed A.A. Califf L.L. Dessolin J. Arnér E.S. Williams D.L. PLoS Med. 2007; 4: e206Crossref PubMed Scopus (295) Google Scholar). TGR possesses a fusion of conventional TR domains with a glutaredoxin (Grx) domain (13Sun Q.-A. Kirnarsky L. Sherman S. Gladyshev V.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3673-3678Crossref PubMed Scopus (242) Google Scholar, 16Sun Q.-A. Yalin W. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). TGR, like GR and TR, is a homodimer, with monomers oriented in a head-to-tail manner. Based on biochemical data, 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 finally 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 (13Sun Q.-A. Kirnarsky L. Sherman S. Gladyshev V.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3673-3678Crossref PubMed Scopus (242) Google Scholar, 17Sun Q.A. Su D. Novoselov S.V. Carlson B.A. Hatfield D.L. Gladyshev V.N. Biochemistry. 2005; 44: 14528-14537Crossref PubMed Scopus (71) Google Scholar). Recently, a crystallographic structure of an S. mansoni C-terminally truncated TGR (GCstop) has been solved. 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 (18Angelucci F. Miele A.E. Boumis G. Dimastrogiovanni D. Brunori M. Bellelli A. Proteins. 2008; (in press)PubMed Google Scholar). In the current study, we have characterized the linked thioredoxin-glutathione system of the platyhelminth E. granulosus, the causative agent of hydatid disease. We demonstrated the occurrence of functional linked systems in both cytosol and mitochondria. The analysis of activities of TGR mutants revealed that the Grx domain is required for the GR activity, but does not affect the TR activity; in contrast, both Trx- and glutathione-dependent activities require selenocysteine (Sec) residue. Our results also indicate that [GSSG]/[GSH] ratio regulates TGR activities and strongly suggest that glutathionylation/deglutathionylation is involved in this regulation. In addition, 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. To analyze the subcellular localization of the putative mitochondrial variants of Trx and TGR, constructs were generated using pEGFP-N2 (Clontech). In the case of Trx, the sequence was retrieved from Partigen (cluster EGC03292), and the entire coding region, including the leader peptide, was cloned as an in-frame fusion to EGFP. In the case of TGR the N-terminal fragment of mitochondrial TGR, containing the leader peptide followed by the Grx domain of TGR, was cloned as an EGFP fusion. In both cases, a Kozak consensus sequence was included in the forward primer for initiation of translation at the first AUG codon. For transient expression, mouse NIH 3T3 cells (ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum in the presence of 100 units/ml penicillin and 50 units/ml nystatin. Transfections were carried out in 35-mm glass bottom culture dishes using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Transfection mix was prepared using 3 μg of plasmid DNA (mitochondrial Trx construct, mitochondrial TGR construct, or pEGFP-N2, used as a control) and 6 μl of Lipofectamine per dish. Transfections were carried out in Opti-MEM (Invitrogen) for 8 h. The transfection medium was replaced with a DMEM culture medium containing MitoTracker Red CM-H2XRos (Molecular Probes), a marker of the mitochondrial compartment; cells were incubated for 30 min and then washed twice with DMEM. Transiently transfected cells were detected by confocal microscopy (Bio-Rad, MRC1024ES laser scanning microscope). Different constructs were made for expression of wild-type TGR (TGRGCUG) (where U is Sec, and GCUG is the C-terminal tetrapeptide) and the following mutants: Sec596 to Cys (TGRGCCG), Sec596 to stop (TGRGC*), Sec596 to Cys, and Cys595 to Sec (TGRGUCG), as well as a mutant lacking the entire Grx domain of TGR (TRGCUG). 