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  • 901.
    Zamotin, Vladimir
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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

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

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

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

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

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  • 902.
    Zamotin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gharibyan, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gibanova, NV
    Lavrikova, MA
    Dolgikh, DA
    Kirpichnikov, MP
    Kostanyan, IA
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 10, p. 2451-2457Article in journal (Refereed)
    Abstract [en]

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

  • 903.
    Zdunek, Janusz
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Martinez, G V
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lycksell, P O
    Yin, Y
    Nilsson, S
    Shen, Y
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Wijmenga, S
    Global structure and dynamics of human apolipoprotein CII in complex with micelles: evidence for increased mobility of the helix involved in the activation of lipoprotein lipase.2003In: Biochemistry, ISSN 0006-2960, Vol. 42, no 7, p. 1872-89Article in journal (Refereed)
  • 904.
    Zhang, Ce
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, Yonggang
    Gilthorpe, Jonathan
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    van der Maarel, Johan RC
    MRP14 (S100A9) protein interacts with alzheimer beta-amyloid peptide and induces its fibrillization2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, p. e32953-Article in journal (Refereed)
    Abstract [en]

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

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  • 905.
    Zhang, Jin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Patrik L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin2016In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 50, no 21, p. 11984-11993Article in journal (Refereed)
    Abstract [en]

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

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  • 906.
    Zhang, Jin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Mikael J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andersson, Patrik L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals2018In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 52, no 20, p. 11865-11874Article in journal (Refereed)
    Abstract [en]

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

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

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

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

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

  • 909. Zhao, X
    et al.
    Georgieva, B
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Ippel, J H
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wijmenga, S
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Rothstein, R
    Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.2000In: Mol Cell Biol, ISSN 0270-7306, Vol. 20, no 23, p. 9076-83Article in journal (Refereed)
  • 910.
    Zheng, Wenjing
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gorre, Nagaraju
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Noda, Tetsuo
    Ogawa, Wataru
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Maternal phosphatidylinositol 3-kinase signalling is crucial for embryonic genome activation and preimplantation embryogenesis2010In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 11, no 11, p. 890-895Article in journal (Refereed)
    Abstract [en]

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

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

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

  • 912.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    The Helicobacter pylori sialic acid binding adhesin SabA is regulated via a network of two-component systemsManuscript (preprint) (Other academic)
    Abstract [en]

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

  • 913.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallström, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öhman, Carina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Engstrand, Lars
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism2014In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 7, article id e1004234Article in journal (Refereed)
    Abstract [en]

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

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  • 914.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ul Mushtaq, Ameeq
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur P. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-22020In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 172, article id 105628Article in journal (Refereed)
    Abstract [en]

    Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

  • 915.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural topology and activation of an initial adenylate kinase-substrate complex2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 6, p. 1055-1061Article in journal (Refereed)
    Abstract [en]

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

  • 916. Åstedt, B
    et al.
    Lecander, I
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The placental type plasminogen activator inhibitor, PAI-2.1987In: Fibrinolysis, Elsevier, 1987, 1, p. 203-208Chapter in book (Refereed)
  • 917.
    Öhman, Carina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallström, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Olofsson, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Christer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aspholm, Marina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Phase variation and expression mechanisms of the sialic acid binding adhesin SabA in Helicobacter pyloriManuscript (preprint) (Other academic)
    Abstract [en]

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

  • 918. Öquist, Mats G.
    et al.
    Erhagen, Björn
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Haei, Mahsa
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ilstedt, Ulrik
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B.
    The effect of temperature and substrate quality on the carbon use efficiency of saprotrophic decomposition2017In: Plant and Soil, ISSN 0032-079X, E-ISSN 1573-5036, Vol. 414, no 1, p. 113-125Article in journal (Refereed)
    Abstract [en]

    Background and aims: Mineralization of soil organic matter (SOM) constitutes a major carbon flux to the atmosphere. The carbon use efficiency (CUE) of the saprotrophic microorganisms mineralizing SOM is integral for soil carbon dynamics. Here we investigate how the CUE is affected by temperature, metabolic conditions, and the molecular complexity of the substrate.

