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  • 1.
    Andersson, M.
    et al.
    Karolinska Institute, Stockholm, Sweden.
    Holmgren, A.
    Karolinska Institute, Stockholm, Sweden.
    Spyrou, Giannis
    Karolinska Institute, Stockholm, Sweden and Karolinska Institute, Novum, Huddinge, Sweden.
    NK-lysin, a disulfide-containing effector peptide of T-lymphocytes, is reduced and inactivated by human thioredoxin reductase. Implication for a protective mechanism against NK-lysin cytotoxicity1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 17, p. 10116-10120Article in journal (Refereed)
    Abstract [en]

    The cytotoxic and antibacterial polypeptide NK-lysin has a molecular mass of approximately 9 kDa and contains three disulfide bonds. The activity was highly dependent on intact disulfides, because the bactericidal effect on Escherichia coli and the cytolytic effect on human 3B6 lymphocytes was inhibited when NK-lysin was treated with dithiothreitol prior to incubation with the cells. NK-lysin was a direct substrate for human or calf thymus thioredoxin reductase and preincubation of the peptide with mammalian thioredoxin reductase, and NADPH abolished its antibacterial and cytolytic activities. The addition of human thioredoxin further enhanced the inhibitory effect of thioredoxin reductase and NADPH. In contrast, e. coli thioredoxin reductase showed no direct disulfide reductase activity with NK-lysin in agreement with previous data showing large differences in structure and substrate specificity between the mammalian and E. coli enzymes. NK-lysin is the first identified macromolecular disulfide substrate for human thioredoxin reductase apart from human thioredoxin. When 3B6 cells were incubated with NADPH, thioredoxin, and thioredoxin reductase prior to addition of NK-lysin, cytotoxicity was markedly reduced. These data suggest that thioredoxin reductase inactivates NK-lysin and provides a mechanism by which the cytotoxic activity of NK-lysin is regulated.

  • 2.
    Arnér, Elias S. J.
    et al.
    Karolinska Institute, Stockholm, Sweden.
    Nakamura, H.
    Kyoto University, Japan.
    Sasada, Tetsuro
    Karolinska Institute, Huddinge, Sweden.
    Yodoi, Junji
    Karolinska Institute, Huddinge, Sweden.
    Holmgren, Arne
    Karolinska Institute, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institute, Huddinge, Sweden.
    Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex2001In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 31, no 10, p. 1170-1178Article in journal (Refereed)
    Abstract [en]

    Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.

  • 3.
    Björnstedt, M.
    et al.
    Karolinska Institute, Stockholm, Sweden.
    Kumar, S.
    Karolinska Institute, Stockholm, Sweden.
    Björkhem, L.
    Karolinska Institute, Stockholm, Sweden.
    Spyrou, Giannis
    Karolinska Institute, Stockholm, Sweden.
    Holmgren, A.
    Karolinska Institute, Stockholm, Sweden.
    Selenium and the thioredoxin and glutaredoxin systems1997In: Biomedical and environmental sciences, ISSN 0895-3988, E-ISSN 2214-0190, Vol. 10, no 2-3, p. 271-279Article in journal (Refereed)
    Abstract [en]

    Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FAD-containing enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a K(m)-value (6 mumol.L-1) and a high turnover number (kappa cat 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH-dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the human plasma glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furthermore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SECIS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of selenium for cell proliferation.

  • 4.
    Bohm, S.
    et al.
    Center for BioTechnology, NOVUM, Huddinge, Sweden.
    Bakke, M.
    Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden.
    Nilsson, M.
    Center for BioTechnology, NOVUM, Huddinge, Sweden.
    Zanger, U. M.
    Department of Pharmacology, Biocenter, Basel, Switzerland.
    Spyrou, Giannis
    Department of Medical Chemistry I, Karolinska Institute, Stockholm, Sweden.
    Lund, J.
    Center for BioTechnology, NOVUM, Huddinge, Sweden.
    Cooperating nonconsensus cAMP-responsive elements are mediators of adrenocorticotropin-induced VL30 transcription in steroidogenic adrenal cells1993In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 268, no 6, p. 3952-3963Article in journal (Refereed)
    Abstract [en]

    Pituitary-derived trophic hormones regulate cell-type-specific expression of VL30 retrotransposons in tissues that are engaged in steroidogenesis. We show that adrenocorticotropic hormone and forskolin induced VL30 transcription in the steroidogenic adrenal cell line Y1 and that the transcriptional activation was cell type- and protein kinase A-dependent. Three novel cAMP-responsive elements (CREs), within the VL30 long terminal repeat, were identified and shown to activate transcription synergistically when templates bearing multiple sites were compared with templates bearing a single site. This type of regulation was evident only in forskolin-treated cells, and the response elements were found to be inactive as mediators of constitutive transcription. In vitro binding analyses indicated that a consensus CRE and the nonconsensus VL30 CREs differ with respect to binding affinity and specificity to a number of nuclear factors that were identified to be related to proteins within the CREB, Jun, and C/EBP families of transcription factors. The relatively low affinity and/or a restricted binding specificity of the VL30 CREs made it possible to detect forskolin-induced binding of CREB- and Jun-related proteins to these sequences. We suggest that cAMP-induced transcription, specific for steroidogenic cells, can be mediated by a novel type of nonconsensus CREs and that the mechanism for this type of gene regulation is distinct from that mediated through a consensus CRE. We also report the identification of a novel factor, distinct from previously characterized CRE-binding proteins, that constitutively binds to the identified CREs.

  • 5.
    Castellazzi, M.
    et al.
    Ecole Normale Supérieure, Lyon, France.
    Spyrou, Giannis
    Institut Pasteur, Paris, France.
    La Vista, N.
    Ecole Normale Supérieure, Lyon, France.
    Dangy, J-P
    Ecole Normale Supérieure, Lyon, France.
    Piu, F.
    Ecole Normale Supérieure, Lyon, France.
    Yaniv, M.
    Institut Pasteur, Paris, France.
    Brun, G.
    Ecole Normale Supérieure, Lyon, France.
    Overexpression of c-jun, junB, or junD affects cell growth differently1991In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 88, no 20, p. 8890-8894Article in journal (Refereed)
    Abstract [en]

    The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.

  • 6.
    Chantzoura, Eleni
    et al.
    Center of Basic Research I, Biochemistry Division, Biomedical Research Foundation, Academy of Athens, Greece.
    Prinarakis, Efthimios
    Center of Basic Research I, Biochemistry Division, Biomedical Research Foundation, Academy of Athens, Greece.
    Panagopoulos, Dimitris
    School of Biology, Aristotle University of Thessaloniki, Greece.
    Mosialos, George
    School of Biology, Aristotle University of Thessaloniki, Greece.
    Spyrou, Giannis
    Center of Basic Research I, Biochemistry Division, Biomedical Research Foundation, Academy of Athens, Greece.
    Glutaredoxin-1 regulates TRAF6 activation and the IL-1 receptor/TLR4 signalling2010In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 403, no 3-4, p. 335-339Article in journal (Refereed)
    Abstract [en]

    Glutaredoxin-1 (GRX-1) is a cytoplasmic enzyme that highly contributes to the antioxidant defense system. It catalyzes the reversible reduction of glutathione-protein mixed disulfides, a process called deglutathionylation. Here, we investigated the role of GRX-1 in the pathway triggered by interleukin-1/Toll-like receptor 4 (IL-1R/TLR4) by using RNA interference (RNAi) in HEK293 and HeLa cells. TNF receptor-associated factor 6 (TRAF6) is an intermediate signalling molecule involved in the signal transduction by members of the interleukin-1/Toll-like receptor (IL-1R/TLR) family. TRAF6 has an E3 ubiquitin ligase activity which depends on the integrity of an amino-terminal really interesting new gene (RING) finger motif. Upon receptor activation, TRAF6 undergoes K63-linked auto-polyubiquitination which mediates protein-protein interactions and signal propagation. Our data showed that IL-1R and TLR4-mediated NF-κB induction was severely reduced in GRX-1 knockdown cells. We found that the RING-finger motif of TRAF6 is S-glutathionylated under normal conditions. Moreover, upon IL-1 stimulation TRAF6 undergoes deglutathionylation catalyzed by GRX-1. The deglutathionylation of TRAF6 is essential for its auto-polyubiquitination and subsequent activation. Taken together, our findings reveal another signalling molecule affected by S-glutathionylation and uncover a crucial role for GRX-1 in the TRAF6-dependent activation of NF-κB by IL-1R/TLRs.