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 (5Agorio A. Chalar C. Cardozo S. Salinas G. J. Biol. Chem. 2003; 278: 12920-12928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In the case of mutant TGRs, the reverse primers were modified appropriately. For Sec-encoding constructs (TGRGCUG, TGRGUCG, and TRGCUG), further engineering of the reverse primers was needed to specify Sec, because the UGASec codon requires recoding by an Sec insertion sequence (SECIS) element present in the selenoprotein mRNAs. Thus, for these constructs, the reverse gene-specific primer contained, at the 5′-end, the SECIS element of Escherichia coli formate dehydrogenase H at a 10-nucleotide distance from the UGASec codon (sequences of primers for every construct are detailed in supplemental Table S1). This strategy with a bacterial-type SECIS has been previously used for C-terminal Sec incorporation in E. coli (19Arnér E.S. Sarioglu H. Lottspeich F. Holmgren A. Bock A. J. Mol. Biol. 1999; 292: 1003-1016Crossref PubMed Scopus (202) Google Scholar). The amplified products were first cloned into pGEM-T-easy (Promega), and the construct sequences were verified prior to subsequent subcloning into pET28a (Novagen). Constructs were used to transform E. coli BL21(DE3) cells, or, in the case of selenoprotein constructs, BL21(DE3) cells previously transformed with pSUABC, a plasmid that supports high level expression of genes involved in Sec synthesis and decoding (selA, selB, and selC) (19Arnér E.S. Sarioglu H. Lottspeich F. Holmgren A. Bock A. J. Mol. Biol. 1999; 292: 1003-1016Crossref PubMed Scopus (202) Google Scholar). Expression of recombinant proteins was carried out following the protocol described in a previous study (20Rengby O. Johansson L. Carlson L.A. Serini E. Vlamis-Gardikas A. Karsnas P. Arnér E.S. Appl. Environ. Microbiol. 2004; 70: 5159-5167Crossref PubMed Scopus (74) Google Scholar), which has been optimized for expression of selenoproteins. Essentially, induction of recombinant proteins was carried out with 100 μm isopropyl 1-thio-β-d-galactopyranoside at late exponential phase (A600 = 2.4), during 24 h at 24 °C. Recombinant clones were grown in modified LB media according to a previous study (21Bar-Noy S. Gorlatov S.N. Stadtman T.C. Free Radic. Biol. Med. 2001; 30: 51-61Crossref PubMed Scopus (37) Google Scholar), supplemented with 0.1 g/liter cysteine and 0.37 g/liter methionine (22Müller S. Heider J. Böck A. Arch. Microbiol. 1997; 168: 421-427Crossref PubMed Scopus (59) Google Scholar), in the presence of kanamycin (50 μg/ml), and chloramphenicol (33 μg/ml); the latter was used only in the case of bacterial cultures harboring the TGRGCUG, TGRGUCG, and TRGCUG constructs. At the time of induction the culture was supplemented with 5 μm sodium selenite, 20 μg/ml riboflavin, 20 μg/ml pyridoxine, and 20 μg/ml niacin according to a previous study (21Bar-Noy S. Gorlatov S.N. Stadtman T.C. Free Radic. Biol. Med. 2001; 30: 51-61Crossref PubMed Scopus (37) Google Scholar). For recombinant TGRs that did not contain Sec (TGRGCCG and TGRGC*) the same protocol was followed, except that the plasmid pSUABC was not used. The bacterial cultures were centrifuged, and the pellets were resuspended in modified nickel-nitrilotriacetic acid lysis buffer (300 mm NaCl, 50 mm sodium phosphate, 20 mm imidazole, pH 7.2) containing 1 mm phenylmethylsulfonyl fluoride and 1 mg/ml lysozyme, and sonicated (10 pulses of 1 min with 1-min pauses). The lysates were centrifuged for 1 h at 30,000 × g, and supernatants were applied to a nickel-nitrilotriacetic acid column (Qiagen), washed with 300 mm NaCl, 50 mm sodium phosphate, 30 mm imidazole, pH 7.2, and eluted with 250 mm imidazole. The protein-containing fractions were applied to PD10 desalting columns (GE Healthcare) using phosphate-buffered saline, 150 mm potassium chloride, 50 mm sodium phosphate, pH 7.2. Fractions containing the recombinant proteins were stored at –70 °C before use. Total protein concentration and FAD content were determined spectrophotometrically at 280 (ϵ = 54.24 mm–1 cm–1) and 460 nm (ϵ = 11.3 mm–1 cm–1), respectively. The selenium content of selenoproteins was determined by atomic absorption using a Plasma Emission Spectrometer (Jarrell-Ash 965 ICP) in Chemical Analysis Laboratory, University of Georgia. The purity of the recombinant proteins was analyzed by running 10% SDS-PAGE gels, under reducing conditions, and by size-exclusion chromatography on a Superose 12 column (GE Healthcare). mRNAs encoding cytosolic and mitochondrial E. granulosus Trxs were amplified by reverse transcription-PCR from total larval worm mRNA as described above. Specific forward and reverse primers for cytosolic and the predicted mature mitochondrial Trx were derived from previously published sequences (23Chalar C. Martinez C. Agorio A. Salinas G. Soto J. Ehrlich R. Biochem. Biophys. Res. Commun. 1999; 262: 302-307Crossref PubMed Scopus (19) Google Scholar) and from Partigene (cluster EGC03292), respectively. The amplified products were first cloned into pGEM-T-easy (Promega), sequenced and subsequently subcloned into pET28a (Novagen) using appropriate restriction enzymes. Constructs were used to transform E. coli BL21(DE3) host cells. Expression of recombinant proteins was carried out following the standard protocol for expression of recombinant proteins. Essentially, recombinant clones were grown on LB in the presence of kanamycin, and induction of recombinant proteins was carried out with 100 μm isopropyl 1-thio-β-d-galactopyranoside at early exponential phase (A600 = 0.5), for 3 h at 37 °C. The bacterial cultures were centrifuged, and the recombinant proteins were purified and desalted as described above for TGR, except that all buffers had pH 7.8. Fractions containing the recombinant proteins were stored at –70 °C prior to use. Protein concentration was determined spectrophotometrically at 280 nm (ϵ = 7.6 and 6.1 mm–1 cm–1 for cytosolic and mitochondrial Trx, respectively). The purity of the recombinant proteins was analyzed by running 15% SDS-PAGE gels under reducing conditions, and by size exclusion chromatography on a Superdex 75 column (GE Healthcare). To label cells with 75Se, E. coli cells carrying the different constructs were grown at 37 °C until A600 reached 0.4, and the culture was supplemented with ∼50 μCi 75Se (as freshly neutralized sodium selenite, specific activity of 1000 Ci/mmol, Research Reactor Facility, University of Missouri, Columbia, MO). After an additional 30 min, isopropyl 1-thio-β-d-galactopyranoside was added to each cell culture at a final concentration of 100 μm. After 3 h of induction at 37 °C, cells were collected, washed, and lysed by boiling in SDS-PAGE sample buffer containing 50 mm 1,4-dithiothreitol (DTT). Cell lysates were then subjected to SDS-PAGE followed by transfer of proteins onto a polyvinylidene difluoride membrane. 75Se signal was visualized with a phosphorimaging device (Fuji). Insulin Reduction Assay for Trx Activity—The efficient reduction of two interchain disulfides of insulin catalyzed by Trx in the presence of DTT was used as a measure of Trx activity, according to a previous study (24Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). The reaction was followed by the increase in absorbance at 650 nm due to the precipitation of free insulin B-chain. The 0.8-ml reaction mixtures contained 0.33 mm DTT, 130 μm insulin, and 2 mm EDTA in 100 mm potassium phosphate buffer, pH 7.0. Runs with DTT alone were performed as controls. DTNB Reduction Assay for TR Activity—The reduction