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  • 51.
    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.

  • 52.
    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.

  • 53.
    Plaza-Menacho, Iván
    et al.
    Department of Genetics, Hanzeplein 1, 9700 RB, Groningen, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands / Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, United Kingdom.
    van der Sluis, Tineke
    Department of Pathology, Hanzeplein 1, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
    Hollema, Harry
    Department of Pathology, Hanzeplein 1, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
    Gimm, Oliver
    Department of Surgery, Martin Luther University, Ernst Grube Strasse 40, 06097 Halle-Wittenberg, Germany.
    Buys, Charles H C M
    Department of Genetics, Hanzeplein 1, 9700 RB, Groningen, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
    Magee, Anthony I
    Section of Molecular and Cellular Medicine, Imperial College, London SW7 2AZ, United Kingdom.
    Isacke, Clare M
    Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, United Kingdom.
    Hofstra, Robert M W
    Department of Genetics, Hanzeplein 1, 9700 RB, Groningen, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
    Eggen, Bart J L
    Developmental Genetics, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands .
    Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation.2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 9, p. 6415-24Article in journal (Refereed)
    Abstract [en]

    The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.

  • 54.
    Qian, Hong
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Le Blanc, Katarina
    Karolinska University Hospital, Sweden.
    Sigvardsson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 31, p. 25795-25807Article in journal (Refereed)
    Abstract [en]

    Despite significant progress in our understanding of mesenchymal stem cell (MSC) biology during recent years, much of the information is based on experiments using in vitro culture-selected stromal progenitor cells. Therefore, the natural cellular identity of MSCs remains poorly defined. Numerous studies have reported that CD44 expression is one of the characteristics of MSCs in both humans and mice; however, we here have prospectively isolated bone marrow stromal cell subsets from both human and mouse bone marrow by flow cytometry and characterized them by gene expression analysis and function assays. Our data provide functional and molecular evidence suggesting that primary mesenchymal stem and progenitor cells of bone marrow reside in the CD44(-) cell fraction in both mice and humans. The finding that these CD44(-) cells acquire CD44 expression after in vitro culture provides an explanation for the previous misconceptions concerning CD44 expression on MSCs. In addition, the other previous reported MSC markers, including CD73, CD146, CD271, and CD106/VCAM1, are also differentially expressed on those two cell types. Our microarray data revealed a distinct gene expression profile of the freshly isolated CD44(-) cells and the cultured MSCs generated from these cells. Thus, we conclude that bone marrow MSCs physiologically lack expression of CD44, highlighting the natural phenotype of MSCs and opening new possibilities to prospectively isolate MSCs from the bone marrow.

  • 55.
    Rajan, Meenu Rohini
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Nyman, Elin
    Linköping University, Department of Biomedical Engineering, Medical Informatics. Linköping University, Faculty of Science & Engineering. Cardiovascular and Metabolic Diseases, Innovative Medicines, and Drug Metabolism and Pharmacokinetics, AstraZeneca Research and Development, Gothenburg, Sweden .
    Kjölhede, Preben
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center of Paediatrics and Gynaecology and Obstetrics, Department of Gynaecology and Obstetrics in Linköping.
    Cedersund, Gunnar
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Science & Engineering. Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Systems-wide Experimental and Modeling Analysis of Insulin Signaling through Forkhead Box Protein O1 (FOXO1) in Human Adipocytes, Normally and in Type 2 Diabetes2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 30, p. 15806-15819Article in journal (Refereed)
    Abstract [en]

