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  • 851.
    Wieloch, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ehlers, Ina
    Frank, David
    Gessler, Arthur
    Grabner, Michael
    Yu, Jun
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tree-ring cellulose exhibits several distinct intramolecular 13C signals2017In: Geophysical Research Abstracts, 2017, Vol. 19, article id EGU2017-14723Conference paper (Refereed)
  • 852.
    Wieloch, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ehlers, Ina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yu, Jun
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Frank, David
    Grabner, Michael
    Gessler, Arthur
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Intramolecular 13C analysis of tree rings provides multiple plant ecophysiology signals covering decades2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5048Article in journal (Refereed)
    Abstract [en]

    Measurements of carbon isotope contents of plant organic matter provide important information in diverse fields such as plant breeding, ecophysiology, biogeochemistry and paleoclimatology. They are currently based on 13C/12C ratios of specific, whole metabolites, but we show here that intramolecular ratios provide higher resolution information. In the glucose units of tree-ring cellulose of 12 tree species, we detected large differences in 13C/12C ratios (>10‰) among carbon atoms, which provide isotopically distinct inputs to major global C pools, including wood and soil organic matter. Thus, considering position-specific differences can improve characterisation of soil-to-atmosphere carbon fluxes and soil metabolism. In a Pinus nigra tree-ring archive formed from 1961 to 1995, we found novel 13C signals, and show that intramolecular analysis enables more comprehensive and precise signal extraction from tree rings, and thus higher resolution reconstruction of plants’ responses to climate change. Moreover, we propose an ecophysiological mechanism for the introduction of a 13C signal, which links an environmental shift to the triggered metabolic shift and its intramolecular 13C signature. In conclusion, intramolecular 13C analyses can provide valuable new information about long-term metabolic dynamics for numerous applications.

  • 853.
    Wieloch, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ehlers, Ina
    Yu, Jun
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Frank, David
    Grabner, Michael
    Gessler, Arthur
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tree-ring cellulose exhibits several interannual 13C signals on the intramolecular level2018In: Geophysical Research Abstracts, 2018, Vol. 20, article id EGU2018-17509-2Conference paper (Refereed)
    Abstract
  • 854.
    Wieloch, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sharkey, Thomas David
    Werner, Roland Anton
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Intramolecular 13C/12C signals reflect carbon allocation in plant leavesManuscript (preprint) (Other academic)
  • 855.
    Wilczynska, Malgorzata
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Fa, M
    Karolin, J
    Ohlsson, P I
    Johansson, L B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins.1997In: Nature Structural Biology, ISSN 1072-8368, Vol. 4, no 5, p. 354-7Article in journal (Refereed)
  • 856.
    Wilczynska, Malgorzata
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Fa, M
    Ohlsson, P I
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex.1995In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 270, no 50, p. 29652-5Article in journal (Refereed)
    Abstract [en]

    Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

  • 857.
    Wilczynska, Malgorzata
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lobov, Sergei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The spontaneous polymerization of plasminogen activator inhibitor type-2 and Z-antitrypsin are due to different molecular aberrations2003In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 537, no 1-3, p. 11-16Article in journal (Refereed)
    Abstract [en]

    The wild-type form of plasminogen activator inhibitor type-2 (PAI-2) and the pathogenic Z-mutant of alpha(1)-antitrypsin (alpha(1)AT) are serpins that spontaneously polymerize by the loop-sheet mechanism. Compared to the consensus serpin sequence, both PAI-2 and Z-alpha(1)AT have deviations in the so-called breach region located at the top of the A beta-sheet. In the case of Z-alpha(1)AT, conformational perturbations caused by a single amino acid substitution result in polymerization in vivo and predisposes to disease. To test whether the polymerization of PAI-2 is due to aberrations in the breach region, we constructed substitution mutants of PAI-2 with conserved residues in this region. Analysis of the mutants revealed that deviations in the breach region modulate but are not the major cause of PAI-2 polymerization. Rather, PAI-2 exists in a highly polymerogenic conformation and does not require conformational rearrangements before polymerization can take place.

  • 858.
    Wilczynska, Malgorzata
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lobov, Sergei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ohlsson, Per-Ingvar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.2003In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 8, p. 1753-1761Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

  • 859.
    Wilhelm, Kristina
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Protein complexes: assembly, structure and function2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

     Most proteins must fold into their native conformations to fulfil their biological functions. Failure of proteins to fold leads to cell pathology and a broad range of human diseases referred to as protein misfolding disease, e.g., Alzheimer’s disease, Parkinson’s disease, and type II diabetes. More than 40 proteins are known to be connected with misfolding diseases. These proteins share no sequence homology but all assemble into cross-b sheet containing insoluble fibrillar aggregates. Despite the pathological conditions that these proteins can induce, living organisms can take advantage of the inherent ability of these proteins to form such structures and to generate novel and diverse biological function, the functional amyloid.

     This thesis examines different aspects of cross-b sheet containing aggregates. The first paper describes the humoral response to aggregated structures of insulin and the astrocytical biomarker S100B in patients suffering from Parkinson’s disease. We show that the patients have an increased immunreactivity towards insulin and S100B in Parkinson’s disease patients compared to a control group.

