FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061018077> ?p ?o ?g. }

• W2061018077 endingPage "27314" @default.
• W2061018077 startingPage "27302" @default.
• W2061018077 abstract "Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (ΔΨm), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of ΔΨm dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against β-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells. Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (ΔΨm), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of ΔΨm dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against β-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells. A drastic alteration of the mitochondrial redox environment, which includes rapid oxidation of NAD(P)H and glutathione, is one of the earliest events of the mitochondrial pathway of apoptosis (1Nieminen A.L. Byrne A.M. Herman B. Lemasters J.J. Am. J. Physiol. 1997; 272: C1286-C1294Crossref PubMed Google Scholar, 2Macho A. Hirsch T. Marzo I. Marchetti P. Dallaporta B. Susin S.A. Zamzami N. Kroemer G. J. Immunol. 1997; 158: 4612-4619PubMed Google Scholar, 3Gendron M.C. Schrantz N. Metivier D. Kroemer G. Maciorowska Z. Sureau F. Koester S. Petit P.X. Biochem. J. 2001; 353: 357-367Crossref PubMed Scopus (98) Google Scholar). This phenomenon is associated with dissipation of the mitochondrial transmembrane potential (ΔΨm) and subsequent induction of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; ANT, adenine nucleotide translocator; BSO, buthionine sulfoximine; DHR123, dihydrorhodamine 123; DiOC6, 3′-dihexyloxacarbocyanine iodide; EGFP, enhanced green fluorescent protein; EtBr, ethidium bromide; etdh-1, ethidium homodimer-1; GPx, glutathione peroxidase; H2O2, hydrogen peroxide; HE, dihydroethidine; MMP, mitochondrial membrane permeabilization; MnSOD, manganese superoxide dismutase; MTS, mitochondrial targeting sequence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ⋅NO and superoxide (O2˙−, superoxide; Prdx, peroxiredoxin; SeCys, selenocysteine; t-BHP, tert-butylhydroperoxide; TMRE, tetramethylrhodamine ethyl ester; Trx, thioredoxin; Trx2, mitochondrial thioredoxin; TrxR2, mitochondrial thioredoxin reductase-2; VP16, etoposide; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter. generation (4Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsch T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1431) Google Scholar). Onset of mitochondrial membrane permeabilization (MMP), a decisive step in the mitochondrial pathway of apoptosis, is regulated by both pyridine nucleotide (NAD/NADH and NADP+/NADPH) and glutathione (GSSG/GSH) redox equilibrium (5Chernyak B.V. Bernardi P. Eur. J. Biochem. 1996; 238: 623-630Crossref PubMed Scopus (210) Google Scholar, 6Costantini P. Chernyak B.V. Petronilli V. Bernardi P. J. Biol. Chem. 1996; 271: 6746-6751Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). Protein thiol (sulfhydryl) groups can be oxidized directly or indirectly by ROS, i.e. via GSH oxidation and mixed disulfide formation, such as glutathiolation. For example, specific thiol groups on the adenine nucleotide translocator (ANT), an inner membrane constituent of the mitochondrial permeability transition pore complex, have been implicated in modulating MMP. Oxidation of Cys-56 and crosslinking of Cys-56 to Cys-159 were shown to convert the ADP/ATP translocase into an opened nonspecific pore, a process causing MMP (7Costantini P. Belzacq A.S. Vieira H.L. Larochette N. de Pablo M.A. Zamzami N. Susin S.A. Brenner C. Kroemer G. Oncogene. 2000; 19: 307-314Crossref PubMed Scopus (261) Google Scholar, 8Vieira H.L. Haouzi D. El Hamel C. Jacotot E. Belzacq A.S. Brenner C. Kroemer G. Cell Death Differ. 