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  • 301. Herrera, Phabiola M
    et al.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Velapatiño, Billie
    Santivañez, Livia
    Balqui, Jacqueline
    Finger, S Alison
    Sherman, Jonathan
    Zimic, Mirko
    Cabrera, Lilia
    Watanabe, Jose
    Rodríguez, Carlos
    Gilman, Robert H
    Berg, Douglas E
    DNA-level diversity and relatedness of Helicobacter pylori strains in shantytown families in Peru and transmission in a developing-country setting.2008In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 12, p. 3912-3918Article in journal (Refereed)
    Abstract [en]

    The efficiency of transmission of a pathogen within families compared with that between unrelated persons can affect both the strategies needed to control or eradicate infection and how the pathogen evolves. In industrialized countries, most cases of transmission of the gastric pathogen Helicobacter pylori seems to be from mother to child. An alternative model, potentially applicable among the very poor in developing countries, where infection is more common and the sanitary infrastructure is often deficient, invokes frequent transmission among unrelated persons, often via environmental sources. In the present study, we compared the genotypes of H. pylori from members of shantytown households in Peru to better understand the transmission of H. pylori in developing-country settings. H. pylori cultures and/or DNAs were obtained with informed consent by the string test (a minimally invasive alternative to endoscopy) from at least one child and one parent from each of 62 families. The random amplified polymorphic DNA fingerprints of 57 of 81 (70%) child-mother strain pairs did not match, nor did the diagnostic gene sequences (>1% DNA sequence difference), independent of the child's age (range, 1 to 39 years). Most strains from siblings or other paired family members were also unrelated. These results suggest that H. pylori infections are often community acquired in the society studied. Transmission between unrelated persons should facilitate the formation of novel recombinant genotypes by interstrain DNA transfer and selection for genotypes that are well suited for individual hosts. It also implies that the effective prevention of H. pylori infection and associated gastroduodenal disease will require anti-H. pylori measures to be applied communitywide.

  • 302. Hoch, Nicolas C
    et al.
    Chen, Eric S-W
    Buckland, Robert
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wang, Shun-Chung
    Fazio, Alessandro
    Hammet, Andrew
    Pellicioli, Achille
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Tsai, Ming-Daw
    Heierhorst, Jörg
    Molecular basis of the essential s phase function of the rad53 checkpoint kinase2013In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 33, no 16, p. 3202-3213Article in journal (Refereed)
    Abstract [en]

    The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

  • 303. Hoernke, M.
    et al.
    Mohan, J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kahra, Dana
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Westenhoff, S.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Schwieger, C.
    Determining membrane bound protein structures by infrared reflection-absorption spectroscopy2017In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S161-S161Article in journal (Other academic)
  • 304. Hoernke, Maria
    et al.
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mohan, Jagan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Westenhoff, Sebastian
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schwieger, Christian
    Structural Mechanism in a Membrane Remodelling ATP-ASE2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 578A-578AArticle in journal (Other academic)
  • 305.
    Hofer, Anders
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Crona, Mikael
    Logan, Derek T
    Sjöberg, Britt-Marie
    DNA building blocks: keeping control of manufacture2012In: Critical reviews in biochemistry and molecular biology, ISSN 1040-9238, E-ISSN 1549-7798, Vol. 47, no 1, p. 50-63Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α(6)β(2) complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli. Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

  • 306.
    Hofer, Anders
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Ekanem, J T
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Allosteric regulation of Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo.1998In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 273, no 51, p. 34098-104Article in journal (Refereed)
  • 307.
    Hofer, Anders
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Schmidt, P P
    Gräslund, Astrid
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei.1997In: Proceedings of the National Academy of Sciences of the U S A, ISSN 0027-8424, Vol. 94, no 13, p. 6959-64Article in journal (Refereed)
  • 308.
    Hofer, Anders
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Steverding, D
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Brun, R
    Thelander, Lars
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness.2001In: Proceedings of the National Academy of Sciences of the U S A, ISSN 0027-8424, Vol. 98, no 11, p. 6412-6Article in journal (Refereed)
  • 309.
    Hogg, Matthew
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA Polymerase ε2012In: The eukaryotic replisome: a guide to protein structure and function / [ed] MacNeill S, Springer Science+Business Media B.V., 2012, Vol. 62, p. 237-57Chapter in book (Refereed)
    Abstract [en]

    DNA polymerase ε (Pol ε) is one of three replicative DNA polymerases in eukaryotic cells. Pol ε is a multi-subunit DNA polymerase with many functions. For example, recent studies in yeast have suggested that Pol ε is essential during the initiation of DNA replication and also participates during leading strand synthesis. In this chapter, we will discuss the structure of Pol ε, the individual subunits and their function.

  • 310.
    Hogg, Matthew
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ganai, Rais Ahmad
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural basis for processive DNA synthesis by yeast DNA polymerase ε2014In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 21, no 1, p. 49-56Article in journal (Refereed)
    Abstract [en]

    DNA polymerase ε (Pol ε) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ε (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ε has the highest fidelity among B-family polymerases despite the absence of an extended b-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ε to encircle the nascent doublestranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes

  • 311.
    Hogg, Matthew
    et al.
    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.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Promiscuous DNA synthesis by human DNA polymerase θ2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 6, p. 2611-2622Article in journal (Refereed)
    Abstract [en]

    The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3'-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.

