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  • 1.
    Aboulaich, Nabila
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Vener, Alexander V
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 17, p. 11446-11449Article in journal (Refereed)
    Abstract [en]

    In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

  • 2.
    Alenius, Mattias
    et al.
    Umeå University.
    Bohm, S
    Umeå University.
    Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 42, p. 26083-26086Article in journal (Refereed)
    Abstract [en]

    We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin Ca-type domains followed by two fibronectin type III domains, Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane, These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II, In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections, The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.

  • 3.
    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.

  • 4.
    Bauer, M. K. A.
    et al.
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen.
    Vogt, M.
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen.
    Los, Marek Jan
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen, Germany.
    Siegel, J.
    Department of Virology, Albrecht-Ludwigs-University, Freiburg, Germany.
    Wesselborg, Sebastian
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen, Germany.
    Schulze-Osthoff, Klaus
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen.
    Role of reactive oxygen intermediates in activation-induced CD95 (APO-1/Fas) ligand expression1998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 14, p. 8048-8055Article in journal (Refereed)
    Abstract [en]

    Activation-induced cell death of T lymphocytes requires the inducible expression of CD95 (APO-1/Fas) ligand, which triggers apoptosis in CD95-bearing target cells by an autocrine or paracrine mechanism. Although execution of the CD95 death pathway is largely independent of reactive oxygen intermediates, activation-induced cell death is blocked by a variety of antioxidants. In the present study, we investigated the involvement of redox processes in the regulation of CD95 ligand (CD95L) expression in Jurkat T cells. We show that various antioxidants potently inhibited the transcriptional activation of CD95L following T cell receptor litigation or stimulation of cells with phorbol ester and ionomycin. Conversely, a prooxidant such as hydrogen peroxide alone was able to increase CD95L expression. As detected by Western blot and cytotoxicity assays, functional expression of CD95L protein was likewise diminished by antioxidants. Inhibition of CD95L expression was associated with a decreased DNA binding activity of nuclear factor (NF)-kappa B, an important redox-controlled transcription factor. Moreover, inhibition of NF-kappa B activity by a transdominant I kappa B mutant attenuated CD95L expression. Our data suggest that, although reactive oxygen intermediates do not act as mediators in the execution phase of CD95-mediated apoptosis, they are involved in the transcriptional regulation of CD95L expression.

  • 5.
    Belogurov, Georgiy A
    et al.
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Penttinen, Anni
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Huopalahti, Saila
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Baykov, Alexander A
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Lahti, Reijo
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 25Article in journal (Refereed)
    Abstract [en]

    H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.

  • 6.
    Bengtson, Per
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Larson, Cecilia
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Lundblad, Arne
    Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Larson, Göran
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Påhlsson, Peter
    Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 34, p. 31575-31582Article in journal (Refereed)
    Abstract [en]

    The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLex) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLex and SLex-related epitopes. One individual showed lower levels of SLex expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg110 → Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. TheFUT7 Arg110 is conserved in all previously cloned vertebrate α1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of ≤1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLex and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

  • 7.
    Bergqvist, Niclas
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    Nyman, Elin
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering. AstraZeneca RandD, Sweden.
    Cedersund, Gunnar
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Stenkula, Karin G.
    Lund University, Sweden.
    A systems biology analysis connects insulin receptor signaling with glucose transporter translocation in rat adipocytes2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 27, p. 11206-11217Article in journal (Refereed)
    Abstract [en]

    Type 2 diabetes is characterized by insulin resistance, which arises from malfunctions in the intracellular insulin signaling network. Knowledge of the insulin signaling network is fragmented, and because of the complexity of this network, little consensus has emerged for the structure and importance of the different branches of the network. To help overcome this complexity, systems biology mathematical models have been generated for predicting both the activation of the insulin receptor (IR) and the redistribution of glucose transporter 4 (GLUT4) to the plasma membrane. Although the insulin signal transduction between IR and GLUT4 has been thoroughly studied with modeling and time-resolved data in human cells, comparable analyses in cells from commonly used model organisms such as rats and mice are lacking. Here, we combined existing data and models for rat adipocytes with new data collected for the signaling network between IR and GLUT4 to create a model also for their interconnections. To describe all data (amp;gt;140 data points), the model needed three distinct pathways from IR to GLUT4: (i) via protein kinase B (PKB) and Akt substrate of 160 kDa (AS160), (ii) via an AS160-independent pathway from PKB, and (iii) via an additional pathway from IR, e.g. affecting the membrane constitution. The developed combined model could describe data not used for training the model and was used to generate predictions of the relative contributions of the pathways from IR to translocation of GLUT4. The combined model provides a systems-level understanding of insulin signaling in rat adipocytes, which, when combined with corresponding models for human adipocytes, may contribute to model-based drug development for diabetes.

  • 8.
    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.

