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  • 201. Fei, Y Y
    et al.
    Schmidt, Alexej
    Helicure AB, Umeå Biotech Incubator, Umeå.
    Bylund, Göran
    Helicure AB, Umeå Biotech Incubator, Umeå.
    Johansson, D X
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lebrilla, C
    Solnick, J V
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Helicure AB, Umeå Biotech Incubator, Umeå.
    Zhu, X D
    Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori2011In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 83, no 16, p. 6336-6341Article in journal (Refereed)
    Abstract [en]

    Infectious diseases are often initiated by microbial adherence that is mediated by the binding of attachment molecules, termed adhesins, to cell surface receptors on host cells. We present an experimental system, oblique-incidence reflectivity difference (OI-RD) microscopy, which allows the detection of novel, low-affinity microbial attachment mechanisms that may be essential for infectious processes. OI-RD microscopy was used to analyze direct binding of the oncopathogen, Helicobacter pylori ( H. pylori ) to immobilized glycoconjugates in real time with no need for labeling tags. The results suggest the presence of additional Lewis b blood group antigen (Le(b)) binding adhesins that have not been detected previously. OI-RD microscopy also confirmed the high-affinity binding of H. pylori outer-membrane protein BabA to Le(b). The OI-RD microscopy method is broadly applicable to real-time characterization of intact microbial binding to host receptors and offers new strategies to elucidate the molecular interactions of infectious agents with human host cells.

  • 202. Feng, P
    et al.
    Ohlsson, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The structure of the TATA-less rat tissue-type plasminogen activator gene. Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.1990In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 265, no 4, p. 2022-7Article in journal (Refereed)
    Abstract [en]

    The genomic region carrying the rat tissue-type plasminogen activator (tPA) gene including its 5'-flanking sequence has been isolated and characterized by restriction enzyme analysis, Southern blotting, and DNA sequencing of all coding parts and the promoter region. The gene is approximately 25 kilobase pairs in size and comprises 14 exons separated by 13 introns. All the exon/intron boundaries agree with the GT-AG rule. The organization of the rat tPA gene is very similar to its human counterpart, and the location of the introns in the protein structure is identical to the human tPA gene. To characterize the promoter region, the transcription initiation site was identified by S1 nuclease protection experiments. A DNA fragment carrying 621 nucleotides of the 5'-flanking sequence was found to confer basal promoter activity and hormone responsiveness to a reporter gene construct in primary cultures of rat granulosa cells. Analysis of the rat tPA promoter sequence and a comparison with the human and mouse counterparts reveal several species-specific differences: the rat and mouse tPA promoters lack typical TATA and CAAT sequences found in the human tPA gene. Furthermore, the rat tPA promoter contains a consensus cAMP-responsive element shown to be required for cAMP responsiveness in eucaryotic genes. At the same position as the cAMP-responsive element in the rat gene, the mouse and human tPA genes have a 12-O-tetradecanoylphorbol-13-acetate-responsive element known to mediate activation by phorbol esters. The differences in the promoter sequences of the rat, mouse, and human tPA genes may have implications for the regulation of the tPA gene in different species.

  • 203.
    Fijolek, Artur
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Salvage and de novo synthesis of nucleotides in Trypanosoma brucei and mammalian cells2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    All living cells are dependent on nucleic acids for their survival. The genetic information stored in DNA is translated into functional proteins via a messenger molecule, the ribonucleic acid (RNA). Since DNA and RNA can be considered as polymers of nucleotides (NTPs), balanced pools of NTPs are crucial to nucleic acid synthesis and repair. The de novo reduction of ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs), the precursors for DNA synthesis, is catalyzed by the enzyme ribonucleotide reductase (RNR). In cycling cells the dominant form of mammalian RNR consists of two proteins called R1 and R2. A proteasome-mediated degradation completely deprives postmitotic cells of R2 protein. The nonproliferating cells use instead a p53 inducible small RNR subunit, called p53R2 to synthesize dNTPs for mitochondrial DNA replication and DNA repair. To address the ongoing controversy regarding the localization and subsequently function and regulation of RNR subunits, the subcellular localization of all the mammalian RNR subunits during the cell cycle and after DNA damage was followed as a part of this thesis. Irrespective of the employed methodology, only a cytosolic localization could be observed leading to a conclusion that the dNTPs are synthesized in the cytosol and transported into the nucleus or mitochondria for DNA synthesis and repair. Thus, our data do not support the suggestion that nuclear translocation is a new additional mechanism regulating ribonucleotide reduction in mammalian cells.

    In an attempt to find a cure for African sleeping sickness, a lethal disease caused by a human pathogen, Trypanosoma brucei, nucleotide metabolism of the parasite was studied. The trypanosomes exhibit strikingly low CTP pools compared with mammalian cells and they also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. Following expression, purification and kinetic studies of the recombinant T. brucei CTP synthetase it was found that the enzyme has a higher Km value for UTP than the mammalian CTP synthetase. In combination with a lower UTP pool the high Km may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase was irreversibly inhibited by the glutamine analog acivicin, a drug extensively tested as an antitumor agent. Daily injections of acivicin to trypanosome-infected mice were sufficient to suppress the parasite infections. The drug was shown to be trypanocidal when added to cultured bloodstream T. brucei for four days at 1 uM concentration. Therefore, acivicin may qualify as a drug with “desirable” properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi. Trypanosomes lack de novo purine biosynthesis and are therefore dependent on exogenous purines such as adenosine that is taken up from the blood by high-affinity transporters. We found that besides the cleavage-dependent pathway, where adenosine is converted to adenine by inosine-adenosine-guanosine-nucleoside hydrolase, T. brucei can also salvage adenosine by adenosine kinase (AK). The efficient adenosine transport combined with a high-affinity AK yields a strong salvage system in T. brucei, but on the other hand makes the parasites highly sensitive to adenosine analogs such as adenine arabinoside (Ara-A). The cleavage-resistant Ara-A was shown to be readily taken up by the parasites and phosphorylated by the TbAK-dependent pathway, inhibiting trypanosome proliferation and survival by incorporation into nucleic acids and by affecting nucleotide levels in the parasite.