  • 7.
    Chen, J.
    et al.
    Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Boston, Massachusetts, USA.
    Stewart, V.
    Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Boston, Massachusetts, USA.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Hilberg, F.
    Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Boston, Massachusetts, USA.
    Wagner, E. F.
    Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Boston, Massachusetts, USA.
    Alt, F. W.
    Howard Hughes Medical Institute, Children's Hospital, Department of Genetics, Boston, Massachusetts, USA.
    Generation of normal T and B lymphocytes by c-jun deficient embryonic stem cells1994In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 1, no 1, p. 65-72Article in journal (Refereed)
    Abstract [en]

    To determine the potential roles of c-jun in lymphocyte development, we generated somatic chimeric mice by injecting homozygous c-jun mutant embryonic stem (ES) cells into blastocysts from recombination activating gene-2 (RAG-2)-deficient mice. Chimeric mice had poor restoration of thymocytes, but contained substantial numbers of mature T and B lymphocytes in the periphery. Stimulation of c-jun-/- B cells resulted in normal levels of proliferation and immunoglobulin secretion. Likewise, stimulation of c-jun-/- T cells resulted in essentially normal levels of IL-2R alpha expression, IL-2 secretion, and proliferation. We further showed that the relatively normal activation responses of the c-jun-/- T cells probably results from the fact that other members of the Jun family contribute to the bulk of the activator protein-1 (AP-1) complexes in normal T cells and, as a result, AP-1 complexes are found at relatively normal levels in c-jun-/- T cells.

  • 8.
    Cunnea, P.
    et al.
    Department of Biosciences and Nutrition at NOVUM, Karolinska Institutet, Stockholm, Sweden.
    Fernandes, AP
    Department of Laboratory Medicine, Pathology Division, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
    Capitanio, A.
    Department of Laboratory Medicine, Pathology Division, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
    Eken, S.
    Department of Laboratory Medicine, Pathology Division, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences and Nutrition at NOVUM, Karolinska Institutet, Stockholm / Foundation for Biomedical Research, Academy of Athens, Athens, Greece.
    Björnstedt, M.
    Department of Laboratory Medicine, Pathology Division, Karolinska University Hospital, Huddinge, Stockholm, Sweden.
    Increased expression of specific thioredoxin family proteins; a pilot immunohistochemical study on human hepatocellular carcinoma2007In: International journal of immunopathology and pharmacology, ISSN 0394-6320, Vol. 20, no 1, p. 17-24Article in journal (Refereed)
    Abstract [en]

    Hepatocellular Carcinoma (HCC) is one of the most frequent cancers worldwide, however, prognosis remains poor following its discovery. We investigate the Thioredoxin superfamily of proteins as diagnostic markers for HCC. Furthermore, we delineate possible roles of the endoplasmic reticulum member of the superfamily, ERdj5, in carcinogenesis. Using antibodies against Thioredoxin 1, Thioredoxin Reductase 1 and ERdj5, we performed immunohistochemistry on paraffin embedded liver biopsy sections from HCC patients. All three redox proteins exhibited elevated expression levels in tumor tissue compared to internal control, with ERdj5 showing a remarkable 3-fold increase. In vitro cell viability experiments using Hepatocellular Carcinoma HuH7 cells treated with ERdj5 small interfering RNA showed that ERdj5 knockdown cells exhibited less resistance to Doxorubicin (chemotherapy drug), but more resistance to Tunicamycin (Endoplasmic Stress inducer), compared to control cells. In conclusion, we introduce members of the Thioredoxin superfamily as possible immunohistochemical markers in the diagnostics of hepatocellular carcinoma and indicate a potential defensive role for ERdj5 in chemotherapeutic drug resistance.

  • 9.
    Cunnea, Paula M
    et al.
    Centre for Biotechnology, Department of Biosciences at Novum, Karolinska Institute, Södertörns Högskola, S-14157 Huddinge, Sweden..
    Miranda-Vizuete, Antonio
    Centre for Biotechnology, Department of Biosciences at Novum, Karolinska Institute, Södertörns Högskola, S-14157 Huddinge, Sweden..
    Bertoli, Gloria
    Università Vita-Salute San Raffaele, 20132 Milan, Italy.
    Simmen, Thomas
    Università Vita-Salute San Raffaele, 20132 Milan, Italy.
    Damdimopoulos, Anastasios E
    Centre for Biotechnology, Department of Biosciences at Novum, Karolinska Institute, Södertörns Högskola, S-14157 Huddinge, Sweden..
    Hermann, Stefan
    Centre for Biotechnology, Department of Biosciences at Novum, Karolinska Institute, Södertörns Högskola, S-14157 Huddinge, Sweden..
    Leinonen, Saku
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, SE-141 57 Huddinge, Sweden.
    Huikko, Markku Pelto
    Department of Pathology, Tampere University Hospital, Fin-33101 Tampere, Finland.
    Gustafsson, Jan-Ake
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, SE-141 57 Huddinge, Sweden.
    Sitia, Roberto
    Department of Biological and Technological Research, San Raffaele Scientific Institute / Università Vita-Salute San Raffaele, 20132 Milan, Italy.
    Spyrou, Giannis
    Centre for Biotechnology, Department of Biosciences at Novum, Karolinska Institute, Södertörns Högskola, S-14157 Huddinge, Sweden..
    ERdj5, an endoplasmic reticulum (ER)-resident protein containing DnaJ and thioredoxin domains, is expressed in secretory cells or following ER stress.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 2, p. 1059-66Article in journal (Refereed)
    Abstract [en]

    A complex array of chaperones and enzymes reside in the endoplasmic reticulum (ER) to assist the folding and assembly of and the disulfide bond formation in nascent secretory proteins. Here we characterize a novel human putative ER co-chaperone (ERdj5) containing domains resembling DnaJ, protein-disulfide isomerase, and thioredoxin domains. Homologs of ERdj5 have been found in Caenorhabditis elegans and Mus musculus. In vitro experiments demonstrated that ERdj5 interacts via its DnaJ domain with BiP in an ATP-dependent manner. ERdj5 is a ubiquitous protein localized in the ER and is particularly abundant in secretory cells. Its transcription is induced during ER stress, suggesting potential roles for ERdj5 in protein folding and translocation across the ER membrane.

  • 10.
    Damdimopoulos, Anastasios E.
    et al.
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden / Foundation for Biomedical Research, Academy of Athens, Athens, Greece.
    Nuclear immobilization of DsRed1 tagged proteins: a novel tool for studying DNA-protein interactions?2007In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1773, no 6, p. 687-690Article in journal (Refereed)
    Abstract [en]

    DsRed1 is a red fluorescent protein that can be used as a fusion partner with other proteins to determine their subcellular localization, similarly to the popular green fluorescent proteins (GFP). Here, we report that fusion of DsRed1 to estrogen receptor alpha (ER alpha) renders the transcription factor immobile within the nucleus. Furthermore, we show that the immobilization is dependent on DNA interaction and that the binding to the DNA can be direct as well as indirect for DsRed to immobilize with its fusion partners. This observation could provide a new tool to be used for the identification of target genes containing low affinity binding sites for several transcription factors including ER alpha. In addition, it could be employed for studies on protein-DNA interactions as well as protein-protein interactions during protein complex formation on chromatin in the event of transcription initiation and regulation.