    Insulin resistance is a major aspect of type 2 diabetes (T2D), which results from impaired insulin signaling in target cells. Signaling to regulate forkhead box protein O1 (FOXO1) may be the most important mechanism for insulin to control transcription. Despite this, little is known about how insulin regulates FOXO1 and how FOXO1 may contribute to insulin resistance in adipocytes, which are the most critical cell type in the development of insulin resistance. We report a detailed mechanistic analysis of insulin control of FOXO1 in human adipocytes obtained from non-diabetic subjects and from patients with T2D. We show that FOXO1 is mainly phosphorylated through mTORC2-mediated phosphorylation of protein kinase B at Ser(473) and that this mechanism is unperturbed in T2D. We also demonstrate a cross-talk from the MAPK branch of insulin signaling to stimulate phosphorylation of FOXO1. The cellular abundance and consequently activity of FOXO1 are halved in T2D. Interestingly, inhibition of mTORC1 with rapamycin reduces the abundance of FOXO1 to the levels in T2D. This suggests that the reduction of the concentration of FOXO1 is a consequence of attenuation of mTORC1, which defines much of the diabetic state in human adipocytes. We integrate insulin control of FOXO1 in a network-wide mathematical model of insulin signaling dynamics based on compatible data from human adipocytes. The diabetic state is network-wide explained by attenuation of an mTORC1-to-insulin receptor substrate-1 (IRS1) feedback and reduced abundances of insulin receptor, GLUT4, AS160, ribosomal protein S6, and FOXO1. The model demonstrates that attenuation of the mTORC1-to-IRS1 feedback is a major mechanism of insulin resistance in the diabetic state.

  • 56.
    Ruiz Pavón, Lorena
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Lundh, Fredrik
    School of Pure and Applied Natural Sciences, Kalmar University, Kalmar, Sweden .
    Lundin, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Mishra, Arti
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Persson, Bengt
    School of Pure and Applied Natural Sciences, Kalmar University, Kalmar, Sweden.
    Spetea (Wiklund), Cornelia
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter -functional characterization in Escherichia coli2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 20, p. 13520-13527Article in journal (Refereed)
    Abstract [en]

    In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na+-dependent inorganic phosphate (Pi) transporters. The function of ANTR1 as a Na+-dependent Pi transporter was demonstrated by heterologous expression and uptake of radioactive Pi into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated Pi uptake in a pH- and Na+-dependent manner as compared with the control cells. Among various tested effectors, Pi was the preferred substrate. Although it competed with the uptake of Pi, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a Pi transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of Pi produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H+ electrochemical gradient storage.

  • 57.
    Sadek, Christine M.
    et al.
    Karolinska Institutet, Huddinge, Sweden.
    Jiménez, Alberto
    Karolinska Institutet, Huddinge, Sweden.
    Damdimopoulos, Anastasios E
    Karolinska Institutet, Huddinge, Sweden.
    Kieselbach, Thomas
    Karolinska Institutet, Huddinge, Sweden.
    Nord, Magnus
    Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Davis, Elaine C.
    McGill University, Montreal, Quebec, Canada.
    Oko, Richard
    Queen's University, Kingston, Ontario, Canada.
    van der Hoorn, Frans A.
    University of Calgary, Alberta, Canada.
    Miranda-Vizuete, Antonio
    Karolinska Institutet, Huddinge, Sweden.
    Characterization of human thioredoxin-like 2. A novel microtubule-binding thioredoxin expressed predominantly in the cilia of lung airway epithelium and spermatid manchette and axoneme2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 15, p. 13133-13142Article in journal (Refereed)
    Abstract [en]

    We describe here the cloning and characterization of a novel member of the thioredoxin family, thioredoxin-like protein 2 (Txl-2). The Txl-2 open reading frame codes for a protein of 330 amino acids consisting of two distinct domains: an N-terminal domain typical of thioredoxins and a C-terminal domain belonging to the nucleoside-diphosphate kinase family, separated by a small interface domain. The Txl-2 gene spans approximately 28 kb, is organized into 11 exons, and maps at locus 3q22.3-q23. A splicing variant lacking exon 5 (Delta 5Txl-2) has also been isolated. By quantitative real time PCR we demonstrate that Txl-2 mRNA is ubiquitously expressed, with testis and lung having the highest levels of expression. Unexpectedly, light and electron microscopy analyses show that the protein is associated with microtubular structures such as lung airway epithelium cilia and the manchette and axoneme of spermatids. Using in vitro translated proteins, we demonstrate that full-length Txl-2 weakly associates with microtubules. In contrast, Delta 5Txl-2 specifically binds with very high affinity brain microtubule preparations containing microtubule-binding proteins. Importantly, Delta 5Txl-2 also binds to pure microtubules, proving that it possesses intrinsic microtubule binding capability. Taken together, Delta 5Txl-2 is the first thioredoxin reported to bind microtubules and might therefore be a novel regulator of microtubule physiology.