  • 9.
    Brannmark, Cecilia
    et al.
    University of Gothenburg, Sweden.
    Lövfors, William
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    Komai, Ali M.
    University of Gothenburg, Sweden.
    Axelsson, Tom
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Science & Engineering.
    El Hachmane, Mickael F.
    University of Gothenburg, Sweden.
    Musovic, Saliha
    University of Gothenburg, Sweden.
    Paul, Alexandra
    Chalmers University of Technology, Sweden.
    Nyman, Elin
    Linköping University, Department of Biomedical Engineering, Division of Biomedical Engineering. Linköping University, Faculty of Science & Engineering. AstraZeneca RandD, Sweden.
    Olofsson, Charlotta S.
    University of Gothenburg, Sweden.
    Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 49, p. 20032-20043Article in journal (Refereed)
    Abstract [en]

    Adiponectin is a hormone secreted from white adipocytes and takes part in the regulation of several metabolic processes. Although the pathophysiological importance of adiponectin has been thoroughly investigated, the mechanisms controlling its release are only partly understood. We have recently shown that adiponectin is secreted via regulated exocytosis of adiponectin-containing vesicles, that adiponectin exocytosis is stimulated by cAMP-dependent mechanisms, and that Ca2+ and ATP augment the cAMP-triggered secretion. However, much remains to be discovered regarding the molecular and cellular regulation of adiponectin release. Here, we have used mathematical modeling to extract detailed information contained within our previously obtained high-resolution patch-clamp time-resolved capacitance recordings to produce the first model of adiponectin exocytosis/secretion that combines all mechanistic knowledge deduced from electrophysiological experimental series. This model demonstrates that our previous understanding of the role of intracellular ATP in the control of adiponectin exocytosis needs to be revised to include an additional ATP-dependent step. Validation of the model by introduction of data of secreted adiponectin yielded a very close resemblance between the simulations and experimental results. Moreover, we could show that Ca2+-dependent adiponectin endocytosis contributes to the measured capacitance signal, and we were able to predict the contribution of endocytosis to the measured exocytotic rate under different experimental conditions. In conclusion, using mathematical modeling of published and newly generated data, we have obtained estimates of adiponectin exo- and endocytosis rates, and we have predicted adiponectin secretion. We believe that our model should have multiple applications in the study of metabolic processes and hormonal control thereof.

  • 10.
    Brannmark, Cecilia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Palmer, Robert
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Glad, Torkel
    Linköping University, Department of Electrical Engineering, Automatic Control. Linköping University, The Institute of Technology.
    Cedersund, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Mass and Information Feedbacks through Receptor Endocytosis Govern Insulin Signaling as Revealed Using a Parameter-free Modeling Framework2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 26, p. 20171-20179Article in journal (Refereed)
    Abstract [en]

    Insulin and other hormones control target cells through a network of signal-mediating molecules. Such networks are extremely complex due to multiple feedback loops in combination with redundancy, shared signal mediators, and cross-talk between signal pathways. We present a novel framework that integrates experimental work and mathematical modeling to quantitatively characterize the role and relation between coexisting submechanisms in complex signaling networks. The approach is independent of knowing or uniquely estimating model parameters because it only relies on (i) rejections and (ii) core predictions (uniquely identified properties in unidentifiable models). The power of our approach is demonstrated through numerous iterations between experiments, model-based data analyses, and theoretical predictions to characterize the relative role of co-existing feedbacks governing insulin signaling. We examined phosphorylation of the insulin receptor and insulin receptor substrate-1 and endocytosis of the receptor in response to various different experimental perturbations in primary human adipocytes. The analysis revealed that receptor endocytosis is necessary for two identified feedback mechanisms involving mass and information transfer, respectively. Experimental findings indicate that interfering with the feedback may substantially increase overall signaling strength, suggesting novel therapeutic targets for insulin resistance and type 2 diabetes. Because the central observations are present in other signaling networks, our results may indicate a general mechanism in hormonal control.

  • 11.
    Brännmark, Cecilia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Nyman, Elin
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Fagerholm, Siri
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Bergenholm, Linnéa
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ekstrand, Eva-Maria
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Cedersund, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Insulin Signaling in Type 2 Diabetes: Experimental and Modeling Analyses Reveal Mechanisms of Insulin Resistance in Human Adipocytes2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 14, p. 9867-9880Article in journal (Refereed)
    Abstract [en]

    Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems-level mechanistic understanding of insulin resistance in humans. We developed a dynamic mathematical model of insulin signaling – normally and in diabetes – based on quantitative steady-state and dynamic time-course data on signaling intermediaries in human mature adipocytes. At the core of insulin resistance is attenuation of a positive feedback from mammalian target of rapamycin in complex with raptor (mTORC1) to the insulin receptor substrate-1 (IRS1), which explains reduced sensitivity and signal strength throughout the signaling network. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.

