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  • 1.
    Aguilar, Ximena
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schleucher, Jurgen
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 5, s. e98575-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.

  • 2.
    Blomberg, Jeanette
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Aguilar, Ximena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rautio, Linn
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 13, s. 5938-5950Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 3.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lee, Cheng Choo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Scanning electron microscopy as a tool for evaluating morphology of amyloid structures formed on surface plasmon resonance chips2018Ingår i: Data in Brief, E-ISSN 2352-3409, Vol. 19, s. 1166-1170Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 4.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lee, Cheng Choo
    Sandblad, Linda
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 13, s. 1940-1949Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 5.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The role of histidines in amyloid β fibril assembly2017Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, nr 8, s. 1167-1175Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 6.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lindhagen Persson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharabyan, A
    Vestling, M
    Brännström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Morozova-Roche, Ludmilla
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Design of oligomer-specific antibodiesManuskript (preprint) (Övrigt vetenskapligt)
  • 7.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lindhagen-Persson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vestling, Monika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sellin, Mikael E.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Brännström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Morozova-Roche, Ludmilla
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Forsgren, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A Generic Method for Design of Oligomer-Specific Antibodies2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 3, s. e90857-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 8.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sellin, Mikael E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmfeldt, Per
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Brattsand, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Dermatologi och venereologi.
    Gullberg, Martin
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.2009Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, nr 3, s. 1144-1154Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

  • 9.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Lindhagen-Persson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ca2+ enhances Aβ polymerization rate and fibrillar stability in a dynamic manner2013Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 450, s. 189-197Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 10.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Nilsson, Lina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pihl, Mathias
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The N-terminal Region of Amyloid β Controls the Aggregation Rate and Fibril Stability at Low pH Through a Gain of Function Mechanism2014Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, nr 31, s. 10956-10964Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 11.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer2011Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, nr 9, s. e25157-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 12.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brattsand, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Dermatologi och venereologi.
    Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis2012Ingår i: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 393, nr 5, s. 369-377Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 13.
    Bugaytsova, Jeanna A.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björnham, Oscar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik. Swedish Defence Research Agency, 906 21 Umeå, Sweden.
    Chernov, Yevgen A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gideonsson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Henriksson, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mendez, Melissa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sjöström, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mahdavi, Jafar
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. School of Life Sciences, CBS, University of Nottingham, NG7 2RD Nottingham, UK.
    Shevtsova, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ilver, Dag
    Moonens, Kristof
    Quintana-Hayashi, Macarena P.
    Moskalenko, Roman
    Aisenbrey, Christopher
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bylund, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schmidt, Alexej
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Åberg, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Koeniger, Verena
    Vikström, Susanne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rakhimova, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ögren, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Avdelningen för medicin.
    Liu, Hui
    Goldman, Matthew D.
    Whitmire, Jeannette M.
    Åden, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Younson, Justine
    Kelly, Charles G.
    Gilman, Robert H.
    Chowdhury, Abhijit
    Mukhopadhyay, Asish K.
    Nair, G. Balakrish
    Papadakos, Konstantinos S.
    Martinez-Gonzalez, Beatriz
    Sgouras, Dionyssios N.
    Engstrand, Lars
    Unemo, Magnus
    Danielsson, Dan
    Suerbaum, Sebastian
    Oscarson, Stefan
    Morozova-Roche, Ludmilla A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Holgersson, Jan
    Esberg, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Strömberg, Nicklas
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Landström, Maréne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Eldridge, Angela M.
    Chromy, Brett A.
    Hansen, Lori M.
    Solnick, Jay V.
    Linden, Sara K.
    Haas, Rainer
    Dubois, Andre
    Merrell, D. Scott
    Schedin, Staffan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Remaut, Han
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berg, Douglas E.
    Boren, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence2017Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, nr 3, s. 376-389Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 14.
    Bugaytsova, Jeanna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björnhamn, Oscar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Henriksson, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Johansson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mendez, Melissa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sjöström, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Aisenbrey, Christopher
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Shevtsova, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bylund, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mahdavi, Jafar
    Ögren, Johan
    Ilver, Dag
    Gilman, Robert H
    Chowdhury, Abhijit
    The Swedish Institute for Control, Solna, Swede.
    Mukhopadhyay, Asish K
    Engstrand, Lars
    Oscarson, Stefan
    Kelly, Charles G
    Younson, Justine S
    Odenbreit, Stefan
    Solnick, Jay
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Haas, Rainer
    Dubois, Andre
    Schedin, Staffan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Berg, Douglas E
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    pH regulated H. pylori adherence: implications for persistent infection and diseaseManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