  • 312. Holland, Petter
    et al.
    Knaevelsrud, Helene
    Soreng, Kristiane
    Mathai, Benan J.
    Lystad, Alf Hakon
    Pankiv, Serhiy
    Bjorndal, Gunnveig T.
    Schultz, Sebastian W.
    Lobert, Viola H.
    Chan, Robin B.
    Zhou, Bowen
    Liestol, Knut
    Carlsson, Sven R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Melia, Thomas J.
    Di Paolo, Gilbert
    Simonsen, Anne
    HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 13889Article in journal (Refereed)
    Abstract [en]

    A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1-and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

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  • 313. Holmberg, M
    et al.
    Leonardsson, G
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The species-specific differences in the cAMP regulation of the tissue-type plasminogen activator gene between rat, mouse and human is caused by a one-nucleotide substitution in the cAMP-responsive element of the promoters.1995In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 231, no 2, p. 466-74Article in journal (Refereed)
    Abstract [en]

    In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species-specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein.

  • 314. Holmfeldt, Per
    et al.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sellin, Mikael E
    Segerman, Bo
    Carlsson, Sven R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gullberg, Martin
    The Schistosoma mansoni protein SM16/SmSLP/SmSPO-1 is a membrane-binding protein that lacks the proposed microtubule-regulatory activity2007In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 156, no 2, p. 225-234Article in journal (Refereed)
  • 315.
    Holst, Mikkel Roland
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Vidal-Quadras, Maite
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Song, Jie
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Hubert, Madlen
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lundborg, Magnus
    Landström, Maréne
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion2017In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 20, no 8, p. 1893-1905Article in journal (Refereed)
    Abstract [en]

    Cellular blebbing, caused by local alterations in cellsurface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrinindependent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension- mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol- interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

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  • 316.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iashchishyn, Igor A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of General Chemistry, Sumy State University, Ukraine.
    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.
    Immunochemical Detection of alpha-Synuclein Autoantibodies in Parkinson's Disease: Correlation between Plasma and Cerebrospinal Fluid Levels2017In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 8, no 6, p. 1170-1176Article in journal (Refereed)
    Abstract [en]

    Autoantibodies to Parkinson's disease (PD) amyloidogenic protein, a-synuclein, were recognized as a prospective biomarker for early disease diagnostics, yet there is inconsistency in previous reports, potentially related to PD status. Therefore, plasma and cerebrospinal fluid (CSF) of the cross-sectional cohort of 60 individuals, including recently diagnosed PD patients with mild and moderate PD and age-matched controls, were examined by enzyme-linked immunosorbent assay (ELISA). Nonparametric statistics was used for data analysis. We found significantly elevated levels of a-synuclein autoantibodies in both plasma and CSF in mild PD compared to controls, followed by some decrease in moderate PD. Receiver operating characteristic and effect size analyses confirmed the diagnostic power of a-synuclein antibodies in both plasma and CSF. For the first time, we showed the correlation between plasma and CSF a-synuclein antibody levels for mild, moderate, and combined PD groups. This indicates the potentiality of a-synuclein antibodies as PD biomarker and the increased diagnostic power of their simultaneous analysis in plasma and CSF.

  • 317.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iashchishyn, Igor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of General Chemistry, Sumy State University, Sumy 40007, Ukraine.
    Moskalenko, Roman A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. 3 Department of Pathology, Sumy State University, Sumy 40007, Ukraine.
    Wang, Chao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Warmlander, Sebastian K. T. S.
    Wallin, Cecilia
    Graslund, Astrid
    Kovacs, Gabor G.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Co-aggregation of pro-inflammatory S100A9 with alpha-synuclein in Parkinson's disease: ex vivo and in vitro studies2018In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 15, article id 172Article in journal (Refereed)
    Abstract [en]

    Background: Chronic neuroinflammation is a hallmark of Parkinson's disease (PD) pathophysiology, associated with increased levels of pro-inflammatory factors in PD brain tissues. The pro-inflammatory mediator and highly amyloidogenic protein S100A9 is involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease. This is the first report on the co-aggregation of alpha-synuclein (alpha-syn) and S100A9 both in vitro and ex vivo in PD brain.

    Methods: Single and sequential immunohistochemistry, immunofluorescence, scanning electron and atomic force (AFM) microscopies were used to analyze the ex vivo PD brain tissues for S100A9 and alpha-syn location and aggregation. In vitro studies revealing S100A9 and alpha-syn interaction and co-aggregation were conducted by NMR, circular dichroism, Thioflavin-T fluorescence, AFM, and surface plasmon resonance methods.

    Results: Co-localized and co-aggregated S100A9 and alpha-syn were found in 20% Lewy bodies and 77% neuronal cells in the substantia nigra; both proteins were also observed in Lewy bodies in PD frontal lobe (Braak stages 4-6). Lewy bodies were characterized by ca. 10-23 mu m outer diameter, with S100A9 and alpha-syn being co-localized in the same lamellar structures. S100A9 was also detected in neurons and blood vessels of the aged patients without PD, but in much lesser extent. In vitro S100A9 and alpha-syn were shown to interact with each other via the alpha-syn C-terminus with an apparent dissociation constant of ca. 5 mu M. Their co-aggregation occurred significantly faster and led to formation of larger amyloid aggregates than the self-assembly of individual proteins. S100A9 amyloid oligomers were more toxic than those of alpha-syn, while co-aggregation of both proteins mitigated the cytotoxicity of S100A9 oligomers.

    Conclusions: We suggest that sustained neuroinflammation promoting the spread of amyloidogenic S100A9 in the brain tissues may trigger the amyloid cascade involving alpha-syn and S100A9 and leading to PD, similar to the effect of S100A9 and A beta co-aggregation in Alzheimer's disease. The finding of S100A9 involvement in PD may open a new avenue for therapeutic interventions targeting S100A9 and preventing its amyloid self-assembly in affected brain tissues.