  • 12.
    Börjesson, Sara I.
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Englund, Ulrika H.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Asif, Muhammad H.
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, The Institute of Technology.
    Willander, Magnus
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    Elinder, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Intracellular potassium (K+) concentration decrease is not obligatory for apoptosis2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 46, p. 39823-39828Article in journal (Refereed)
    Abstract [en]

    K+ efflux is observed as an early event in the apoptotic process in various cell types. Loss of intracellular K+ and subsequent reduction in ionic strength is suggested to release the inhibition of proapoptotic caspases. In this work, a new K+-specific microelectrode was used to study possible alterations in intracellular K+ in Xenopus laevis oocytes during chemically induced apoptosis. The accuracy of the microelectrode to detect changes in intracellular K+ was verified with parallel electrophysiological measurements within the same cells. In concordance with previous studies on other cell types, apoptotic stimuli reduced the intracellular K+ concentration in Xenopus oocytes and increased caspase-3 activity. The reduction in intracellular K+ was prevented by dense expression of voltage-gated K (Kv) channels. Despite this, the caspase-3 activity was increased similarly in Kv channel expressing oocytes as in oocytes not expressing Kv channels. Thus, in Xenopus oocytes caspase-3 activity is not dependent on the intracellular concentration of K+.

  • 13.
    Chilkova, O.
    et al.
    Dept. of Med. Biochem./Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Johansson, E.
    Dept. of Med. Biochem./Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
    The quaternary structure of DNA polymerase e from Saccharomyces cerevisiae2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 16, p. 14082-14086Article in journal (Refereed)
    Abstract [en]

    DNA polymerase e (Pol e) trom Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol e have been cumbersome due to protease sensitivity and the limited amounts of Pol e in cells. We have developed a protocol for overexpression and purification of Pol e from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol e was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 Å), a molecular mass for Pol e of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol e. Thus, both DNA polymerase d and Pol e are purified as monomeric complexes, in agreement with accumulating evidence that Pol d and Pol e are located on opposite strands of the eukaryotic replication fork.

  • 14.
    Civitelli, Livia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Sandin, Linnea
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Nelson, Erin
    Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science.
    Iqbal Khattak, Sikander
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Kågedal, Katarina
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    The Luminescent Oligothiophene p-FTAA Converts Toxic A beta(1-42) Species into Nontoxic Amyloid Fibers with Altered Properties2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 17, p. 9233-9243Article in journal (Refereed)
    Abstract [en]

    Aggregation of the amyloid-(beta) peptide (A beta) in the brain leads to the formation of extracellular amyloid plaques, which is one of the pathological hallmarks of Alzheimer disease (AD). It is a general hypothesis that soluble prefibrillar assemblies of the A beta peptide, rather than mature amyloid fibrils, cause neuronal dysfunction and memory impairment in AD. Thus, reducing the level of these prefibrillar species by using molecules that can interfere with the A beta fibrillation pathway may be a valid approach to reduce A beta cytotoxicity. Luminescent-conjugated oligothiophenes (LCOs) have amyloid binding properties and spectral properties that differ when they bind to protein aggregates with different morphologies and can therefore be used to visualize protein aggregates. In this study, cell toxicity experiments and biophysical studies demonstrated that the LCO p-FTAA was able to reduce the pool of soluble toxic A beta species in favor of the formation of larger insoluble nontoxic amyloid fibrils, there by counteracting A beta-mediated cytotoxicity. Moreover, p-FTAA bound to early formed A beta species and induced a rapid formation of beta-sheet structures. These p-FTAA generated amyloid fibrils were less hydrophobic and more resistant to proteolysis by proteinase K. In summary, our data show that p-FTAA promoted the formation of insoluble and stable A beta species that were nontoxic which indicates that p-FTAA might have therapeutic potential.

  • 15.
    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.

  • 16.
    Czubryt, Michael P.
    et al.
    Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, University of Manitoba, Winnipeg, Canada .
    Lamoureux, Lise
    University of Manitoba, Canada .
    Ramjiawan, Angela
    University of Manitoba, Canada .
    Abrenica, Bernard
    University of Manitoba, Canada .
    Jangamreddy, Jaganmohan Reddy
    University of Manitoba, Canada .
    Swan, Kristin
    University of Manitoba, Canada .
    Regulation of Cardiomyocyte Glut4 Expression by ZAC12010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 22, p. 16942-16950Article in journal (Refereed)
    Abstract [en]