     The second part of this work focuses on a functional amyloid. HAMLET (human a-lactalbumin made lethal for tumour cells) is a complex of a-lactalbumin and oleic acid, which kills tumour cells but not healthy differentiated cells. We wish to expand the concept of HAMLET to a structurally related protein and therefore create and characterize a complex of equine lysozyme and oleic acid (Paper II). We chose equine lysozyme because both proteins (equine lysozyme and a-lactalbumin) share common ancestors and are spatially related. The newly designed complex was named ELOA, for equine lysozyme with oleic acid. ELOA represents a functional oligomer due to its multimeric state and its ability to bind amyloid specific dyes. In the third paper, we investigate the interaction of the cytotoxic ELOA with live cells in real time to find a mechanistic model (Paper III).

     It is known that HAMLET is not only tumouricidal but is also toxic towards many bacteria. Therefore in the last part of the thesis, we investigated the effects of ELOA on different bacterial strains and focused on its interplay Streptococcus pneumoniae (Paper IV).

     These studies have added significantly to many aspects of protein folding and misfolding from its involvement in Parkinson’s disease to the newly gained functions and structural aspects of de novo produced ELOA.

  • 860.
    Wilhelm, Kristina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Darinskas, A
    Noppe, W
    Duchardt, E
    Mok, KH
    Vukojevic, V
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Protein oligomerization induced by oleic acid at the solidliquid interface: equine lysozyme cytotoxic complexes2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 15, p. 3975-3989Article in journal (Refereed)
    Abstract [en]

    Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid–liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein α-lactalbumin, known as humanα-lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4–30 lysozyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical morphology, assembles into ring-shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well-populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

  • 861.
    Wilhelm, Kristina R
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gruden, M A
    P.K. Anokhin Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow, Russia.
    Zamotin, V
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Malisauskas, M
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Casaite, V
    Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius, Lithuania.
    Darinskas, A
    Institute of Immunology, Vilnius University, Vilnius, Lithuania.
    Forsgren, Lars
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology.
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients2007In: European Journal of Neurology, ISSN 1351-5101, E-ISSN 1468-1331, Vol. 14, no 3, p. 327-334Article in journal (Refereed)
    Abstract [en]

    Peripheral immune responses can be sensitive indicators of disease pathology. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of 26 Parkinson's disease (PD) patients compared with controls by using ELISA. We found a statistically significant increase of the autoimmune responses to both antigens in PD patients compared with controls with a mean increase of 70% and 50% in the autoimmune reactions towards insulin and S100B, respectively. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in PD patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type, while sequential epitope of native insulin is hidden within the amyloid structures. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.

  • 862. Williams, Jessica S
    et al.
    Clausen, Anders R
    Lujan, Scott A
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Clark, Alan B
    Burgers, Peter M
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kunkel, Thomas A
    Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific2015In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 22, no 4, p. 291-U35Article in journal (Refereed)
    Abstract [en]

    Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA-damage phenotypes. Here we show that these phenotypes are incurred by a high level of ribonucleotides incorporated by a leading strand-replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension.

  • 863. Williams, Jessica S
    et al.
    Clausen, Anders R
    Nick McElhinny, Stephanie A
    Watts, Brian E
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kunkel, Thomas A
    Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase ɛ.2012In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 11, no 8, p. 649-656Article in journal (Refereed)
    Abstract [en]

    We have investigated the ability of the 3' exonuclease activity of Saccharomyces cerevisiae DNA polymerase ɛ (Pol ɛ) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ɛ proofreads ribonucleotides with apparent efficiencies that vary from none at some locations to more than 90% at others, with rA and rU being more efficiently proofread than rC and rG. Previous studies show that failure to repair ribonucleotides in the genome of rnh201Δ strains that lack RNase H2 activity elevates the rate of short deletions in tandem repeat sequences. Here we show that this rate is increased by 2-4-fold in pol2-4 rnh201Δ strains that are also defective in Pol ɛ proofreading. In comparison, defective proofreading in these same strains increases the rate of base substitutions by more than 100-fold. Collectively, the results indicate that although proofreading of an 'incorrect' sugar is less efficient than is proofreading of an incorrect base, Pol ɛ does proofread newly inserted rNMPs to enhance genome stability.

  • 864. Williams, Jessica S.
    et al.
    Smith, Dana J.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lujan, Scott A.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kunkel, Thomas A.
    Topoisomerase 1-Mediated Removal of Ribonucleotides from Nascent Leading-Strand DNA2013In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 49, no 5, p. 1010-1015Article in journal (Refereed)
    Abstract [en]

    RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.