2000; 7: 1146-1154Crossref PubMed Scopus (203) Google Scholar, 9Halestrap A.P. McStay G.P. Clarke S.J. Biochimie (Paris). 2002; 84: 153-166Crossref PubMed Scopus (629) Google Scholar). More recently, ANT was also shown to be a critical target of apoptosis induction by nitric oxide, peroxynitrite, and 4-hydroxynonenal (10Vieira H.L. Belzacq A.S. Haouzi D. Bernassola F. Cohen I. Jacotot E. Ferri K.F. El Hamel C. Bartle L.M. Melino G. Brenner C. Goldmacher V. Kroemer G. Oncogene. 2001; 20: 4305-4316Crossref PubMed Scopus (228) Google Scholar). The cascade of events leading to MMP includes rapid NAD(P)H oxidation/depletion and tightly linked dissipation of ΔΨm, decreased oxidative phosphorylation, enhanced generation of superoxide, and mitochondrial osmotic swelling (4Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsch T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1431) Google Scholar, 11Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar). The consequent rupture of the outer mitochondrial membrane culminates with the release of soluble intermembrane death effector proteins including the caspase-9 activator cytochrome c (12Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), apoptosis-inducing factor (13Joza N. Susin S.A. Daugas E. Stanford W.L. Cho S.K. Li C.Y. Sasaki T. Elia A.J. Cheng H.Y. Ravagnan L. Ferri K.F. Zamzami N. Wakeham A. Hakem R. Yoshida H. Kong Y.Y. Mak T.W. Zuniga-Pflucker J.C. Kroemer G. Penninger J.M. Nature. 2001; 410: 549-554Crossref PubMed Scopus (1153) Google Scholar), and endonuclease G (14Parrish J. Li L. Klotz K. Ledwich D. Wang X. Xue D. Nature. 2001; 412: 90-94Crossref PubMed Scopus (353) Google Scholar, 15Li L.Y. Luo X. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1399) Google Scholar), which induce nuclear DNA cleavage. MMP can be the rate-limiting step of the mitochondrial pathway of apoptosis, regulated by the redox status of critical thiol groups in ANT and by interaction of the permeability transition pore complex with several factors including adenine nucleotides, Ca2+, and both anti-apoptotic and proapoptotic Bcl-2 family of proteins (reviewed in Refs. 8Vieira H.L. Haouzi D. El Hamel C. Jacotot E. Belzacq A.S. Brenner C. Kroemer G. Cell Death Differ. 2000; 7: 1146-1154Crossref PubMed Scopus (203) Google Scholar and 9Halestrap A.P. McStay G.P. Clarke S.J. Biochimie (Paris). 2002; 84: 153-166Crossref PubMed Scopus (629) Google Scholar). The mitochondrial redox environment depends on both total cellular redox environment and compartmentalized mitochondrial reduction capacity (16Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3655) Google Scholar). This reduction capacity depends on the concentration of electron donor molecules such as NADPH, NADH, and glutathione (GSH) acting as antioxidant buffers, ROS detoxification enzymes, and protein-disulfide reductases. In mammalian mitochondria, ROS detoxification is achieved by successive conversion of ⋅NO and superoxide (O2˙− into H2O2 (and O2) by manganese superoxide dismutase (MnSOD) and peroxide reduction by glutathione peroxidases (GPx1 and GPx4) and potentially by the mitochondrial thioredoxin cycle system. Because of its elevated intracellular concentration (1–10 mm), GSH is largely responsible for low redox potential and free thiol level inside cells and organelles (16Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3655) Google Scholar, 17Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar). Experimentally induced glutathione deficiency in newborn rats has been shown to result in striking enlargement and degeneration of mitochondria (18Jain A. Martensson J. Stole E. Auld P.A. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1913-1917Crossref PubMed Scopus (410) Google Scholar). A second antioxidant defense, the mitochondrial thioredoxin system, which includes Trx2, thioredoxin reductase-2, and NADPH (see below), is a potential source of disulfide reductase activity required for maintaining mitochondrial proteins in their reduced state; thioredoxins catalyze reduction of protein disulfides at much higher rates than GSH (19Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 20Arner E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2001) Google Scholar). An additional mitochondrial thiol/disulfide oxidoreductase, glutaredoxin-2 (Grx2), was recently discovered, which relies on GSH and not Trx as electron donor (21Lundberg M. Johansson C. Chandra J. Enoksson M. Jacobsson G. Ljung J. Johansson M. Holmgren A. J. Biol. Chem. 2001; 276: 26269-26275Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 22Gladyshev V.N. Liu A. Novoselov S.V. Krysan K. Sun Q.A. Kryukov V.M. Kryukov G.V. Lou M.F. J. Biol. Chem. 2001; 276: 30374-30380Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Little is known on the function(s) of the mitochondrial thioredoxin system and its potential role in the regulation of cell survival. Recent studies suggest that the mitochondrial Trx system is essential for mammalian development because disruption of the Trx2 gene in the mouse confers a lethal embryonic phenotype associated with massive apoptosis during early embryogenesis (23Nonn L. Williams R.R. Erickson R.P. Powis G. Mol. Cell. Biol. 2003; 23: 916-922Crossref PubMed Scopus (359) Google Scholar). Chicken cells (DT40) conditionally deficient for Trx2 expression have been reported to undergo apoptosis in the absence of exogenous stress (24Tanaka T. Hosoi F. Yamaguchi-Iwai Y. Nakamura H. Masutani H. Ueda S. Nishiyama A. Takeda S. Wada H. Spyrou G. Yodoi J. EMBO J. 2002; 21: 1695-1703Crossref PubMed Scopus (280) Google Scholar). In addition, glutathione depletion, serum withdrawal, and exposure to the prooxidant mitochondrial toxin, antimycin A, were all shown to enhance apoptosis in Trx2-deficient DT40 cells (24Tanaka T. Hosoi F. Yamaguchi-Iwai Y. Nakamura H. Masutani H. Ueda S. Nishiyama A. Takeda S. Wada H. Spyrou G. Yodoi J. EMBO J. 2002; 21: 1695-1703Crossref PubMed Scopus (280) Google Scholar, 25Tanaka T. Nakamura H. Nishiyama A. Hosoi F. Masutani H. Wada H. Yodoi J. Free Radic. Res. 2000; 33: 851-855Crossref PubMed Scopus (130) Google Scholar). On the other hand, overexpression of Trx2 was reported to enhance basal ΔΨm and protect human HEK-293 Trx2-transfected cells against etoposide-mediated cytotoxicity. In contrast, these cells were not protected against antimycin A cytotoxicity and turned out to be more sensitive than control cells to rotenone, another prooxidant mitochondrial toxin (26Damdimopoulos A.E. Miranda-Vizuete A. Pelto-Huikko M. Gustafsson J.A. Spyrou G. J. Biol. Chem. 2002; 277: 33249-33257Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Another study reported that Trx2 overexpression conferred increased oxidoresistance to human osteosarcoma cells exposed to tert-butylhydroperoxide (t-BHP) (26Damdimopoulos A.E. Miranda-Vizuete A. Pelto-Huikko M. Gustafsson J.A. Spyrou G. J. Biol. Chem. 2002; 277: 33249-33257Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 27Chen Y. Cai J. Murphy T.J. Jones D.P. J. Biol. Chem. 2002; 277: 33242-33248Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Thus, the effects of Trx2 overexpression appear to be complex and to depend on unidentified variables including the possibility that endogenous Trx2 may not be a limiting factor in some experimental systems. Peroxiredoxins (Prdx) form a new family of thiol-specific peroxidases that rely on Trx as the hydrogen donor for the reduction of H2O2 and lipid hydroperoxides (28Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (202) Google Scholar, 29Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar). Two mitochondrial Prdx isoforms have been identified so far. Prdx3, originally cloned from murine erythroleukemia cells (30Yamamoto T. Matsui Y. Natori S. Obinata M. Gene (Amst.). 