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  • 204.
    Fijolek, Artur
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.2007In: Journal of biological chemistry, ISSN 0021-9258, Vol. 282, no 16, p. 11858-11865Article in journal (Refereed)
  • 205. Filatov, D
    et al.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Induction of the mouse ribonucleotide reductase R1 and R2 genes in response to DNA damage by UV light.1996In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 271, no 39, p. 23698-704Article in journal (Refereed)
  • 206.
    Fjellström, Mona
    et al.
    Umeå University, Umeå University Library, Centre for teaching and learning (UPL).
    Larsson, Malin
    Planeringsenheten, Umeå universitet.
    Edlund, Ann-Catrine
    Umeå University, Faculty of Arts, Department of language studies.
    Kjellsson Lind, Annika
    Umeå University, Faculty of Science and Technology, Department of Science and Mathematics Education. Planeringsenheten, Umeå universitet.
    Ågren, Per-Olof
    Umeå University, Faculty of Social Sciences, Department of Informatics.
    Eriksson, Nils
    Umeå University, Faculty of Social Sciences, Department of Sociology.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arvidsson, Erik
    Umeå Studentkår.
    Fakultetsaudit: Intern bedömning av kvalitetsarbete för utbildning på grund- och avancerad nivå vid Teknisk-naturvetenskaplig fakultet hösten 20162017Report (Other (popular science, discussion, etc.))
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  • 207.
    Flodell, Sara
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure and Dynamics of the Hepatitis B Virus Encapsidation Signal Revealed by NMR Spectroscopy2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the study of the three-dimensional structure and dynamics of the hepatitis B virus (HBV) encapsidation signal, epsilon, by means of nuclear magnetic resonance (NMR) and mutational data. HBV replicates by reverse transcription of an RNA pregenome into the viral DNA genome, which becomes enclosed in viral particles (encapsidation). Epsilon is a stem-loop structure within the RNA pregenome and both the primary sequence and secondary structure of epsilon are strongly conserved, in agreement with its essential function of propagating HBV. Epsilon is therefore a potential target for drug design. Studying the structure of epsilon requires development of new methods in the field of structural biology, as it is such a large RNA. Knowing the structure of epsilon will help to better understand the encapsidation mechanism and priming step of reverse transcription. This will help us in the search for antiviral drugs that block epsilon and prevent the viral reverse transcriptase from binding.

    NMR spectroscopy is a method that provides detailed structural and dynamical data in solution under natural conditions. However, the size of the molecules that can be studied with NMR is limited. NMR spectra become more and more difficult to interpret as the size of the molecule increases. To circumvent this problem, large RNA molecules can be divided into smaller parts and only the parts essential for NMR studies are selected. The information obtained from these smaller fragments can then be used to determine the structure of the larger molecule. Furthermore, a new method of enzymatically synthesizing nucleoside triphosphates with isotopes suitable for NMR has made it possible to specifically label the RNA molecules. Using this method it is possible to derive highly detailed molecular structures of RNA up to a size of 150 nucleotides. The method of selective isotope labelling was applied to different parts of HBV epsilon. Three RNA fragments of 27 (apical loop), 36 (internal bulge) and 61 (whole epsilon) nucleotides (nt) were synthesized in the unlabelled form. The 27-nt and 36-nt RNAs were also synthesized with (13C, 15N, 1', 3', 4', 5', 5"-2H5)-labelled uridines. The 61-nt sequence was (13C, 15N)-guanidine labelled. This labelling allowed unambiguous assignment of otherwise inaccessible parameters. The unlabelled and labelled RNA sequences provided the necessary data for structure derivation of the whole epsilon.

    The apical loop of epsilon forms a pseudo-triloop motif. There is only one conformation of the loop that fulfils all the restraints, including experimental chemical shifts. However, the loop adopts several structures that fulfil the experimental distance, torsion angle and residual dipolar coupling restraints. This may reflect true flexibility. Indeed, relaxation studies on the unlabelled and labelled 27-nt sequences show that the residues that show multiple conformations are flexible. This can be an important feature for the recognition and subsequent binding of epsilon to the viral polymerase.

    The information gained on the HBV encapsidation signal is useful in our understanding of the initiation of replication of the virus. This can in turn contribute to the search for drugs against HBV.

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  • 208.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Cromsigt, Jenny
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kidd-Ljunggren, Karin
    Wijmenga, Sybren
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs.2002In: Journal of Biomolecular Structure and Dynamics, ISSN 0739-1102, E-ISSN 1538-0254, Vol. 19, no 4, p. 627-636Article in journal (Other academic)
    Abstract [en]

    Hepatitis B virus (HBV) HBV is DNA virus with a unique replication strategy, which involves reverse transcription of its pregenomic RNA. Essential for this reverse transcription are the 5'- and 3'-ends of its pregenomic RNA (5'-RT-RNA and 3'-RT-RNA, respectively) which form conserved bulged stem-loop structures. The 5'-RT-RNA consists of a 67 nucleotide bulged stem-loop structure, epsilon, which constitutes the signal for encapsidation of the pregenomic RNA and subsequent reverse transcription. The reverse transcriptase (RT) initially binds to the completely conserved apical loop of epsilon and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. Structural studies of epsilon can provide important parameters required for the design of RNA targeted anti- viral drugs directed against Hepatitis B virus. NMR studies of large RNA systems (> ca. 50 nucleotides) require novel approaches, e.g., different labeling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing (13)C/(15)N/(2)H labeled nucleotides has been developed based on converting specifically labeled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway and nucleotide and salvage pathways. These NTPs give a large freedom in designing different labeling patterns in in vitro synthesized RNAs under study for NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides). Here this new technique is applied for structural studies on 27, 36 and 61 nucleotides long RNA fragments, mimicking different regions of epsilon.

  • 209.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kidd-Ljunggren, K
    Wijmenga, S
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mobile nucleotides mediate binding of HBV reverse transcriptase to its RNA targetManuscript (preprint) (Other academic)
  • 210.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Petersen, Michael
    Girard, Frederic
    Zdunek, Janusz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kidd-Ljunggren, Karin
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wijmenga, Sybren
    Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 16, p. 4449-4457Article in journal (Refereed)
    Abstract [en]

    Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

  • 211.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Cromsigt, Jenny
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Ippel, Hans
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kidd-Ljunggren, Karin
    Wijmenga, Sybren
    The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges.2002In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 21, p. 4803-4811Article in journal (Refereed)
    Abstract [en]

    Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (epsilon), situated near the 5' end of the pregenome. epsilon has been predicted to form a bulged stem-loop with the apical stem capped by a hexa- loop. After the initial binding to this apical stem- loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem-loop of epsilon. Application of new isotope-labeling techniques (13C/15N/2H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa-loop, the apical stem is capped by a stable UGU tri-loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3' neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of epsilon. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase.

  • 212. Fodera, Vito
    et al.
    Vetri, Valeria
    Wind, Thea S.
    Noppe, Wim
    Cornett, Claus
    Donald, Athene M.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vestergaard, Bente
    Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures2014In: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 5, no 18, p. 3254-3258Article in journal (Refereed)
    Abstract [en]

    Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.