  • 58.
    Schmidt, Gudula
    et al.
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Germany.
    Goehring, Udo-Michael
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Germany.
    Schirmer, Jörg
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Germany.
    Lerm, Maria
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Germany.
    Aktories, Klaus
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Germany.
    Identification of the C-terminal part of Bordetella dermonecrotic toxin as a transglutaminase for rho GTPases1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 45, p. 31875-31881Article in journal (Refereed)
    Abstract [en]

    Bordetella dermonecrotic toxin (DNT) causes the deamidation of glutamine 63 of Rho. Here we identified the region of DNT harboring the enzyme activity and compared the toxin with the cytotoxic necrotizing factor 1, which also deamidates Rho. The DNT fragment (DeltaDNT) covering amino acid residues 1136-1451 caused deamidation of RhoA at glutamine 63 as determined by mass spectrometric analysis and by the release of ammonia. In the presence of dansylcadaverine or ethylenediamine, DeltaDNT caused transglutamination of Rho. Deamidase and transglutaminase activities were blocked in the mutant proteins Cys(1292) --> Ala, His(1307) --> Ala, and Lys(1310) --> Ala of DeltaDNT. Deamidation and transglutamination induced by DeltaDNT blocked intrinsic and Rho- GTPase-activating protein-stimulated GTPase activity of RhoA. DeltaDNT deamidated and transglutaminated Rac and Cdc42 in the absence and presence of ethylenediamine, respectively. Modification of Rho proteins by DeltaDNT was nucleotide-dependent and did not occur with GTPgammaS-loaded GTPases. In contrast to cytotoxic necrotizing factor, which caused the same kinetics of ammonia release in the absence and presence of ethylenediamine, ammonia release by DeltaDNT was largely increased in the presence of ethylenediamine, indicating that DeltaDNT acts primarily as a transglutaminase.

  • 59.
    Schmidt, Gudula
    et al.
    Albert-Ludwigs-Universität Freiburg, Germany.
    Selzer, Jörg
    Albert-Ludwigs-Universität Freiburg, Germany.
    Lerm, Maria
    Albert-Ludwigs-Universität Freiburg, Germany.
    Aktories, Klaus
    Albert-Ludwigs-Universität Freiburg, Germany.
    The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity1998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 22, p. 13669-13674Article in journal (Refereed)
    Abstract [en]

    Recently, it has been reported that cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli induces formation of stress fibers by deamidation of glutamine 63 of RhoA (Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725-729); Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., and Boquet, P. (1997) Nature 387, 729-733). By using mass spectrometric analysis, we show now that the toxin transfers ethylenediamine, putrescine, and dansylcadaverine specifically onto glutamine 63 of RhoA. RhoA was also a substrate for guinea pig liver transglutaminase, which modified not only glutamine 63, but also glutamine residues at positions 52 and 136. Treatment of the fully active N-terminal fragment of CNF1 (amino acid residues 709-1014) with iodoacetamide inhibited both deamidation and transglutamination activities. Moreover, exchange of cysteine 866 with serine blocked the enzyme activity of the N-terminal CNF1 fragment. In addition, we identified histidine 881 to be essential for the enzyme activity of CNF1. The data indicate that CNF1 shares a catalytic dyad of cysteine and histidine residues with eukaryotic transglutaminases and cysteine proteases.