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

  • 15.
    Bugaytsova, Jeanna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chernov, Yevgen A
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gideonsson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mendez, Melissa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Henriksson, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Mahdavi, Jafar
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. School of Life Sciences, CBS, University of Nottingham, Nottingham, UK..
    Quintana-Hayashi, Macarena
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden..
    Shevtsova, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sjöström, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Moskalenko, Roman
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Department of Pathology, Medical Institute, State University, Sumy, Ukraine.
    Aisenbrey, Christopher
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Université de Strasbourg, Institut de Chimie, Strasbourg, France.
    Moonens, Kristof
    Structural and Molecular Microbiology, VIB Department of Structural Biology, Belgium.
    Björnham, Oscar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik. FOI Totalförsvarets Forskningsinstitut, Umeå, Sweden..
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bylund, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Königer, Verena
    Max von Pettenkofer Institute of Hygiene and Medical Microbiology, LMU, Munich, Germany.
    Vikström, Susanne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schmidt, Alexej
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Rakhimova, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ögren, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Ilver, Dag
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Liu, Hui
    Department of Medicine, USUHS, Bethesda, MD, USA.
    Goldman, Matthew
    Department of Pediatrics, USUHS, Bethesda, MD, USA.
    Whitmire, Jeannette M
    Department of Microbiology and Immunology, USUHS, Bethesda, MD USA.
    Kelly, Charles G
    King's College London, Dental Institute, London, UK.
    Gilman, Robert H
    Department of International Health, John Hopkins School of Public Health, Baltimore, MD, USA.
    Chowdhury, Abhijit
    Centre for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education & Research, Kolkata, India.
    Mukhopadhyay, Asish K
    Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Kolkata, India.
    Nair, Balakrish G
    Translational Health Science and Technology Institute, Haryana, India.
    Papadakos, Konstantinos S
    Hellenic Pasteur Institute, Athens, Greece.
    Martinez-Gonzalez, Beatriz
    Hellenic Pasteur Institute, Athens, Greece.
    Sgouras, Dionyssios N
    Hellenic Pasteur Institute, Athens, Greece.
    Engstrand, Lars
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Unemo, Magnus
    Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
    Danielsson, Dan
    Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
    Sebastian, Suerbaum
    Institute for Medical Microbiology and Hospital Epidemiology Hannover Medical School, Hannover, Germany.
    Oscarson, Stefan
    Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Dublin, Ireland.
    Morozova-Roche, Ludmilla
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Holgersson, Jan
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden.
    Strömberg, Nicklas
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Esberg, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Eldridge, Angela
    Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.
    Chromy, Brett A
    Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.
    Hansen, Lori
    Departments of Medical Microbiology and Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA.
    Solnick, Jay
    Departments of Medical Microbiology and Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA.
    Haas, Rainer
    Max von Pettenkofer Institute of Hygiene and Medical Microbiology, LMU Munich, Munich, Germany.
    Schedin, Staffan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Lindén, Sara K
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Dubois, Andre
    Department of Medicine, USUHS, Bethesda, MD, USA.
    Merrell, D. Scott
    Department of Microbiology and Immunology, USUHS, Bethesda, MD, USA.
    Remaut, Han
    Structural and Molecular Microbiology, VIB Department of Structural Biology, VIB, Brussels, Belgium.
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berg, Douglas E
    Department of Medicine, University of California San Diego, La Jolla, CA, USA.
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Acid Responsive Helicobacter pylori Adherence: Implications for Chronic Infection and DiseaseManuskript (preprint) (Övrigt vetenskapligt)
  • 16.
    Elfving, Nils
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Davoine, Céline
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Benlloch, Reyes
    Blomberg, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Müller, Dörte
    Nilsson, Anders
    Ulfstedt, Mikael
    Ronne, Hans
    Wingsle, Gunnar
    Nilsson, Ove
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development2011Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 20, s. 8245-8250Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

  • 17.
    Hainzl, Tobias
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Meriläinen, Gitte
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structural basis of signal-sequence recognition by the signal recognition particle 2011Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 18, nr 3, s. 389-391Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain—structural changes that ensure a hierarchical succession of events during protein targeting.