    The transcription factor ZAC1 is expressed in a variety of tissues including the developing heart, but its physiological role is unclear. We examined the role of ZAC1 in regulating expression of the insulin-responsive glucose transporter GLUT4 and whether ZAC1 expression is altered in cardiomyocyte hypertrophy. We demonstrated expression of Zac1 mRNA and protein in rat cardiomyocytes by PCR and Western blotting, respectively. Using a combination of chromatin immunoprecipitation and luciferase assays, we showed that ZAC1 regulates Glut4 expression via a specific binding site in the Glut4 promoter. Overexpression of ZAC1 increased Glut4 mRNA and protein expression and resulted in increased glucose uptake in cardiomyocytes as determined by a fluorescent analog uptake assay. Induction of hypertrophy by phenylephrine or isoproterenol resulted in increased Zac1 expression. We identified a novel putative promoter in the Zac1 gene and demonstrated increased binding of MEF2 to this promoter in response to hypertrophic stimulation. MEF2 regulated transactivation of the Zac1 promoter and ZAC1 protein expression. This work identifies ZAC1 as a novel and previously unknown regulator of cardiomyocyte Glut4 expression and glucose uptake. Our results also implicate MEF2 as a regulator of ZAC1 expression in response to induction of hypertrophy.

  • 17.
    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.

  • 18.
    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.

  • 19.
    Espinosa, Alexander
    et al.
    Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ambrosi, Aurélie
    Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
    Anandapadamanaban, Madhanagopal
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Sandberg Abelius, Martina
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Sheng, Yi
    Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada.
    Nyberg, Filippa
    Department of Clinical Sciences at Danderyd Hospital, Karolinska Institute, Stockholm, Sweden.
    Arrowsmith, Cheryl H.
    Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Wahren-Herlenius, Marie
    Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
    Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, p. 36478-36491Article in journal (Refereed)
    Abstract [en]

    Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

  • 20.
    Fernandes, Aristi Potamitou
    et al.
    Karolinska Institutet, Stockholm.
    Fladvad, Malin
    Karolinska Institutet, Stockholm.
    Berndt, Carsten
    Karolinska Institutet, Stockholm.
    Andrésen, Cecilia
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Lillig, Christopher Horst
    Karolinska Institutet, Stockholm.
    Neubauer, Peter
    University of Oulu, Finland.
    Sunnerhagen, Maria
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Holmgren, Arne
    Karolinska Institutet, Stockholm.
    Vlamis-Gardikas, Alexios
    Karolinska Institutet, Stockholm.
    A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 26, p. 24544-24552Article in journal (Refereed)
    Abstract [en]

    Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750–2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3′,5′-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

  • 21.
    Fladvad, Malin
    et al.
    Karolinska Institutet, Stockholm.
    Bellanda, Massimo
    University of Padova, Italy.
    Fernandes, Aristi Potamitou
    Karolinska Institutet, Stockholm.
    Mammi, Stefano
    University of Padova, Italy.
    Vlamis-Gardikas, Alexios
    Karolinska Institutet, Stockholm.
    Holmgren, Arne
    Karolinska Institutet, Stockholm.
    Sunnerhagen, Maria
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Molecular Mapping of Functionalities in the Solution Structure of Reduced Grx4, a Monothiol Glutaredoxin from Escherichia coli2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 26, p. 24553-24561Article in journal (Refereed)
  • 22.
    Gasser, Andreas
    et al.
    University Medical Center Hamburg-Eppendorf, Germany .
    Bruhn, Sören
    University Medical Center Hamburg-Eppendorf, Germany.
    Guse, Andreas
    University Medical Center Hamburg-Eppendorf, Germany.
    Second Messenger Function of Nicotinic Acid Adenine Dinucleotide Phosphate Revealed by an Improved Enzymatic Cycling Assay2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 25, p. 16906-16913Article in journal (Refereed)
    Abstract [en]

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nm (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation.

  • 23.
    Gjesing Welinder, Karen
    et al.
    Aalborg University, Denmark.
    Hansen, Rasmus
    Aalborg University, Denmark; Wellspring, USA.
    Toft Overgaard, Michael
    Aalborg University, Denmark.
    Brohus, Malene
    Aalborg University, Denmark.
    Sonderkaer, Mads
    Aalborg University, Denmark.
    von Bergen, Martin
    Aalborg University, Denmark; UFZ Helmholtz Centre Environm Research, Germany; UFZ Helmholtz Centre Environm Research, Germany.
    Rolle-Kampczyk, Ulrike
    UFZ Helmholtz Centre Environm Research, Germany.
    Otto, Wolfgang
    UFZ Helmholtz Centre Environm Research, Germany.
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
    Arinell, Karin
    University of Örebro, Sweden.
    Evans, Alina L.
    Hedmark University of Coll, Norway.
    Swenson, Jon E.
    Norwegian University of Life Science, Norway; Norwegian Institute Nat Research, Norway.
    Revsbech, Inge G.
    Aarhus University, Denmark.
    Frobert, Ole
    University of Örebro, Sweden.
    Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 43, p. 22509-22523Article in journal (Refereed)
    Abstract [en]

    Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 degrees C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