    Methods: We incubated O-horizon soil samples (with either 13C–glucose or 13C–cellulose) from a boreal coniferous forest at 4, 9, 14, and 19 °C, and calculated CUEs based on the amount of 13C–CO2and 13C–labelled microbial biomass produced. The effects of substrate, temperature, and metabolic conditions (representing unlimited substrate supply and substrate limitation) on CUE were evaluated.

    Results: CUE from metabolizing glucose was higher as compared to cellulose. A slight decrease in CUE with increasing temperature was observed in glucose amended samples (but only in the range 9–19 °C), but not in cellulose amended samples. CUE differed significantly with metabolic conditions, i.e. CUE was higher during unlimited growth conditions as compared to conditions with substrate limitation.

    Conclusions: We conclude that it is integral to account for both differences in CUE during different metabolic phases, as well as complexity of substrate, when interpreting temperature dependence on CUE in incubation studies.

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  • 919. Öquist, Mats
    et al.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Klemedtsson, Leif
    Harrysson Drotz, Stina
    Grip, Harald
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats
    Water availability controls microbial temperature responses in frozen soil CO2 production2009In: Global Change Biology, ISSN 1354-1013, E-ISSN 1365-2486, Vol. 15, no 11, p. 2715-22Article in journal (Refereed)
    Abstract [en]

    Soil processes in high-latitude regions during winter are important contributors to global carbon circulation, but our understanding of the mechanisms controlling these processes is poor and observed temperature response coefficients of CO2 production in frozen soils deviate markedly from thermodynamically predicted responses (sometimes by several orders of magnitude). We investigated the temperature response of CO2 production in 23 unfrozen and frozen surface soil samples from various types of boreal forests and peatland ecosystems and also measured changes in water content in them after freezing. We demonstrate that deviations in temperature responses at subzero temperatures primarily emanates from water deficiency caused by freezing of the soil water, and that the amount of unfrozen water is mainly determined by the quality of the soil organic matter, which is linked to the vegetation cover. Factoring out the contribution of water limitation to the CO2 temperature responses yields response coefficients that agree well with expectations based on thermodynamic theory concerning biochemical temperature responses. This partitioning between a pure temperature response and the effect of water availability on the response of soil CO2 production at low temperatures is crucial for a thorough understanding of low-temperature soil processes and for accurate predictions of C-balances in northern terrestrial ecosystems.

  • 920.
    Östberg, Yngve
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Berg, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Comstedt, Pär
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wieslander, Åke
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Functional analysis of a lipid galactosyltransferase synthesizing the major envelope lipid in the Lyme disease spirochete Borrelia burgdorferi2007In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 272, no 1, p. 22-29Article in journal (Refereed)
    Abstract [en]

    One of the major lipids in the membranes of Borrelia burgdorferi is monogalactosyl diacylglycerol (MGalDAG), a glycolipid recently shown to carry antigenic potency. Herein, it is shown that the gene mgs (TIGR designation bb0454) of B. burgdorferi encodes for the protein bbMGS that, when expressed in Escherichia coli, catalyzes the glycosylation of 1,2-diacylglycerol with specificity for the donor substrate UDP-Gal yielding MGalDAG. Related lipid enzymes were found in many Gram-positive bacteria. The presence of this galactosyltransferase activity and synthesis of a cholesteryl galactoside by another enzyme were verified in B. burgdorferi cell extract. Besides MGalDAG, phosphatidylcholine, phosphatidylglycerol, and cholesterol were also found as major lipids in the cell envelope. The high isoelectric point of bbMGS and clustered basic residues in its amino acid sequence suggest that the enzyme interacts with acidic lipids in the plasma membrane, in agreement with strong enzymatic activation of bbMGS by phosphatidylglycerol. The membrane packing and immunological properties of MGalDAG are likely to be of great importance in vivo.

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