  • 60.
    Sjöstrand, Dan
    et al.
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Paratcha, Gustavo
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
    Persson, Bengt
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Ibanez, Carlos
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
    Disruption of the GDNF Binding Site in NCAM DissociatesLigand Binding and Homophilic Cell Adhesion2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 17, p. 12734-12740Article in journal (Refereed)
    Abstract [en]

    Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent towhich this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM tomediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor α1, shedding light on the molecular architecture of a multicomponent GDNF receptor.

  • 61.
    Spetea, Cornelia
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Keren, Nir
    Hundal, Torill
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Doan, Jean-Michel
    Ohad, Itzhak
    Andersson, Bertil
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    GTP enhances the degradation of the photosystem II D1 protein irrespective of its conformational heterogeneity at the Q(B) site2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 10, p. 7205-7211Article in journal (Refereed)
    Abstract [en]

    The light exposure history and/or binding of different herbicides at the Q(B) site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea C., Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6547-6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38-44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photo-system II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosystem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.

  • 62.
    Spyrou, Giannis
    et al.
    Novum, Karolinska Institutet, Huddinge, Sweden.
    Enmark, E.
    Novum, Karolinska Institutet, Huddinge, Sweden.
    Miranda-Vizuete, A.
    Novum, Karolinska Institutet, Huddinge, Sweden.
    Gustafsson, Jan-Åke
    Novum, Karolinska Institutet, Huddinge, Sweden.
    Cloning and expression of a novel mammalian thioredoxin1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 5, p. 2936-2941Article in journal (Refereed)
    Abstract [en]

    We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.

  • 63.
    Spyrou, Giannis
    et al.
    Department of Biochemistry, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
    Reichard, Peter
    Department of Biochemistry, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
    Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis1987In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 262, no 34, p. 16425-16432Article in journal (Refereed)
    Abstract [en]

    The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides. In permeabilized cells both deoxyliponucleotides were formed from dCTP. In intact cells they could be labeled from [5-3H] deoxycytidine or cytidine via transformation of the nucleosides to dCTP. Their turnover was slow compared to that of dCTP. When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions. Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine. Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine. Earlier work (Nicander, B., and Reichard, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine. Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.

  • 64.
    Spyrou, Giannis
    et al.
    Medical Nobel Institute, Karolinska Institutet, Stockholm. Sweden .
    Reichard, Peter
    Medical Nobel Institute, Karolinska Institutet, Stockholm. Sweden .
    Intracellular compartmentation of deoxycytidine nucleotide pools in S phase mouse 3T3 fibroblasts1989In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 264, no 2, p. 960-964Article in journal (Refereed)
    Abstract [en]

    We labeled mouse 3T3 fibroblasts, synchronized in G0 or S phase, from [3H]cytidine or [3H]deoxycytidine and measured the flow of isotope into and through deoxycytidine nucleotide pools, including the two deoxyliponucleotides dCDP choline and dCDP ethanolamine. Compared to G0 cells, S phase cells had much larger pools with a 20-40-fold faster turnover. The dCTP pool of S phase cells during steady state conditions attained a 6-fold higher specific activity than the pool of G0 cells when labeled from cytidine but a 10-fold lower specific activity when labeled from deoxycytidine. The dCTP pool of G0 cells showed a slow but measurable turnover indicating a limited amount of de novo synthesis also in resting cells. The labeling pattern of dCTP and deoxyliponucleotides of G0 cells was compatible with a simple precursor-product relationship. In S phase cells, however, dCDP choline had a 4-6 times higher specific activity during steady state conditions than dCTP and dCMP when the cells were labeled with [3H]deoxycytidine. We suggest that 3T3 cells contain two distinct intracellular dCTP pools, one labeled preferentially from cytidine and used for DNA replication, the other labeled from deoxycytidine and used for deoxyliponucleotide synthesis. We speculate that the latter pool during S phase may be temporarily sequestered in the cell's membrane fraction before equilibration with the much larger dCTP pool originating in S phase cells from the reduction of CDP.