  • 213. Fonseca, Luis Vazquez
    et al.
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Clinical Genetics Unit, Department of Women and Children's Health, IRP Città della Speranza, University of Padova, Padova, Italy.
    Calderan, Cristina
    Desbats, Maria Andrea
    Acosta, Manuel J.
    Cerqua, Cristina
    Cassina, Matteo
    Ashraf, Shazia
    Hildebrandt, Friedhelm
    Sartori, Geppo
    Navas, Placido
    Trevisson, Eva
    Salviati, Leonardo
    Mutations in COQ8B (ADCK4) found in patients with steroid-resistant nephrotic syndrome alter COQ8B function2018In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 39, no 3, p. 406-414Article in journal (Refereed)
    Abstract [en]

    Mutations in COQ8B cause steroid-resistant nephrotic syndrome with variable neurological involvement. In yeast, COQ8 encodes a protein required for coenzyme Q (CoQ) biosynthesis, whose precise role is not clear. Humans harbor two paralog genes: COQ8A and COQ8B (previously termed ADCK3 and ADCK4). We have found that COQ8B is a mitochondrial matrix protein peripherally associated with the inner membrane. COQ8B can complement a Delta COQ8 yeast strain when its mitochondrial targeting sequence (MTS) is replaced by a yeast MTS. This model was employed to validate COQ8B mutations, and to establish genotype-phenotype correlations. All mutations affected respiratory growth, but there was no correlation between mutation type and the severity of the phenotype. In fact, contrary to the case of COQ2, where residual CoQ biosynthesis correlates with clinical severity, patients harboring hypomorphic COQ8B alleles did not display a different phenotype compared with those with null mutations. These data also suggest that the system is redundant, and that other proteins (probably COQ8A) may partially compensate for the absence of COQ8B. Finally, a COQ8B polymorphism, present in 50% of the European population (NM_024876.3:c.521A > G, p.His174Arg), affects stability of the protein and could represent a risk factor for secondary CoQ deficiencies or for other complex traits.

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  • 214. Forey, Romain
    et al.
    Poveda, Ana
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Barthe, Antoine
    Padioleau, Ismael
    Renard, Claire
    Lambert, Robin
    Skrzypczak, Magdalena
    Ginalski, Krzysztof
    Lengronne, Armelle
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Pardo, Benjamin
    Pasero, Philippe
    Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication2020In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164Article in journal (Refereed)
    Abstract [en]

    The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

  • 215.
    Forslund, Josefin M. E.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pfeiffer, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stojkovič, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Pauline H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The presence of rNTPs decreases the speed of mitochondrial DNA replication2018In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 3, article id e1007315Article in journal (Refereed)
    Abstract [en]

    Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondnal DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol gamma) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol gamma exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol gamma and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol gamma reported here could have implications for forms of rntDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTPidNIP ratios that are expected to prevail in such disease states, Pol gamma enters a polymerasetexonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.

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  • 216. Fortune, John M
    et al.
    Pavlov, Youri I
    Welch, Carrie M
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M J
    Kunkel, Thomas A
    Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions.2005In: Journal of biological chemistry, ISSN 0021-9258, Vol. 280, no 33, p. 29980-7Article in journal (Refereed)
  • 217.
    Francis, Monika K.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulation of GRAF1 membrane sculpting function during cell movement2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    All eukaryotic cells rely on endocytic events to satisfy a constant need for nutrient and fluid uptake from their surroundings. Endocytosis-dependent turnover of cell surface constituents also serves to control signal transduction and establish morphological changes in response to extracellular stimuli. During endocytosis, distinct protein machineries re-sculpt the plasma membrane into vesicular carriers that enclose molecules that are to be taken up into the cell. Besides those produced from the canonical clathrin-mediated endocytic machinery, it is becoming increasingly clear that other membrane carriers exist. The indisputable connection between the function of these uptake systems and various disease states, highlights why it is so important to increase our knowledge about the underlying molecular machineries.

    The aim of this thesis was therefore to characterise the function of GRAF1, a protein suggested to be a tumour suppressor due to that the gene has been found to be mutated in certain cancer patients. My work focused on understanding how this protein operates during formation of clathrin-independent carriers, with possible implications for disease development.

    Previous in vitro studies showed that GRAF1 harbours a GTPase activating domain to inactivate Rho GTPase Cdc42, a major actin cytoskeleton regulator. Herein, microscopy based approaches used to analyse HeLa cells demonstrated the importance of a transient interaction between GRAF1 and Cdc42 for proper processing of GRAF1-decorated carriers. Although GRAF1-mediated inactivation of Cdc42 was not vital for the budding of carriers from the plasma membrane, it was important for carrier maturation. In addition, studies of purified GRAF1 and its association with lipid bilayers identified a membrane scaffolding-dependent oligomerisation mechanism, with the ability to sculpt membranes. This was consistent with the assumption that GRAF1 possesses an inherent banana shaped membrane binding domain. Remarkably, this function was autoinhibited and in direct competition with the Cdc42 interaction domain.

    Finally, other novel GRAF1 interaction partners were identified in this study. Interestingly, many of these partners are known to be associated with protein complexes involved in cell adherence, spreading and migration. Although never actually seen localising to mature focal adhesions that anchor cells to their growth surface, dynamic GRAF1 carriers were captured travelling to and from such locations. Moreover, GRAF1 was recruited specifically to smaller podosome-like structures. Consistent with this, the tracking of GRAF1 in live cells uncovered a clear pattern of dynamic carrier formation at sites of active membrane turnover – notably protrusions at the cell periphery. Furthermore, the silencing of GRAF1 gave rise to cells defective in spreading and migration, indicating a targeting of GRAF1-mediated endocytosis to aid in rapid plasma membrane turnover needed for morphological changes that are a prerequisite for cell movement. Since these cells exhibited an increase in active Rab8, a GTPase responsible for polarised vesicle transport, the phenotype could also be explained by a defect in Rab8 trafficking that results in hyperpolarisation.

    Taken together, the spatial and temporal regulation of GRAF1 membrane sculpting function is likely to be accomplished via its membrane binding propensity, in concert with various protein interactions. The importance of GRAF1 in aiding membrane turnover during cell movement spans different functional levels – from its local coordination of membrane and actin dynamics by interacting with Cdc42, to its global role in membrane lipid trafficking.