1989; 80: 337-343Crossref PubMed Scopus (110) Google Scholar), is exclusively detected in mitochondria (31Watabe S. Hiroi T. Yamamoto Y. Fujioka Y. Hasegawa H. Yago N. Takahashi S.Y. Eur. J. Biochem. 1997; 249: 52-60Crossref PubMed Scopus (132) Google Scholar). Prdx3 expression can be induced in response to oxidant treatments, and antisense-mediated inhibition of Prdx3 expression was shown to sensitize bovine aortic endothelial cells to oxidative challenges (32Araki M. Nanri H. Ejima K. Murasato Y. Fujiwara T. Nakashima Y. Ikeda M. J. Biol. Chem. 1999; 274: 2271-2278Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Prdx3 was identified as a target gene of nuclear c-Myc and shown to be essential for maintaining mitochondrial mass and transmembrane potential in transformed rat and human cells (33Wonsey D.R. Zeller K.I. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6649-6654Crossref PubMed Scopus (154) Google Scholar). In addition, overexpression of Prdx3 was found to inhibit apoptosis induced by oxidative insults in various cancer cell lines (34Nonn L. Berggren M. Powis G. Mol. Cancer Res. 2003; 1: 682-689PubMed Google Scholar). Prdx5 is a second peroxiredoxin isoform that is located in the mitochondria when expressed as a long form, whereas a short form was found associated with peroxisomes (35Seo M.S. Kang S.W. Kim K. Baines I.C. Lee T.H. Rhee S.G. J. Biol. Chem. 2000; 275: 20346-20354Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Recently, Prdx5 overexpression was reported to be protective against ibotenate-mediated excitotoxicity (36Plaisant F. Clippe A. Vander Stricht D. Knoops B. Gressens P. Free Radic. Biol. Med. 2003; 34: 862-872Crossref PubMed Scopus (82) Google Scholar). Mammalian TrxR isoforms are flavin homodimeric oxidoreductases with an essential redox-active cysteine-selenocysteine (SeCys) conserved in their C-terminal part, which use NADPH to reduce the redox-active disulfide in oxidized Trx (37Zhong L. Arner E.S. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854-5859Crossref PubMed Scopus (410) Google Scholar, 38Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (765) Google Scholar). These enzymes can also reduce a variety of nondisulfide substrates including selenite and peroxides. The broad substrate specificity of TrxR appears to be restricted to the mammalian isoforms because yeast and bacterial TrxR isoenzymes can only reduce Trx (39Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (817) Google Scholar). A mitochondrial TrxR isoform, TrxR2, has been cloned from various mammals including mouse, cow, rat, and human (40Gasdaska P.Y. Gasdaska J.R. Cochran S. Powis G. FEBS Lett. 1995; 373: 5-9Crossref PubMed Scopus (177) Google Scholar, 41Kawai H. Ota T. Suzuki F. Tatsuka M. Gene (Amst.). 2000; 242: 321-330Crossref PubMed Scopus (20) Google Scholar, 42Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 43Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Biochim. Biophys. Acta. 1999; 1447: 113-118Crossref PubMed Scopus (38) Google Scholar, 44Miranda-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, 45Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Little is known about TrxR2 functions. The specific co-localization of TrxR2, Trx2, and Prdx3 in mitochondria, presumed to function together, may suggest a defense system against H2O2 produced by the mitochondrial respiratory chain (45Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). However, the relative contribution of this system to peroxide detoxification, as compared with the role of the GSH system, is not known. With regard to apoptosis, it was proposed that the mitochondrial thioredoxin/thioredoxin reductase system can play a role in redox regulation of the mitochondrial membrane permeability (46Wudarczyk J. Debska G. Lenartowicz E. Arch. Biochem. Biophys. 