  • 11.
    Damdimopoulos, Anastasios E.
    et al.
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Miranda-Vizuete, Antonio
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Pelto-Huikko, Markku
    Department of Developmental Biology, Tampere University Hospital, Finland.
    Gustafsson, Jan-Åke
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 36, p. 33249-33257Article in journal (Refereed)
    Abstract [en]

    Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, DeltaPsi(m). Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.

  • 12.
    Damdimopoulos, Anastasios E.
    et al.
    Department of Biosciences, Novum, Karolinska Institute, Huddinge, Sweden.
    Miranda-Vizuete, Antonio
    Department of Biosciences, Novum, Karolinska Institute, Huddinge, Sweden.
    Treuter, Eckardt
    Department of Biosciences, Novum, Karolinska Institute, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Department of Biosciences, Novum, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences, Novum, Karolinska Institute, Huddinge, Sweden.
    An alternative splicing variant of the selenoprotein thioredoxin reductase is a modulator of estrogen signaling2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 37, p. 38721-38729Article in journal (Refereed)
    Abstract [en]

    The selenoprotein thioredoxin reductase (TrxR1) is an integral part of the thioredoxin system. It serves to transfer electrons from NADPH to thioredoxin leading to its reduction. Interestingly, recent work has indicated that thioredoxin reductase can regulate the activity of transcription factors such as p53, hypoxia-inducible factor, and AP-1. Here, we describe that an alternative splicing variant of thioredoxin reductase (TrxR1b) containing an LXXLL peptide motif, is implicated in direct binding to nuclear receptors. In vitro interaction studies revealed direct interaction of the TrxR1b with the estrogen receptors alpha and beta. Confocal microscopy analysis showed nuclear colocalization of the TrxR1b with both estrogen receptor alpha and beta in estradiol-17beta-treated cells. Transcriptional studies demonstrated that TrxR1b can affect estrogen-dependent gene activation differentially at classical estrogen response elements as compared with AP-1 response elements. Based on these results, we propose a model where thioredoxin reductase directly influences the estrogen receptor-coactivator complex assembly on non-classical estrogen response elements such as AP-1. In summary, our results suggest that TrxR1b is an important modulator of estrogen signaling.

  • 13.
    Damdimopoulos, Anastasios E.
    et al.
    Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden / Foundation for Biomedical Research, Academy of Athens, Athens, Greece.
    Gustafsson, Jan-Åke
    Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden.
    Ligands differentially modify the nuclear mobility of estrogen receptors alpha and beta2008In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 149, no 1, p. 339-345Article in journal (Refereed)
    Abstract [en]

    Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor alpha (ERalpha) and ERbeta mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERalpha, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERalpha. Interestingly, we found that for ERbeta such a classification was not possible because ERbeta ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERalpha, whereas ERbeta retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERbeta retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.

  • 14.
    Diamanti, E.
    et al.
    Departments of Endodontics and Basic Sciences, Dental School, University of Athens, Athens, Greece / Biochemistry Division, Foundation for Biomedical Research, Academy of Athens, Athens, Greece.
    Mathieu, S.
    INSERM UMR 911, CR02, Aix-Marseille Université, Marseille, France.
    Jeanneau, C.
    Aix-Marseille Université, CNRS, ISM UMR 7287, Marseille cedex 09, France.
    Kitraki, E.
    Departments of Endodontics and Basic Sciences, Dental School, University of Athens, Athens, Greece.
    Panopoulos, P.
    Departments of Endodontics and Basic Sciences, Dental School, University of Athens, Athens, Greece.
    Spyrou, G.
    Biochemistry Division, Foundation for Biomedical Research, Academy of Athens, Athens, Greece.
    About, I.
    Aix-Marseille Université, CNRS, ISM UMR 7287, Marseille cedex 09, France.
    Endoplasmic reticulum stress and mineralization inhibition mechanism by the resinous monomer HEMA2013In: International Endodontic Journal, ISSN 0143-2885, E-ISSN 1365-2591, Vol. 46, no 2, p. 160-168Article in journal (Refereed)
    Abstract [en]

    AIM: To investigate the expression of two endoplasmic reticulum (ER)-resident key chaperone proteins, ERdj5 and BiP, under the influence of resinous monomers and its relationship with the inhibition of mineralization caused by the monomer 2-hydroxyethyl methacrylate (HEMA).

    METHODOLOGY: The ERdj5 and BiP expression was studied in vitro, in primary human pulp cell cultures after treatment with three different HEMA concentrations at different time periods. Subsequently, the expression of both the odontoblast markers dentine sialoprotein (DSP) and osteonectin (OSN) was studied in human pulp cells under the same conditions.

    RESULTS: The ERdj5 and BiP expression was upregulated in the pulp cells. DSP and OSN were largely dispersed in the cytoplasm in control cell cultures but accumulated in a perinuclear area after exposure to HEMA. Their expression levels were not affected.

    CONCLUSIONS: The increased expression of ERdj5 and BiP may reflect activation of ER stress. DSP and OSN accumulation into the cells may lead to their secretion arrest and inhibition of dentine matrix formation. These events may elucidate the mechanism by which HEMA inhibits the mineralization process.

  • 15.
    Doucas, V.
    et al.
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Spyrou, Giannis
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Yaniv, M.
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Unregulated expression of c-Jun or c-Fos proteins but not Jun D inhibits oestrogen receptor activity in human breast cancer derived cells1991In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 10, no 8, p. 2237-2245Article in journal (Refereed)
    Abstract [en]

    We present evidence that oestrogen receptor activity in human MCF-7 breast cancer cells is reduced by over-expression of c-Jun or c-Fos proteins and to a lesser extent by Jun B overexpression. In contrast, overexpression of Jun D protein does not affect the activity of the oestrogen receptor. A region of c-Jun found to be required for repression of oestrogen receptor activity is located outside the DNA binding domain and is not conserved among the three Jun proteins. Finally, we suggest that c-Jun and c-Fos act independently to inactivate the oestrogen receptor.

  • 16.
    Hirai, S.
    et al.
    Yokohama City University School of Medicine, Japan.
    Izawa, M.
    Yokohama City University School of Medicine, Japan.
    Osada, S.
    Yokohama City University School of Medicine, Japan.
    Spyrou, Giannis
    Karolinska Institute, Stockholm, Sweden.
    Ohno, S.
    Yokohama City University School of Medicine, Japan.
    Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK1996In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 12, no 3, p. 641-650Article in journal (Refereed)
    Abstract [en]

    JNK/SAPKs are identified as new members of the MAPK family; they phosphorylate c-Jun protein in response to several cellular stimuli including ultraviolet irradiation, TNF and osmotic shock. We have identified a protein kinase, MUK, as an activator of the JNK-pathway, whose kinase domain shows significant homology to MAPKKK-related proteins such as c-Raf and MEKK. The over-expression of MUK or MEK kinase (MEKK) in NIH3T3 or COS1 cells results in the activation of JNK1 and the accumulation of a hyper-phosphorylated form of c-Jun. While MEKK also activates the ERK pathway, MUK is a rather selective activator of the JNK pathway. On the other hand, c-Raf activates the JNK pathway only slightly despite its remarkable ability to activate the ERK pathway. Even though we originally identified MUK as a MAPKKK-related protein kinase, a greater similarity to mixed lineage kinase (MLK) is found not only in the catalytic domain but also in the 'leucine-zipper'-like motifs located at the C-terminal side of the catalytic domain. The structural divergence between MUK and MEKK reveals the multiplicity of signaling pathways that activate JNK/SAPKs.