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  • 218.
    Francis, Monika K.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Vidal-Quadras, Maite
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Santarella-Mellwig, Rachel
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF12015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 22, p. 4183-4195Article in journal (Refereed)
    Abstract [en]

    Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase activating protein (GAP) GRAF1, facilitate rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, distinct from clathrin coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane resulting in a corresponding decrease in Cdc42 microdomain association. However, Cdc42 captured in its active state was, via a GAP domain mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers affected in their endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

  • 219.
    Francis, Monika K.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Krupp, Nikolai
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Behrmann, Elmar
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    GRAF1 sculpts membrane through a regulated oligomerisation reactionManuscript (preprint) (Other academic)
  • 220. Fritz, Günter
    et al.
    Botelho, Hugo M
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gomes, Cláudio M
    Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 22, p. 4578-4590Article in journal (Refereed)
    Abstract [en]

    The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

  • 221. Frontini, M
    et al.
    Kotova, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mantovani, R
    Mechanisms of transcriptional activation of the AdML promoter by NF-Y.Manuscript (Other academic)
  • 222. Fujimoto, Saori
    et al.
    Olaniyi Ojo, Olabisi
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wu, Jeng Yih
    Odenbreit, Stefan
    Haas, Rainer
    Graham, David Y
    Yamaoka, Yoshio
    Helicobacter pylori BabA expression, gastric mucosal injury, and clinical outcome.2007In: Clin Gastroenterol Hepatol, ISSN 1542-7714, Vol. 5, no 1, p. 49-58Article in journal (Refereed)
  • 223. Galway, A B
    et al.
    Oikawa, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hsueh, A J
    Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C.1989In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 125, no 1, p. 126-35Article in journal (Refereed)
    Abstract [en]

    Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.

  • 224. Gan, Haiyun
    et al.
    Yu, Chuanhe
    Devbhandari, Sujan
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Han, Junhong
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Remus, Dirk
    Zhang, Zhiguo
    Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress2017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 2, p. 446-455Article in journal (Refereed)
    Abstract [en]

    The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.

  • 225.
    Ganai, Rais A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, USA.
    Zhang, Xiao-Ping
    Heyer, Wolf-Dietrich
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Strand displacement synthesis by yeast DNA polymerase epsilon2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 17, p. 8229-8240Article in journal (Refereed)
    Abstract [en]

    DNA polymerase epsilon (Pol epsilon) is a replicative DNA polymerase with an associated 3'aEuro"5' exonuclease activity. Here, we explored the capacity of Pol epsilon to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol epsilon is unable to carry out extended strand displacement synthesis unless its 3'aEuro"5' exonuclease activity is removed. However, the wild-type Pol epsilon holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol epsilon from synthesizing DNA up to the fork junction. This inhibition was observed for Pol epsilon but not with Pol delta, RB69 gp43 or Pol eta. Neither was Pol epsilon able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol epsilon is distributive. Our results suggest that Pol epsilon is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol epsilon participates in short-patch base excision repair and ribonucleotide excision repair.

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  • 226.
    Ganai, Rais Ahmad
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural and biochemical basis for the high fidelity and processivity of DNA polymerase ε2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA polymerase epsilon (Pol ε) is a multi-subunit B-family DNA polymerase that is involved in leading strand DNA replication in eukaryotes. DNA Pol ε in yeast consists of four subunits, Pol2, Dpb2, Dpb3, and Dpb4. Pol2 is the catalytic subunit and Dpb2, Dpb3, and Dpb4 are the accessory subunits. Pol2 can be further divided into an N-terminal catalytic core (Pol2core) containing both the polymerase and exonuclease active sites and a C-terminus domain. We determined the X-ray crystal structure of Pol2core at 2.2 Å bound to DNA and with an incoming dATP. Pol ε has typical fingers, palm, thumb, exonuclease, and N-terminal domains in common with all other B-family DNA polymerases. However, we also identified a seemingly novel domain we named the P-domain that only appears to be present in Pol ε. This domain partially encircles the nascent duplex DNA as it leaves the active site and contributes to the high intrinsic processivity of Pol ε.

    To ask if the crystal structure of Pol2core can serve as a model for catalysis by Pol ε, we investigated how the C-terminus of Pol2 and the accessory subunits of Pol ε influence the enzymatic mechanism by which Pol ε builds new DNA efficiently and with high fidelity. Pre-steady state kinetics revealed that the exonuclease and polymerization rates were comparable between Pol2core and Pol ε. However, a global fit of the data over five nucleotide-incorporation events revealed that Pol ε is slightly more processive than Pol2 core. The largest differences were observed when measuring the time for loading the polymerase onto a 3' primer-terminus and the subsequent incorporation of one nucleotide. We found that Pol ε needed less than a second to incorporate the first nucleotide, but it took several seconds for Pol2core to incorporate similar amounts of the first nucleotide.

    B-family polymerases have evolved an extended β-hairpin loop that is important for switching the primer terminus between the polymerase and exonuclease active sites. The high-resolution structure of Pol2core revealed that Pol ε does not possess an extended β-hairpin loop. Here, we show that Pol ε can processively transfer a mismatched 3' primer-terminus between the polymerase and exonuclease active sites despite the absence of a β-hairpin loop. Additionally we have characterized a series of amino acid substitutions in Pol ε that lead to altered partitioning of the 3'primer-terminus between the two active sites.

    In a final set of experiments, we investigated the ability of Pol ε to displace the downstream double-stranded DNA while carrying out DNA synthesis. Pol ε displaced only one base pair when encountering double-stranded DNA after filling a gap or a nick. However, exonuclease deficient Pol ε carries out robust strand displacement synthesis and can reach the end of the templates tested here. Similarly, an abasic site or a ribonucleotide on the 5'-end of the downstream primer was efficiently displaced but still only by one nucleotide. However, a flap on the 5'-end of the blocking primer resembling a D-loop inhibited Pol ε before it could reach the double-stranded junction. Our results are in agreement with the possible involvement of Pol ε in short-patch base excision repair and ribonucleotide excision repair but not in D-loop extension or long-patch base excision repair.

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  • 227.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Switching between polymerase and exonuclease sites in DNA polymerase ε2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 2, p. 932-942Article in journal (Refereed)
    Abstract [en]

    The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.

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  • 228.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NY 10016, USA.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA Replication - A Matter of Fidelity2016In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 62, no 5, p. 745-755Article, review/survey (Refereed)
    Abstract [en]

    The fidelity of DNA replication is determined by many factors, here simplified as the contribution of the DNA polymerase (nucleotide selectivity and proofreading), mismatch repair, a balanced supply of nucleotides, and the condition of the DNA template (both in terms of sequence context and the presence of DNA lesions). This review discusses the contribution and interplay between these factors to the overall fidelity of DNA replication.