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  • 318.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jia, Xueen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Department of Physics.
    Johansson, Per
    Wang, Chao
    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, Sumy State University, Sumy 40000, Ukraine.
    Steinau, Andreas
    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.
    Wågberg, Thomas
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Svensson, Johan
    Zetterberg, Henrik
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pro-inflammatory S100A9 Protein as a Robust Biomarker Differentiating Early Stages of Cognitive Impairment in Alzheimer's Disease2016In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 1, p. 34-39Article in journal (Refereed)
    Abstract [en]

    Pro-inflammatory protein S100A9 was established as a biomarker of dementia progression and compared with others such as Aβ1-42 and tau-proteins. CSF samples from 104 stringently diagnosed individuals divided into five subgroups were analyzed, including nondemented controls, stable mild cognitive impairment (SMCI), mild cognitive impairment due to Alzheimer's disease (MCI-AD), Alzheimer's disease (AD), and vascular dementia (VaD) patients. ELISA, dot-blotting, and electrochemical impedance spectroscopy were used as research methods. The S100A9 and Aβ1-42 levels correlated with each other: their CSF content decreased already at the SMCI stage and declined further under MCI-AD, AD, and VaD conditions. Immunohistochemical analysis also revealed involvement of both Aβ1-42 and S100A9 in the amyloid-neuroinflammatory cascade already during SMCI. Tau proteins were not yet altered in SMCI; however their contents increased during MCI-AD and AD, diagnosing later dementia stages. Thus, four biomarkers together, reflecting different underlying pathological causes, can accurately differentiate dementia progression and also distinguish AD from VaD.

  • 319.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordvall, Lina-Maria
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Golla, Krishna Prasad
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Modulation of α-synuclein fibrillization by ring-fused 2-pyridones: templation and inhibition involve oligomers with different structure2013In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 532, no 2, p. 84-90Article in journal (Refereed)
    Abstract [en]

    In a recent study we discovered that a ring-fused 2-pyridone compound triggered fibrillization of a key protein in Parkinson's disease, α-synuclein. To reveal how variations in compound structure affect protein aggregation, we now prepared a number of strategic analogs and tested their effects on α-synuclein amyloid fiber formation in vitro. We find that, in contrast to the earlier templating effect, some analogs inhibit α-synuclein fibrillization. For both templating and inhibiting compounds, the key species formed in the reactions are α-synuclein oligomers that contain compound. Despite similar macroscopic appearance, the templating and inhibiting oligomers are distinctly different in secondary structure content. When the inhibitory oligomers are added in seed amounts, they inhibit fresh α-synuclein aggregation reactions. Our study demonstrates that small chemical changes to the same central fragment can result in opposite effects on protein aggregation.

  • 320.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph F
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Emma K
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hultgren, Scott J
    Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, United States.
    Chapman, Matthew
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mechanisms of Protein Oligomerization: Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 7, p. 3439-3444Article in journal (Refereed)
    Abstract [en]

    Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on (15)N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.

  • 321.
    Hosokawa, K
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Ottander, Ulrika
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Wahlberg, P
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cajander, S
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Olofsson, Jan I
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum2001In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 7, no 2, p. 137-145Article in journal (Refereed)
    Abstract [en]

    To investigate the potential importance of oestrogen as a local regulator of human corpus luteum function, the mRNA expression pattern and cellular localization of oestrogen receptors (ERs), ER-alpha and ER-beta, were studied in corpora lutea grouped according to age, where days 2-5 post-LH rise were designated as the early luteal phase, days 6-10 as mid-luteal and days 11-14 as the late luteal phase respectively. Northern blot analysis using an ER-beta probe in samples from whole ovarian tissue and isolated corpora lutea, revealed a major band at 7.5 kb and several minor bands between 4-10 kb, while no signals for ER-alpha mRNA were obtained. However, using a semi-quantitative reverse transcription-polymerase chain reaction followed by Southern blotting, ER-beta mRNA levels were found to be 63% lower (P: < 0.05, n = 39) in the mid-luteal phase compared with the early luteal phase, while ER-alpha mRNA expression showed no statistical differences between the different age groups. Using in-situ hybridization, ER-beta mRNA expression was localized to the steroidogenic luteal cells as well as perivascular cells and fibroblasts in the corpus luteum. Immunohistochemistry confirmed the localization of ER-beta protein, but no clear staining of luteal cells was found using antibodies against ER-alpha. Collectively, the findings of low to moderate expression of ER-beta mRNA and protein in the steroidogenic cells, and also in vascular endothelial cells of the corpus luteum, as opposed to diminutive amounts of ER-alpha mRNA, suggest that oestrogen activity is primarily transduced via ER-beta in the human corpus luteum.

  • 322.
    Hosseinzadeh, Ava
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rofougaran, Reza
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Vodnala, Munender
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kötemann, A
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Adenosine is a drugable negative regulator of neutrophil activity during Candida albicans infectionManuscript (preprint) (Other academic)
  • 323. Howes, Mark T
    et al.
    Kirkham, Matthew
    Riches, James
    Cortese, Katia
    Walser, Piers J
    Simpson, Fiona
    Hill, Michelle M
    Jones, Alun
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. MRC Laboratory of Molecular Biology, Cambridge, England.
    Lindsay, Margaret R
    Hernandez-Deviez, Delia J
    Hadzic, Gordana
    McCluskey, Adam
    Bashir, Rumasia
    Liu, Libin
    Pilch, Paul
    McMahon, Harvey
    Robinson, Phillip J
    Hancock, John F
    Mayor, Satyajit
    Parton, Robert G
    Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells2010In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 190, no 4, p. 675-691Article in journal (Refereed)
    Abstract [en]

    Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

  • 324. Hsueh, A J
    et al.
    Liu, Y X
    Cajander, S B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Molecular mechanisms in the hormonal regulation of plasminogen activator activity in ovarian granulosa cells and cumulus-oocyte complexes.1988In: Progress in clinical and biological research, ISSN 0361-7742, Vol. 267, p. 227-57Article in journal (Refereed)
  • 325. Hsueh, A J
    et al.
    Liu, Y X
    Cajander, S
    Peng, X R
    Dahl, K
    Kristensen, P
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization.1988In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 122, no 4, p. 1486-95Article in journal (Refereed)
    Abstract [en]

    GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.