  • 865.
    Williams, Lindsey N
    et al.
    Department of Pathology, University of Washington, Seattle, WA 98195, USA.
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Knowels, Gary M
    Department of Pathology, University of Washington, Seattle, WA 98195, USA.
    Schultz, Eric M
    Department of Pathology, University of Washington, Seattle, WA 98195, USA.
    Fox, Edward J
    Department of Pathology, University of Washington, Seattle, WA 98195, USA.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Herr, Alan J
    Department of Pathology, University of Washington, Seattle, WA 98195, USA.
    dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 19, p. E2457-E2466Article in journal (Refereed)
    Abstract [en]

    Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

  • 866. Williamson, Philip T. F.
    et al.
    Horrocks, Jack
    Maheswaran, Luckshi
    Concistre, Maria
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Unravelling the Role of S100A9 in the Development of Neurodegenerative Disease2019In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 338A-338AArticle in journal (Other academic)
  • 867. Wu, Bin
    et al.
    Girard, Frederic
    van Buuren, Bernd
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tessari, Marco
    Wijmenga, Sybren
    Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold.2004In: Nucleic Acids Research, ISSN 1362-4962, Vol. 32, no 10, p. 3228-39Article in journal (Refereed)
  • 868.
    Wu, Junfang
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Domellöf, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Zivkovic, Angela M.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Nording, Malin L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation2016In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 469, no 3, p. 626-632Article in journal (Refereed)
    Abstract [en]

    Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol water (MeOH/H2O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H2O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9-24 days after delivery) and late (31-87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant.

  • 869.
    Wu, Zhihong
    et al.
    Department of Traditional Chinese Integrated Western Medicine, Qi Lu Hospital, Shandong University, Jinan, 250012, People’s Republic of China.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gong, Kebo
    Qi Lu Children’s Hospital, Shandong University, Jinan, 250022, People’s Republic of China.
    Wu, Zhihua
    Qi Lu Children’s Hospital, Shandong University, Jinan, 250022, People’s Republic of China.
    Zhang, Tingguo
    Department of Pathology, Qi Lu Hospital, Shandong University, Jinan, 250012, People’s Republic of China.
    Zhang, Xiaodan
    Department of Traditional Chinese Integrated Western Medicine, Qi Lu Hospital, Shandong University, Jinan, 250012, People’s Republic of China.
    Li, Shuling
    Department of Traditional Chinese Integrated Western Medicine, Qi Lu Hospital, Shandong University, Jinan, 250012, People’s Republic of China.
    Increased osteopontin expression is associated with progression from vulvar precancerous lesions to vulvar squamous cell carcinoma2014In: Archives of Gynecology and Obstetrics, ISSN 0932-0067, E-ISSN 1432-0711, Vol. 289, no 3, p. 637-644Article in journal (Refereed)
    Abstract [en]

    Vulvar squamous cell carcinoma (VSCC) contributes to about 3-5 % of all gynecological cancers. Vulvar intraepithelial neoplasia (VIN) and vulvar lichen sclerosus (VLS) are regarded as precancerous lesions. Early detection and treatment of precancerous lesions may prevent development of VSCC. Osteopontin (OPN) has been shown to be involved in many physiological and pathological processes, such as tumor progression, by promoting cancer cell invasion and metastasis. As a result of these findings, OPN has been described as a potential marker for tumor progression in some malignancies. In this study, we investigated the expression of OPN in vulvar tissue specimens and compared its expression between different histopathological grades. In the present study, the expression patterns of OPN in 80 paraffin-embedded tissue specimens, including 25 VSCC samples, 21 VIN lesions and 21 VLS, in addition to 13 normal vulvar samples, were examined by the immunohistochemical method and chromogenic in situ hybridization. The intensity of OPN expression steadily increased according to the pathological grades. In addition, OPN staining was found in the extracellular matrix in VSCC. Expression levels of OPN increased from VLS and VIN to VSCC, and steadily increased with the pathological stage of VSCC. Our results suggest that OPN may be associated with the progression of VSCC.

  • 870.
    Wåhlin, Anders
    et al.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Ambarki, Khalid
    Umeå University, Faculty of Medicine, Department of Radiation Sciences. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Birgander, Richard
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Malm, Jan
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Eklund, Anders
    Umeå University, Faculty of Medicine, Department of Radiation Sciences. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Intracranial pulsatility is associated with regional brain volume in elderly individuals2014In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 35, no 2, p. 365-372Article in journal (Refereed)
    Abstract [en]

    Excessive intracranial pulsatility is thought to damage the cerebral microcirculation, causing cognitive decline in elderly individuals. We investigated relationships between brain structure and measures related to intracranial pulsatility among healthy elderly. Thirty-seven stroke-free, non-demented individuals (62-82 years of age) were included. We assessed brain structure, invasively measured cerebrospinal fluid (CSF) pulse pressure, and magnetic resonance-quantified arterial and CSF flow pulsatility, as well as arterial pulse pressure. Using both multivariate partial least squares and ordinary regression analyses, we identified a significant pattern of negative relationships between the volume of several brain regions and measures of intracranial pulsatility. The strongest relationships concerned the temporal lobe cortex and hippocampus. These findings were also coherent with observations of positive relationships between intracranial pulsatility and ventricular volume. In conclusion, elderly subjects with high intracranial pulsatility display smaller brain volume and larger ventricles, supporting the notion that excessive cerebral arterial pulsatility harms the brain. This calls for research investigating altered intracranial cardiac-related pulsatile stress as a potential risk factor that may cause or worsen the prognosis in subjects developing cognitive impairment and dementia.