1996; 327: 215-221Crossref PubMed Scopus (34) Google Scholar, 47Rigobello M.P. Callegaro M.T. Barzon E. Benetti M. Bindoli A. Free Radic. Biol. Med. 1998; 24: 370-376Crossref PubMed Scopus (119) Google Scholar). In support of this suggestion, auranofin, a potent inhibitor of mitochondrial TrxR, was reported to induce MMP and loss of ΔΨ in isolated mitochondria, two alterations that were completely inhibited by cyclosporin A, a specific inhibitor of mitochondrial permeability transition (48Rigobello M.P. Scutari G. Boscolo R. Bindoli A. Br. J. Pharmacol. 2002; 136: 1162-1168Crossref PubMed Scopus (151) Google Scholar). Here we report the effect of transfection-mediated overexpression of TrxR2 or EGFP-tagged TrxR2 on mouse Neuro2A cell growth, mitochondrial transmembrane potential, ROS production, and viability, in response to a variety of prooxidant and non-oxidant inducers of apoptosis. Cell Culture and Reagents—Mouse Neuro2A (N2A), African green monkey COS-7, and human HeLa cell lines were purchased from ATCC. Cells were cultured at 37 °C in 5% CO2 atmosphere in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 0.1 μm sodium selenite. The fluorescent probes 3′-dihexyloxacarbocyanine iodide (DiOC6), dihydrorhodamine 123 (DHR123), dihydroethidine (HE), and tetramethylrhodamine ethyl ester (TMRE) were purchased from Molecular Probes. Unless specified otherwise, all reagents were from Sigma. Construction of Trx2, TrxR2, Mito-EGFP, and Mito-EGFP-TrxR2 Expression Plasmids—Total RNA was extracted from mouse liver using Trizol Reagent (Invitrogen). Full-length Trx2 and TrxR2 cDNAs were generated by reverse transcriptase-PCR with oligo(dT) using the RET-ROscript kit (Ambion). TrxR2 and Trx2 cDNAs were amplified by PCR with Pfu turbo polymerase using primers 83/84 and 143/144, respectively (Table I). PCR products were then isolated from agarose gels with the Qiagen gel extraction kit and subcloned in the pCR®-Blunt II-TOPO® vector (Invitrogen) before cloning into the EcoRI site of plasmid pCAGGS, i.e. downstream of a cytomegalovirus enhancer/actin promoter (49Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4597) Google Scholar), to generate pCAGGS-TrxR2 or pCAGGS-Trx2. For the construction of mito-EGFP, EGFP and the mitochondrial targeting sequence (MTS) of TrxR2 were amplified separately, MTS from pCAGGS-TrxR2 with primers 83/216 and EGFP from pEGFP-N1 (Clontech) with primers 217/219 including a stop codon. The amplified MTS and EGFP DNAs were digested with NheI, ligated together, and digested by EcoRI. Following separation of ligation products in agarose gels, DNA was isolated from the band of correct size (0.87 kb) and inserted in the EcoRI site of pCAGGS. For the construction of mito-EGFP-TrxR2, MTS was amplified from pCAGGS-TrxR2 with primers 83/216, EGFP from pEGFP-N1 with primers 217/218, and TrxR2 excluding MTS from pCAGGS-TrxR2 with primers 215/84. Amplified MTS and EGFP were digested by NheI; EGFP and TrxR2 DNA products were digested by SacI, and the three DNA preparations were mixed together for ligation. Following separation of ligation products in agarose gels, DNA was isolated from the band of correct size (2.6 kb) and inserted in the EcoRI site of