  • 24.
    Grenegård, Magnus
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Vretenbrant-Öberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Nylander, Martina
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Désilets, Stéphanie
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Lindström, Eva G
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Larsson, Anders
    Department of Medical Sciences, University Hospital, Uppsala SE-75105, Sweden.
    Ramström, Ida
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Ramström, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Lindahl, Tomas L
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
    The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 27, p. 18493-18504Article in journal (Refereed)
    Abstract [en]

    Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

  • 25.
    Hakizimana, Pierre
    et al.
    Université Libre de Bruxelles, Belgium.
    Masureel, Matthieu
    Université Libre de Bruxelles, Belgium.
    Gbaguidi, Bénédicte
    Université Libre de Bruxelles, Belgium.
    Ruysschaert, Jean-Marie
    Université Libre de Bruxelles, Belgium.
    Govaerts, Cédric
    Université Libre de Bruxelles, Belgium.
    Interactions between phosphatidylethanolamine headgroup and LmrP, a multidrug transporter: a conserved mechanism for proton gradient sensing?2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 14, p. 9369-9376Article in journal (Refereed)
    Abstract [en]

    In a number of cases, the function of membrane proteins appears to require the presence of specific lipid species in the bilayer. We have shown that the secondary multidrug transporter LmrP requires the presence of phosphatidylethanolamine (PE), as its replacement by phosphatidylcholine (PC) inhibits transport activity and directly affects its structure, although the underlying mechanism was unknown. Here, we show that the effect of PE on the structure and the function of LmrP is mediated by interactions between the lipid headgroup and the protein. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by PC, including energy transfer between the protein tryptophan residues and the lipid headgroups. We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.

  • 26.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Protein compactness measured by fluorescence resonance energy transfer - Human carbonic anhydrase II Is considerably expanded by the interaction of GroEL2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 24, p. 21765-21775Article in journal (Refereed)
    Abstract [en]

    Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence in fluorescence resonance energy transfer. The ratio between the fluorescence intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid (AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nn (direct AEDANS absorption) was used to estimate the average distances between the seven Trp residues in human carbonic anhydrase II (HCA II) and the AEDANS label, Guanidine HCl denaturation of the HCA II variants was also performed to obtain a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the protein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL, It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA II to bind to GroEL, and the volume of the molten globule intermediate increased similar to2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (to similar to 117,000 Angstrom (3)), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity, Recently, we found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarstrom, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), thereby facilitating rearrangement of misfolded structure.

  • 27.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Andersson, D.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Structural mapping of an aggregation nucleation site in a molten-globule intermediate1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, p. 32897-32903Article in journal (Refereed)
  • 28.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Owenius, Rikard
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Protein substrate binding induces conformational changes in the chaperonin GroEL: A suggested mechanism for unfoldase activity2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 30, p. 22832-22838Article in journal (Refereed)
    Abstract [en]

    Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxy1-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.

  • 29.
    Hennig, Janosch
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Ottosson, L.
    Department of Medicine, CMM L8:04, Karolinska Hospital, 5-171 76 Stockholm, Sweden.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Horvath, L.
    Department of Medicine, CMM L8:04, Karolinska Hospital, 5-171 76 Stockholm, Sweden.
    Kuchroo, V.K.
    Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, United States.
    Broo, K.
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy, Göteborg University, S-412 66 Göteborg, Sweden.
    Wahren-Herlenius, M.
    Department of Medicine, CMM L8:04, Karolinska Hospital, 5-171 76 Stockholm, Sweden.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Structural organization and Zn2+ -dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro522005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 39, p. 33250-33261Article in journal (Refereed)
    Abstract [en]

    Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn 2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 30.
    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.

  • 31.
    Karlsson, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Ekeroth, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Elwing, Hans
    Department of Cell and Molecular Biology, Göteborg University, Sweden.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 27, p. 25558-25564Article in journal (Refereed)
    Abstract [en]

    The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.

  • 32.
    Lagergren, Anna
    et al.
    Department for Hematopoetic Stem Cell Biology, Lund Stemcell Center, Lund University, Lund, Sweden.
    Månsson, Robert
    Department for Hematopoetic Stem Cell Biology, Lund Stemcell Center, Lund University, Lund, Sweden.
    Zetterblad, Jenny
    Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Linköping University, Faculty of Health Sciences.
    Smith, Emma
    Department for Hematopoetic Stem Cell Biology, Lund Stemcell Center, Lund University, Lund, Sweden.
    Basta, Barbro
    Department of Molecular Pharmacology, AstraZeneca R&D Mölndal, Sweden.
    Bryder, David
    Department for Hematopoetic Stem Cell Biology, Lund Stemcell Center, Lund University, Lund, Sweden.
    Åkerblad, Peter
    Department of Molecular Pharmacology, AstraZeneca R&D Mölndal, Sweden.
    Sigvardsson, Mikael
    Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Linköping University, Faculty of Health Sciences.
    The Cxcl12, Periostin, and Ccl9 genes are direct targets for early B-cell factor in OP-9 stroma cells2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 19, p. 14454-14462Article in journal (Refereed)
    Abstract [en]