  • 871. Xing, Xuanxuan
    et al.
    Kane, Daniel P.
    Bulock, Chelsea R.
    Moore, Elizabeth A.
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Shcherbakova, Polina V.
    A recurrent cancer-associated substitution in DNA polymerase ε produces a hyperactive enzyme2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 374Article in journal (Refereed)
    Abstract [en]

    Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Severe mutator effects of the most common variant, Polε-P286R, modeled in yeast suggested that its pathogenicity involves yet unknown mechanisms beyond simple proofreading deficiency. We show that, despite producing a catastrophic amount of replication errors in vivo, the yeast Polε-P286R analog retains partial exonuclease activity and is more accurate than exonuclease-dead Polε. The major consequence of the arginine substitution is a dramatically increased DNA polymerase activity. This is manifested as a superior ability to copy synthetic and natural templates, extend mismatched primer termini, and bypass secondary DNA structures. We discuss a model wherein the cancer-associated substitution limits access of the 3'-terminus to the exonuclease site and promotes binding at the polymerase site, thus stimulating polymerization. We propose that the ultramutator effect results from increased polymerase activity amplifying the contribution of Polε errors to the genomic mutation rate.

  • 872. Xu, Weixin
    et al.
    Zhang, Ce
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Derreumaux, Philippe
    Graslund, Astrid
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mu, Yuguang
    Intrinsic determinants of A beta(12-24) pH-dependent self-assembly revealed by combined computational and experimental studies2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e24329-Article in journal (Refereed)
    Abstract [en]

    The propensity of amyloid-beta (A beta) peptide to self-assemble into highly ordered amyloid structures lies at the core of their accumulation in the brain during Alzheimer's disease. By using all-atom explicit solvent replica exchange molecular dynamics simulations, we elucidated at the atomic level the intrinsic determinants of the pH-dependent dimerization of the central hydrophobic segment A beta(12-24) and related these with the propensity to form amyloid fibrils measured by experimental tools such as atomic force microscopy and fluorescence. The process of A beta(12-24) dimerization was evaluated in terms of free energy landscape, side-chain two-dimensional contact probability maps, beta-sheet registries, potential mean force as a function of inter-chain distances, secondary structure development and radial solvation distributions. We showed that dimerization is a key event in A beta(12-24) amyloid formation; it is highly prompted in the order of pH 5.0 > 2.9 > > 8.4 and determines further amyloid growth. The dimerization is governed by a dynamic interplay of hydrophobic, electrostatic and solvation interactions permitting some variability of beta-sheets at each pH. These results provide atomistic insight into the complex process of molecular recognition detrimental for amyloid growth and pave the way for better understanding of the molecular basis of amyloid diseases.

  • 873.
    Xu, Weixin
    et al.
    East China Normal University, Shanghai, China.
    Zhang, Ce
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhang, John Z H
    East China Normal University, Shanghai, China.
    Mu, Yuguang
    School of Biological Sciences, Nanyang Technological University, Singapore.
    pH-Dependent Conformational Ensemble and Polymorphism of Amyloid-beta Core Fragment2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 28, p. 8392-9Article in journal (Refereed)
    Abstract [en]

    Characterization of amyloid oligomeric species is important due to its possible responsibility for the toxicity of amyloid proteins, whereas it is difficult to detect by current spectroscopic techniques. The pH-dependent tetramerization and fibrillation of the central hydrophobic segment of Alzheimer amyloid β-peptide (Aβ(12-24)) were respectively explored by all-atom replica exchange molecular dynamics simulations and by fluorescence and atomic force microscopy measurements. Our combined study shows that more β-sheet structures in the early event of tetramerization is linked directly to the high propensity to form amyloid fibrils in the consequent fibrillation. Both tetramerization and fibrillation are strongly regulated by pH. At pH 5.0, Aβ(12-24) has two opposite terminal charges. The electrostatic attraction between the side-chains of His13/His14 and Glu22/Asp23 thus acts as a "pattern keeper", resulting in high propensity of amyloid formation. These results suggest that pH effects most likely by affecting the ionization properties of the Aβ(12-24) peptide. Specifically, the pH-dependent equilibrium conformational distribution of different aggregate species are well-investigated in detail. Our findings also give hints to other experimental findings that the kinetics and morphologies of Aβ fibril formation are strongly pH-dependent.

  • 874. Yamaoka, Y
    et al.
    Ojo, O
    Fujimoto, S
    Odenbreit, S
    Haas, R
    Gutierrez, O
    El-Zimaity, H M T
    Reddy, R
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Graham, D Y
    Helicobacter pylori outer membrane proteins and gastroduodenal disease.2006In: Gut, ISSN 0017-5749, Vol. 55, no 6, p. 775-81Article in journal (Refereed)
  • 875.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Studies of in vivo prostate amyloidosis and autoimmune responses towards amyloid structures in neurodegeneration2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    By using multidisciplinary analysis of CA inclusions in prostate glands of patients diagnosed with prostate cancer, we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We have found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate.

    We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of PD patients compared with healthy controls. Peripheral immune responses can be sensitive indicators of disease pathology. We found a statistically significant increase of the autoimmune responses to both antigens in patients compared with controls. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.

    Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies - a-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance analyses. We found significantly higher antibody levels towards monomeric a-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards a-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression. Pooled IgGs from PD patients and controls interacted also with amyloid fibrils of Ab (1-40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to amyloid conformational epitope, though displaying higher specificity towards human amyloid species associated with neurodegeneration. The findings suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein - a-synuclein can be used in treatment strategies and in diagnostics, especially in identifying early disease.

  • 876.
    Yanamandra, Kiran
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Alexeyev, Oleg
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Srivastava, Vaibhav
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Shchukarev, Andrey
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brorsson, Ann-Christin
    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
    Tartaglia, Gian Gaetano
    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
    Vogl, Thomas
    Institute of Immunology, University of Münster, Münster, Germany.
    Kayed, Rakez
    Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America.
    Wingsle, Gunnar
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Olsson, Jan
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Dobson, Christopher M
    Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
    Bergh, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Elgh, Fredrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate2009In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 4, no 5, p. e5562-Article in journal (Refereed)
    Abstract [en]

    Background The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer's disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon - prostate tissue remodelling in middle-aged and elderly men.

    Methodology/Principal Findings By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions.

    Conclusions/Significance These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.

  • 877.
    Yanamandra, Kiran
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gruden, Marina A
    Institute of Normal Physiology, Moscow.
    Casaite, Vida
    Institute of Biochemistry, Vilnius .
    Meskys, Rolandas
    Institute of Biochemistry, Vilnius .
    Forsgren, Lars
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Alpha-Synuclein Reactive Antibodies as Diagnostic Biomarkers in Blood Sera of Parkinson's Disease Patients2011In: PLoS One, ISSN 1932-6203, Vol. 6, no 4, p. e18513-Article in journal (Refereed)
    Abstract [en]

    Background

    Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinson's (PD) and Alzheimer's diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimer's disease and potentially other neurodegeneration ailments.

    Methodology/Principal Findings

    Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies – α-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance. We found significantly higher antibody levels towards monomeric α-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression (P<0.0001). This indicates potential protective role of autoimmunity in maintaining the body homeostasis and clearing protein species whose disbalance may lead to amyloid assembly. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards α-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression (P<0.0001). Pooled IgGs from PD patients and controls interacted also with the amyloid fibrils of Aβ (1–40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to the generic amyloid conformational epitope, displaying higher specificity towards human amyloid species associated with neurodegeneration.

    Conclusions/Significance

    Our findings may suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein – α-synuclein can be of value in the development of treatment and diagnostic strategies, especially during the early disease stages.

  • 878.
    Yau, Wai-Lok
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nguyen-Dinh, Van
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lindquist, Richard
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication2019In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 93, no 18, article id e00292-19Article in journal (Refereed)
    Abstract [en]

    Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required. IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level.

  • 879. Yoshida, Kazumasa
    et al.
    Bacal, Julien
    Desmarais, Damien
    Padioleau, Ismaël
    Tsaponina, Olga
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pantesco, Véronique
    Dubois, Emeric
    Parrinello, Hugues
    Skrzypczak, Magdalena
    Ginalski, Krzysztof
    Lengronne, Armelle
    Pasero, Philippe
    The histone deacetylases sir2 and rpd3 act on ribosomal DNA to control the replication program in budding yeast2014In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 54, no 4, p. 691-697Article in journal (Refereed)
    Abstract [en]

    In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins, whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3Δ cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors.

  • 880. Yousefzadeh, M. J.
    et al.
    Wyatt, D. W.
    Takata, K.
    Mu, Y.
    Hensley, S. C.
    Tomida, J.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doublie, S.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ramsden, D. A.
    McBride, K. M.
    Wood, R. D.
    Mammalian POLQ, Chromosome Stability and DNA Double-Strand Break Repair2015In: Environmental and Molecular Mutagenesis, ISSN 0893-6692, E-ISSN 1098-2280, Vol. 56, p. S48-S48Article in journal (Other academic)
  • 881. Yousefzadeh, Matthew J
    et al.
    Wyatt, David W
    Takata, Kei-Ichi
    Mu, Yunxiang
    Hensley, Sean C
    Tomida, Junya
    Bylund, Göran O
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Doublié, Sylvie
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ramsden, Dale A
    McBride, Kevin M
    Wood, Richard D
    Mechanism of suppression of chromosomal instability by DNA polymerase POLQ2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 10, p. e1004654-Article in journal (Refereed)
    Abstract [en]

    Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3' single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.

  • 882.
    Yu, Chuanhe
    et al.
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, USA.
    Gan, Haiyun
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, USA.
    Han, Junhong
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, USA.
    Zhou, Zhi-Xiong
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, USA.
    Jia, Shaodong
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Farrugia, Gianrico
    Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, USA.
    Ordog, Tamas
    Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, USA.
    Zhang, Zhiguo
    Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, USA.
    Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall2014In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 56, no 4, p. 551-563Article in journal (Refereed)
    Abstract [en]

    In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication. Surprisingly, at stalled forks, PCNA is unloaded specifically from lagging strands. PCNA unloading depends on the Elg1-containing alternative RFC complex, ubiquitination of PCNA, and the checkpoint kinases Mec1 and Rad53. Cells deficient in PCNA unloading exhibit increased chromosome breaks. Our studies provide a tool for studying replication-related processes and reveal a mechanism whereby checkpoint kinases regulate strand-specific unloading of PCNA from stalled replication forks to maintain genome stability.