  • 17.
    Ingelsson, Björn
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Söderberg, Daniel
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Strid, Tobias
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Söderberg, Anita
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Bergh, Ann-Charlotte
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Loitto, Vesa-Matti
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Lotfi, Kourosh
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Haematology.
    Segelmark, Mårten
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Heart and Medicine Center, Department of Nephrology.
    Spyrou, Giannis
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Rosén, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 3, p. E478-E487Article in journal (Refereed)
    Abstract [en]

    Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

  • 18.
    Jiang, Kai
    et al.
    Karolinska Institutet, Stockholm, Sweden.
    Spyrou, Giannis
    Karolinska Institutet, Stockholm, Sweden.
    Rökaeus, Åke
    Karolinska Institutet, Stockholm, Sweden.
    Characterization of phorbolester-inducible human neuronal factors involved in trans-activation of the galanin gene1998In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 246, no 1, p. 192-198Article in journal (Refereed)
    Abstract [en]

    The expression of the neuropeptide galanin (GAL) is elevated in vivo upon nerve stimulation, injury, and in vitro by phorbol 12-myristate-13-acetate (PMA), suggesting that a signal pathway involving protein kinase C activation may be involved in GAL-gene activation. When plasmids containing a different length of the bovine GAL-promoter fused to luciferase were transfected into the human neuroblastoma cell line (SK-N-SH subclone SH-SY5Y), a PMA-responsive element was identified in the promoter-region -68 to -46 base pairs (bp). Co-transfection experiments with plasmids expressing cJun and cFos revealed that they could act alone, as well as synergistically with PMA to induce luciferase activity. Electrical mobility shift assays revealed that a cAMP response element (CRE)-like sequence (TGACGCGG; -59 to -52 bp) bound PMA-inducible nuclear proteins present in SH-SY5Y cells. These proteins appear to bind mainly as CRE-binding protein/activating-transcription-factor (CREB/ATF) and Jun/ATF heterodimers. In addition, an apparent PMA-inducible protein(s) not recognized by CREB/ATF and Jun antibodies bound to the CRE-like containing probe.

  • 19.
    Jiménez, Alberto
    et al.
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Oko, Richard
    Queen's University, Kingston, Ontario, Canada.
    Gustafsson, Jan-Åke
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Pelto-Huikko, Markku
    Tampere University Medical School and Tampere University Hospital, Finland.
    Miranda-Vizuete, Antonio
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Cloning, expression and characterization of mouse spermatid specific thioredoxin-1 gene and protein2002In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 8, no 8, p. 710-718Article in journal (Refereed)
    Abstract [en]

    Thioredoxins are proteins that participate in different cellular processes via redox-mediated reactions. For humans, we have recently described two novel members of this family that display a male germ cell specific expression pattern, named spermatid specific thioredoxin (Sptrx-1 and Sptrx-2 respectively). We report here the cloning and characterization of the mouse Sptrx-1 gene and protein, which are similar to those described for the human orthologue. The mouse Sptrx-1 open reading frame encodes for a protein of 462 aa composed of an N-terminal repetitive domain of a 15 residue motif followed by a C-terminal domain typical of thioredoxins. The mouse Sptrx-1 gene sequence is interrupted by only one intron of 525 bp located in the 5'-UTR, and using fluorescence in-situ hybridization we have mapped its chromosomal location to 17E1.2-1.3. Northern blot analysis identified the testis as the only tissue expressing mouse Sptrx-1 mRNA, and by in-situ hybridization we found a strong labelling in the testicular seminiferous tubules, mostly in the round spermatids. Affinity purified antibodies against human Sptrx-1 crossreacted well with the mouse protein confirming its expression in seminiferous tubules at the later stages of spermiogenesis. Recombinant mouse Sptrx-1 displayed protein disulphide reducing activity in an enzymatic assay coupled to NADPH and thioredoxin reductase. The availability of the mouse Sptrx-1 gene sequence is the first step towards the generation of knock-out mice, whose characterization will provide significant information regarding the in-vivo function of Sptrx-1 and its possible implication in several sperm anomalies.

  • 20.
    Johansson, L.
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Thomsen, J. S.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, A. E.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Treuter, E.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    The orphan nuclear receptor SHP inhibits agonist-dependent transcriptional activity of estrogen receptors ERalpha and ERbeta1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 1, p. 345-353Article in journal (Refereed)
    Abstract [en]

    SHP (short heterodimer partner) is an unusual orphan nuclear receptor that contains a putative ligand-binding domain but lacks a conserved DNA-binding domain. Although no conventional receptor function has yet been identified, SHP has been proposed to act as a negative regulator of nuclear receptor signaling pathways, because it interacts with and inhibits DNA binding and transcriptional activity of various nonsteroid receptors, including thyroid hormone and retinoid receptors. We show here that SHP interacts directly with agonist-bound estrogen receptors, ERalpha and ERbeta, and inhibits ER-mediated transcriptional activation. SHP specifically targets the ligand-regulated activation domain AF-2 and competes for binding of coactivators such as TIF2. Thus, SHP may represent a new category of negative coregulators for ligand-activated nuclear receptors. SHP mRNA is widely expressed in rat tissues including certain estrogen target tissues, and subcellular localization studies demonstrate that SHP is a nuclear protein, suggesting a biological significance of the SHP interactions with ERs. Taken together, these results identify ERs as novel SHP targets and suggest that competition for coactivator-binding is a novel mechanism by which SHP may inhibit nuclear receptor activation.

  • 21.
    Lallemand, D.
    et al.
    Institut Pasteur, Paris cedex 15, France.
    Spyrou, Giannis
    Novum, Karolinska Institute, Huddinge, Sweden.
    Yaniv, M.
    Institut Pasteur, Paris cedex 15, France.
    Pfarr, C. M.
    Institut Pasteur, Paris cedex 15, France.
    Variations in Jun and Fos protein expression and AP-1 activity in cycling, resting and stimulated fibroblasts1997In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 14, no 7, p. 819-830Article in journal (Refereed)
    Abstract [en]

    We have analysed the different Jun and Fos proteins as NIH3T3 fibroblasts pass from exponential growth to quiescence and during the first 24 h after their re-entry into the cell cycle following serum stimulation. We show that these proteins can be divided into 3 subgroups based on their pattern of expression. The first contains c-Jun, Jun-D and Fra-2 which are expressed at high level in cycling cells and are only mildly induced by serum. The second contains Jun-B, c-Fos, Fos-B and deltaFos-B whose levels are low in cycling cells but increase strongly and rapidly after stimulation by serum. The third group contains only Fra-1, which is absent from cycling cells and behaves as a delayed early response protein after serum stimulation. AP-1 binding activity is low both in cycling and quiescent fibroblasts but increases after stimulation by serum with kinetics matching the induction of the various Jun and Fos proteins. Antibody supershift analyses demonstrate that the composition of AP-1 binding activity reflects the relative abundance of each Jun and Fos protein. Furthermore, the state of post-translational modification varies continuously for all of the AP-1 proteins as growth conditions change. These data indicate that AP-1 activity during the G0-G1 transition is finely regulated and complex, involving changes both in protein expression and in posttranslational modification.

  • 22.
    Madeja, Zbigniew
    et al.
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden / Jagiellonian University, Kraków, Poland.
    Sroka, Jolanta
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden / Jagiellonian University, Kraków, Poland.
    Nyström, Christina
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Björkhem-Bergman, Linda
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Nordman, Tomas
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Damdimopoulos, Anastasios
    Karolinska Institutet, Huddinge, Sweden.
    Nalvarte, Ivan
    Karolinska Institutet, Huddinge, Sweden.
    Eriksson, Lennart C.
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Spyrou, Giannis
    Foundation of Biomedical Research, Academy of Athens, Greece / Karolinska Institutet, Huddinge, Sweden.
    Olsson, Jerker M.
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    Björnstedt, Mikael
    Karolinska Institutet, Karolinska University Hospital in Huddinge, Stockholm, Sweden.
    The role of thioredoxin reductase activity in selenium-induced cytotoxicity2005In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 69, no 12, p. 1765-1772Article in journal (Refereed)
    Abstract [en]

    The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.