of 5,5′-dithiobis (2-dinitrobenzoic acid) (DTNB) with concomitant NADPH oxidation was determined by the increase in absorbance at 412 nm due to formation of 5′-thionitrobenzoic acid at 25 °C (25Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (284) Google Scholar). The 0.8-ml reaction mixtures contained 0.2 mm NADPH, 5 mm DTNB, and 10 mm EDTA in 100 mm potassium phosphate buffer, pH 7.0. Insulin Reduction Assay for TR Activity—The Trx-coupled assay of TR activity takes advantage of the NADPH-dependent reduction of Trx by TR, which is followed by the decrease in absorbance at 340 nm; in this assay, excess of insulin is used as an electron sink to maintain a constant concentration of oxidized Trx (25Arnér E.S. Zhong L. Holmgren A. Methods Enzymol. 1999; 300: 226-239Crossref PubMed Scopus (284) Google Scholar). The 0.8-ml reaction mixtures contained 0.2 mm NADPH, 1 mm EDTA, 0.5 mg/ml insulin, and E. granulosus cytosolic or mitochondrial Trx (concentrations ranged from 0 to 80 μm and from 0 to 140μm, respectively), in 50 mm potassium phosphate buffer, pH 7.0. The kinetic parameters of TGR with its physiological substrates, cytosolic and mitochondrial Trx, were determined from Michaelis-Menten plots of vo (derived from time-course experiments) against substrate concentration. GR Assay—The GR activity was assayed as NADPH-dependent reduction of oxidized glutathione (GSSG), which is followed as the decrease in absorbance at 340 nm (26Carlberg I. Mannervik B. Methods Enzymol. 1985; 113: 484-490Crossref PubMed Scopus (2620) Google Scholar). The 0.8-ml reaction mixture contained 0.125 mm NADPH, 1 mm GSSG, 1 mm EDTA in 100 mm potassium phosphate buffer, pH 7.0. All enzymatic assays were carried out in a Cary 50 (Varian) spectrophotometer at 25 °C. Analyses of the kinetic data were performed using ORIGIN software (OriginLab). Mass Spectrometry Analysis—Wild-type TGR samples (10 nm concentration) were incubated with GSSG in the presence or absence of 0.125 mm NADPH at molar ratios under which hysteresis was or was not observed (1 mm or 30 μm GSSG, respectively) in 100 mm potassium phosphate buffer, pH 7.0, containing 1 mm EDTA, and immediately passed through a PD10 desalting column (GE Healthcare). Protein-containing fractions were digested with trypsin and subjected to analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (4800 Analyzer, Applied Biosystems). The mass spectrometry analysis was carried out at the Institut Pasteur, Montevideo. In Vitro Culture of Larval Worms—50,000 protoscoleces, obtained from asceptical punction of a single hydatid cyst from bovine lung, were washed several times with phosphate-buffered saline and then incubated at 37 °C, 5% CO2, in DMEM supplemented with antibiotics and 20 mm HEPES, pH 7.4. Cultured protoscoleces were treated with 1, 2.5, 5, and 10 μm auranofin or with the vehicle (DMSO), in the presence or absence of 100 μm hydrogen peroxide. Protoscoleces were observed under the microscope, and viability was assessed by exclusion of the vital dye eosin. Mitochondrial Localization of Trx and TGR Forms—Sequence analyses suggested the occurrence of both cytosolic and mitochondrial forms of TGR and Trx, with TGR forms generated from a single gene, and two genes for Trx forms. The results of transient expression in mammalian NIH 3T3 cells of the predicted mitochondrial forms of Trx and TGR are shown in Fig. 1. Both EGFP fusion proteins co-localized with MitoTracker, indicating that the signal peptides of these proteins direct the fusions to the mitochondrial compartment. No obvious staining of the cytosol or other subcellular compartments was observed. TGR has been previously shown to be present in the mitochondrial subcellular fraction of a larval worm aqueous extract (5Agorio A. Chalar C. Cardozo S. Salinas G. J. Biol. Chem. 2003; 278: 12920-12928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar); however, the mitochondrial location of Trx was previously limited to in silico predictions in platyhelminths (7Salinas G. Selkirk M.E. Chalar C. Maizels R.M. Fernández C. Trends Parasitol. 