  • 883. Yu, Chuanhe
    et al.
    Gan, Haiyun
    Serra-Cardona, Albert
    Zhang, Lin
    Gan, Songlin
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Xu, Rui-Ming
    Zhang, Zhiguo
    A mechanism for preventing asymmetric histone segregation onto replicating DNA strands2018In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 361, no 6409, p. 1386-+-, article id eaat8849Article in journal (Refereed)
    Abstract [en]

    How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging strand preference increases markedly in cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, due to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading vs lagging strands, and that Dbp3-Dpb4 plays a significant role in this poorly understood process.

  • 884. Yu, Ling-Zhu
    et al.
    Xiong, Bo
    Gao, Wen-Xue
    Wang, Chun-Min
    Zhong, Zhi-Sheng
    Huo, Li-Jun
    Wang, Qiang
    Hou, Yi
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, X Johné
    Schatten, Heide
    Chen, Da-Yuan
    Sun, Qing-Yuan
    MEK1/2 regulates microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte meiotic maturation.2007In: Cell cycle (Georgetown, Tex.), ISSN 1551-4005, Vol. 6, no 3, p. 330-8Article in journal (Refereed)
    Abstract [en]

    It is well known that MAPK plays pivotal roles in oocyte maturation, but the function of MEK (MAPK kinase) remains unknown. We have studied the expression, subcellular localization and functional roles of MEK during meiotic maturation of mouse oocytes. Firstly, we found that MEK1/2 phoshorylation (p-MEK1/2, indicative of MEK activation) was low in GV (germinal vesicle) stage, increased 2h after GVBD (germinal vesicle breakdown), and reached the maximum at metaphase II. Secondly, we found that P-MEK1/2 was restricted in the GV prior to GVBD. In prometaphase I and metaphase I, P-MEK1/2 was mainly associated with the spindle, especially with the spindle poles. At anaphase I and telophase I, p-MEK1/2 became diffusely distributed in the region between the separating chromosomes, and then became associated with the midbody. The association of p-MEK1/2 with spindle poles was further confirmed by its colocalization with the centrosomal proteins, gamma-tubulin and NuMA. Thirdly, we have investigated the possible functional role of MEK1/2 activation by intravenous administration and intrabursal injection of a specific MEK inhibitor, U0126, and by microinjection of MEK siRNA into oocytes. All these manipulations cause disorganized spindle poles and spindle structure, misaligned chromosomes and larger than normal polar bodies. Our results suggest that MEK1/2 may function as a centrosomal protein and may have roles in microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte maturation.

  • 885.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A number of diseases are linked to protein folding problems which lead to the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains.

    The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin.

    Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence.

    Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner.

    We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity.

    Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death.

    These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.

  • 886.
    Zamotin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharibyan, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gibanova, NV
    Lavrikova, MA
    Dolgikh, DA
    Kirpichnikov, MP
    Kostanyan, IA
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 10, p. 2451-2457Article in journal (Refereed)
    Abstract [en]

    Prefibrillar cytotoxicity was suggested as a common amyloid characteristic. We showed two types of albebetin prefibrillar oligomers are formed during incubation at pH 7.3. Initial round-shaped oligomers consist of 10–15 molecules determined by atomic force microscopy, do not bind thioflavin-T and do not affect viability of granular neurons and SH-SY5Y cells. They are converted into ca. 30–40-mers possessing cross-β-sheet and reducing viability of neuronal cells. Neither monomers nor fibrils possess cytotoxicity. We suggest that oligomeric size is important for stabilising cross-β-sheet core critical for cytotoxicity. As albebetin was used as a carrier-protein for drug delivery, examination of amyloidogenicity is required prior polypeptide biomedical applications.

  • 887.
    Zdunek, Janusz
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Martinez, G V
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lycksell, P O
    Yin, Y
    Nilsson, S
    Shen, Y
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Wijmenga, S
    Global structure and dynamics of human apolipoprotein CII in complex with micelles: evidence for increased mobility of the helix involved in the activation of lipoprotein lipase.2003In: Biochemistry, ISSN 0006-2960, Vol. 42, no 7, p. 1872-89Article in journal (Refereed)
  • 888.
    Zhang, Ce
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, Yonggang
    Gilthorpe, Jonathan
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    van der Maarel, Johan RC
    MRP14 (S100A9) protein interacts with alzheimer beta-amyloid peptide and induces its fibrillization2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, p. e32953-Article in journal (Refereed)
    Abstract [en]

    Increasing evidence supports the contribution of local inflammation to the development of Alzheimer's disease (AD) pathology, although the precise mechanisms are not clear. In this study, we demonstrate that the pro-inflammatory protein S100A9 interacts with the A beta 1-40 peptide and promotes the formation of fibrillar beta-amyloid structures. This interaction also results in reduced S100A9 cytotoxicity by the binding of S100A9 toxic species to A beta 1-40 amyloid structures. These results suggest that secretion of S100A9 during inflammation promotes the formation of amyloid plaques. By acting as a sink for toxic species, plaque formation may be the result of a protective response within the brain of AD patients, in part mediated by S100A9.