pCAGGS. Recombinant plasmids with the required correct insert orientation were identified by restriction enzyme digestion. This was confirmed by DNA sequencing of whole recombinant DNAs inserted in pCAGGS.Table IPCR primers used for cloning and construction of chimeric gene recombinants Nucleotide sequences not complementary to genomic DNA are shown in lowercase letters. Restriction sites used for cloning are underlined, and the enzymes are indicated in parentheses. Letters in boldface show nucleotide changes introduced in order to generate NheI (primer 217) or SacI (primer 218) restriction sites. Lowercase italic letters represent nucleotides added to reconstitute the EGFP coding frame.PrimerPrimer sequence835′-atatgaattcAGCCCTGACCGCACTGAGGTG-3′ (EcoRI)845′-atatgaattcGTAAAACATCCTCTTTATTAGTGCTCTAG-3′ (EcoRI)1435′-atatctcgagCAGGCCGGCTGGG-3′ (XhoI)1445′-ttttctcgagGGCAGTGCAAACCTGG-3′ (XhoI)2155′-atatgagctctacaaaAGTGCAGCGGGAGGG-3′ (SacI)2165′-atatatatagctagccatACTCGCCGCGCCCC-3′ (NheI)2175′-atatATGGctagcAAGGGCGAGGAG-3′ (NheI)2185′-GACTTGTAgAGCTCGTCCATGCC-3′ (SacI)2195′-atatgaattcaCTTGTACAGCTCGTCCATGCC-3′ (EcoRI) Open table in a new tab Transfections—Neuro2A transfectant cells expressing exogenous TrxR2, Trx2, or EGFP fusion proteins to various extents were obtained by calcium phosphate transfections (50Maniatis T. Sambrook J. Fritsch E.F. 2nd Ed. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 33-36Google Scholar) using increasing ratios of pCAGGS constructs to pINDsp1-Hygro plasmid (for selection with hygromycin-B, Invitrogen). The basal amount of pINDsp1-Hygro was always 1 μg per 100-mm culture dish (Sarstedt). Stable transfectant populations were generated with the following pCAGGS constructs using pCAGGS/pINDsp1-Hygro μg DNA ratios as indicated in parentheses: CtrlH63, pCAGGS (30/1); TrxR2H56, pCAGGS-TrxR2 (10/1); TrxR2H64, pCAGGS-TrxR2 (30/1); Trx2H161, pCAGGS-Trx2 (30/1); Trx2/TrxR2H162, pCAGGS-Trx2 (30/1) and pCAGGS-TrxR2 (30/1); TrxR2H163, pCAGGS-TrxR2 (60/1); mito-EGFPH143, pCAGGS-mito-EGFP (30/1); and EGFP-TrxR2H144, pCAGGS-EGFP-TrxR2 (30/1). Selection for hygromycin-B (500 μg/ml)-resistant cells started 48 h post-transfection for at least 2 weeks. Resistant colonies from each transfection were pooled to produce a heterogeneous population of transfected cells. The clone TrxR2c169 derives from one colony isolated from a pCAGGS-TrxR2 transfectant population. FuGENE 6 reagent (Roche Applied Science) was used for transient transfections according to the procedure recommended by the manufacturer. In brief, a ratio of 3/1 for FuGENE reagent (μl)/plasmid (μg) was incubated for 30 min at room temperature in incomplete medium before addition to 50% subconfluent cells in complete medium for 24 h. Cell Fractionation—Cell fractionation was carried out by differential centrifugation mainly as described previously (51Jana N.R. Zemskov E.A. Wang G. Nukina N. Hum. Mol. Genet. 2001; 10: 1049-1059Crossref PubMed Scopus (384) Google Scholar). In brief, cells (2–4 × 107) were lysed with 60 Dounce strokes with a tight fitting pestle in buffered sucrose (BS): 20 mm Hepes, pH 7.5, 250 mm sucrose, 1 mm EDTA, 1 mm EGTA, 10 mm KCl, and a tablet/10 ml of complete mix protease inhibitors (Roche Applied Science). After two centrifugations at 1,000 × g to discard nuclei, mitochondria were pelleted at 10,000 × g, washed once, and resuspended in BS before storage at –80 °C. The cytosolic fraction was obtained by centrifugation of the post-mitochondrial supernatant at 100,000 × g for 1 h. Protein contents were determined by Bradford assay (Bio-Rad). Antibodies and Western Blot Analysis—Polyclonal antibodies against mouse Trx2, Prdx3, and TrxR2 were produced by injecting rabbits with