  • 229.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University.
    Modulation of strand displacement synthesis of DNA polymerase ε byprocessive 3'- 5' exonuclease activity.Manuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    DNA polymerase epilson (Pol ε) is a replicative DNA polymerase with a processive 3'-5' exonuclease activity. Here we ask if Pol ε is capable of performing strand displacement synthesis, which influence many DNA processes in vivo. We found that Polε is unable to carry out extended strand displacement synthesis unless the proofreading is inactivated. However, Pol ε efficiently displaced one nucleotide when encountering double stranded DNA after filling a gap of 8 nucleotides. An abasic moiety at the 5'-end of the downstream primer was as efficiently displaced and still only with one nucleotide. Pol ε also efficiently recognized the 3'-OH innicked DNA and displaced the 5'- nucleotide, regardless if it was a normal phosphorylated deoxyribonucleotide or a ribonucleotide. A flap, mimicking a D-loop or a hairpin structure, on the 5'-end of the blocking primer inhibited Pol ε, and did not allow Pol ε to efficiently synthesize DNA up to the junction with double-stranded DNA. Finally, we show that strand displacement synthesis is limited by the processive 3'–5' exonuclease activity in Pol ε. Our results suggests that Pol ε is unable to extend D-loops during homologous recombination or participate in long-patch base excision repair based on the inhibition by the 5'–flap of the downstream primer. Our results do, however, support that Pol ε may participate in short patch base excision repair and ribonucleotide excision repair.

  • 230.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA Polymerase epsilon Catalytic Core and Holoenzyme Have Comparable Catalytic Rates2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3825-3835Article in journal (Refereed)
    Abstract [en]

    The holoenzyme of yeast DNApolymerase ε (Pol ε) consists of four subunits– Pol2, Dpb2, Dpb3, and Dpb4. A proteasesensitivesite results in a N-terminalproteolytic fragment of Pol2, called Pol2core,that consists of the catalytic core of Pol ε andretains both polymerase and exonucleaseactivities. Pre-steady-state kinetics showedthat the exonuclease rates on single-stranded,double-stranded, and mismatched DNA werecomparable between Pol ε and Pol2core. Singleturnover pre-steady-state kinetics alsoshowed that the kpol of Pol ε and Pol2core werecomparable when pre-loading the polymeraseonto the primer-template before adding Mg2+and dTTP. However, a global fit of the dataover six sequential nucleotide incorporationsrevealed that the overall polymerization rateand processivity was higher for Pol ε than forPol2core. The largest difference was observedwhen challenged for the formation of aternary complex and incorporation of thefirst nucleotide. Pol ε needed less than asecond to incorporate a nucleotide, butseveral seconds passed before Pol2coreincorporated detectable levels of the firstnucleotide. We conclude that the accessorysubunits and the C-terminus of Pol2 do notinfluence the catalytic rate of Pol ε butfacilitate the loading and incorporation of thefirst nucleotide by Pol ε.

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  • 231. Gangabadage, Chinthaka Saneth
    et al.
    Zdunek, Janusz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tessari, Marco
    Nilsson, Solveig
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Wijmenga, Sybren Sipke
    Structure and dynamics of human apolipoprotein CIII2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 25, p. 17416-17427Article in journal (Refereed)
    Abstract [en]

    Human apolipoprotein CIII (apoCIII) is a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins. ApoCIII inhibits lipoprotein lipase as well as binding of lipoproteins to cell surface heparan sulfate proteoglycans and receptors. High levels of apoCIII are often correlated with elevated levels of blood lipids (hypertriglyceridemia). Here, we report the three-dimensional NMR structure and dynamics of human apo-CIII in complex with SDS micelles, mimicking its natural lipid-bound state. Thanks to residual dipolar coupling data, the first detailed view is obtained of the structure and dynamics of an intact apolipoprotein in its lipid-bound state. ApoCIII wraps around the micelle surface as a necklace of six approximately 10-residue amphipathic helices, which are curved and connected via semiflexible hinges. Three positively charged (Lys) residues line the polar faces of helices 1 and 2. Interestingly, their three-dimensional conformation is similar to that of the low density lipoprotein receptor binding motifs of apoE/B and the receptor-associated protein. At the C-terminal side of apoCIII, an array of negatively charged residues lines the polar faces of helices 4 and 5 and the adjacent flexible loop. Sequence comparison shows that this asymmetric charge distribution along the solvent-exposed face of apoCIII as well as other structural features are conserved among mammals. This structure provides a template for exploration of molecular mechanisms by which human apoCIII inhibits lipoprotein lipase and receptor binding.

  • 232. Garbacz, Marta A.
    et al.
    Cox, Phillip B.
    Sharma, Sushma
    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.
    Kunkel, Thomas A.
    The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity2019In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 8, p. 3986-3995Article in journal (Refereed)
    Abstract [en]

    The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.

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  • 233. Garg, Parie
    et al.
    Stith, Carrie M
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Burgers, Peter M
    Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication.2004In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 18, no 22, p. 2764-2773Article in journal (Refereed)
  • 234. Garzón, J.
    et al.
    Rodríguez, R.
    Kong, Ziqing
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rodríguez-Acebes, S.
    Méndez, J.
    Moreno, S.
    García-Higuera, I.
    Shortage of dNTPs underlies altered replication dynamics and DNA breakage in the absence of the APC/C cofactor Cdh12017In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 36, no 42, p. 5808-5818Article in journal (Refereed)
    Abstract [en]

    The APC/C-Cdh1 ubiquitin-ligase complex targets cell cycle regulators for proteosomal degradation and helps prevent tumor development and accumulation of chromosomal aberrations. Replication stress has been proposed to be the main driver of genomic instability in the absence of Cdh1, but the real contribution of APC/C-Cdh1 to efficient replication, especially in normal cells, remains unclear. Here we show that, in primary MEFs, acute depletion or permanent ablation of Cdh1 slowed down replication fork movement and increased origin activity. Partial inhibition of origin firing does not accelerate replication forks, suggesting that fork progression is intrinsically limited in the absence of Cdh1. Moreover, exogenous supply of nucleotide precursors, or ectopic overexpression of RRM2, the regulatory subunit of Ribonucleotide Reductase, restore replication efficiency, indicating that dNTP availability could be impaired upon Cdh1 loss. Indeed, we found reduced dNTP levels in Cdh1-deficient MEFs. Importantly, DNA breakage is also significantly alleviated by increasing intracellular dNTP pools, strongly suggesting that genomic instability is the result of aberrant replication. These observations highlight the relevance of APC/C-Cdh1 activity during G1 to ensure an adequate supply of dNTPs to the replisome, prevent replication stress and the resulting chromosomal breaks and, ultimately, suppress tumorigenesis.