  • 889.
    Zhang, Jin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Patrik L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin2016In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 50, no 21, p. 11984-11993Article in journal (Refereed)
    Abstract [en]

    Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the interactions between TTR and THDCs, we determined the crystallographic structures of human TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and 2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and TTR were further characterized using molecular dynamics simulations. A structure-based virtual screening (VS) protocol was developed with the intention of providing an efficient tool for the discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders, 12 representatives were selected, and their TTR binding affinities were studied with isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26 and 100 mu M. To elucidate structural details in their binding to TTR, crystal structures were determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-cresol, bisphenol S, clonixin, and triclopyr. The compounds were found to bind in the TTR hormone binding sites as predicted. Our results show that the developed VS protocol is able to successfully identify potential THDCs, and we suggest that it can be used to propose THDCs for future toxicological evaluations.

  • 890.
    Zhang, Jin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Mikael J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andersson, Patrik L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals2018In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 52, no 20, p. 11865-11874Article in journal (Refereed)
    Abstract [en]

    Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream (Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (>500-fold lower affinity). Crystal structures of the seabream TTR TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.

  • 891. Zhang, Sicai
    et al.
    Berntsson, Ronnie P. A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.
    Tepp, William H.
    Tao, Liang
    Johnson, Eric A.
    Stenmark, Pal
    Dong, Min
    Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 1637Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs), the most potent toxins known, are potential bioterrorism agents. It is well established that all seven serotypes of BoNTs (BoNT/A-G) require complex gangliosides as co-receptors. Here, we report that BoNT/DC, a presumed mosaic toxin between BoNT/D and BoNT/C1, binds and enters efficiently into neurons lacking complex gangliosides and shows no reduction in toxicity in mice deficient in complex gangliosides. The co-crystal structure of BoNT/DC with sialyl-Thomsen-Friedenreich antigen (Sialyl-T) suggests that BoNT/DC recognizes only the sialic acid, but not other moieties in gangliosides. Using liposome flotation assays, we demonstrate that an extended loop in BoNT/DC directly interacts with lipid membranes, and the co-occurring sialic acid binding and loop-membrane interactions mediate the recognition of gangliosides in membranes by BoNT/DC. These findings reveal a unique mechanism for cell membrane recognition and demonstrate that BoNT/DC can use a broad range of sialic acid-containing moieties as co-receptors.

  • 892. Zhao, Li Na
    et al.
    Zhang, Tong
    Zhang, Ce
    Wang, Chao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chew, Lock Yue
    Mu, Yuguang
    S100A9 induces aggregation-prone conformation in Abeta peptides: a combined experimental and simulation study2013In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 3, no 46, p. 24081-24089Article in journal (Refereed)
    Abstract [en]

    Inflammation is one of the prominent pathological features in Alzheimer's disease (AD). Recently, there have been various proposed roles of neuroinflammation, such as the driving forces, bystander, byproduct or the neuroprotective response. Notwithstanding these diverse possible mechanisms, experiments have found that S100A9 is one of the pro-inflammatory proteins abundant and over-expressed in the inflammation sites of AD. In this paper, we examine the role of S100A9 in the oligomerization process of A beta peptides by means of replica exchange molecular dynamics simulation and experimental investigations. Our experiments, based on atomic force microscopy and Thioflavin T spectroscopic assays, have clearly indicated that the close interaction between S100A9 and A beta has significantly enhanced the A beta oligomerization. In line with the experimental observation, our simulation studies have revealed that the pro-inflammatory S100A9 protein interacts with the A beta peptides directly, mainly through hydrophobic interactions with the A beta central hydrophobic core region. In addition, the formation of hydrogen bonds between the residues of the S100A9 homodimer and the two ends of the A beta peptides is found to cause a straightening of the A beta(12-24) peptides. A more straight A beta(12-24) peptide with a higher beta-content then may function as a template to induce the folding of new incoming A beta peptides, which leads to the formation of aggregation-prone oligomers.

  • 893. Zhao, X
    et al.
    Georgieva, B
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Ippel, J H
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wijmenga, S
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Rothstein, R
    Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.2000In: Mol Cell Biol, ISSN 0270-7306, Vol. 20, no 23, p. 9076-83Article in journal (Refereed)
  • 894.
    Zheng, Wenjing
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gorre, Nagaraju
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Noda, Tetsuo
    Ogawa, Wataru
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Maternal phosphatidylinositol 3-kinase signalling is crucial for embryonic genome activation and preimplantation embryogenesis2010In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 11, no 11, p. 890-895Article in journal (Refereed)
    Abstract [en]

    Maternal effect factors derived from oocytes are important for sustaining early embryonic development before the major wave of embryonic genome activation (EGA). In this study, we report a two-cell-stage arrest of embryos lacking maternal 3-phosphoinositide-dependent protein kinase 1 as a result of suppressed EGA. Concurrent deletion of maternal Pten completely rescued the suppressed EGA and embryonic progression through restored AKT signalling, which fully restored the fertility of double-mutant females. Our study identifies maternal phosphatidylinositol 3-kinase signalling as a new maternal effect factor that regulates EGA and preimplantation embryogenesis in mice.

