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  • 51.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gustafsson, Claes M
    The mediator complex.2004In: Advances in Protein Chemistry, ISSN 0065-3233, E-ISSN 1557-8941, Vol. 67, p. 43-65Article in journal (Refereed)
  • 52.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Gustafsson, Claes M
    The yeast Mediator complex and its regulation.2005In: Trends in Biochemical Sciences, ISSN 0968-0004, Vol. 30, no 5, p. 240-4Article in journal (Refereed)
  • 53.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Hjortsberg, K
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase.1993In: Proceedings of the National Academy of Science U S A, ISSN 0027-8424, Vol. 90, no 23, p. 11322-6Article in journal (Refereed)
  • 54.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Skog, Sven
    Tribukait, Bernard
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs1990In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 29, no 23, p. 5452-5458Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.

  • 55.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Skogman, E
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit1992In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 11, no 13, p. 4953-4959Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.

  • 56. Björnberg, O
    et al.
    Vodnala, Munender
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rasmussen, A
    Andersen, G
    Piskur, J
    Ribosylurea accumulates in yeast urc4 mutants2010In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 29, no 4-6, p. 433-437Article in journal (Refereed)
    Abstract [en]

    Yeast Saccharomyces (Lachancea) kluyveri urc4 mutants, unable to grow on uracil, biotransformed (14)C(2)-uracil into two labeled compounds, as detected by high performance liquid chromatography (HPLC). These two compounds could also be obtained following organic synthesis of ribosylurea. This finding demonstrates that in the URC pyrimidine degradation pathway, the opening of the uracil ring takes place when uracil is attached to the ribose moiety. Ribosylurea has not been reported in the cell metabolism before and the two observed compounds likely represent an equilibrium mixture of the pyranosyl and furanosyl forms.

  • 57.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Bugaytsova, Jeanna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Dynamic force spectroscopy of the Helicobacter pylori BabA-Lewis b binding2009In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 143, no 1-2, p. 102-105Article in journal (Refereed)
    Abstract [en]

    The binding strength of the Helicobacter pylori adhesin–receptor complex BabA-ABO/Lewis b has been analyzed by means of dynamic force pectroscopy. High-resolution measurements of rupture forces were performed in situ on single bacterial cells, expressing the high-affinity binding BabA adhesin, by the use of force measuring optical tweezers. The resulting force spectra revealed the mechanical properties of a single BabA–Leb bond. It was found that the bond is dominated by one single energy barrier and that it is a slipbond. The bond length and thermal off-rate were assessed to be 0.86±0.07 nm and 0.015±0.006 s−1, respectively.

  • 58.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Fällman, Erik
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Ohlsson, J.
    Nilsson, U.J.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers2005In: Journal of Biomedical Optics, ISSN 1560-2281 (Online), Vol. 10, no 4, p. 044024-Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of approximately 100 pNs, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25+/-1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.

  • 59.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aguilar, Ximena
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rautio, Linn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, p. 5938-5950Article in journal (Refereed)
    Abstract [en]

    Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

  • 60.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Höglund, Andreas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, David
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Jacobsson, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line2011In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 16, no 8, p. 783-794Article in journal (Refereed)
    Abstract [en]

    Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

  • 61.
    Bodén, Ida
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The roles of the plasminogen activator and matrix metalloproteinase systems in ovulation and corpus luteum formation2004Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems in gonadotropin-induced ovulation, and in CL formation and function.

    A mouse model for gonadotropin-induced CL formation was developed in order to control the timing of CL formation. In this model, immature mice were induced to ovulate by administrating gonadotropins and the endogenous prolactin surges were mimicked by administration of prolactin twice daily from day 2 of CL development. We observed that steroidogenic acute regulatory protein (StAR) mRNA was highly expressed at days 3 and day 6 of CL development and the levels remained high until late stages of CL regression.

    Since mice lacking plasminogen (plg-/-) only have a 14% reduction of ovulation efficiency, our hypothesis was that the MMP system could compensate for the loss of plasminogen. When administrating the MMP-inhibitor galardin to gonadotropin-primed ovulating mice, we found that wild-type mice (plg+/+ and C67BL/J6) and heterozygous mice (plg+/-) had an 18-20% reduction in ovulation efficiency as compared to untreated mice.

    Two models for CL formation, the adult pseudopregnant (psp) mouse model and a model whereby immature gonadotropin-primed mice were treated with prolactin, were used to study the formation and function of the CL in plg-/- mice treated with galardin. At day 3 of CL development, we found no alterations other than a slightly lower number of CL in plg-/- mice. This is most likely a secondary effect of the lower ovulation efficiency found in these mice. On the other hand, we found a 54% reduction in serum progesterone levels in plg-/- mice and a 37% reduction in the plg+/- mice as compared to wild type mice. At day 6 of CL development we saw a 45 % reduction of serum progesterone level in the plg-/- mice and a 22 % reduction in the plg+/- mice. A similar trend was observed at day 3 of CL development in immature gonadotropinprimed mice treated with prolactin. Galardin treatment did not alter the results significantly and the CLs were healthy and viable in these mice.