hemocyanin-conjugated peptides, CLEAFLKKLIG for Trx2, CSPTASKEYFEKVHQ for Prdx3, and KRSGLEPTVTGCCG for TrxR2, respectively. The peptides were injected in complete Freund's adjuvant for the first immunization and with incomplete adjuvant 6 weeks later for three immunization boosts administered every 3 weeks. Affinity-purified GPx1 antibody was prepared from a rabbit antiserum produced by repeated immunization with recombinant human GPx1 expressed in bacteria (52Mirault M.E. Tremblay A. Beaudoin N. Tremblay M. J. Biol. Chem. 1991; 266: 20752-20760Abstract Full Text PDF PubMed Google Scholar), with three boosts at 2-week intervals followed by three boosts at 1-month intervals using bovine GPx protein (Sigma G-6137). The antibody was purified by affinity for bovine GPx isolated by SDS-PAGE and electrotransfer on polyvinylidene difluoride membrane. All antibodies were stored at –20 °C after addition of 50% glycerol. SDS-PAGE (10%) was performed with equal amounts of protein (10 μg per track) separated in mini gels (Mini-Protean II, Bio-Rad) and electrotransferred onto polyvinylidene difluoride membranes. The membranes were saturated with TBS, 0.1% Tween 5% before incubation with antibody. The antibodies were used at the following dilutions: 1/5,000 for TrxR2, Trx2 (second boost), and Cox IV (Molecular Probes A-6431); 1/20,000 for Prdx3 (second boost) and β-actin (Sigma A5441); 1/10,000 for MnSOD (Upstate Biotechnology, Inc., 06-984); and 1/500 for purified GPx1 antibody. Antibody incubations were done at room temperature for 2–3 h in TBS, 0.1% Tween + 1% skim milk powder, except for MnSOD and GPx1, which were incubated overnight at 4 °C and 2 h at 37 °C, respectively, and without milk for GPx1 antibody. Enzyme Assays—TrxR and Trx activities were measured using the insulin reduction assay as described previously (39Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (817) Google Scholar) with minor modifications. Cytosolic or sonicated (30 s) total extract or mitochondrial fractions (25 μg of protein) in BS buffer (30 μl) were added to 20 μl of enzymatic assay mixture to the final concentrations as follows: 85 mm Hepes, pH 7.5, 3.4 mm EDTA, 0.68 mm NADPH, 5 μm human Trx (Sigma), and 2.14 mg/ml insulin in a 96-well microplates. The 50-μl reaction mixtures were incubated at 37 °C for 20 min, and the reaction was stopped by adding 200 μl of 6 m guanidine HCl in 0.1 m Tris, pH 8, containing 0.4 mg/ml 5,5′-dithiobis(2-nitrobenzoic) acid. After 10 min at room temperature, the absorbance was read at 405 nm, and TrxR specific activity was calculated from the relation: units/g = A/20 min/27.2/g protein. Backgrounds from reactions without Trx reflecting the contribution of endogenous sulfhydryl groups in the extracts were subtracted. Trx activity was determined in the insulin reduction assay as described above except that bovine TrxR (American Diagnostica, Greenwich, CT) was used instead of human Trx. Glutathione Assay—Total glutathione equivalents (GSH + GSSG) were determ" @default.
• W2061018077 created "2016-06-24" @default.
• W2061018077 creator A5024868883 @default.
• W2061018077 creator A5084126110 @default.
• W2061018077 creator A5086242516 @default.
• W2061018077 date "2004-06-01" @default.
• W2061018077 modified "2023-10-18" @default.
• W2061018077 title "Mitochondrial Thioredoxin System" @default.
• W2061018077 cites W1199623702 @default.
• W2061018077 cites W121254590 @default.
• W2061018077 cites W1480948328 @default.
• W2061018077 cites W1510054089 @default.
• W2061018077 cites W1536961425 @default.
• W2061018077 cites W1550007121 @default.
• W2061018077 cites W1574486032 @default.
• W2061018077 cites W1591929009 @default.
• W2061018077 cites W1595716614 @default.