  • 895. Zhou, Yizhou
    et al.
    Smith, Daniel
    Leong, Bryan J
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chapman, Matthew R
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Promiscuous cross-seeding between bacterial amyloids promotes interspecies biofilms2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 42, p. 35092-35103Article in journal (Refereed)
    Abstract [en]

    Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimer's, Parkinson's and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2 and Citrobacter koseri were able to cross-seed in vitro. The polymerization of E. coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA secreted from E. coli csgB- mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA secreted by S. typhimurium csgB- mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.

  • 896.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    The Helicobacter pylori sialic acid binding adhesin SabA is regulated via a network of two-component systemsManuscript (preprint) (Other academic)
    Abstract [en]

    The acid-responsive signaling system ArsRS plays a key role in regulating factors important for survival in acidic conditions during infection of the human stomach by Helicobacter pylori. In addition, ArsRS was suggested to control the disease-associated attachment protein SabA, however, mechanistic data is still lacking. We show that the repressing effect of the ArsRS system on SabA expression occurs both at acidic and neutral conditions and is mediated at the transcriptional level. Purified His6-ArsR binds PsabA DNA at several sites, with varying affinity and independent of phosphorylation status and H. pylori strains showed unique cognate PsabA sequences to tweak the ArsR binding ability, resulting in strain-dependent repression of SabA expression. By site-directed mutagenesis we reveal key amino acids for the binding activity of ArsR. Finally, we show that that ArsR binds to A/T-rich DNA as dimers or larger multimers, suggesting that ArsR has affinity for DNA structures rather than to a specific promoter DNA sequence. SabA expression is further influenced by the FlgRS and CrdRS two-component systems, illustrating a complicated crosstalk among regulatory networks in H. pylori.

  • 897.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallström, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Carina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Engstrand, Lars
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism2014In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 7, article id e1004234Article in journal (Refereed)
    Abstract [en]

    During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

  • 898.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural topology and activation of an initial adenylate kinase-substrate complex2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 6, p. 1055-1061Article in journal (Refereed)
    Abstract [en]

    Enzymatic activity is ultimately defined by the structure, chemistry and dynamics of the Michaelis complex. There exist a large number of experimentally determined structures between enzymes and substrates or substrate analogues or inhibitors. However, transient, short-lived encounter and equilibrium structures also play fundamental roles during enzymatic reaction cycles. Such structures are inherently difficult to study with conventional experimental techniques. The enzyme adenylate kinase undergoes major conformational rearrangements in response to binding of its substrates ATP and AMP. ATP is sandwiched between two binding surfaces in the closed and active enzyme conformation. Thus, ade-nylate kinase harbors two spatially distant surfaces in the substrate free open conformation of which one is responsible for the initial interaction with ATP. Here, we have performed primarily nuclear magnetic resonance experiments on Escherichia coli adenylate kinase (AKeco) variants that enabled identification of the site responsible for the initial ATP interaction. This allowed a characterization of the structural topology of an initial equilibrium complex between AKeco and ATP. Based on the results it is suggested that the ATP binding mechanism to AKeco is a mixture between "induced fit" and "conformational selection" models. It is shown that ATP is activated in the initial enzyme bound complex since it displays an appreciable rate of non-productive ATP hydrolysis. In summary our results provide novel structural and functional insights into adenylate kinase catalysis.

  • 899. Åstedt, B
    et al.
    Lecander, I
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The placental type plasminogen activator inhibitor, PAI-2.1987In: Fibrinolysis, Elsevier, 1987, 1, p. 203-208Chapter in book (Refereed)
  • 900.
    Öhman, Carina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallström, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Olofsson, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Christer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aspholm, Marina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Phase variation and expression mechanisms of the sialic acid binding adhesin SabA in Helicobacter pyloriManuscript (preprint) (Other academic)
    Abstract [en]

    Bacterial attachment to host epithelial surfaces by means of bacterial adhesion proteins is a key event in colonization. Phase variation is a mechanism used by bacteria that mediates frequent and reversible gains and losses in expression of proteins. In the inflamed stomach, H. pylori adherence to sialyl Lewis antigens (sLex) is mediated by the sialic acid binding adhesin (SabA). Instability in sLex-binding was previously reported and here we show that this is caused by the high frequency of ON/OFF switching in SabA expression. Our data shows that SabA phase variation is due to slippages in the number of CT repeat sequences in the 5’ end of the sabA gene (i.e. slipped strand mispairing). The sabA operon was defined and the sabA transcriptional start site was determined. Changes in the number of thymine bases present in a mononucleotide stretch upstream of the sabA gene and in close proximity to a -35-like promoter element did not affect the ON/OFF phase variation. Instead, we show that changes in intrinsic DNA properties are likely to influence SabA expression. The effect of growth phase on sLex-binding properties and SabA expression was also analyzed. SabA expression and sLex-binding increased as H. pylori entered late logarithmic phase. Our data show the ability of H. pylori to cycle between an adherent and non-adherent phenotype by phase variation mechanisms and adjustment of receptor binding activity. These data increase our understanding of how H. pylori adjust adherence properties during persistent infection.

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