  • 326. Hsueh, A.J.W.
    et al.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    GnRH as an ovulation-inducing agent: Regulation of plasminogen activator activity1985In: Proceedings of the 11th World Congress of Gynecology and Obstetrics, Springer-Verlag New York, 1985Conference paper (Refereed)
  • 327. Hu, Z Y
    et al.
    Liu, Y X
    Liu, K
    Byrne, S
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Feng, Q
    Ockleford, C D
    Expression of tissue type and urokinase type plasminogen activators as well as plasminogen activator inhibitor type-1 and type-2 in human and rhesus monkey placenta.1999In: Journal of Anatomy, ISSN 0021-8782, E-ISSN 1469-7580, Vol. 194 ( Pt 2), p. 183-95Article in journal (Refereed)
    Abstract [en]

    The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI-1 and PAI-2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI-1 and PAI-2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI-2 was noted in villous trophoblast whereas tPA and PAI-1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI-1 and PAI-2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI-1 and PAI-2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI-1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI-2 appears mainly to play a role in degradation of trophoblast cell-associated extracellular matrix, and thus may be of greatest importance during early stages of placentation.

  • 328. Huang, Qin
    et al.
    Sun, Dan
    Hussain, Muhammad Zubair
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Zoology, Government Emerson College, Multan, Pakistan.
    Liu, Yonggang
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Laboratory of Stem Cell and Tissue Engineering, Chongqing Medical University, Chongqing, China.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhang, Ce
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. State Key Laboratory of Cultivation Base for Photoelectric Technology and Functional Materials, Institute of Photonics and Photon-Technology, Northwest University, Xi’an, China.
    HEWL interacts with dissipated oleic acid micelles, and decreases oleic acid cytotoxicity2019In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 2, article id e0212648Article in journal (Refereed)
    Abstract [en]

    Senile plaques are well-known hallmarks of Alzheimer's Diseases (AD). However, drugs targeting tangles of the protein tau and plaques of beta-amyloid have no significant effect on disease progression, and the studies on the underlying mechanism of AD remain in high demand. Growing evidence supports the protective role of senile plaques in local inflammation driven by S100A9. We herein demonstrate that oleic acid (OA) micelles interact with hen egg white lysozyme (HEWL) and promote its amyloid formation. Consequently, SH-SY5Y cell line and mouse neural stem cells are rescued from OA toxicity by co-aggregation of OA and HEWL. Using atomic force microscopy in combination with fluorescence microscopy, we revealed that HEWL forms round-shaped aggregates in the presence of OA micelles instead of protofibrils of HEWL alone. These HEWL amyloids act as a sink for toxic OA micelles and their co-aggregate form large clumps, suggesting a protective function in amyloid and OA cytotoxicity.

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  • 329. Hulpia, Fabian
    et al.
    Mabille, Dorien
    Campagnaro, Gustavo D.
    Schumann, Gabriela
    Maes, Louis
    Roditi, Isabel
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    de Koning, Harry P.
    Caljon, Guy
    Van Calenbergh, Serge
    Combining tubercidin and cordycepin scaffolds results in highly active candidates to treat late-stage sleeping sickness2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 5564Article in journal (Refereed)
    Abstract [en]

    African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3'-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3'-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3'-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.

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  • 330.
    Hultin, Magnus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Turnover of chylomicrons in the rat1995Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mechanisms involved in the clearance of chylomicrons and aspects of the interactions at the vascular endothelium were studied in the rat.

    The poly-anion heparin, known to release lipoprotein lipase (LPL) from the vascular endothelium, enhanced the clearance of chylomicrons. Five minutes after heparin injection, the clearance of chylomicron triglycerides and retinyl esters was markedly accelerated. The rapid initial clearance was followed by a slower clearance of heavily lipolyzed chylomicrons. In contrast, one hour after heparin the clearance of both triglycerides and retinyl esters was retarded. This decreased removal of chylomicrons coincided with a decrease in the heparin releasable LPL activity, indicating that the previous release to plasma by heparin had resulted in net loss of functional LPL in the tissues.

    The poly-cation protamine released hepatic lipase and some LPL from their binding sites to plasma. One hour after protamine, plasma triglyceride levels were increased, indicating that chylomicron removal was impeded. It has been speculated that protamine inactivates LPL in vivo, but this was not the case. Ten minutes after injection of protamine normal amounts of LPL could be released by heparin. Thus, the accumulation of plasma triglycerides was not due to a rapid inactivation of LPL by protamine.

    LPL has specificity for sn-1,3-ester bonds. To investigate if this specificity is important in vivo, a lipid emulsion containing medium-chain fatty acids (MCFA) in the sn-1,3-position and long-chain fatty acids (LCFA) in the sn-2-position was synthesized, as well as an emulsion containing MCFA-TG mixed with LCFA-TGs (MMM/LLL). In vitro experiments showed large differences in the hydrolysis of the emulsions, but in vivo there were only small differences in the metabolism.