    In conclusion, our data suggest that both plasminogen and MMPs, alone or in combination, are dispensable for ovulation and for the formation of a viable CL under the conditions used in this study. The reduced serum progesterone levels observed in the plg-/- mice did not appear to be a result of defective CL formation. Instead, plasmin may have a novel role in the maintenance of luteal function. StAR expression may also be a good marker for CL development and regression in mice.

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  • 62. Boldinova, Elizaveta O.
    et al.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Khairullin, Rafil
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Kazan Volga Reg Fed Univ, Russia.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, article id e0184489Article in journal (Refereed)
    Abstract [en]

    Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37 degrees C. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

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  • 63. Boldinova, Elizaveta O.
    et al.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shilkin, Evgeniy S.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol2017In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 18, no 7, article id 1584Article, review/survey (Refereed)
    Abstract [en]

    PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.

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  • 64. Borekci, Gulay
    et al.
    Lee, Natuschka M.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rapid Microscope Based Identification Method for Tuberculosis and Other Mycobacteria: Fluorescence In Situ Hybridization (FISH) - Current State of The Art and Future Research Needs2016In: Tuberculosis, SM Group , 2016Chapter in book (Refereed)
    Abstract [en]

    Tuberculosis (TB) is one of the major increasing causes of illness and death worldwide, especially in Asia and Africa. Rapid and accurate diagnosis of mycobacteria is important in the prevention and effective treatment of tuberculosis. Today, conventional culture methods are still accepted as the gold standard for the identification of mycobacteria in routine mycobacteriology laboratories. However, even if these methods are highly efficient and useful, these methods are time-consuming and labor-laborious. In recent years, several novel DNA-based and non-invasive techniques, such as RAMAN spectroscopy and microcalorimetry have been developed for a more rapid and reliable identification. Unfortunately, these methods are not capable of visualizing the cells in their natural environment such as in tissues. Visualization of cells may however provide fundamental, complementary information for the overall understanding of the molecular and microbial ecology of mycobacteria in disease processes. Here, we present a current state of the art review of the Fluorescence In Situ Hybridization (FISH) methods which can be used to identify, visualize and quantify whole cells of different species of mycobacteria, especially the tuberculosis complex, and their associates in their natural environment without prior cultivation. Although this method also allows for an easy, rapid and cost-efficient identification (~1-3 hours) 2Tuberculosis | www.smgebooks.comCopyright  Borekci G.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited. and simultaneous in situ visualization of different microbial species, it has so far only been used to a limited extent. Here, we will discuss both the potentials as well as limitations of FISH for the detection of mycobacteria and other relevant associates, and suggest some future research needs.

  • 65.
    Boren, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    HELICOBACTER PYLORI; MULTITALENTED ADAPTATION OF BINDING PROPERTIES2014In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 34, no 10, p. 5840-5841Article in journal (Other academic)
  • 66. Borg, H
    et al.
    Nordensson, I
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundgren, E
    Evidence for IFN-beta heterogeneity in a substrain of Namalwa cells.1985In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, E-ISSN 1559-0291, Vol. 11, no 2, p. 111-22Article in journal (Refereed)
    Abstract [en]

    A substrain of Namalwa cells, denoted substrain B, was grown in fermentors up to the 100-L scale, and was induced with Sendai virus to produce interferon (IFN). The titer of the crude IFN varied extensively between different batches; part of the variation was caused by a differential expression of IFN-alpha and IFN-beta. More than 80% of the IFN activity was IFN-beta by several criteria. A two-step purification procedure was developed and the resulting preparation had a specific activity of approximately 10(6) U/mg protein. The IFN-beta type was found to be heterogeneous, and could be separated into several components, which probably represented post-translational modifications of one molecule.

  • 67.
    Botelho, Hugo M.
    et al.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    Leal, Sonia S.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    Cardoso, Isabel
    Molecular Neurobiology Unit, Instituto de Biologia Molecular e Celular, Porto, Portugal.
    Yanamandra, Kiran
    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.
    Fritz, Günter
    Department of Neuropathology, University of Freiburg, Germany.
    Gomes, Claudio M.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    S100A6 Amyloid Fibril formation is Calcium-modulated and enhances Superoxide Dismutase-1 (SOD1) aggregation2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 50, p. 42233-42242Article in journal (Refereed)
    Abstract [en]

    S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel beta-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metalmodulated aggregation propensity may be a key aspect in their physiology and function.