    The development of blood cells from hematopoietic stem cells in the bone marrow is dependent on communication with bone marrow stroma cells, making these cells central for the appropriate regulation of hematopoiesis. To identify transcription factors that may play a role in gene regulation in stroma cells, we performed comparative gene expression analysis of fibroblastic NIH3T3 cells, unable to support hematopoiesis in vitro, and OP-9 stroma cells, highly efficient in this regard. These experiments revealed that transcription factors of the early B cell factor (EBF) family were highly expressed in OP-9 cells as compared with the NIH3T3 cells. To identify potential targets genes for EBF proteins in stroma cells, we overexpressed EBF in fibroblasts and analyzed the pattern of induced genes by microarray analysis. This revealed that EBF was able to up-regulate expression of among others the Cxcl12, Ccl9, and Periostin genes. The identification of relevant promoters revealed that they all contained functional EBF binding sites able to interact with EBF in OP-9 cells. Furthermore, ectopic expression of a dominant negative EBF protein or antisense EBF-1 RNA in OP-9 stroma cells resulted in reduced expression of these target genes. These data suggest that EBF proteins might have dual roles in hematopoiesis acting both as intrinsic regulators of B-lymphopoiesis and as regulators of genes in bone marrow stroma cells. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 33.
    Lerm, Maria
    et al.
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Germany.
    Schmidt, Gudula
    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, Germany.
    Aktories, Klaus
    Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Germany.
    Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1)1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 41, p. 28999-29004Article in journal (Refereed)
    Abstract [en]

    The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.

  • 34.
    Lopes, Fernando
    et al.
    Univ Calgary, Canada.
    Keita, Åsa
    Linköping University, Department of Clinical and Experimental Medicine, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
    Saxena, Alpana
    Univ Calgary, Canada.
    Reyes, Jose Luis
    Univ Calgary, Canada.
    Mancini, Nicole L.
    Univ Calgary, Canada.
    Al Rajabi, Ala
    Univ Calgary, Canada.
    Wang, Arthur
    Univ Calgary, Canada.
    Baggio, Cristiane H.
    Univ Calgary, Canada.
    Dicay, Michael
    Univ Calgary, Canada.
    van Dalen, Rob
    Univ Toronto, Canada.
    Ahn, Younghee
    Univ Calgary, Canada.
    Carneiro, Matheus B. H.
    Univ Calgary, Canada.
    Peters, Nathan C.
    Univ Calgary, Canada.
    Rho, Jong M.
    Univ Calgary, Canada.
    MacNaughton, Wallace K.
    Univ Calgary, Canada.
    Girardin, Stephen E.
    Univ Toronto, Canada.
    Jijon, Humberto
    Univ Calgary, Canada.
    Philpott, Dana J.
    Univ Toronto, Canada.
    Söderholm, Johan D
    Linköping University, Department of Clinical and Experimental Medicine, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Linköping.
    McKay, Derek M.
    Univ Calgary, Canada.
    ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 9, p. 3073-3087Article in journal (Refereed)
    Abstract [en]

    The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAP K1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohns disease susceptibility mutation in the autophagy gene ATG16L.1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress ATF6 DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

  • 35.
    Löfstedt, T
    et al.
    Lund University.
    Jögi, A
    Lund University.
    Sigvardsson, Mikael
    Lund University.
    Gradin, K
    Karolinska Institute.
    Poellinger, L
    Karolinska Institute.
    Påhlman, S
    Lund University.
    Axelson, H
    Lund University.
    Induction of ID2 expression by hypoxia-inducible factor-1 - A role in dedifferentiation of hypoxic neuroblastoma cells2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 38, p. 39223-39231Article in journal (Refereed)
    Abstract [en]

    ID ( inhibitor of differentiation/DNA binding) proteins, frequently deregulated in advanced human malignancies, can participate in multiple fundamental traits of cancer, such as block of differentiation, increased proliferation, tissue invasiveness, and angiogenesis. We have previously demonstrated that hypoxia decreases expression of neuronal marker genes in neuroblastoma, but induces genes expressed in the neural crest, such as ID2. Because of its involvement in normal neural crest development and its ability to inhibit proneuronal bHLH proteins, the hypoxic induction of ID2 was of particular interest. Here we report fast induction kinetics of ID2 expression in hypoxic neuroblastoma cells. The up-regulation of ID2 was abolished by addition of actinomycin D, implicating a hypoxia-driven transcriptional mechanism. Analyzing the ID2 promoter revealed several potential binding sites for hypoxia-inducible factors. Subsequent electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated two functional HIF-1 binding sites within ID2 gene regulatory sequences located at -725 and -1893 relative to the transcriptional initiation point. In transfection assays, DNA constructs of the ID2 promoter, including the functional HIF-1 binding sites, induced luciferase reporter activity in a HIF-1-specific manner. These observations demonstrate that ID2 is actively engaged by hypoxia and represents a novel HIF-1 target. Hypoxia-induced ID2 expression could play a significant role in the previously observed dedifferentiation of hypoxic neuroblastoma cells, which in a clinical setting could lead to less mature and more aggressive tumors.