    To further study if lipid emulsions are cleared by the same mechanisms as chylomicrons, an emulsion was made by the same formulation as Intralipid® with addition of 3H-triolein and ,4C-cholesteryl ester. As measured by the removal of cholesteryl esters, the emulsion was cleared at the same rate as was chylomicrons. The triglyceride label was, however, removed more slowly from the emulsion droplets than from chylomicrons. Together with the lower recirculation of labeled free fatty acids (FFA) in plasma, this suggests that there was less lipolysis of the emulsion. The current view that removal of lipid emulsions in vivo is mainly dependent on LPL-mediated hydrolysis might thus not be correct.

    To further analyze the metabolism of chylomicrons, a compartmental model was developed. In this process, the distribution volume for chylomicrons was shown to be larger than the blood volume, a model for the metabolism of FFA in the rat was validated, and the full tissue distribution of injected chylomicrons was determined. According to the model, about half of the triglyceride label was removed from the circulation together with the core label while for the emulsion this number was about 80 %. In fasted rats all labeled fatty acids appeared to mix with the plasma FFA pool, while in fed rats about one-fifth of the fatty acids did not mix with the FFA but was apparently channeled directly to tissue metabolism.

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  • 331. Hägglund, A C
    et al.
    Basset, P
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stromelysin-3 is induced in mouse ovarian follicles undergoing hormonally controlled apoptosis, but this metalloproteinase is not required for follicular atresia.2001In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 64, no 2, p. 457-63Article in journal (Refereed)
    Abstract [en]

    Apoptotic processes are often associated with an intense proteolytic remodeling of the extracellular matrix (ECM). Proteolytic degradation of the ECM can also be a signal that induces apoptosis. Here, we have investigated the expression pattern and functional role of the matrix metalloproteinase stromelysin-3 in follicular atresia. Twenty-four hours after the treatment of immature female mice with a low dose of eCG, both apoptosis and the stromelysin-3 mRNA expression were suppressed approximately threefold. However, the initial suppression of apoptosis and stromelysin-3 expression was followed by a time-dependent increase, and 96 h after eCG treatment, the levels were similar to those of untreated control mice. In 15- to 16-day-old juvenile mice, the ovary consisted of relatively undeveloped follicles, and almost no apoptosis and only low stromelysin-3 mRNA expression were observed. However, at the age of 21 days, when several antral follicles were present, a fivefold induction in both apoptosis and stromelysin-3 mRNA expression was detected. For both models, in situ analysis revealed that the expression of stromelysin-3 mRNA was localized to the granulosa cells of atretic follicles. To address the functional role of stromelysin-3 in follicular atresia, stromelysin-3-deficient mice were studied. However, no difference in the pattern of apoptotic DNA fragmentation and no apparent morphological differences were observed when ovaries from wild-type and stromelysin-3-deficient mice were compared. Taken together, our data indicate that stromelysin-3 is induced during follicular atresia, but that this protease is not obligatory for initiation or completion of the atretic process.

  • 332. Hägglund, A C
    et al.
    Ny, A
    Leonardsson, G
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulation and localization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse ovary during gonadotropin-induced ovulation.1999In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 140, no 9, p. 4351-8Article in journal (Refereed)
    Abstract [en]

    At the time of ovulation, proteolytic degradation of the follicular wall is required to release the mature oocyte. Extracellular proteases, such as serine proteases and matrix metalloproteinases (MMPs), are thought to play important roles in this process. In this study we have examined the regulation of 11 MMPs and 3 tissue inhibitors of metalloproteinases (TIMPs) during gonadotropin-induced ovulation in the mouse. Northern blot hybridization showed that messenger RNA for several MMPs and TIMPs, including gelatinase A, MT1-MMP, stromelysin-3, MMP-19, TIMP-1, TIMP-2, and TIMP-3, were present at detectable levels in the mouse ovary. In addition, ovarian extracts contained gelatinolytic activities corresponding to the inactive proforms of gelatinase A and gelatinase B. Most of the MMPs and TIMPs were expressed at a constitutive level throughout the periovulatory period. However, MMP-19 and TIMP-1 revealed a different expression pattern; they were both induced 5-10 times by hCG and reached their maximum levels at 12 h after hCG treatment, corresponding to the time of ovulation. At this time point, MMP-19 and TIMP-1 messenger RNA were localized to the granulosa and thecal-interstitial cells of large preovulatory and ovulating follicles. This temporal and spatial regulation pattern suggests that MMP-19 might be involved in the tissue degradation that occurs during follicular rupture and that TIMP-1 could have a role in terminating MMP activity after ovulation.

  • 333. Hägglund, A C
    et al.
    Ny, A
    Liu, K
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation.1996In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 137, no 12, p. 5671-7Article in journal (Refereed)
    Abstract [en]

    Several lines of indirect evidence indicate that plasmin-mediated proteolysis plays a role in the breakdown of the follicle wall during ovulation. Consistent with this, the ovulation efficiency of mice lacking the two known physiological plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), is reduced by 26%. Surprisingly, mice with a single deficiency of either tPA or uPA gene function were normal in their capacity to ovulate. In this study we used in situ hybridization and casein in situ zymography to localize the expression of messenger RNAs (mRNAs) encoding PAs and PA inhibitors and to examine the net PA activity in the mouse ovary at the time of ovulation. Although uPA mRNA expressed by granulosa cells is the most abundant and dramatically up-regulated PA before ovulation, a previously unnoticed coordinated induction oftPA mRNA was found in thecal-interstitial tissue. The existence of redundant mechanisms for plasmin production in the ovary may be the cause of the normal ovulation efficiency in single deficient mice lacking tPA or uPA. The expression of mRNAs for PA inhibitors, types 1 and 2, was low in the ovary, with minor inductions at restricted time points. In contrast, expression of protease nexin-1 (PN-1) by granulosa cells was high during the entire periovulatory period. Among subpopulations of granulosa cells, the expression of PN-1 and uPA was heterogeneous and complementary. Cumulus cells expressed high levels of PN-1 mRNA and low levels of uPA mRNA, thereby providing an inhibitory activity that may protect the mucified matrix of the cumulus oocyte complex from proteolytic degradation.