  • 68. Bourbon, Henri-Marc
    et al.
    Aguilera, Andres
    Ansari, Aseem Z
    Asturias, Francisco J
    Berk, Arnold J
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blackwell, T Keith
    Borggrefe, Tilman
    Carey, Michael
    Carlson, Marian
    Conaway, Joan W
    Conaway, Ronald C
    Emmons, Scott W
    Fondell, Joseph D
    Freedman, Leonard P
    Fukasawa, Toshio
    Gustafsson, Claes M
    Han, Min
    He, Xi
    Herman, Paul K
    Hinnebusch, Alan G
    Holmberg, Steen
    Holstege, Frank C
    Jaehning, Judith A
    Kim, Young-Joon
    Kuras, Laurent
    Leutz, Achim
    Lis, John T
    Meisterernest, Michael
    Naar, Anders M
    Nasmyth, Kim
    Parvin, Jeffrey D
    Ptashne, Mark
    Reinberg, Danny
    Ronne, Hans
    Sadowski, Ivan
    Sakurai, Hiroshi
    Sipiczki, Matthias
    Sternberg, Paul W
    Stillman, David J
    Strich, Randy
    Struhl, Kevin
    Svejstrup, Jasper Q
    Tuck, Simon
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Winston, Fred
    Roeder, Robert G
    Kornberg, Roger D
    A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II.2004In: Molecular Cell, ISSN 1097-2765, Vol. 14, no 5, p. 553-7Article in journal (Refereed)
  • 69. Broach, James R
    et al.
    Bharatula, Vasudha
    Chereji, Razvan
    Elfving, Nils
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozov, Alexandre
    The Msn2 mediated stress response: Survival based on "hedging your bet" and a dynamic interplay of transcription factor binding and nucleosome occupancy2015In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, no Suppl. 1, p. S221-S222Article in journal (Other academic)
    Abstract [en]

    Yeast cell subjected to many different stresses elicit an acute transcriptional stress response mediated by the Msn2 transcription factor, which alters expression of both a stress specific-cohort of genes as well as a common cohort of genes that changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have shown by dynamic single cell analysis that stresses regulate Msn2 activity through cytoplasm to nuclear relocalization but do so in an unusual way: stresses induce increased frequency of bursts of short-lived, recurrent periods of Msn2 nuclear localization with different stresses eliciting different patterns of bursts. Moreover, genetically identical cells subject to an identical stress can behave quite differently, with some cells mounting a robust nuclear occupancy of Msn2 while others show no nuclear localization at all. We have proposed that this idiosyncratic behavior allows populations of cells to “hedge their bet” as to what will be the optimum strategy for surviving the ensuing stress. We have used computational modeling and single cell analysis to determine that bursting is a consequence of noise in the stress signaling pathways amplified by the small number of Msn2 molecules in the cell. Moreover, we have applied genome wide chromatin immunoprecipitation and nucleosome profiling to address how different stresses determine where Msn2 binds under a particular stressful conditions, and thus what genes are regulated by that stress, and how that binding affects, and is affected by, nucleosome positioning and other transcription factor binding. These results provide in vivo validation of Widon's model of indirect cooperativity of transcription factor binding, mediated by partial unwinding of nucleosomes by one transcription factor to allow access for a second transcription factor to a previously occluded binding site. Finally, we have addressed the “bet hedging” hypothesis by showing that persistence of the Msn2-mediated stress response yields cell growth arrest and have identified the targets responsible for that growth arrest. We have applied experimental evolution paradigms to address the relative fitness of cells exhibiting stochastic stress responses versus those with a uniform response. In short, our results indicate that the stress response is complex and that complexity is critical for cell survival.

  • 70. Bru, Samuel
    et al.
    Marc Martinez-Lainez, Joan
    Hernandez-Ortega, Sara
    Quandt, Eva
    Torres-Torronteras, Javier
    Marti, Ramon
    Canadell, David
    Arino, Joaquin
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jimenez, Javier
    Clotet, Josep
    Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 101, no 3, p. 367-380Article in journal (Refereed)
    Abstract [en]

    Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4 Delta) or hydrolyse polyP (ppn1 Delta, ppx1 Delta) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

  • 71. Bryan, Thomas
    et al.
    Luo, Xiliang
    Forsgren, Lars
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Davis, Jason J.
    The robust electrochemical detection of a Parkinson's disease marker in whole blood sera2012In: Chemical Science, ISSN 2041-6520, Vol. 3, no 12, p. 3468-3473Article in journal (Refereed)
    Abstract [en]