  • 235. Geisler, Matt
    et al.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Karpinski, Stanislaw
    Kleczkowski, Leszek
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.2004In: Plant Molecular Biology, ISSN 0167-4412, Vol. 56, no 5, p. 783-94Article in journal (Refereed)
    Abstract [en]

    UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

  • 236.
    Gennebäck, Nina
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Hellman, Urban
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Malm, Linus
    Institutionen för skoglig genetik och växtfysiologi, Sveriges lantbruks universitet.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rongquist, Gunnar
    Institutionen för medicinska vetenskaper, Klinisk kemi.
    Waldenström, Anders
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Mörner, Stellan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Characterisation of exosomes derived from cultured cardiomyocytes treated with different growth factorsArticle in journal (Refereed)
  • 237.
    Gennebäck, Nina
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Cardiology.
    Hellman, Urban
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Cardiology.
    Malm, Linus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ronquist, Gunnar
    Waldenström, Anders
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Cardiology.
    Mörner, Stellan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Cardiology.
    Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes2013In: Journal of Extracellular Vesicles, ISSN 2001-3078, E-ISSN 2001-3078, Vol. 2Article in journal (Refereed)
    Abstract [en]

    Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes. Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression. The exosomes were rounded in shape and had an average size of 50-90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups. We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.

  • 238.
    Gharibyan, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Amyloids here, amyloids there…What’s wrong with them?2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloid formation is inherent property of proteins which under certain circumstances can become a pathologic feature of a group of diseases called amyloidosis. There are about 30 known human amyloidosis and more than 27 identified proteins involved in these pathologies.  Besides these proteins, there are a growing number of proteins non-related to diseases shown to form amyloid-like structures in vitro, which make them excellent tools for studying amyloid formation mechanisms, physicochemical properties of different amyloid species and the nature of their influence on tissues and cells.  It is important to understand the mechanisms by which amyloids interact with different types of cells, as the leading hypothesis in amyloid field suggests that amyloids and especially their intermediate states are the main harmful, toxic species causing tissue and cell degeneration.

    Using de-novo synthesized protein albebetin as a model of amyloidogenic protein, we demonstrated that it forms amyloid-like structures under physiological conditions (pH 7 and 37°C). During aggregation it forms 2 different types of intermediate oligomers — cross-b sheet containing and lacking β-sheet oligomers. Only the former induces cellular toxicity in a dose dependent manner. Further aggregation leads to the formation of fully mature amyloid-like fibrils, which are not toxic to the cells during studied period of incubation.

    Another model protein in our studies was hen egg white lysozyme, which readily forms amyloid under denaturing conditions (pH 2,2 and 57°C). In contrast to albebetin and many other proteins reported in the literature, we showed that both oligomers and mature fibrils from hen lysozyme affect cell viability. Targeting different mechanisms involved in cellular death, we revealed that oligomers induce slow and apoptotic-like cell death, while mature fibrils cause rapid and mainly necrotic-like cellular death.   

    One of the important aspects of amyloid studies is to develop measures for inhibiting or re-directing the process of amyloid formation to abolish or neutralize toxic amyloid species. Among the agents having inhibitory or modulatory properties small, phenol containing molecules are widely studied. We investigated the effect of the novel nootropic drug noopept on amyloid formation process of α-synuclein, as this drug is a small dipeptide containing a phenol ring. We showed that noopept is able to modulate amyloid formation process by accelerating it to rapid conversion of α-synuclein into fully mature fibrils, thus eliminating the stage of population of toxic oligomeric species.  Using wide range of cytotoxicity assays we showed that amyloid-like fibrils formed in the presence of noopept have no cytotoxic properties.  As this medicine is becoming popular and freely available in some countries as a cognitive enhancer, neuroprotective and nootropic agent, further detailed investigations and clinical trials are needed to assess the safety and benefit of noopept in particular for the patients with amyloid related neurodegenerative diseases (such as Parkinson’s or Alzheimer’s diseases).    

    While in vitro models are useful to study some specific aspects of protein aggregation, their properties and effects on cell viability, it is very difficult or practically impossible to create an absolutely accurate model of in vivo situation. Therefore, it is important to turn to in vivo/ex vivo studies to relate the knowledge accumulated from in vitro studies to the real situation in the body.

    Using human brain hippocampus tissues from individuals with Alzheimer’s disease, we found that besides well-known and widely accepted main pathological hallmark — Ab peptide deposition, S100A9 and S100A8 pro-inflammatory calcium-binding proteins are also localized in the plaques and in surrounding tissues and very explicitly co-localized with Ab. Moreover, we found the presence of S100A9 within the neuronal cells, which has not been reported before and can be an important clue for understanding the mechanisms of neurodegeneration. In vitro cytotoxicity studies showed that S100A9 protein can efficiently induce cytotoxicity when added exogenously to the neuronal cell culture. These findings suggest that S100A8 and S100A9 proteins play an important role in Alzheimer’s pathology, and potentially can be candidates for the amyloid plaque formation and neurodegeneration. Whether they are associated with inflammatory processes underlying the early onset of disease or produced and accumulated as a consequence of A-beta induced pathology remain to be clarified.

    We found that Alzheimer’s disease is not the only pathology associated with A-beta and S100A9 deposition in a form of plaques. Immunohistochemical studies of an aortic valve surgically removed from a patient with aortic stenosis revealed plaque-like structures positively stained with A-beta and S100A9 proteins. These areas are also positively stained with fibril-specific antibodies as well as with Congo red, which also shows very distinct apple-green birefringence under the polarized light. Besides, there is intracellular localization and co-localization of both proteins in interstitial cells throughout the whole fibrous tissue of the valve. The presented case report is the first finding suggesting inflammatory protein S100A9 as well as A-beta peptide as potential candidates for amyloid formation in aortic stenosis valves.  We suggest that there is a specific interaction between A-beta and S100A9 during amyloid formation, which can be involved in amyloid-associated pathology in various tissues and organs in the body, which can potentially be caused by inflammatory processes, particularly by its chronic, long lasting forms.