  • 23.
    Miranda-Vizuete, A.
    et al.
    Novum, Karolinska Institute, Huddinge, Sweden.
    Damdimopoulos, A. E.
    Novum, Karolinska Institute, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Novum, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Novum, Karolinska Institute, Huddinge, Sweden.
    Cloning, expression, and characterization of a novel Escherichia coli thioredoxin1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 49, p. 30841-30847Article in journal (Refereed)
    Abstract [en]

    Thioredoxin (Trx) is a small ubiquitous protein that displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel thioredoxin in Escherichia coli as well as the expression and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2 contains two distinct domains: an N-terminal domain of 32 amino acids including two CXXC motifs and a C-terminal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce the interchain disulfide bridges of insulin. The apparent Km value of Trx2 for thioredoxin reductase is similar to that of the previously characterized E. coli thioredoxin (Trx1). The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with dithiothreitol, suggesting that oxidation of cysteine residues other than the ones in the active site might regulate its activity. A truncated form of the protein, lacking the N-terminal domain, is insensitive to the presence of dithiothreitol, further confirming the involvement of the additional cysteine residues in modulating Trx2 activity. In addition, the presence of the N-terminal domain appears to confer heat sensitivity to Trx2, unlike Trx1. Finally, Trx2 is present normally in growing E. coli cells as shown by Western blot analysis.

  • 24.
    Miranda-Vizuete, A.
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, A. E.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Pedrajas, J. R.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 261, no 2, p. 405-412Article in journal (Refereed)
    Abstract [en]

    We have isolated a 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa. It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD-binding and NADPH-binding domains. However, human TrxR2 differs from human TrxR1 by the presence of a 33-amino acid extension at the N-terminus which has properties characteristic of a mitochondrial translocation signal. Northern-blot analysis identified one mRNA species of 2.2 kb with highest expression in prostate, testis and liver. We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria. Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation. Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q11.2.

  • 25.
    Miranda-Vizuete, A
    et al.
    Karolinska Institute, Huddinge, Sweden.
    Gustafsson, Jan-.Åke
    Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Karolinska Institute, Huddinge, Sweden.
    Molecular cloning and expression of a cDNA encoding a human thioredoxin-like protein1998In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 243, no 1, p. 284-288Article in journal (Refereed)
    Abstract [en]

    This report describes the cloning of a human cDNA that encodes a new protein (Txl, Thioredoxin-like) that belongs to the expanding family of thioredoxins based on sequence comparison of the deduced amino acid sequence. This cDNA, with a total length of 1,278 bp, consists of 205 bp of 5'-untranslated sequence (including an in frame stop codon), an open reading frame of 870 bp and a 203 bp fragment of 3'-untranslated sequence. The coding sequence predicts a protein of 289 amino acids with two distinct domains: an N-terminal domain of 105 residues homologous to the rest of mammalian thioredoxins containing the conserved active site (CGPC) and a C-terminal domain of 184 residues with no homology with any other protein in the database. Northern blot analysis indicates that the txl probe hybridizes to a 1.3 Kb mRNA and is ubiquitously expressed in human tissues with the highest expression in stomach, testis and bone marrow.

  • 26.
    Miranda-Vizuete, A.
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Pedrajas, J. R.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, A. E.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Cloning and sequencing of mouse glutaredoxin (grx) cDNA1999In: Mitochondrial DNA, ISSN 1940-1736, E-ISSN 1940-1744, Vol. 10, no 3, p. 179-182Article in journal (Refereed)
    Abstract [en]

    Glutaredoxins are small proteins (12 kDa) with a conserved active sequence Cys-Pro-Tyr(-Phe)-Cys that catalyse GSH-disulfide oxidoreduction reactions in the presence of NADPH and glutathione reductase. Many mammalian glutaredoxins have been characterized and human and pig cDNA sequence determined. However, no mouse glutaredoxin cDNA or protein sequence has yet been reported. We have cloned a cDNA from a mouse liver library that encodes the putative mouse glutaredoxin homologue. The deduced polypeptide sequence encodes a 107 amino acid protein displaying a high degree of homology with other members of the glutaredoxin family.

  • 27.
    Miranda-Vizuete, A.
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Identification of the first human glutaredoxin pseudogene localized to human chromosome 20q11.2.2001In: DNA Sequence, ISSN 1042-5179, E-ISSN 1029-2365, Vol. 11, no 6, p. 535-539Article in journal (Refereed)
  • 28.
    Miranda-Vizuete, A.
    et al.
    Novum, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Novum, Karolinska Institutet, Huddinge, Sweden.
    The novel oxidoreductase KDRF (KM-102-derived reductase-like factor) is identical with human thioredoxin reductase1997In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 325 ( Pt 1), p. 287-288Article in journal (Refereed)
  • 29.
    Miranda-Vizuete, Antonio
    et al.
    Karolinska Institutet, Novum, Huddinge, Sweden.
    Damdimopoulos, Anastasios E.
    Karolinska Institutet, Novum, Huddinge, Sweden.
    Spyrou, Giannis
    Karolinska Institutet, Novum, Huddinge, Sweden.
    cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1)1999In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1447, no 1, p. 113-118Article in journal (Refereed)
    Abstract [en]

    Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.

  • 30.
    Miranda-Vizuete, Antonio
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, Anastasios E.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    The mitochondrial thioredoxin system2000In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 2, no 4, p. 801-810Article in journal (Refereed)
    Abstract [en]

    Eukaryotic organisms from yeast to human possess a mitochondrial thioredoxin system composed of thioredoxin and thioredoxin reductase, similar to the cytosolic thioredoxin system that exists in the same cells. Yeast and mammalian mitochondrial thioredoxins are monomers of approximately 12 kDa and contain the typical conserved active site WCGPC. However, there are important differences between yeast and mammalian mitochondrial thioredoxin reductases that resemble the differences between their cytosolic counterparts. Mammalian mitochondrial thioredoxin reductase is a selenoprotein that forms a homodimer of 55 kDa/subunit; while yeast mitochondrial thioredoxin reductase is a homodimer of 37 kDa/subunit and does not contain selenocysteine. A function of the mitochondrial thioredoxin system is as electron donor for a mitochondrial peroxiredoxin, an enzyme that detoxifies the hydrogen peroxide generated by the mitochondrial metabolism. Experiments with yeast mutants lacking both the mitochondrial thioredoxin system as well as the mitochondrial peroxiredoxin system suggest an important role for mitochondrial thioredoxin, thioredoxin reductase, and peroxiredoxin in the protection against oxidative stress.

  • 31.
    Miranda-Vizuete, Antonio
    et al.
    Karolinska Institutet, Huddinge, Sweden.
    Ljung, Johanna
    Karolinska Institute, Stockholm, Sweden.
    Damdimopoulos, Anastasios E.
    Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Karolinska Institutet, Huddinge, Sweden.
    Oko, Richard
    Queen's University, Kingston, Ontario, Canada.
    Pelto-Huikko, Markku
    Tampere University Medical School and Tampere University Hospital, Finland.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 34, p. 31567-31574Article in journal (Refereed)
    Abstract [en]

    Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member of the thioredoxin family in humans, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx open reading frame encodes for a protein of 486 amino acids composed of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx mRNA is only expressed in human testis, specifically in round and elongating spermatids. Immunostaining of human testis sections identified Sptrx protein in spermatids, while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the postmeiotic phase of spermatogenesis, when the sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.