    Protein aggregation, leading to amyloid deposition in the brain, is implicated in the pathology of a number of increasingly prevalent neurodegeneration states such as Parkinson's disease (PD), Alzheimer's disease and prion diseases. The body's protective response to the formation of such deposits is to generate specific autoimmune antibodies. Alpha-synuclein, a natively unfolded protein relatively abundant in the brain, is the main constituent of Lewy body amyloid dispositions in PD. Previous assays determining content of alpha-synuclein in bodily fluids have proven to be largely inconclusive. Here we have taken a novel approach in utilising alpha-synuclein modified electrodes to sample the autoantibodies generated as the body responds to changes in its homeostasis. We show that these electroanalytical assays not only robustly distinguish between disease state and control individuals but also map out disease progression with unprecedented sensitivity and clarity. The impedimetric electrode surfaces are highly specific, reusable, exhibit a linear range from 0.5 to 10 nM and a detection limit of 55 +/- 3 pM. We believe electroanalyses such as these, possible with less than 10 microlitres of fluid and a total assay time of only a few minutes, to be of value for early diagnosis of PD and possibly other alpha-synucleinopathies, and for monitoring disease progression and effects of possible disease modifying interventions.

  • 72.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharibyan, Anna L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Islam, Tohidul
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Lina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lee, Cheng Choo
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pamrén, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Scanning electron microscopy as a tool for evaluating morphology of amyloid structures formed on surface plasmon resonance chips2018In: Data in Brief, E-ISSN 2352-3409, Vol. 19, p. 1166-1170Article in journal (Refereed)
    Abstract [en]

    We demonstrate the use of Scanning Electron microscopy (SEM) in combination with Surface Plasmon Resonance (SPR) to probe and verify the formation of amyloid and its morphology on an SPR chip. SPR is a technique that measures changes in the immobilized weight on the chip surface and is frequently used to probe the formation and biophysical properties of amyloid structures. In this context it is of interest to also monitor the morphology of the formed structures. The SPR chip surface is made of a layer of gold, which represent a suitable material for direct analysis of the surface using SEM. The standard SPR chip used here (CM5-chip, GE Healthcare, Uppsala, Sweden) can easily be disassembled and directly analyzed by SEM. In order to verify the formation of amyloid fibrils in our experimental conditions we analyzed also in-solution produced structures by using Transmission Electron Microscopy (TEM). For further details and experimental findings, please refer to the article published in Journal of Molecular Biology, (Brännström K. et al., 2018) [1].

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  • 73.
    Brännström, Kristoffer
    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.
    Gharibyan, Anna L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Lina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lee, Cheng Choo
    Sandblad, Linda
    Pamrén, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 13, p. 1940-1949Article in journal (Refereed)
    Abstract [en]

    Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1-40 and Aβ1-42 are the dominant forms. The fibril architectures of Aβ1-40 and Aβ1-42 differ and Aβ1-42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1-42 can be cross-templated and incorporated into the ends of Aβ1-40 fibrils, while incorporation of Aβ1-40 monomers into Aβ1-42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1-40 to incorporate into the ends of Aβ1-42 fibrils and the capacity of Aβ1-42 monomers to adopt the properties of Aβ1-40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1-42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1-40 from adopting the fibrillar properties of Aβ1-42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.

  • 74.
    Brännström, Kristoffer
    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.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The role of histidines in amyloid β fibril assembly2017In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 8, p. 1167-1175Article in journal (Refereed)
    Abstract [en]

    Low pH has a strong stabilising effect on the fibrillar assembly of amyloid β, which is associated with Alzheimer's disease. The stabilising effect is already pronounced at pH 6.0, suggesting that protonation of histidines might mediate this effect. Through the systematic substitution of the three native histidines in Aβ for alanines, we have evaluated their role in fibril stability. Using surface plasmon resonance, we show that at neutral pH the fibrillar forms of all His-Ala variants are destabilised by a factor of 4-12 compared to wild-type Aβ. However, none of the His-Ala Aβ variants impair the stabilising effect of the fibril at low pH.

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  • 75.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lindhagen Persson, Malin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharabyan, A
    Vestling, M
    Brännström, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Design of oligomer-specific antibodiesManuscript (preprint) (Other academic)
  • 76.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lindhagen-Persson, Malin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharibyan, Anna L.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vestling, Monika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sellin, Mikael E.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Brännström, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Forsgren, Lars
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Generic Method for Design of Oligomer-Specific Antibodies2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 3, p. e90857-Article in journal (Refereed)
    Abstract [en]

    Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e. g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the A beta peptide and alpha-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.