  • 334.
    Hägglund, Gunnar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morgenstern, Ralf
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    1915-Pos 195 Pt Nmr: Interactions Of The Cancer Drug Cis-platin With Membranes And Mgst1, A Integral Membrane Detoxification Protein2008In: Biophysical Journal, 2008, p. 1915-Conference paper (Other academic)
    Abstract [en]

    The microsomal Glutathione-transferase (MGST1) is an integral membrane protein, which catalyses the conjugation of glutathione (tripeptide GSH) with xenobiotics; a process essential for cells to remove and detoxify e. g. carcinogens. While this glutathione system plays an essential role in healthy cell survival, glutathione has been shown to have a pivotal role in the development of acquired drug resistance. It prevents successful chemotherapies against a range of cancer types, therapies often based on cisplatin based drugs. These Pt compounds are initially quite effective, they become non-effective e.g. during the treatment of prostate cancer (very common 10000 new cases/a in Sweden) which progresses into a non-curable form during therapy. To understand the molecular mechanism behind the activity of Pt drugs and their inhibition by the human defense system, we use an solid state NMR approach (complemented by liquid NMR) to elucidate for cis-platin (diamino-dichloroplatinat II):

    conversion of cis-platin complex into an diamino-diaqua-complex, essential for its membrane passage into the cell interior.

    lipid membrane - drug interactions: binding to cell membrane surface, solubility and membrane transport.

    Pt drug binding to MGST1 enzyme, followed by glutathione conjugation into more water soluble compounds.

  • 335.
    Hägglöf, Peter
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen activator inhibitor type-1: structure-function studies and its use as a reference for intramolecular distance measurements2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A.

    There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form.

    The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography.

    The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

    To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form.

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  • 336.
    Hägglöf, Peter
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 3, p. 823-832Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

  • 337.
    Hällman, Peter
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Influence by DNA polymerase epsilon on DNA replication fidelity.2014Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
  • 338.
    Håberg, Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Membrane-remodeling by SNX18 in endosomal transport and autophagy2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The intracellular space of eukaryotic cells is subdivided into functionally distinct membrane-enclosed organelles. Regulation of these intracellular membranes requires an intricate network of specialized lipids and proteins that maintain organellar integrity and mediate transport between organelles. Proteins of the sorting nexin (SNX) family are membrane-binding regulators of transport events within the endomembrane system. The endomembrane system includes organelles associated with endocytic, secretory and degradative processes in the cell. The aims of this thesis were to functionally characterize SNX18 and SNX33, members of the SNX9-subfamily of sorting nexins, and to elucidate the role of SNX18 in autophagy.

    We demonstrated that all three proteins in the SNX9-family are capable of both membrane binding and remodeling, and interact with the membrane scission enzyme dynamin. We found that SNX18 localizes to endosomal structures in the endomembrane system, together with several identified factors previously described as regulators of endosomal transport. These results indicate that SNX18 mediates budding of membrane carriers in endosomal trafficking. In addition to this, knockdown of SNX18 in cultured cells was found to inhibit autophagy. Autophagy is a catabolic process by which cells degrade and recycle cellular components. It is a cellular response to various stress conditions such as oxidative stress, nutrient deprivation and infections. The components destined for degradation by autophagy are sequestered into a double-membrane structure called the autophagosome in which they are delivered to the lysosome. SNX18 interacts directly with proteins connected to autophagosome formation. Moreover, we demonstrated that the membrane-remodeling capability of SNX18 is a prerequisite for autophagosome formation.

    Taken together, our results lead to the conclusions that SNX18 remodels cellular membranes during formation of carriers for endosomal transport and that it is a positive regulator of autophagy and autophagosome formation.

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    Membrane-remodeling by SNX18 in endosomal transport and autophagy
  • 339.
    Håberg, Karin
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    SNX18 is an SNX9 paralog that acts as a membrane tubulator in AP-1-positive endosomal trafficking.2008In: Journal of Cell Science, ISSN 0021-9533, Vol. 121, no Pt 9, p. 1495-505Article in journal (Refereed)
  • 340.
    Håkansson, Pelle
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ribonucleotide reductase and DNA damage2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A prerequisite for a multicellular organism to survive is the ability to correctly replicate and repair DNA while minimizing the number of heritable mutations. To achieve this, cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs), the precursors for DNA synthesis. The rate-limiting step in de novo biosynthesis of dNTPs is catalyzed by the enzyme ribonucleotide reductase (RNR).

    The classic eukaryotic RNR enzyme consists of a large and a small subunit. Together, these subunits form a heterotetrameric RNR complex. The larger subunit harbours active sites whereas the smaller subunit contains a stable tyrosyl free radical. Both subunits are required for RNR activity.

    Since failure to correctly regulate de novo dNTP biosynthesis can lead to misincorporation of nucleotides into DNA, genetic abnormalities and cell death, RNR activity is tightly regulated. The regulation of RNR activity involves cell cycle-specific expression and degradation of the RNR proteins, as well as binding of allosteric effectors to the large RNR subunit.