  • 18. Hammer, Neal D
    et al.
    McGuffie, Bryan A
    Zhou, Yizhou
    Badtke, Matthew P
    Reineke, Ashley A
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gestwicki, Jason E
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chapman, Matthew R
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation2012Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 422, nr 3, s. 376-389Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1st, 2nd or 3rd repeating units were able to convert CsgA into fibers. However, mutants missing either the 4th or 5th repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

  • 19.
    Henriksson, Sara
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mendez, Melissa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bugaytsova, Jeanna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nordén, Jenny
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berg, Douglas E
    Blixt, Ola
    Teneberg, Susann
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Clinical isolates of Helicobacter pylori demonstrates alternative BabA-mediated adherence to human gastric mucosaManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Helicobacter pylori infection is life-long and can cause peptic ulcer disease and gastric cancer. The H. pylori BabA adhesin binds the ABO/Leb blood group (bg) antigens (Leb), which mediates attachment to the gastric epithelium. The prevalence of ABO binding is high worldwide and also in northern Europe. However, prevalence is reduced by 50% in Germany and is further reduced in Spain and Portugal. An inventory of strains from different European populations resulted in strains with high level of BabA expression but very little or no binding to Leb. The majority of such strains could not bind to human gastric mucosa in vitro. We further characterized a Spanish isolates, strain 812, that binds only weakly to soluble Leb-conjugate but still adheres firmly to gastric mucosa indicative of that it might bind to an alternative set of receptor. Receptor analysis by glycan arrays revealed higher binding of strain 812 to ALeb and Bleb glycans than to Leb, indicating that BabA from strain 812 has shifted its binding epitope somewhat away from the central Fuca1.2Gal bg domain and closer to the very terminal bg A and B determinants, i.e. GalNAca1.3Gal (bgA) or the Gala1.3Gal (bgB). By a colony screening approach we identified a subpopulation of 812 clones adapted for stronger Leb binding. Such affinity shifts comes from replacement of distinguishing amino acids by mechanisms of recombination with a BabA-related outer membrane protein.

  • 20.
    Holmfeldt, Per
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Brännström, Kristoffer
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Stenmark, Sonja
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Gullberg, Martin
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18-->e mutation in leukemic cells.2006Ingår i: Mol Biol Cell, ISSN 1059-1524, Vol. 17, nr 7, s. 2921-2930Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Op18/stathmin (Op18) is a phosphorylation-regulated microtubule destabilizer that is frequently overexpressed in tumors. The importance of Op18 in malignancy was recently suggested by identification of a somatic Q18-->E mutation of Op18 in an adenocarcinoma. We addressed the functional consequences of aberrant Op18 expression in leukemias by analyzing the cell cycle of K562 cells either depleted of Op18 by expression of interfering hairpin RNA or induced to express wild-type or Q18E substituted Op18. We show here that although Op18 depletion increases microtubule density during interphase, the density of mitotic spindles is essentially unaltered and cells divide normally. This is consistent with phosphorylation-inactivation of Op18 during mitosis. Overexpression of wild-type Op18 results in aneugenic activities, manifest as aberrant mitosis, polyploidization, and chromosome loss. One particularly significant finding was that the aneugenic activity of Op18 was dramatically increased by the Q18-->E mutation. The hyperactivity of mutant Op18 is apparent in its unphosphorylated state, and this mutation also suppresses phosphorylation-inactivation of the microtubule-destabilizing activity of Op18 without any apparent effect on its phosphorylation status. Thus, although Op18 is dispensable for mitosis, the hyperactive Q18-->E mutant, or overexpressed wild-type Op18, exerts aneugenic effects that are likely to contribute to chromosomal instability in tumors.

  • 21.
    Iakovleva, Irina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wijsekera, Alexandra
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Zhang, Jin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, Patrik L.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer2016Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, nr 4, artikel-id e0153529Artikel i tidskrift (Refereegranskat)
    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.

  • 22.
    Iakovleva, Irina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gharibyan, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Intissar, Anan
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Walfridsson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Enthalpic Forces Correlate with Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma2015Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 58, nr 16, s. 6507-6515Artikel i tidskrift (Refereegranskat)
    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.