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  • 77.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Lindhagen-Persson, Malin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ca2+ enhances Aβ polymerization rate and fibrillar stability in a dynamic manner2013In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 450, p. 189-197Article in journal (Refereed)
    Abstract [en]

    Identifying factors that affect the self-assembly of the amyloid-β peptide (Aβ) is of utmost importance in the quest to understand the molecular mechanisms causing Alzheimer's disease (AD). Ca2+ has previously been shown to accelerate both Aβ fibril nucleation and maturation, and a dysregulated Ca2+ homeostasis frequently correlates with development of AD. The mechanisms regarding Ca2+ binding as well as its effect on fibril kinetics are not fully understood. Using a polymerization assay we show that Ca2+ in a dynamic and reversible manner enhances both the elongation rate and fibrillar stability, where specifically the "dock and lock" phase mechanism is enhanced. Through NMR analysis we found that Ca2+ affects the fibrillar architecture. In addition, and unexpectedly, we found that Ca2+ does not bind the free Aβ monomer. This implies that Ca2+ binding requires an architecture adopted by assembled peptides, and consequently is mediated through intermolecular interactions between adjacent peptides. This gives a mechanistic explanation to the enhancing effect on fibril maturation and indicates structural similarities between prefibrillar structures and mature amyloid. Taken together we expose how Ca2+ levels affect the delicate equilibrium between the monomeric and assembled Aβ and how fluctuations in vivo may contribute to development and progression of the disease.

  • 78.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Nilsson, Lina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pihl, Mathias
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The N-terminal Region of Amyloid β Controls the Aggregation Rate and Fibril Stability at Low pH Through a Gain of Function Mechanism2014In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, no 31, p. 10956-10964Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease is linked to a pathological polymerization of the endogenous amyloid β-peptide (Aβ) that ultimately forms amyloid plaques within the human brain. We used surface plasmon resonance (SPR) to measure the kinetic properties of Aβ fibril formation under different conditions during the polymerization process. For all polymerization processes, a critical concentration of free monomers, as defined by the dissociation equilibrium constant (KD), is required for the buildup of the polymer, for example, amyloid fibrils. At concentrations below the KD, polymerization cannot occur. However, the KD for Aβ has previously been shown to be several orders of magnitude higher than the concentrations found in the cerebrospinal and interstitial fluids of the human brain, and the mechanism by which Aβ amyloid forms in vivo has been a matter of debate. Using SPR, we found that the KD of Aβ dramatically decreases as a result of lowering the pH. Importantly, this effect enables Aβ to polymerize within a picomolar concentration range that is close to the physiological Aβ concentration within the human brain. The stabilizing effect is dynamic, fully reversible, and notably pronounced within the pH range found within the endosomal and lysosomal pathways. Through sequential truncation, we show that the N-terminal region of Aβ contributes to the enhanced fibrillar stability due to a gain of function mechanism at low pH. Our results present a possible route for amyloid formation at very low Aβ concentrations and raise the question of whether amyloid formation in vivo is restricted to a low pH environment. These results have general implications for the development of therapeutic interventions.

  • 79.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e25157-Article in journal (Refereed)
    Abstract [en]

    Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided "dock and lock" mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1-40 Glu22Gly (Aβ(40Arc)) and wild type Aβ1-40 (Aβ(40WT)). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ(40WT) is significantly impaired to acquire the fibrillar architecture of Aβ(40Arc). A detailed characterisation demonstrated that Aβ(40WT) has a restricted ability to dock and isomerise with high binding affinity onto Aβ(40Arc) fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.

  • 80.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brattsand, Maria
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.
    Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis2012In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 393, no 5, p. 369-377Article in journal (Refereed)
    Abstract [en]

    SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.

  • 81.
    Buckland, Robert
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA precursor asymmetries, Mismatch Repair and their effect on mutation specificity2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed.

    In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

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  • 82.
    Buckland, Robert J
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Watt, Danielle L
    Chittoor, Balasubramanyam
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kunkel, Thomas A
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, article id e1004846Article in journal (Refereed)
    Abstract [en]

    The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

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  • 83.
    Bugaytsova, Jeanna A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björnham, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics. Swedish Defence Research Agency, 906 21 Umeå, Sweden.
    Chernov, Yevgen A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mahdavi, Jafar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. School of Life Sciences, CBS, University of Nottingham, NG7 2RD Nottingham, UK.
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ilver, Dag
    Moonens, Kristof
    Quintana-Hayashi, Macarena P.
    Moskalenko, Roman
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmidt, Alexej
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Åberg, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Koeniger, Verena
    Vikström, Susanne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ögren, Johan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Section of Medicine.
    Liu, Hui
    Goldman, Matthew D.
    Whitmire, Jeannette M.
    Åden, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Younson, Justine
    Kelly, Charles G.
    Gilman, Robert H.
    Chowdhury, Abhijit
    Mukhopadhyay, Asish K.
    Nair, G. Balakrish
    Papadakos, Konstantinos S.
    Martinez-Gonzalez, Beatriz
    Sgouras, Dionyssios N.
    Engstrand, Lars
    Unemo, Magnus
    Danielsson, Dan
    Suerbaum, Sebastian
    Oscarson, Stefan
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Holgersson, Jan
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Landström, Maréne
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Eldridge, Angela M.
    Chromy, Brett A.
    Hansen, Lori M.
    Solnick, Jay V.
    Linden, Sara K.
    Haas, Rainer
    Dubois, Andre
    Merrell, D. Scott
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Remaut, Han
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berg, Douglas E.
    Boren, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence2017In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, no 3, p. 376-389Article in journal (Refereed)
    Abstract [en]