  • 65.
    Thomas, Christophoros G
    et al.
    Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Foundation for Biomedical Research, Academy of Athens, Greece.
    ERdj5 sensitizes neuroblastoma cells to endoplasmic reticulum stress-induced apoptosis2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 10, p. 6282-6290Article in journal (Refereed)
    Abstract [en]

    Down-regulation of the unfolded protein response (UPR) can be therapeutically valuable in cancer treatment, and endoplasmic reticulum (ER)-resident chaperone proteins may thus be targets for developing novel chemotherapeutic strategies. ERdj5 is a novel ER chaperone that regulates the ER-associated degradation of misfolded proteins through its associations with EDEM and the ER stress sensor BiP. To investigate whether ERdj5 can regulate ER stress signaling pathways, we exposed neuroblastoma cells overexpressing ERdj5 to ER stress inducers. ERdj5 promoted apoptosis in tunicamycin, thapsigargin, and bortezomib-treated cells. To provide further evidence that ERdj5 induces ER stress-regulated apoptosis, we targeted Bcl-2 to ER of ERdj5-overexpressing cells. Targeting the Bcl-2 to ER prevented the apoptosis induced by ER stress inducers but not by non-ER stress apoptotic stimuli, suggesting induction of ER stress-regulated apoptosis by ERdj5. ERdj5 enhanced apoptosis by abolishing the ER stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) and the subsequent translational repression. ERdj5 was found to inhibit the eIF2alpha phosphorylation under ER stress through inactivating the pancreatic endoplasmic reticulum kinase. The compromised integrated stress response observed in ERdj5-overexpressing ER-stressed cells due to repressed eIF2alpha phosphorylation correlated with impaired neuroblastoma cell resistance under ER stress. These results demonstrate that ERdj5 decreases neuroblastoma cell survival by down-regulating the UPR, raising the possibility that this protein could be a target for anti-tumor approaches.

  • 66.
    Thuswaldner, Sophie
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Lagerstedt, Jens O
    Rojas-Stütz, Marc
    Bouhidel, Karim
    Der, Christophe
    Leborgne-Castel, Nathalie
    Mishra, Arti
    Marty, Francis
    Schoefs, Benoit
    Adamska, Iwona
    Persson, Bengt L
    Spetea Wiklund, Cornelia
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 12, p. 8848-8859Article in journal (Refereed)
    Abstract [en]

    In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis. (i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 67.
    Treuter, E.
    et al.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Johansson, Lotta
    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.
    Wärnmark, A.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Leers, J.
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Pelto-Huikko, M.
    Tampere University Medical School and Tampere University Hospital, Finland..
    Sjöberg, Maria
    Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
    Wright, A. P.
    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.
    Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 10, p. 6667-6677Article in journal (Refereed)
    Abstract [en]

    Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

  • 68.
    Törnkvist, Åsa
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Parpal, Santiago
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Gustavsson, Johanna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Inhibition of Raf-1 kinase expression abolishes insulin stimulation of DNA synthesis in H4IIE hepatoma cells1994In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 269, no 19, p. 13919-13921Article in journal (Refereed)
    Abstract [en]

    The involvement of Raf-1 kinase in the insulin signal transduction chain leading to control of cell proliferation was studied in the H4IIE rat hepatoma cell line by inhibiting expression of the kinase with antisense oligodeoxyribonucleotide directed against Raf-1 mRNA. Antisense oligonucleotide was found to reduce (at 2 microM) or completely block (at 15 microM) the stimulation by insulin of DNA synthesis, measured as thymidine incorporation. The residual DNA synthesis seen in the absence of insulin stimulation was also inhibited by the Raf-1 kinase antisense oligonucleotide.