  • 32.
    Miranda-Vizuete, Antonio
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Genomic organization and identification of a novel alternative splicing variant of mouse mitochondrial thioredoxin reductase (TrxR2) gene2002In: Molecules and Cells, ISSN 1016-8478, E-ISSN 0219-1032, Vol. 13, no 3, p. 488-492Article in journal (Refereed)
    Abstract [en]

    Eukaryotic mitochondria are equipped with a complete thioredoxin system, composed of thioredoxin and thioredoxin reductase, which has been implicated in the protection against the reactive oxygen intermdiates generated during the respiratory process in this organelle. Like its cytosolic counterpart, mammalian mitochondrial thioredoxin reductase is a homodimeric selenoprotein. We report here the genomic organization of the mouse mitochondrial thioredoxin gene (TrxR2) that spans 53 kb and consists of 18 exons ranging from 20 to 210 bp. All splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly at the same position as the human TrxR2 gene, the only mammalian mitochondrial thioredoxin reductase gene whose genomic structure has been elucidated to date. In addition, we have identified a novel mRNA splicing variant lacking intron 14 resulting in a protein subunit with a shorter interface domain. This new splicing variant provides a framework for further analysis of this important enzyme as its predicted homodimeric conformation can now be expanded to a putative heterodimeric structure as well as a small subunit homodimer with the obvious implications at the regulatory level.

  • 33.
    Miranda-Vizuete, Antonio
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Genomic structure and chromosomal localization of human thioredoxin-like protein gene (txl)2000In: Mitochondrial DNA, ISSN 1940-1736, E-ISSN 1940-1744, Vol. 10, no 6, p. 419-424Article in journal (Refereed)
    Abstract [en]

    Human thioredoxin-like protein (txl) is a novel member of the expanding thioredoxin superfamily whose main characteristic is the presence, after the thioredoxin domain, of a C-terminal extension of 184 residues with no homology with any other protein in the databases. Txl is a cytosolic ubiquitously expressed protein and it has been copurified with a kinase of the STE20 family, which is proteolytically activated by caspases in apoptosis. However, no cellular function has yet been assigned to this protein. In the present study we report the genomic organization of the txl gene which encompasses approximately 36 kb organized in eight exons ranging from 96 bp to 303 bp. In contrast, intron sizes are much bigger ranging from 1.5 kb to 12 kb. Chromosomal localization of txl gene revealed that it maps at position 18q21, a region frequently affected in different human tumours. Furthermore, we have identified the putative homologues of txl in both Drosophila melanogaster and Caenorhabditis elegans that display much closer homology to the known thioredoxins than the human txl protein. Indeed, critical residues for optimal thioredoxin activity are present in both Drosophila and Caenorhabditis txl but absent in the human protein suggesting that txl might have evolved to carry out a function different from the general disulfide reductase typical of thioredoxins.

  • 34.
    Miranda-Vizuete, Antonio
    et al.
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Identification of a novel thioredoxin-1 pseudogene on human chromosome 102000In: Mitochondrial DNA, ISSN 1940-1736, E-ISSN 1940-1744, Vol. 10, no 6, p. 411-414Article in journal (Refereed)
    Abstract [en]

    Thioredoxin (Trx) is a small, ubiquitous protein of 12 kDa that acts as general dithiol-disulfide oxidoreductase via its conserved redox active site Trp-Cys-Gly-Pro-Cys which is located in a protrusion of the protein (Holmgren 1985). Mammalian cells contain two forms of thioredoxin: Trx1, a cytosolic enzyme able to translocate to the nucleus under certain stimuli and be secreted from the cells thus displaying cytokine-like properties and Trx2, a mitochondrial enzyme (Tagaya et al. 1989; Spyrou et al 1997). While many functions have been described for Trx1 for example antioxidant enzyme, modulator of transcription factors, electron donor for enzymes like ribonucleotide reductase and PAPS reductase, etc. (see introduction Spyrou et al. 1997), only an antioxidant function has been assigned to Trx2. Trx2 acts as a reductant of a mitochondrial thioredoxin-dependent peroxidase which protects cells against hydrogen peroxide treatment (Araki et al. 1999). Human Trx1 gene maps at chromosome 9q31 and several bands hybridize with a Trx1 probe in Southern blots suggesting the existence of Trx1 derived sequences in the human genome (Heppell-Parton et al. 1995; Kaghad et al. 1994). As only one active Trx1 gene has been described, the other hybridizing bands might correspond to different inactive pseudogenes (Kaghad et al. 1994), but only one Trx1 pseudogene has been described so far (GenBank accession number: AF146023 (Tonissen and Wells 1991)). We report here the sequence of a second Trx1 retrotranscribed pseudogene and propose the nomenclature wTrx1-1 and wTrx1-2.

  • 35.
    Nalvarte, Ivan
    et al.
    Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, Anastasios E
    Karolinska Institutet, Huddinge, Sweden.
    Nystöm, Christina
    Department of Laboratory Medicine, Division of Pathology, F46, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden.
    Nordman, Tomas
    Department of Laboratory Medicine, Division of Pathology, F46, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden.
    Miranda-Vizuete, Antonio
    Karolinska Institutet, Huddinge, Sweden.
    Olsson, Jerker M.
    Department of Laboratory Medicine, Division of Pathology, F46, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden.
    Eriksson, Lennart
    Department of Laboratory Medicine, Division of Pathology, F46, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden.
    Björnstedt, Mikael
    Department of Laboratory Medicine, Division of Pathology, F46, Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden.
    Arnér, Elias S.J.
    Karolinska Institutet, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden / Institute of Biomedical Research, Academy of Athens, Greece.
    Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells: Effect on cell growth and differentiation2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 52, p. 54510-54517Article in journal (Refereed)
    Abstract [en]

    The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.

  • 36.
    Nalvarte, Ivan
    et al.
    Karolinska Institute, Sweden.
    Damdimopoulos, Anastasios E.
    Karolinska Institute, Sweden.
    Ruegg, Joelle
    Karolinska Institute, Sweden; Swedish Toxicol Science Research Centre Swetox, Sweden.
    Spyrou, Ioannis
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion2015In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 35, no e00269Article in journal (Refereed)
    Abstract [en]

    The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510-54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.

  • 37.
    Nalvarte, Ivan
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, Anastasios E
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition2004In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 36, no 10, p. 1270-1278Article in journal (Refereed)
    Abstract [en]

    The ubiquitously expressed mammalian thioredoxin reductases are selenoproteins that together with NADPH regenerate active reduced thioredoxins and are involved in diverse actions mediated by redox control. Two main forms of mammalian thioredoxin reductases have been isolated, one cytosolic (TrxR1) and one present in mitochondria (TrxR2). Although the principal target for TrxRs is thioredoxin, the cytosolic form can regenerate several important antioxidants such as ascorbic acid, lipoic acid, and ubiquinone. In this study we demonstrate that cytochrome c is a substrate for both TrxR1 and TrxR2. In addition, cells overexpressing TrxR2 are more resistant to impairment of complex III in the mitochondrial respiratory chain upon both antimycin A and myxothiazol treatments, suggesting a complex III bypassing function of TrxR2. Furthermore, we show that cytochrome c is reduced by TrxR2 in vitro, not only by using NADPH as an electron donor but also by using NADH, pointing at TrxR2 as an important redox protein on complex III impairment. These findings may be valuable in understanding respiratory disorders in mitochondrial diseases.