    The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

  • 84.
    Bugaytsova, Jeanna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björnhamn, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mahdavi, Jafar
    Ögren, Johan
    Ilver, Dag
    Gilman, Robert H
    Chowdhury, Abhijit
    The Swedish Institute for Control, Solna, Swede.
    Mukhopadhyay, Asish K
    Engstrand, Lars
    Oscarson, Stefan
    Kelly, Charles G
    Younson, Justine S
    Odenbreit, Stefan
    Solnick, Jay
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Haas, Rainer
    Dubois, Andre
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Berg, Douglas E
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    pH regulated H. pylori adherence: implications for persistent infection and diseaseManuscript (preprint) (Other academic)
    Abstract [en]

    Helicobacter pylori’s BabA adhesin binds strongly to gastric mucosal ABH/Leb glycans on the stomach epithelium and overlying mucus, materials continuously shed into the acidic gastric lumen. Here we report that this binding is acid labile, acid inactivation is fully reversible; and acid lability profiles vary with BabA sequence and correlate with disease patterns. Isogenic H. pylori strains from the gastric antrum and more acidic corpus were identified that differed in acid lability of receptor binding and in sequence near BabA’s carbohydrate binding domain. We propose that reversible acid inactivation of receptor binding helps H. pylori avoid clearance by mucosal shedding, and that strain differences in acid lability affect tissue tropism and the spectrum of associated gastric diseases.

  • 85.
    Bugaytsova, Jeanna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chernov, Yevgen A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    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).
    Mahdavi, Jafar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. School of Life Sciences, CBS, University of Nottingham, Nottingham, UK..
    Quintana-Hayashi, Macarena
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden..
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Moskalenko, Roman
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Pathology, Medical Institute, State University, Sumy, Ukraine.
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Université de Strasbourg, Institut de Chimie, Strasbourg, France.
    Moonens, Kristof
    Structural and Molecular Microbiology, VIB Department of Structural Biology, Belgium.
    Björnham, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics. FOI Totalförsvarets Forskningsinstitut, Umeå, Sweden..
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Königer, Verena
    Max von Pettenkofer Institute of Hygiene and Medical Microbiology, LMU, Munich, Germany.
    Vikström, Susanne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmidt, Alexej
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ögren, Johan
    Umeå University, Faculty of Medicine, Department of Odontology.
    Ilver, Dag
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Liu, Hui
    Department of Medicine, USUHS, Bethesda, MD, USA.
    Goldman, Matthew
    Department of Pediatrics, USUHS, Bethesda, MD, USA.
    Whitmire, Jeannette M
    Department of Microbiology and Immunology, USUHS, Bethesda, MD USA.
    Kelly, Charles G
    King's College London, Dental Institute, London, UK.
    Gilman, Robert H
    Department of International Health, John Hopkins School of Public Health, Baltimore, MD, USA.
    Chowdhury, Abhijit
    Centre for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education & Research, Kolkata, India.
    Mukhopadhyay, Asish K
    Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Kolkata, India.
    Nair, Balakrish G
    Translational Health Science and Technology Institute, Haryana, India.
    Papadakos, Konstantinos S
    Hellenic Pasteur Institute, Athens, Greece.
    Martinez-Gonzalez, Beatriz
    Hellenic Pasteur Institute, Athens, Greece.
    Sgouras, Dionyssios N
    Hellenic Pasteur Institute, Athens, Greece.
    Engstrand, Lars
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Unemo, Magnus
    Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
    Danielsson, Dan
    Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
    Sebastian, Suerbaum
    Institute for Medical Microbiology and Hospital Epidemiology Hannover Medical School, Hannover, Germany.
    Oscarson, Stefan
    Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Dublin, Ireland.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Holgersson, Jan
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Eldridge, Angela
    Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.
    Chromy, Brett A
    Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.
    Hansen, Lori
    Departments of Medical Microbiology and Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA.
    Solnick, Jay
    Departments of Medical Microbiology and Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA.
    Haas, Rainer
    Max von Pettenkofer Institute of Hygiene and Medical Microbiology, LMU Munich, Munich, Germany.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Lindén, Sara K
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Dubois, Andre
    Department of Medicine, USUHS, Bethesda, MD, USA.
    Merrell, D. Scott
    Department of Microbiology and Immunology, USUHS, Bethesda, MD, USA.
    Remaut, Han
    Structural and Molecular Microbiology, VIB Department of Structural Biology, VIB, Brussels, Belgium.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berg, Douglas E
    Department of Medicine, University of California San Diego, La Jolla, CA, USA.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Acid Responsive Helicobacter pylori Adherence: Implications for Chronic Infection and DiseaseManuscript (preprint) (Other academic)
  • 86.
    Bylund, Göran O
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nord, Stefan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Lövgren, J Mattias
    Wikström, P Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Alterations in the β flap and β' dock domains of the RNA polymerase abolish NusA-mediated feedback regulation of the metY-nusA-infB operon2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 16, p. 4113-4122Article in journal (Refereed)
    Abstract [en]