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  • 239.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Islam, Tohidul
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pettersson, Nina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Golchin, Solmaz A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundgren, Johanna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Gabriella
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Genot, Melany
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schultz, Nina
    Wennström, Malin
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Apolipoprotein E Interferes with IAPP Aggregation and Protects Pericytes from IAPP-Induced Toxicity2020In: Biomolecules, E-ISSN 2218-273X, Vol. 10, no 1, article id 134Article in journal (Refereed)
    Abstract [en]

    Apolipoprotein E (ApoE) has become a primary focus of research after the discovery of its strong linkage to Alzheimer’s disease (AD), where the ApoE4 variant is the highest genetic risk factor for this disease. ApoE is commonly found in amyloid deposits of different origins, and its interaction with amyloid-β peptide (Aβ), the hallmark of AD, is well known. However, studies on the interaction of ApoEs with other amyloid-forming proteins are limited. Islet amyloid polypeptide (IAPP) is an amyloid-forming peptide linked to the development of type-2 diabetes and has also been shown to be involved in AD pathology and vascular dementia. Here we studied the impact of ApoE on IAPP aggregation and IAPP-induced toxicity on blood vessel pericytes. Using both in vitro and cell-based assays, we show that ApoE efficiently inhibits the amyloid formation of IAPP at highly substoichiometric ratios and that it interferes with both nucleation and elongation. We also show that ApoE protects the pericytes against IAPP-induced toxicity, however, the ApoE4 variant displays the weakest protective potential. Taken together, our results suggest that ApoE has a generic amyloid-interfering property and can be protective against amyloid-induced cytotoxicity, but there is a loss of function for the ApoE4 variant.

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  • 240.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Narayana, Vinod
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ankarcrona, Maria
    Karolinska Institute.
    Brännström, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Olofsson, Anders
    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.
    Emerging role of inflammatory S100A9 in Alzheimer’s disease amyloid growth and neurodegenerationManuscript (preprint) (Other academic)
  • 241.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Narayana, Vinod
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Habib, Ahsan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sulniute, Rima
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Henein, Michael
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Inflammatory S100A9 and Aβ amyloids in heart valve of patient with aortic stenosisManuscript (preprint) (Other academic)
  • 242.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Raveh, Dina
    Department of Life Sciences, Ben Gurion University of the Negev, Israel.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    S100A8/A9 amyloidosis in the ageing prostate: relating ex vivo and in vitro studies2012In: Amyloid Proteins: Methods and Protocols / [ed] Einar M. Sigurdsson, Miguel Calero, María Gasset, Springer Science+Business Media B.V., 2012, Vol. 849, p. 387-401Chapter in book (Refereed)
    Abstract [en]

    The family of S100 proteins encompasses more than 20 members characterized by remarkable conformational and functional diversity. S100 proteins act as central regulators of various cellular processes, including cell survival, proliferation, differentiation, and motility. Many S100 proteins are implicated in various types of cancer as well as neurodegenerative, inflammatory, and autoimmune diseases. Recently, we have found that S100A8⁄A9 proteins are involved in amyloidogenic process in the ageing prostate, contributing to the formation of calcified corpora amylacea (CA) inclusions, which commonly accompany age-dependent prostate tissue remodelling and cancer. Amyloid formation by S100A8/A9 proteins can also be modelled in vitro. Amyloid assembly of S100A8/A9 proteins into oligomeric and fibrillar complexes is modulated by metal ions such as calcium and zinc. Here, we provide insights into the extraction procedures and review the common structural features of ex vivo and in vitro S100A8/A9 amyloids, showing that they share the same generic origin.

  • 243.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Moskaleva, Olesya S
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Margulis, BA
    Kostanyan, IA
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 5, p. 1337-1349Article in journal (Refereed)
    Abstract [en]

    Among the newly discovered amyloid properties, its cytotoxicity plays a key role. Lysozyme is a ubiquitous protein involved in systemic amyloidoses in vivo and forming amyloid under destabilising conditions in vitro. We characterized both oligomers and fibrils of hen lysozyme by atomic force microscopy and demonstrated their dose (5–50 μM) and time-dependent (6–48 h) effect on neuroblastoma SH-SY5Y cell viability. We revealed that fibrils induce a decrease of cell viability after 6 h due to membrane damage shown by inhibition of WST-1 reduction, early lactate dehydrogenase release, and propidium iodide intake; by contrast, oligomers activate caspases after 6 h but cause the cell viability to decline only after 48 h, as shown by fluorescent-labelled annexin V binding to externalized phosphatidylserine, propidium iodide DNA staining, lactate dehydrogenase release, and by typical apoptotic shrinking of cells. We conclude that oligomers induce apoptosis-like cell death, while the fibrils lead to necrosis-like death. As polymorphism is a common property of an amyloid, we demonstrated that it is not a single uniform species but rather a continuum of cross-β-sheet-containing amyloids that are cytotoxic. An abundance of lysozyme highlights a universal feature of this phenomenon, indicating that amyloid toxicity should be assessed in all clinical applications involving proteinaceous materials.

  • 244.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori: molecular insights into regulation of adhesion properties2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Helicobacter pylori infects the human stomach and triggers an inflammatory response that damages the gastric tissue. This host-pathogen interplay has dire consequences as up to 20 % of infected individuals develop peptic ulcer disease or gastric cancer. Given that half of the world’s population is infected, the number of afflicted humans is staggering and also tells that H. pylori is extremely efficient in spreading and maintaining infection. To enable persistent infection many factors play a role, but one important feature of H. pylori is its impressive ability to adhere to the slimy gastric mucus layer and the underlying epithelial cells. This occurs mainly via the BabA and SabA proteins that bind ABO/Leb- and sLex/sLea-antigens. I have in my thesis studied how these two proteins are utilized and regulated.

    H. pylori transcription is in part controlled by two-component systems (TCSs) that use a sensor protein and a DNA-binding response regulator. We have studied how these systems control sabA and to some extent babA and indeed found a better map of how sabA and babA is regulated at the transcriptional level. We also found that variations in a polynucleotide T-tract located in the sabA promotor could fine-tune SabA expression/ sLex-binding. Thus we have exposed how strict regulation by TCSs combined with stochastic processes together shapes attachment in the bacterial population.

    As the buffering mucus layer is constantly exfoliated, placing H. pylori in bactericidal acid, we hypothesized that low pH should abrogate adhesion. SabA expression was indeed repressed in low pH, however BabA expression remained unaffected. The BabA/ Leb-binding was instead directly reversibly hampered by low pH and the degree of pH sensitivity was strain dependent and encoded in the BabA sequence. We believe that the pH dependent loss of binding is one key factor H. pylori utilizes to maintain persistent infection.

    BabA is divided in generalists that bind ABO antigens and specialists that only bind blood group (bg) O. We co-crystalized BabA bound to these receptors and established the structural basis for generalist vs. specialist discrimination. We furthermore found a disulfide-clasped loop (CL2) in the center of the binding domain crucial for binding. Breaking CL2 with N-Acetylcysteine (NAC) disrupted binding and H. pylori infection mice experiments revealed inflammatory reduction upon NAC-treatment.