  • 38.
    Nikitovic, D.
    et al.
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden.
    Holmgren, A.
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
    Inhibition of AP-1 DNA binding by nitric oxide involving conserved cysteine residues in Jun and Fos1998In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 242, no 1, p. 109-112Article in journal (Refereed)
    Abstract [en]

    Nitric oxide (NO), which has diverse biological effects, can modulate AP-1 activity. Since DNA binding of Jun-Jun and Jun-Fos dimers is regulated in vitro by redox control involving conserved cysteines, we hypothesized that the action of NO is mediated via these residues. We performed electrophoretic mobility-shift analyses using Jun and Fos recombinant proteins and NO solutions. Cysteine-to-serine mutants showed that the inhibition of AP-1 activity following NO treatment was dependent on the presence of Cys7272 and Cys154 in the DNA binding domain of Jun and Fos, respectively. The inhibitory effect of NO was reversed by DTT and the thioredoxin system. Our results demonstrate that NO mediates its inhibitory effect by reacting specifically with the conserved cysteine residues in Jun and Fos.

  • 39.
    Nordman, Tomas
    et al.
    Department of Laboratory Medicine, F 46, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Xia, Ling
    Department of Laboratory Medicine, F 46, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Björkhem-Bergman, Linda
    Department of Laboratory Medicine, F 46, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Damdimopoulos, Anastassios
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Nalvarte, Ivan
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Arnér, Elias S.J.
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Eriksson, Lennart C.
    Department of Laboratory Medicine, F 46, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Björnstedt, Mikael
    Department of Laboratory Medicine, F 46, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Olsson, Jerker M.
    Department of Laboratory Medicine, F 46, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase2003In: Biofactors, ISSN 0951-6433, E-ISSN 1872-8081, Vol. 18, no 1-4, p. 45-50Article in journal (Refereed)
    Abstract [en]

    Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.

  • 40.
    Padilla, C. A.
    et al.
    Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm/Sweden.
    Martínez-Galisteo, E.
    Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm/Sweden.
    Bárcena, J. A.
    Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm/Sweden.
    Spyrou, Giannis
    Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm/Sweden.
    Holmgren, A.
    Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm/Sweden.
    Purification from placenta, amino acid sequence, structure comparisons and cDNA cloning of human glutaredoxin1995In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 227, no 1-2, p. 27-34Article in journal (Refereed)
    Abstract [en]

    Glutaredoxin is generally a glutathione-dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)-disulfide-oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH-dependent hydrogen-donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three-dimensional structure in solution to identify conserved residues and predict a structure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.

  • 41.
    Padilla, C. A.
    et al.
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Holmgren, A.
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein1996In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 378, no 1, p. 69-73Article in journal (Refereed)
    Abstract [en]

    Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.

  • 42.
    Pedrajas, José R.
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Kosmidou, Effie
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Miranda-Vizuete, Antonio
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Wright, Anthony P. H.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 10, p. 6366-6373Article in journal (Refereed)
    Abstract [en]

    The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The Deltatrr2 mutant was more sensitive to H2O2, whereas the Deltatrx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae.

  • 43.
    Pedrajas, José Rafael
    et al.
    Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden.
    Miranda-Vizuete, Antonio
    Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden.
    Javanmardy, Negin
    Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden.
    Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 21, p. 16296-16301Article in journal (Refereed)
    Abstract [en]

    Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors. Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues. Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor. However, the biological reducing agent for the 1-Cys Prx has not yet been identified. We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p. Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions. We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor. In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity. This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity. Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.

  • 44.
    Pfarr, C. M.
    et al.
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Mechta, F.
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Spyrou, Giannis
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Lallemand, D.
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Carillo, S.
    Institut de Genetique Moleculaire de Montpellier Unité Mixte de Recherche, Montpellier, France.
    Yaniv, M.
    Département des Biotechnologies, Institut Pasteur, Paris, France.
    Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras1994In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 76, no 4, p. 747-760Article in journal (Refereed)
    Abstract [en]

    As NIH 3T3 fibroblasts become quiescent, the level of c-Jun protein decreases while JunD accumulates. When resting cells are stimulated with fresh serum, nuclear-localized JunD is rapidly degraded, followed by resynthesis of both c-Jun and JunD later in G1. Overexpression of JunD results in slower growth and an increase in the percentage of cells in G0/G1 while c-Jun overexpression produces larger S/G2 and M phase populations. In addition, JunD partially suppresses transformation by an activated ras gene whereas c-Jun cooperates with ras to transform cells. These data indicate that two closely related transcription factors can function in an opposing manner.

  • 45.
    Prinarakis, Efthimios
    et al.
    Center of Basic Research I—Biochemistry Division, Biomedical Research Foundation, Academy of Athens, Athens, Greece.
    Chantzoura, Eleni
    Center of Basic Research I—Biochemistry Division, Biomedical Research Foundation, Academy of Athens, Athens, Greece.
    Thanos, Dimitris
    Center of Basic Research II—Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Athens, Greece.
    Spyrou, Giannis
    Center of Basic Research I—Biochemistry Division, Biomedical Research Foundation, Academy of Athens, Athens, Greece.
    S-glutathionylation of IRF3 regulates IRF3-CBP interaction and activation of the IFN beta pathway2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 6, p. 865-875Article in journal (Refereed)
    Abstract [en]

    Interferon regulatory factor 3 (IRF3) is an essential transcriptional regulator of the interferon genes. IRF3 is constitutively present in a latent conformation in the cell cytoplasm. In cells infected by Sendai virus, IRF3 becomes phosphorylated, homodimerizes, translocates to the nucleus, binds to target genes and activates transcription by interacting with CBP/p300 co-activators. In this study, we report that in non-infected cells IRF3 is post-translationally modified by S-glutathionylation. Upon viral-infection, it undergoes a deglutathionylation step that is controlled by the cytoplasmic enzyme glutaredoxin-1 (GRX-1). In virus-infected GRX-1 knockdown cells, phosphorylation, homodimerization and nuclear translocation of IRF3 were not affected, but the transcriptional activity of IRF3 and the expression of interferon-beta (IFNbeta), were severely reduced. We show that deglutathionylation of IRF3 is necessary for efficient interaction of IRF3 with CBP, an event essential for transcriptional activation of the interferon genes. Taken together, these findings reveal a crucial role for S-glutathionylation and GRX-1 in controlling the activation of IRF3 and IFNbeta gene expression.

  • 46.
    Psarra, Anna-Maria G
    et al.
    Biomedical Research Foundation, Academy of Athens, Center of Basic Research, Athens, Greece.
    Hermann, Stefan
    Biomedical Research Foundation, Academy of Athens, Center of Basic Research, Athens, Greece.
    Panayotou, George
    Biomedical Sciences Research Center Alexander Fleming, Laboratory of Protein Chemistry, Vari, Greece.
    Spyrou, Giannis
    Biomedical Research Foundation, Academy of Athens, Center of Basic Research, Athens, Greece.
    Interaction of mitochondrial thioredoxin with glucocorticoid receptor and NF-κB modulates glucocorticoid receptor and NF-κB signalling in HEK-293 cells2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 422, no 3, p. 521-531Article in journal (Refereed)
    Abstract [en]

    Trx2 (mitochondrial thioredoxin) is an antioxidant and anti-apoptotic factor essential for cell viability. Trx1 (cytoplasmic thioredoxin) is a co-factor and regulator of redox-sensitive transcription factors such as the GR (glucocorticoid receptor) and NF-kappaB (nuclear factor kappaB). Both transcription factors have been detected in mitochondria and a role in mitochondrial transcription regulation and apoptosis has been proposed. In the present study, we show using SPR (surface plasmon resonance) and immunoprecepitation that GR and the p65 subunit of NF-kappaB are Trx2-interacting proteins. The interaction of Trx2 with GR is independent of the presence of GR ligand and of redox conditions. The p65 subunit of NF-kappaB can interact with Trx2 in the oxidized, but not the reduced, form. Using HEK (human embryonic kidney)-293 cell lines with increased or decreased expression of Trx2, we show that Trx2 modulates transcription of GR and NF-kappaB reporter genes. Moreover, Trx2 overexpression modulates the mRNA levels of the COX1 (cytochrome oxidase subunit I) and Cytb (cytochrome b), which are known to be regulated by GR and NF-kappaB. Increased expression of Trx2 differentially affects the expression of Cytb. The glucocorticoid dexamethasone potentiates the expression of Cytb, whereas TNFalpha (tumour necrosis factor alpha) down-regulates it. These results suggest a regulatory role for Trx2 in GR and NF-kappaB signalling pathways.