2004; 20: 340-346Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). TGR Can Provide Electrons to Both Cytosolic and Mitochondrial Trx Forms—Prior to determining the enzymatic parameters of TGR with its physiological substrates, the quality of every recombinant protein was assessed in several ways. First, the purity of TGR and its mutants, and of cytosolic and mitochondrial Trx forms, was determined by SDS-PAGE under reducing conditions (Fig. 2A) and by size exclusion chromatography (data not shown). In the case of selenoproteins, Sec incorporation was evaluated by metabolic labeling of the bacterial cultures with 75Se. The results are shown in Fig. 2 (B and C); specific labeling at the expected molecular weight was observed exclusively in the bacterial lysates expressing selenoproteins, indicating that full-length selenoproteins were synthesized. In addition, the selenium content of recombinant selenoproteins was determined. Sec incorporation was close to 10% in all recombinant selenoproteins (9.2% for wild-type TGRGCUG, 7.4% for inverted TGRGUCG, and 8.7% for TRGCUG). Taken together, all these data indicated that the strategy was successful to produce full-length TGR in a bacterial host, although higher percentages of Sec incorporation (up to 50%) have been previously reported using this methodology for other selenoproteins (19Arnér E.S. Sarioglu H. Lottspeich F. Holmgren A. Bock A. J. Mol. Biol. 1999; 292: 1003-1016Crossref PubMed Scopus (202) Google Scholar). Because only a fraction of the TGR molecules incorporate Sec (due to prevalent termination of translation at Sec UGA codons), active protein concentrations of selenoproteins were corrected according to their selenium content. The activities of recombinant TGR and Trx were initially assessed independently of each other, using the DTNB and insulin reduction assays (see "Experimental Procedures"), respectively. Both recombinant enzymes displayed activity in these independent assays (data not shown). Then, using the insulin coupled assay, the kinetic parameters of TGR with its physiological substrates, cytosolic and mitochondrial Trx, were determined from Michaelis-Menten plots of vo against substrate concentration (Fig. 3). Km and kcat values were of the same order for both Trxs (Fig. 3, Table 1). The catalytic efficiency of TGR was (13.8 ± 0.9) × 106, and (5.8 ± 0.4) × 106 m–1 s–1 for mitochondrial and cytosolic Trx, respectively.TABLE 1Kinetic parameters of wild-type TGR and its mutantsParameterSubstrateTGRGCUGTRGCUGTGRGUCGTGRGCCGApparent Km (μm)mtTrx9.5 ± 0.513.0 ± 0.612.0 ± 0.614.3 ± 0.6cTrx34 ± 2Apparent kcat (s-1)mtTrx131 ± 280 ± 26.4 ± 0.20.530 ± 0.007cTrx197 ± 3DTNB118 ± 360 ± 36.0 ± 0.50.63 ± 0.03Apparent kcat/Km (μm-1s-1)mtTrx13.8 ± 0.96.1 ± 0.40.27 ± 0.030.037 ± 0.002cTrx5.8 ± 0.4 Open table in a new tab Sec but Not the Grx Domain Is Essential for TR Activity—To assess the role of the Grx domain and of Sec at the GCUG C-terminal redox center of TGR in the catalysis, we generated a set of TGR forms: wild-type TGR (TGRGCUG), TRGCUG (without the Grx domain), Sec596 to stop mutant (TGRGC*), Sec596 to Cys mutant (TGRGCCG), and Cys595 to Sec and Sec596 to Cys double mutant (TGRGUCG). Analysis of TR activity with the DTNB assay (shown in Fig. 4 and summarized in Table 1) revealed that TRGCUG and wild-type TGR have similar kcat. TGRGC* had negligible activity even at 500 nm enzyme concentration (data not shown). TGRGCCG had a kcat more than two orders
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