    The RimM protein in Escherichia coli is important for the in vivo maturation of 30S ribosomal subunits and a ΔrimM mutant grows poorly due to assembly and translational defects. These deficiencies are suppressed partially by mutations that increase the synthesis of another assembly protein, RbfA, encoded by the metY-nusA-infB operon. Among these suppressors are mutations in nusA that impair the NusA-mediated negative-feedback regulation at internal intrinsic transcriptional terminators of the metY-nusA-infB operon. We describe here the isolation of two new mutations, one in rpoB and one in rpoC (encoding the β and β' subunits of the RNA polymerase, respectively), that increase the synthesis of RbfA by preventing NusA from stimulating termination at the internal intrinsic transcriptional terminators of the metY-nusA-infB operon. The rpoB2063 mutation changed the isoleucine in position 905 of the β flap-tip helix to a serine, while the rpoC2064 mutation duplicated positions 415 to 416 (valine-isoleucine) at the base of the β' dock domain. These findings support previously published in vitro results, which have suggested that the β flap-tip helix and β' dock domain at either side of the RNA exit tunnel mediate the binding to NusA during transcriptional pausing and termination.

  • 87.
    Bäckström, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundberg, Carina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kersulyte, Dangeruta
    Berg, Douglas E
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Metastability of Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 48, p. 16923-16928Article in journal (Refereed)
  • 88.
    Bäckström, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Elfving, Nils
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Robert
    Wingsle, Gunnar
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit2007In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 26, no 5, p. 717-729Article in journal (Refereed)
  • 89. Calounova, Gabriela
    et al.
    Livera, Gabriel
    Zhang, Xiao-Qun
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gosden, Roger G.
    Welsh, Michael
    The Src Homology 2 Domain-Containing Adapter Protein B (SHB) Regulates Mouse Oocyte Maturation2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 6, article id e11155Article in journal (Refereed)
    Abstract [en]

    SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb(-/-) mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/- and -/- mice revealed impaired early embryo development in the -/- embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development.

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  • 90.
    Carlsson, Sven R
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Simonsen, Anne
    Membrane dynamics in autophagosome biogenesis2015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 2, p. 193-205Article in journal (Other academic)
    Abstract [en]

    Bilayered phospholipid membranes are vital to the organization of the living cell. Based on fundamental principles of polarity, membranes create borders allowing defined spaces to be encapsulated. This compartmentalization is a prerequisite for the complex functional design of the eukaryotic cell, yielding localities that can differ in composition and operation. During macroautophagy, cytoplasmic components become enclosed by a growing double bilayered membrane, which upon closure creates a separate compartment, the autophagosome. The autophagosome is then primed for fusion with endosomal and lysosomal compartments, leading to degradation of the captured material. A large number of proteins have been found to be essential for autophagy, but little is known about the specific lipids that constitute the autophagic membranes and the membrane modeling events that are responsible for regulation of autophagosome shape and size. In this Commentary, we review the recent progress in our understanding of the membrane shaping and remodeling events that are required at different steps of the autophagy pathway.

  • 91.
    Carlsson, Sven R
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Simonsen, Anne
    Recycling endosomes and autophagy2015In: Cell Technology (Saibou Kougaku), Vol. 34, no 2, p. 138-142Article in journal (Other academic)
  • 92. Casaite, Vida
    et al.
    Bruzyte, Simona
    Bukauskas, Virginijus
    Setkus, Arunas
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Meskys, Rolandas
    Expression and purification of active recombinant equine lysozyme in Escherichia coli2009In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 22, no 11, p. 649-654Article in journal (Refereed)
    Abstract [en]

    Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.

  • 93. Casslén, B
    et al.
    Urano, S
    Lecander, I
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen activators in the human endometrium, cellular origin and hormonal regulation.1992In: Blood Coagulation and Fibrinolysis, ISSN 0957-5235, E-ISSN 1473-5733, Vol. 3, no 2, p. 133-8Article in journal (Refereed)
    Abstract [en]

    Endometrial tissue explants in culture were found to release urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). In order to identify their cellular origin and possible hormonal regulation, enriched cultures of glandular epithelial cells and stromal cells were prepared from fresh endometrium, and the cultures treated with hormones. Both epithelial and stromal cell cultures were found to secrete u-PA and t-PA. Treatment of epithelial cell cultures with oestradiol, progesterone and DH-testosterone had no effect on the secretion of t-PA or u-PA. In stromal cell cultures, on the other hand, the secretion of u-PA was significantly reduced after treatment with progesterone, whereas oestradiol and DH-testosterone had no effect. This reduction of u-PA antigen in the tissue culture medium did not result from a reduction of the relative level of u-PA mRNA in the cells, suggesting that the synthesis of u-PA was not reduced. Alternatively, an increased clearance of u-PA by the cells from the medium may explain the reduction. This in vitro observation probably reflects the in vivo reduction of u-PA in endometrial secretion during the secretory phase.