  • 69.
    Van Bogaert, Tom
    et al.
    Ghent University, Belgium.
    Vandevyver, Sofie
    Ghent University, Belgium.
    Dejager, Lien
    Ghent University, Belgium.
    Van Hauwermeiren, Filip
    Ghent University, Belgium.
    Pinheiro, Iris
    Ghent University, Belgium.
    Petta, Ioanna
    Ghent University, Belgium.
    Engblom, David
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kleyman, Anna
    Fritz-Lipmann-Institute e.V., Jena, Germany.
    Schuetz, Guenther
    German Cancer Research Center, Heidelberg.
    Tuckermann, Jan
    Fritz-Lipmann-Institute e.V., Jena, Germany.
    Libert, Claude
    Ghent University, Belgium.
    Tumor Necrosis Factor Inhibits Glucocorticoid Receptor Function in Mice A STRONG SIGNAL TOWARD LETHAL SHOCK2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 30, p. 26555-26567Article in journal (Refereed)
    Abstract [en]

    As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNF alpha, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNF alpha-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNF alpha lethality was completely abolished when it was administered after TNF alpha stimulation, indicating compromised GR function upon TNF alpha challenge. TNF alpha-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNF alpha down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNF alpha also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNF alpha-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNF alpha inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNF alpha is intimately involved.

  • 70.
    Vener, Alexander
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Harms, A.
    Univ. of Wisconsin Biotech. Center, University of Wisconsin, Madison, WI 53706, United States.
    Sussman, M.R.
    Cell. and Molecular Biology Program, Department of Horticulture, University of Wisconsin, Madison, WI 53706, United States, Univ. of Wisconsin Biotech. Center, University of Wisconsin, Madison, WI 53706, United States.
    Vierstra, R.D.
    Cell. and Molecular Biology Program, Department of Horticulture, University of Wisconsin, Madison, WI 53706, United States, Dept. of Horticulture, University of Wisconsin, 1575 Linden Dr., Madison, WI 53706, United States.
    Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes of Arabidopsis thaliana2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 10, p. 6959-6966Article in journal (Refereed)
    Abstract [en]

    The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the Dl, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the Dl, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/ dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.

  • 71.
    Vener, Alexander
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Harms, Amy
    Sussmann, Michael R
    Vierstra, Richard
    Mass spectrometric resolution of reversible protein phosphorylation in photosynthetic membranes of arabidopsis thaliana.2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, p. 6959-6966Article in journal (Refereed)
  • 72.
    Vlamis-Gardikas, A.
    et al.
    Karolinska Institutet, Stockholm, Sweden .
    Åslund, F.
    Karolinska Institutet, Stockholm, Sweden .
    Spyrou, Giannis
    Karolinska Institutet, Stockholm, Sweden .
    Bergman, T.
    Karolinska Institutet, Stockholm, Sweden.
    Holmgren, A.
    Karolinska Institutet, Stockholm, Sweden.
    Cloning, overexpression, and characterization of glutaredoxin 2, an atypical glutaredoxin from Escherichia coli1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 17, p. 11236-11243Article in journal (Refereed)
    Abstract [en]