    In this thesis, in vitro assays based on purified recombinant RNR proteins, in combination with in vivo assays, have been used successfully to study the regulation of RNR activity in response to DNA damage. I present new findings regarding the function of an alternative mammalian RNR small subunit, and on the role of a small RNR inhibitor protein of fission yeast, during normal growth and after DNA damage. I also show conclusively that there are fundamental differences in the regulation of dNTP biosynthesis between the cells of higher and lower eukaryotes after DNA damage.

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  • 341.
    Håkansson, Pelle
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Dahl, Lina
    Chilkova, Olga
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Domkin, Vladimir
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit.2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 3, p. 1778-1783Article in journal (Refereed)
  • 342.
    Håkansson, Pelle
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 12, p. 7834-7841Article in journal (Refereed)
  • 343.
    Håkansson, Pär
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Westlund, Per-Olof
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lindahl, Erik
    Edholm, Olle
    A direct simulation of EPR slow-motion spectra of spin labelled phospholipids in liquid crystalline bilayers based on a molecular dynamics simulation of the lipid dynamics2001In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 3, no 23, p. 5311-5319Article in journal (Refereed)
    Abstract [en]

    EPR line shapes can be calculated from the stochastic Liouville equation assuming a stochastic model for the reorientation of the spin probe. Here we use instead and for the first time a detailed molecular dynamics (MD) simulation to generate the stochastic input to the Langevin form of the Liouville equation. A 0.1 μs MD simulation at T = 50°C of a small lipid bilayer formed by 64 dipalmitoylphosphatidylcholine (DPPC) molecules at the water content of 23 water molecules per lipid was used. In addition, a 10 ns simulation of a 16 times larger system consisting of 32 DPPC molecules with a nitroxide spin moiety attached at the sixth position of the sn2 chain and 992 ordinary DPPC molecules, was used to investigate the extent of the perturbation caused by the spin probe. Order parameters, reorientational dynamics and the EPR FID curve were calculated for spin probe molecules and ordinary DPPC molecules. The timescale of the electron spin relaxation for a spin-moiety attached at the sixth carbon position of a DPPC lipid molecule is 11.9 × 107 rad s−1 and for an unperturbed DPPC molecule it is 3.5 × 107 rad s−1.

  • 344.
    Hörnberg, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Wikström Hultdin, Ulrika
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The effect of iodide and chloride on transthyretin structure and stability2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 26, p. 9290-9299Article in journal (Other academic)
    Abstract [en]

    Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.

  • 345.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Selection of transthyretin amyloid inhibitors2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloidosis is a group of clinical disorders caused by the aggregation of specific proteins into abnormal extracellular deposits. Today, 31 different proteins have been linked to amyloid diseases including transthyretin-related amyloidosis (ATTR). ATTR occurs through the aggregation of either wild-type plasma protein transthyretin (TTR) or a mutated form. TTR is a homotetramer that under normal circumstances functions as a carrier of thyroxine and retinol binding protein. The aggregation cascade requires dissociation of the tetramer into monomers, and preventing this dissociation represents a potential mode of intervention. Interestingly, small molecules, referred as kinetic stabilizers, can bind to TTR’s thyroxine-binding site (TBS) and such molecules are currently being used as a therapeutic approach to impair tetramer dissociation.

    The efficacy of TTR stabilization is directly correlated to the binding affinity of the ligand to TBS. However, the binding of the ligand to TTR in vivo can be affected by other plasma components resulting in poor efficacy. Thus, the selectivity of ligands is an important parameter. We have designed an assay where the ability to stabilize TTR can be directly evaluated in plasma and we have investigated the stabilizing effect of nine potential TTR binders (Paper I). The results, surprisingly, revealed that the binding affinity of molecules has a poor correlation to its selectivity. However, the nature of protein-ligand complex formation can also be described by enthalpic (∆H) and entropic (∆S) energy contributions. ∆H represents the change in chemical bonds and frequently requires a higher order of orientation compared to the ∆S component, which mainly represents the hydrophobic effect via the exclusion of water. We hypothesized that ligands possessing high ΔH in binding to their co-partner would also be more specific in a complex environment such as plasma. By applying a thermodynamic analysis using isothermal titration calorimetry, we found that the selectivity in plasma correlates well with the ∆H contribution and might, therefore, be a better predictor for selectivity.

    Luteolin was found to be a highly selective stabilizer of TTR and was investigated further (Paper II). The ligand displayed a significant rescuing effect in both cell culture and animal models. However, luteolin undergoes rapid enzymatic degradation in the liver and this impairs its use as a potential therapeutic drug. To attempt to circumvent this issue, we modified the most exposed hydroxyl group thus rendering the molecule inert towards glucuronidation (Paper III). The substitutions resulted in higher stability in the face of hepatic degradation molecules, but they also affected the selectivity in a negative manner.

    The screening for new TTR stabilizers resulted in the discovery of tetrabromobisphenol A, which displayed a very high selectivity (Paper IV). This study also included a comparison with the drug Vyndaqel™ which currently is in clinically use, and showed how the dosage could be altered to acquire a better level of saturation and possibly also a better clinical effect.

    Taken together we present new molecules with the ability to stabilize TTR, and these can serve as scaffolds for the design of new drugs. We present a method to measure the efficacy of a TTR-stabilizing drugs in a complex matrix and as well as a way to adjust the dosage of existing drugs. We also show that the selectivity of a drug is affected by the relative proportion of ∆H and ∆S, and this is of interest for drug design in general.