• W2061018077 cites W1604304380 @default.
• W2061018077 cites W1670885862 @default.
• W2061018077 cites W17855639 @default.
• W2061018077 cites W187982100 @default.
• W2061018077 cites W1966920910 @default.
• W2061018077 cites W1968797108 @default.
• W2061018077 cites W1970294266 @default.
• W2061018077 cites W1970519591 @default.
• W2061018077 cites W1972801090 @default.
• W2061018077 cites W1973085272 @default.
• W2061018077 cites W1983397667 @default.
• W2061018077 cites W1988943327 @default.
• W2061018077 cites W1991220156 @default.
• W2061018077 cites W1994977897 @default.
• W2061018077 cites W1996311437 @default.
• W2061018077 cites W1997385369 @default.
• W2061018077 cites W2002593979 @default.
• W2061018077 cites W2005480061 @default.
• W2061018077 cites W2005904769 @default.
• W2061018077 cites W2008757391 @default.
• W2061018077 cites W2009691812 @default.
• W2061018077 cites W2013305709 @default.
• W2061018077 cites W2016927494 @default.
• W2061018077 cites W2018832587 @default.
• W2061018077 cites W2019666203 @default.
• W2061018077 cites W2020400942 @default.
• W2061018077 cites W2024067398 @default.
• W2061018077 cites W2028000670 @default.
• W2061018077 cites W2031060436 @default.
• W2061018077 cites W2034327787 @default.
• W2061018077 cites W2042584010 @default.
• W2061018077 cites W2048162805 @default.
• W2061018077 cites W2048971811 @default.
• W2061018077 cites W2053090763 @default.
• W2061018077 cites W2056743747 @default.
• W2061018077 cites W2056813366 @default.
• W2061018077 cites W2060833911 @default.
• W2061018077 cites W2061958603 @default.
• W2061018077 cites W2063385957 @default.
• W2061018077 cites W2063605230 @default.
• W2061018077 cites W2065020384 @default.
• W2061018077 cites W2065619292 @default.
• W2061018077 cites W2070497052 @default.
• W2061018077 cites W2071497993 @default.
• W2061018077 cites W2072623280 @default.
• W2061018077 cites W2074482029 @default.
• W2061018077 cites W2076528366 @default.
• W2061018077 cites W2088304415 @default.
• W2061018077 cites W2088688594 @default.
• W2061018077 cites W2092317255 @default.
• W2061018077 cites W2098644206 @default.
• W2061018077 cites W2101575450 @default.
• W2061018077 cites W2102259132 @default.
• W2061018077 cites W2103650142 @default.
• W2061018077 cites W2105988137 @default.
• W2061018077 cites W2108102646 @default.
• W2061018077 cites W2113999599 @default.
• W2061018077 cites W2123671636 @default.
• W2061018077 cites W2132615347 @default.
• W2061018077 cites W2132826785 @default.
• W2061018077 cites W2132878122 @default.
• W2061018077 cites W2140513457 @default.
• W2061018077 cites W2148294732 @default.
• W2061018077 cites W2149441947 @default.
• W2061018077 cites W2158748165 @default.
• W2061018077 cites W2163005382 @default.
• W2061018077 cites W2271272206 @default.
• W2061018077 cites W280716631 @default.
• W2061018077 cites W4247896860 @default.
• W2061018077 cites W4251161705 @default.
• W2061018077 cites W758260955 @default.
• W2061018077 doi "https://doi.org/10.1074/jbc.m402496200" @default.
• W2061018077 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15082714" @default.
• W2061018077 hasPublicationYear "2004" @default.
• W2061018077 type Work @default.
• W2061018077 sameAs 2061018077 @default.
• W2061018077 citedByCount "102" @default.
• W2061018077 countsByYear W20610180772012 @default.
• W2061018077 countsByYear W20610180772013 @default.
• W2061018077 countsByYear W20610180772014 @default.
• W2061018077 countsByYear W20610180772015 @default.

• next