    Glutaredoxin 2 (Grx2) from Escherichia coli catalyzes GSH-disulfide oxidoreductions via two redox-active cysteine residues, but in contrast to glutaredoxin 1 (Grx1) and glutaredoxin 3 (Grx3), is not a hydrogen donor for ribonucleotide reductase. To characterize Grx2, a chromosomal fragment containing the E. coli Grx2 gene (grxB) was cloned and sequenced. grxB (645 base pairs) is located between the rimJ and pyrC genes while an open reading frame immediately upstream grxB encodes a novel transmembrane protein of 402 amino acids potentially belonging to class II of substrate export transporters. The deduced amino acid sequence for Grx2 comprises 215 residues with a molecular mass of 24.3 kDa. There is almost no similarity between the amino acid sequence of Grx2 and Grx1 or Grx3 (both 9-kDa proteins) with the exception of the active site which is identical in all three glutaredoxins (C9PYC12 for Grx2). Only limited similarities were noted to glutathione S-transferases (Grx2 amino acids 16-72), and protein disulfide isomerases from different organisms (Grx2 amino acids 70-180). Grx2 was overexpressed and purified to homogeneity and its activity was compared with those of Grx1 and Grx3 using GSH, NADPH, and glutathione reductase in the reduction of 0.7 mM beta-hydroxyethyl disulfide. The three glutaredoxins had similar apparent Km values for GSH (2-3 mM) but Grx2 had the highest apparent kcat (554 s-1). Expression of two truncated forms of Grx2 (1-114 and 1-133) which have predicted secondary structures similar to Grx1 (betaalphabetaalphabetabetaalpha) gave rise to inclusion bodies. The mutant proteins were resolubilized and purified but lacked GSH-disulfide oxidoreductase activity. The latter should therefore require the participation of amino acid residues from the COOH-terminal half of the molecule and is probably not confined to a Grx1-like NH2-terminal subdomain. Grx2 being radically different from the presently known glutaredoxins in terms of molecular weight, amino acid sequence, catalytic activity, and lack of a consensus GSH-binding site is the first member of a novel class of glutaredoxins.

  • 73.
    Xia, Ling
    et al.
    Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Nordman, Tomas
    Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Olsson, Jerker M.
    Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Damdimopoulos, Anastassios
    Karolinska Institutet, Huddinge, Sweden.
    Björkhem-Bergman, Linda
    Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Nalvarte, Ivan
    Karolinska Institutet, Huddinge, Sweden.
    Eriksson, Lennart C.
    Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    Arnér, Elias S. J.
    Karolinska Institutet, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, Huddinge, Sweden.
    Björnstedt, Mikael
    Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
    The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 4, p. 2141-2146Article in journal (Refereed)
    Abstract [en]

    The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.

  • 74. Yokohama, T
    et al.
    Kaya, S
    Abe, K
    Taniguchi, K
    Katoh, T
    Yazawa, M
    Hayashi, Y
    Mårdh, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Acid-labile ATP and/or ADP/Pi binding to the tetraprotomeric form ofNa/K-ATPase accompanying catalytic phosphorylation-dephosphorylation cycle.1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, p. 31792-31794Article in journal (Refereed)
  • 75. Yutaha, K
    et al.
    Zhao, Ming
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Morikawa, A
    Sugiyama, T
    Chakravortty, D
    Koide, N
    Yoshida, T
    Tapping, RI
    Yang, Y
    Yokochi, T
    Lee, JD
    Big mitogen-activated kinase regulates multiple members of the MEF2 protein family.2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, p. 18534-18540Article in journal (Refereed)
  • 76.
    Zhou, Jie
    et al.
    Johann Wolfgang Goethe-University Frankfurt, Germany.
    Damdimopoulos, Anastassios E.
    Novum, Karolinska Institute, Huddinge, Sweden.
    Spyrou, Giannis
    Novum, Karolinska Institute, Huddinge, Sweden and Foundation of Biomedical Research, Academy of Athens, Greece.
    Brüne, Bernhard
    Johann Wolfgang Goethe-University Frankfurt, Germany.
    Thioredoxin 1 and Thioredoxin 2 Have Opposed Regulatory Functions on Hypoxia-inducible Factor-1α*2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 10, p. 7482-7490Article in journal (Refereed)
    Abstract [en]

    Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.

  • 77.
    Åhsberg, Josefine
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Ungerbäck, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Strid, Tobias
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Welinder, Eva
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Stjernberg, Jenny
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Larsson, Malin
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Qian, Hong
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Sigvardsson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 46, p. 33449-33461Article in journal (Refereed)
    Abstract [en]

    Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2R-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

  • 78.
    Öst, Anita
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Örtegren, Unn
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Gustavsson, Johanna
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Nyström, Fredrik H
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 1, p. 5-8Article in journal (Refereed)
    Abstract [en]

    A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

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