    In sum, I have in my thesis dissected how H. pylori controls its adhesive abilities and how intrinsic properties in binding can be exploited for therapeutic purposes.

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  • 245. Glise, Lars
    et al.
    Larsson, Pia
    Jern, Sverker
    Borén, Jan
    Levin, Malin
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Fogelstrand, Per
    Bergh, Niklas
    Disturbed Laminar Blood Flow Causes Impaired Fibrinolysis and Endothelial Fibrin Deposition In Vivo2019In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 119, no 2, p. 223-233Article in journal (Refereed)
    Abstract [en]

    Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature.

  • 246. Goldberg, Emily L.
    et al.
    Asher, Jennifer L.
    Molony, Ryan D.
    Shaw, Albert C.
    Zeiss, Caroline J.
    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.
    Herzog, Raimund I.
    Iwasaki, Akiko
    Dixit, Vishwa Deep
    beta-Hydroxybutyrate deactivates Neutrophil NLRP3 inflammasome to relieve gout flares2017In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 18, no 9, p. 2077-2087Article in journal (Refereed)
    Abstract [en]

    Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1 beta (IL-1 beta) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases beta-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1 beta in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.

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  • 247.
    Greule, Markus
    et al.
    Max Planck Inst for Chemistry.
    Tumino, Luisa Dana
    Inst Lebensmittelchem, JW Goethe Univ Frankfurt.
    Kronewald, Tatjana
    Hener, Uwe
    Inst Lebensmittelchem, JW Goethe Univ Frankfurt.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mosandl, Armin
    Max Planck Inst for Chemistry.
    Keppler, Frank
    Max Planck Inst for Chemistry.
    Improved rapid authentication of vanillin using δ13C and δ2H values2010In: European Food Research and Technology, ISSN 1438-2377, E-ISSN 1438-2385, Vol. 231, no 6, p. 933-941Article in journal (Refereed)
    Abstract [en]

    Vanilla still remains one of the most important and widely used flavours in the food industry and is also extensively employed by fragrance and pharmaceutical manufacturing companies. Natural vanilla flavour, extracted from the pods of the tropic orchid vanilla, is considerably more expensive than synthetic vanillin. The disparity of prices between natural vanillin and that derived from other sources has given rise to many cases of fraudulent adulteration, and for more than 30 years, strenuous efforts have been made to authenticate sources of vanillin. Stable isotope analysis is one of the most powerful analytical tools to distinguish between natural vanillin and that originating from other sources. Recently, a rapid and precise method for analysis of both δ13C and δ2H values of plant methoxyl groups has been published. Here, we report an application of the method for the control of authenticity of vanillin. Carbon and hydrogen stable isotope values of the vanillin molecule and vanillin methoxyl groups of vanillin samples of different origins including authentic and synthetic samples were measured. The results clearly show that use of this approach provides a rapid and reliable authenticity assessment of vanillin. The technique used for these studies is robust and rapid, involves minimum sample preparation and requires only a small amount of vanillin sample, usually 1 mg for stable carbon and 4 mg for stable hydrogen analysis.

  • 248.
    Grimsholm, Ola
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Guo, Yongzhi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Expression patterns of neurotrophins and neurotrophin receptors in articular chondrocytes and inflammatory infiltrates in knee joint arthritis.2008In: Cells, tissues, organs, ISSN 1422-6421, Vol. 188, no 3, p. 299-309Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: It is likely that neurotrophins (NTs) are of great importance for the articular cartilage and the inflammation process in arthritis. METHODS: The immunohistochemical expression of the NTs nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and the associated receptors p75, TrkA and TrkB was examined in the knee joint of arthritic and healthy mice. RESULTS: Immunoreactions for NGF and BDNF were detected in cells and nerve fiber varicosities in the inflammatory infiltrates of the synovial tissue of arthritic joints but not in synovial tissue of controls. p75-immunoreactive nerve fiber-like strands were detected in inflammatory infiltrates. Immunostaining for NGF, BDNF, p75, TrkA and TrkB was noted in articular chondrocytes. There was a statistically significant decrease in reactions for NGF (p < 0.001), TrkA (p = 0.001) and p75 (p < 0.001) in articular chondrocytes in joints exhibiting severe arthritis. CONCLUSION: The findings show that an NT system develops in inflammatory infiltrates of the synovial tissue. Furthermore, most interestingly, autocrine/paracrine effects appear to exist concerning NTs for the articular chondrocytes. The downregulated expression of NGF and NT receptors in articular chondrocytes in arthritis is a new aspect concerning the involvement of NTs in cartilage.

  • 249.
    Grimsholm, Ola
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Guo, Yongzhi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rantapää-Dahlqvist, Solbritt
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Are neuropeptides important in arthritis? Studies on the importance of bombesin/GRP and substance P in a murine arthritis model.2007In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1110, p. 525-538Article in journal (Refereed)
    Abstract [en]

    Interference with the effects of neuropeptides may be of potential therapeutic value for the treatment of rheumatoid arthritis (RA). Two neuropeptides that can be discussed in this context are bombesin/gastrin-releasing peptide (BN/GRP) and substance P (SP). In order to obtain new information on the possible importance of these two peptides, the patterns of immunohistochemical expression of BN/GRP and SP and their related receptors in the mouse knee joint from healthy and arthritic mice were examined. Positive staining for GRP receptor and the SP preferred receptor (the neurokinin-1 receptor [NK-1 R]) was observed in articular chondrocytes. On the whole, there was a decrease in immunoreactions for both the GRP- and the NK-1 receptors in the articular chondrocytes in joints exhibiting severe arthritis. Staining for BN/GRP and GRP receptor was seen in the inflammatory infiltrates of the arthritic joints. New evidence for the occurrence of marked effects of BN/GRP concerning both the articular chondrocytes and the inflammatory process is obtained in this study. With these findings and previous observations of neuropeptide expression patterns and functions we discuss the possibility that interventions with the effects of BN/GRP, SP, and other neuropeptides might be worthwhile in RA.

  • 250. Grinberg, Inna
    et al.
    McGann, Matthew
    Lundin, Daniel
    Crona, Mikael
    Hasan, Mahmudal
    Jonna, Venkateswara Rao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Loderer, Christoph
    Sahlin, Margareta
    Markova, Natalia
    Stenson, John
    Borovok, Ilya
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Logan, Derek
    Sjöberg, Britt-Marie
    Novel ATP-Cone-Driven Allosteric Regulation of Ribonucleotide Reductase Via the Radical-Generating Subunit2018In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 27, p. 87-88Article in journal (Other academic)
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