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  • 346.
    Iakovleva, Irina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    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.
    Wijsekera, Alexandra
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Lina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Zhang, Jin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    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.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0153529Article in journal (Refereed)
    Abstract [en]

    Amyloid formation of the human plasma protein transthyretin (TTR) is associated with several human disorders, including familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Dissociation of TTR’s native tetrameric assembly is the rate-limiting step in the conversion into amyloid, and this feature presents an avenue for intervention because binding of an appropriate ligand to the thyroxin hormone binding sites of TTR stabilizes the native tetrameric assembly and impairs conversion into amyloid. The desired features for an effective TTR stabilizer include high affinity for TTR, high selectivity in the presence of other proteins, no adverse side effects at the effective concentrations, and a long half-life in the body. In this study we show that the commonly used flame retardant tetrabromobisphenol A (TBBPA) efficiently stabilizes the tetrameric structure of TTR. The X-ray crystal structure shows TBBPA binding in the thyroxine binding pocket with bromines occupying two of the three halogen binding sites. Interestingly, TBBPA binds TTR with an extremely high selectivity in human plasma, and the effect is equal to the recently approved drug tafamidis and better than diflunisal, both of which have shown therapeutic effects against FAP. TBBPA consequently present an interesting scaffold for drug design. Its absorption, metabolism, and potential side-effects are discussed.

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  • 347.
    Iakovleva, Irina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokrzywa, Malgorzata
    Walfridsson, Malin
    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.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The flavonoid luteolin, but not luteolin-7-o-glucoside, prevents a transthyretin mediated toxic response2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 5, article id e0128222Article in journal (Refereed)
    Abstract [en]

    Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.

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  • 348.
    Iakovleva, Irina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Lina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gharibyan, Anna
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Intissar, Anan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Walfridsson, Malin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enthalpic Forces Correlate with Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma2015In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 58, no 16, p. 6507-6515Article in journal (Refereed)
    Abstract [en]

    The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an almost universally desired feature within drug design and the finding might have wide-ranging implications for drug design.

  • 349.
    Iashchishyn, Igor A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of General Chemistry, Sumy State University, Sumy, Ukraine.
    Gruden, Marina A.
    Moskalenko, Roman A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Pathology, Sumy State University, Sumy, Ukraine .
    Davydova, Tatiana, V
    Wang, Chao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sewell, Robert D. E.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Intranasally Administered S100A9 Amyloids Induced Cellular Stress, Amyloid Seeding, and Behavioral Impairment in Aged Mice2018In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 9, no 6, p. 1338-1348Article in journal (Refereed)
    Abstract [en]

    Amyloid formation and neuroinflammation are major features of Alzheimer's disease pathology. Proinflammatory mediator S100A9 was shown to act as a link between the amyloid and neuroinflammatory cascades in Alzheimer's disease, leading together with Aβ to plaque formation, neuronal loss and memory impairment. In order to examine if S100A9 alone in its native and amyloid states can induce neuronal stress and memory impairment, we have administered S100A9 species intranasally to aged mice. Single and sequential immunohistochemistry and passive avoidance behavioral test were conducted to evaluate the consequences. Administered S100A9 species induced widespread cellular stress responses in cerebral structures, including frontal lobe, hippocampus and cerebellum. These were manifested by increased levels of S100A9, Box, and to a lesser extent activated caspase-3 immunopositive cells. Upon administration of S100A9 fibrils, the amyloid oligomerization was observed in the brain tissues, which can further exacerbate cellular stress. The cellular stress responses correlated with significantly increased training and decreased retention latencies measured in the passive avoidance test for the SI00A9 treated animal groups. Remarkably, the effect size in the behavioral tests was moderate already in the group treated with native S100A9, while the effect sizes were large in the groups administered S100A9 amyloid oligomers or fibrils. The findings demonstrate the brain susceptibility to neurotoxic damage of S100A9 species leading to behavioral and memory impairments. Intranasal administration of S100A9 species proved to be an effective method to study amyloid induced brain dysfunctions, and 5100A9 itself may be postulated as a target to allay early stage neurodegenerative and neuroinflammatory processes.

  • 350.
    Iashchishyn, Igor A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of General Chemistry, Sumy State University, Sumy, Ukraine.
    Sulskis, Darius
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
    Ngoc, Mai Nguyen
    Smirnovas, Vytautas
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Finke-Watzky Two-Step Nucleation-Autocatalysis Model of S100A9 Amyloid Formation: Protein Misfolding as "Nucleation" Event2017In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 8, no 10, p. 2152-2158Article in journal (Refereed)
    Abstract [en]

    Quantitative kinetic analysis is critical for understanding amyloid mechanisms. Here we demonstrate the application of generic Finke-Watzky (F-W) two-step nucleation-autocatalytic growth model to the concentration-dependent amyloid kinetics of proinflammatory alpha-helical S100A9 protein at pH 7.4 and at 37 and 42 degrees C. The model is based on two pseudoelementary reaction steps applied without further analytical constraints, and its treatment of S100A9 amyloid self-assembly demonstrates that initial misfolding and beta-sheet formation, defined as "nucleation" step, spontaneously takes place within individual S100A9 molecules at higher rate than the subsequent fibrillar growth. The latter, described as an autocatalytic process, will proceed if misfolded amyloid-prone S100A9 is populated on a macroscopic time scale. Short lengths of S100A9 fibrils are consistent with the F-W model. The analysis of fibrillar length distribution by the Beker-Doring model demonstrates independently that such distribution is solely determined by slow fibril growth and there is no fragmentation or secondary pathways decreasing fibrillar length.

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