  • 47.
    Psarra, Anna-Maria G.
    et al.
    Foundation for Biomedical Research of the Academy of Athens, Center for Basic Research, Athens, Greece.
    Solakidi, Sylvia
    National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Laboratory of Molecular Endocrinology, Athens, Greece.
    Trougakos, Ioannis P.
    University of Athens, Greece.
    Margaritis, Loukas H.
    University of Athens, Greece.
    Spyrou, Giannis
    Foundation for Biomedical Research of the Academy of Athens, Center for Basic Research, Athens, Greece.
    Sekeris, Constantine E.
    National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Laboratory of Molecular Endocrinology, Athens, Greece.
    Glucocorticoid receptor isoforms in human hepatocarcinoma HepG2 and SaOS-2 osteosarcoma cells: presence of glucocorticoid receptor alpha in mitochondria and of glucocorticoid receptor beta in nucleoli2005In: International Journal of Biochemistry and Cell Biology, ISSN 1357-2725, E-ISSN 1878-5875, Vol. 37, no 12, p. 2544-2558Article in journal (Refereed)
    Abstract [en]

    In the context of a possible direct action of glucocorticosteroids on mitochondrial transcription and/or apoptosis by way of cognate mitochondrial receptors, the possible localization of glucocorticoid receptors alpha and beta (GRalpha and GRbeta) in mitochondria was explored in human hepatocarcinoma HepG2 and osteosarcoma SaOS-2 cells, in which glucocorticoids exert an anabolic and apoptotic effect, respectively. In both cell types, GRalpha was detected in mitochondria, in nuclei and in cytosol by immunofluorescence labeling and confocal scanning microscopy, by immunogold electron microscopy and by Western blotting. GRbeta was shown to be almost exclusively restricted to the nucleus of the two cell types, being particularly concentrated in nucleoli, pointing to a solely nuclear role of this receptor isoform and to a possible function in nucleoli related processes. Computer analysis identified a putative internal mitochondrial targeting sequence within the glucocorticoid receptor. The demonstration of mitochondrially localized GRalpha in HepG2 and SaOS-2 cells corroborates previous findings in other cell types and further supports a direct role of this receptor in mitochondrial functions.

  • 48.
    Rybnikova, Elena
    et al.
    Pavlov Institute of Physiology RAS, St. Petersburg, Russia .
    Damdimopoulos, A. E.
    Department of Biosciences, Karolinska Institute, Huddinge, Sweden .
    Gustafsson, Jan-Åke
    Department of Biosciences, Karolinska Institute, Huddinge, Sweden .
    Spyrou, Giannis
    Department of Biosciences, Karolinska Institute, Huddinge, Sweden .
    Pelto-Huikko, M.
    Tampere University Medical School and Department of Pathology, Tampere University Hospital, Finland.
    Expression of novel antioxidant thioredoxin-2 in the rat brain2000In: European Journal of Neuroscience, ISSN 0953-816X, E-ISSN 1460-9568, Vol. 12, no 5, p. 1669-1678Article in journal (Refereed)
    Abstract [en]

    Thioredoxins are a class of small redox-regulating proteins that have been implicated in the control of various aspects of cellular functions and seem to be one of the key regulators of signalling in the cellular responses to various stresses. Thioredoxin-2 (Trx2) is a novel mammalian thioredoxin which, in contrast to previously known cytosolic thioredoxin (Trx1), has been localized to the mitochondria. Trx2 is abundantly expressed in skeletal muscle, heart and adrenal gland, as well as in some other peripheral tissues with high metabolic activity. Using in situ hybridization and immunohistochemistry, we have studied the distribution and regulation of Trx2 expression in the rat brain. Trx2 mRNA and protein are highly expressed in the neurons in several brain regions, including the olfactory bulb, frontal cortex, hippocampus, some hypothalamic and thalamic nuclei, cerebellum and numerous brainstem nuclei. In addition, the Trx2 mRNA expression in paraventricular hypothalamic nucleus and reticular thalamic nucleus was found to be sensitive to peripheral glucocorticoids, as dexamethasone treatment caused significant elevation of Trx2 mRNA level in this area. No changes in other brain areas were observed after dexamethasone treatment. These findings implicate a significant regulatory and/or protective function of Trx2 in the nervous system.

  • 49.
    Rökaeus, Åke
    et al.
    Karolinska Institutet, Stockholm, Sweden.
    Jiang, Kai
    Karolinska Institutet, NOVUM, Huddinge, Sweden.
    Spyrou, Giannis
    Karolinska Institutet, NOVUM, Huddinge, Sweden.
    Waschek, James A.
    University of California, Los Angeles, USA.
    Transcriptional control of the galanin gene. Tissue-specific expression and induction by NGF, protein kinase C, and estrogen1998In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 863, p. 1-13Article in journal (Refereed)
    Abstract [en]

    Galanin is a neuropeptide widely expressed in the central and peripheral nervous system where it acts as a neurotransmitter/neuromodulator and possibly an immunoregulator and growth factor. Galanin gene expression is highly regulated during development and by certain hormones and injury situations. We have examined transcriptional control mechanisms for this gene using chimeric bovine galanin/luciferase reporter genes. These were analyzed in cultured cells and in transgenic mice. The studies reveal that enhancer and silencer sequences are involved in conferring cell- and tissue-specific expression, and that specific elements close to the promoter are responsible for nerve growth factor and protein kinase C induction. So far, the studies have not revealed sequences on the bovine gene that mediate the action of estrogen.

  • 50.
    Sadek, C. M.
    et al.
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, A. E.
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Pelto-Huikko, M.
    Tampere University Medical School and Tampere University Hospital, Finland.
    Gustafsson, J. A.
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, G
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Miranda-Vizuete, A
    Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, Huddinge, Sweden.
    Sptrx-2, a fusion protein composed of one thioredoxin and three tandemly repeated NDP-kinase domains is expressed in human testis germ cells2001In: Genes to Cells, ISSN 1356-9597, E-ISSN 1365-2443, Vol. 6, no 12, p. 1077-1090Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Thioredoxins (Trx) are small redox proteins that function as general protein disulphide reductases and regulate several cellular processes such as transcription factor DNA binding activity, apoptosis and DNA synthesis. In mammalian organisms, thioredoxins are generally ubiquitously expressed in all tissues, with the exception of Sptrx-1 which is specifically expressed in sperm cells.

    RESULTS: We report here the identification and characterization of a novel member of the thioredoxin family, the second with a tissue-specific distribution in human sperm, termed Sptrx-2. The Sptrx-2 ORF (open reading frame) encodes for a protein of 588 amino acids with two different domains: an N-terminal thioredoxin domain encompassing the first 105 residues and a C-terminal domain composed of three repeats of a NDP kinase domain. The Sptrx-2 gene spans about 51 kb organized in 17 exons and maps at locus 7p13-14. Sptrx-2 mRNA is exclusively expressed in human testis, mainly in primary spermatocytes, while Sptrx-2 protein expression is detected from the pachytene spermatocytes stage onwards, peaking at round spermatids stage. Recombinant full-length Sptrx-2 expressed in bacteria displayed neither thioredoxin nor NDP kinase enzymatic activity.

    CONCLUSIONS: The sperm specific expression of Sptrx-2, together with its chromosomal assignment to a position reported as a potential locus for flagellar anomalies and male infertility phenotypes such as primary ciliary dyskinesia, suggests that it might be a novel component of the human sperm axonemal organization.

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