  • 36.
    Mansson, R
    et al.
    Lund University.
    Tsapogas, P
    Lund University.
    Akerlund, M
    Lund University.
    Lagergren, A
    Lund University.
    Gisler, R
    Lund University.
    Sigvardsson, Mikael
    Lund University.
    Pearson correlation analysis of microarray data allows for the identification of genetic targets for early B-cell factor2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 17, p. 17905-17913Article in journal (Refereed)
    Abstract [en]

    B lymphocyte development is a complex biological process critically dependent on the transcription factor early B cell factor (EBF). To deepen understanding of the roles for EBF in this process, we have used Pearson correlation analysis to evaluate microarray data from a set of mouse B lymphoid cell lines representing different stages of development. Comparing the expression pattern of EBF to that of the other genes in the data set revealed that VpreB1, mb-1, and lambda5, all known target genes, presented high correlation values to EBF. High correlations were also seen for the VpreB3 and CD19 genes and biochemical as well as functional data supported that they are target genes for EBF even though the expression of CD19 was critically dependent of Pax-5. We also obtained evidence for extensive collaborative actions of EBF and E47 even though microarray analysis of hematopoetic progenitor cells ectopically expressing these proteins suggested that they activated only a subset of pre-B cell restricted genes.

  • 37.
    Margalith, Ilan
    et al.
    University of Zurich Hospital, Switzerland .
    Suter, Carlo
    University of Zurich Hospital, Switzerland .
    Ballmer, Boris
    University of Zurich Hospital, Switzerland .
    Schwarz, Petra
    University of Zurich Hospital, Switzerland .
    Tiberi, Cinzia
    University of Zurich Hospital, Switzerland .
    Sonati, Tiziana
    University of Zurich Hospital, Switzerland .
    Falsig, Jeppe
    University of Zurich Hospital, Switzerland .
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Åslund, Andreas
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Yam, Alice
    Novartis Diagnost, USA .
    Whitters, Eric
    Novartis Diagnost, USA .
    Hornemann, Simone
    University of Zurich Hospital, Switzerland .
    Aguzzi, Adriano
    University of Zurich Hospital, Switzerland .
    Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 23, p. 18872-18887Article in journal (Refereed)
    Abstract [en]

    Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits.

  • 38.
    Merluzzi, S
    et al.
    University of Udine, Italy.
    Moretti, M
    University of Udine, Italy.
    Altamura, S
    University of Udine, Italy.
    Zwollo, P
    The College of William and Mary, Williamsburg, Virginia .
    Sigvardsson, Mikael
    Lund University.
    Vitale, G
    University of Udine, Italy.
    Pucillo, C
    University of Udine, Italy.
    CD40 stimulation induces Pax5/BSAP and EBF activation through a APE/ref-1-dependent redox mechanism2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 3, p. 1777-1786Article in journal (Refereed)
    Abstract [en]

    CD40 is a member of the growing tumor necrosis factor receptor family that has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154, mainly expressed on activated T cells, stimulates B cell proliferation, differentiation, isotype switching, up-regulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. In this study we demonstrate that the redox factor APE/Ref-1 acts as a key signaling intermediate in response to CD40-mediated B cell activation. The transcription factors Pax5a or BSAP ( B cell lineage-specific activator protein) and EBF ( early B cell factor) are constitutively expressed in spleen B cells and CD40 cross-linking induces increases in Pax5a and EBF binding activity compared with nonstimulated B cells. We show that upon CD40 antibody-mediated cross-linking, APE/Ref-1 translocates from the cytoplasm to the nucleus of activated B cells, where it modulates the DNA binding activity of both Pax5a and EBF. Moreover, we show that the repression of APE/Ref-1 protein production is able to block CD40-mediated Pax5a activation. We also provide evidence that APE/Ref-1 can modulate the cooperative activation of the blk promoter operated by Pax5a and EBF and that APE/Ref-1 might directly regulate EBF functional activity. Finally, we show that the interaction between Pax5a and EBF enhances EBF binding activity to its consensus sequence, suggesting that Pax5a can physically interact with EBF and modulate its DNA binding activity.

  • 39.
    Meruvu, Sunitha
    et al.
    Avd för Cellbiologi IBK.
    Walther, Matthias
    Ivanov, Igor
    Hammarström, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Fürstenberger, Gerhard
    Krieg, Peter
    Reddanna, Pallu
    Kuhn, Hartmut
    Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: Targeted substrate modification and site-directed mutagenesis2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 44, p. 36633-36641Article in journal (Refereed)
    Abstract [en]

    Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe390, Gly441, Ala455, and Val631 align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe390 and Ala455 did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly441 to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val631, which aligns with the positional determinant He593 of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val631 for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val 631 as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 40.
    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.