  • 94. Casslén, B
    et al.
    Urano, S
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Progesterone regulation of plasminogen activator inhibitor 1 (PAI-1) antigen and mRNA levels in human endometrial stromal cells.1992In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 66, no 1, p. 75-87Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator activity decreases in the endometrium in the secretory phase of the menstrual cycle. This is partly due to decreased release of urokinase plasminogen activator in response to progesterone. Plasminogen activator inhibitor type 1 (PAI-1) is an efficient inhibitor of both tissue-type and urokinase-type plasminogen activators, and may therefore be instrumental for the control of plasminogen activation. In this study we examined the effects of steroid hormones on PAI-1 release and PAI-1 mRNA levels in primary cultures of human endometrial stromal cells. In these cells the secretion of PAI-1 was increased by progesterone in a dose and time dependent way, but was not affected by estradiol. The progesterone induction of PAI-1 secretion was preceded by a 7-8 fold increase of the steady state level of PAI-1 mRNA in the cells, suggesting that progesterone activates PAI-1 gene expression. Cultured endometrial glandular epithelial cells were found to release only insignificant amounts of PAI-1 with or without hormone treatment. The effect of progesterone on endometrial stromal cells was mimicked by DH-testosterone. However, while the response to progesterone was completely blocked by ZK112993, a potent antagonist of the progesterone receptor, the response to DH-testosterone was partially blocked by ZK112993, and partially by OH-flutamide, a potent antagonist of the androgen receptor. This suggests that a secretory response on PAI-1 expression is mediated via androgen receptors in endometrial tissue.

  • 95. Cerqua, Cristina
    et al.
    Morbidoni, Valeria
    Desbats, Maria Andrea
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Clinical Genetics Unit, Department of Women and Children's Health, University of Padova, Padova, Italy.
    Frasson, Chiara
    Sacconi, Sabrina
    Baldoin, Maria Cristina
    Sartori, Geppo
    Basso, Giuseppe
    Salviati, Leonardo
    Trevisson, Eva
    COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX22018In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, no 4, p. 244-252Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits.

    We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant.

    Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

  • 96. Cerritelli, Susana M
    et al.
    Iranzo, Jaime
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Crouch, Robert J
    Tollervey, David
    El Hage, Aziz
    High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase2020In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 8, p. 4274-4297Article in journal (Refereed)
    Abstract [en]

    Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

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  • 97.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    dNTPs and maintenance of genome stability2019In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 9, p. 21-21Article in journal (Other academic)
  • 98.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, Aimin
    Gräslund, Astrid
    Wijmenga, Sybren S
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit2000In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 97, no 6, p. 2474-2479Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an alpha(2)beta(2) form of RNR consisting of two homodimeric subunits, proteins R1 (alpha(2)) and R2 (beta(2)). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (beta') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex. This complex sediments at 5.6 S as a betabeta' heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an alpha(2)betabeta' heterotetramer, where Rnr4p plays an important role in the interaction between the alpha(2) and the betabeta ' subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit.

  • 99.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Georgieva, Bilyana
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhao, Xiaolan
    Rothstein, Rodney
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase.2003In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 112, no 3, p. 391-401Article in journal (Refereed)
    Abstract [en]

    In eukaryotes, DNA damage elicits a multifaceted response that includes cell cycle arrest, transcriptional activation of DNA repair genes, and, in multicellular organisms, apoptosis. We demonstrate that in Saccharomyces cerevisiae, DNA damage leads to a 6- to 8-fold increase in dNTP levels. This increase is conferred by an unusual, relaxed dATP feedback inhibition of ribonucleotide reductase (RNR). Complete elimination of dATP feedback inhibition by mutation of the allosteric activity site in RNR results in 1.6-2 times higher dNTP pools under normal growth conditions, and the pools increase an additional 11- to 17-fold during DNA damage. The increase in dNTP pools dramatically improves survival following DNA damage, but at the same time leads to higher mutation rates. We propose that increased survival and mutation rates result from more efficient translesion DNA synthesis at elevated dNTP concentrations.

  • 100.
    Chabes, Andrei
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Stillman, Bruce
    Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.2007In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 104, no 4, p. 1183-8Article in journal (Refereed)
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