  • 41.
    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.

  • 42.
    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.

  • 43.
    Nilsson, M.
    et al.
    Department of Clinical Chemistry, University Hospital, S-22185 Lund, Sweden.
    Wang, X.
    Department of Clinical Chemistry, University Hospital, S-22185 Lund, Sweden.
    Rodziewicz-Motowidlo, S.
    Department of Organic Chemistry, Faculty of Chemistry, University of Gdansk, J. Sobieskiego 18, 80-952 Gdansk, Poland.
    Janowski, R.
    Department of Crystallography, A. Mickiewicz University, Polish Academy of Sciences, Grunwaldzka 6, 60-780 Poznan, Poland.
    Lindstrom, V.
    Lindström, V., Department of Clinical Chemistry, University Hospital, S-22185 Lund, Sweden.
    Onnerfjord, P.
    Önnerfjord, P., Dept. of Cell and Molecular Biology, University of Lund, S-22184 Lund, Sweden.
    Westermark, Gunilla
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Grzonka, Z.
    Department of Organic Chemistry, Faculty of Chemistry, University of Gdansk, J. Sobieskiego 18, 80-952 Gdansk, Poland.
    Jaskolski, M.
    Department of Crystallography, A. Mickiewicz University, Polish Academy of Sciences, Grunwaldzka 6, 60-780 Poznan, Poland.
    Grubb, A.
    Department of Clinical Chemistry, University Hospital, S-22185 Lund, Sweden.
    Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C. Use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 23, p. 24236-24245Article in journal (Refereed)
    Abstract [en]

    Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.

  • 44. Nilsson, Maria
    et al.
    Wang, Xin
    Rodziewicz-Motowidlo, Sylwia
    Janowski, Robert
    Lindström, Veronica
    Önnerfjord, Patrik
    Westermark, Gunilla
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Grzonka, Zbigniew
    Jaskolski, Mariusz
    Grubb, Anders
    Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, p. 24236-24245Article in journal (Refereed)
  • 45.
    Nyman, Elin
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Brännmark, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Palmér, Robert
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Brugård, Jan
    MathCore Engn.
    Nyström, Fredrik
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Cedersund, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    A Hierarchical Whole-body Modeling Approach Elucidates the Link between in Vitro Insulin Signaling and in Vivo Glucose Homeostasis2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 29, p. 26028-26041Article in journal (Refereed)
    Abstract [en]

    Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.

  • 46.
    Nyman, Elin
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Health Sciences.
    Rohini Rajan, Meenu
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Fagerholm, Siri
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Brännmark, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Cedersund, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedical Engineering.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    A Single Mechanism Can Explain Network-wide Insulin Resistance in Adipocytes from Obese Patients with Type 2 Diabetes2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 48, p. 33215-33230Article in journal (Refereed)
    Abstract [en]

    The response to insulin is impaired in type 2 diabetes. Much information is available about insulin signaling, but understanding of the cellular mechanisms causing impaired signaling and insulin resistance is hampered by fragmented data, mainly obtained from different cell lines and animals. We have collected quantitative and systems-wide dynamic data on insulin signaling in primary adipocytes and compared cells isolated from healthy and diabetic individuals. Mathematical modeling and experimental verification identified mechanisms of insulin control of the MAPKs ERK1/2. We found that in human adipocytes, insulin stimulates phosphorylation of the ribosomal protein S6 and hence protein synthesis about equally via ERK1/2 and mTORC1. Using mathematical modeling, we examined the signaling network as a whole and show that a single mechanism can explain the insulin resistance of type 2 diabetes throughout the network, involving signaling both through IRS1, PKB, and mTOR and via ERK1/2 to the nuclear transcription factor Elk1. The most important part of the insulin resistance mechanism is an attenuated feedback from the protein kinase mTORC1 to IRS1, which spreads signal attenuation to all parts of the insulin signaling network. Experimental inhibition of mTORC1 using rapamycin in adipocytes from non-diabetic individuals induced and thus confirmed the predicted network-wide insulin resistance.

  • 47.
    Nyström, Sofie
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 31, p. 25975-25984Article in journal (Refereed)
    Abstract [en]

    The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met129 (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90–231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, Tm = 65–66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3–4 °C. The most destabilizing substitution was M129P, which lowered the Tm by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.

  • 48.
    Parpal, Santiago
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Karlsson, Margareta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Thorn, Hans
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 13, p. 9670-9678Article in journal (Refereed)
    Abstract [en]

    Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Strålfors, P. (1999) FASEB. J. 13, 1961–1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with β-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by 125I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.

  • 49.
    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.

  • 50.
    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.

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