  • 23.
    Islam, Tohidul
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Golchin, Solmaz A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pettersson, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hedberg, Isabell
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Virta, Merit-Miriam
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Linnea
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Mr..
    Apolipoprotein E impairs amyloid-β fibril elongation and maturation2019Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease (AD) is strongly linked to amyloid depositions of the Aβ peptide (Aβ). The lipid-binding protein apolipoprotein E (ApoE) has been found to interfere with Aβ amyloid formation and to exert a strong clinical impact to the pathology of AD. The APOE gene exists in three allelic isoforms represented by APOE ε2, APOE ε3, and APOE ε4. Carriers of the APOE ε4 variant display a gene dose-dependent increased risk of developing the disease. Aβ amyloids are formed via a nucleation-dependent mechanism where free monomers are added onto a nucleus in a template-dependent manner. Using a combination of surface plasmon resonance and thioflavin-T assays, we here show that ApoE can target the process of fibril elongation and that its interference effectively prevents amyloid maturation. We expose a complex equilibrium where the concentration of ApoE, Aβ monomers, and the amount of already formed Aβ fibrils will affect the relative proportion and formation rate of mature amyloids versus alternative assemblies. The result illustrates a mechanism which may affect both the clearance rate of Aβ assemblies in vivo and the population of cytotoxic Aβ assemblies.

  • 24.
    Jamroskovic, Jan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Doimo, Mara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chand, Karam
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Obi, Ikenna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kumar, Rajendra
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hedenström, Mattias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Das, Rabindra Nath
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Akhunzianov, Almaz
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia.
    Deiana, Marco
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kasho, Kazutoshi
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sulis Sato, Sebastian
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pourbozorgi-Langroudi, Parham
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mason, James E.
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Medini, Paolo
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Öhlund, Daniel
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper.
    Wanrooij, Sjoerd
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sabouri, Nasim
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization2020Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, nr 6, s. 2876-2888Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

  • 25.
    Jonna, Venkateswara Rao
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Crona, Mikael
    Rofougaran, Reza
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lundin, Daniel
    Johansson, Samuel
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sjöberg, Britt-Marie
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Diversity in Overall Activity Regulation of Ribonucleotide Reductase2015Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 28, s. 17339-17348Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.

  • 26.
    Kulén, Martina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Marie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hansen, Sabine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    van der Lingen, Ingeborg
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hall, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Structure-based design of inhibitors targeting PrfA, the master virulence regulator of Listeria monocytogenes2018Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, nr 9, s. 4165-4175Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein "tunnel" as the main inhibitor binding site (A1), where the compounds participate in an extensive hydrophobic network that restricts the protein's ability to form functional DNA-binding helix−turn−helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-A1 selective PrfA inhibitors with potent antivirulence properties.

  • 27.
    Lindhagen-Persson, Malin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vestling, Monika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Steinitz, Michael
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Amyloid-β oligomer specificity mediated by the IgM isotype: implications for a specific protective mechanism exerted by endogenous auto-antibodies2010Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, nr 11, s. e13928-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-β peptide (Aβ). The correlation between clinical symptoms of AD and Aβ depositions is, however, weak. Instead small and soluble Aβ oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aβ oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aβ deposits. Consequently a specific immunological targeting of Aβ oligomers is of high therapeutic interest.

    Methodology/Principal Findings Previously the generation of conformational-dependent oligomer specific anti-Aβ antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aβ antibody (OMAB). OMAB only demonstrates a weak interaction with Aβ monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aβ-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aβ oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aβ(1-42) at highly substoichiometric ratios. Anti-Aβ auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti-Aβ IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aβ.

    Conclusions/Significance Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aβ auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated.

  • 28. Mahdavi, Jafar
    et al.
    Royer, Pierre-Joseph
    Sjölinder, Hong S.
    Azimi, Sheyda
    Self, Tim
    Stoof, Jeroen
    Wheldon, Lee M.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilson, Raymond
    Moreton, Joanna
    Moir, James W. B.
    Sihlbom, Carina
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Jonsson, Ann-Beth
    Soultanas, Panos
    Ala'aldeen, Dlawer A. A.
    Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence2013Ingår i: Open Biology, ISSN 2046-2441, E-ISSN 2046-2441, Vol. 3, nr 10, artikel-id 130048Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

  • 29. Moonens, Kristof
    et al.
    Gideonsson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Subedi, Suresh
    Bugaytsova, Jeanna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Romao, Ema
    Mendez, Melissa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nordén, Jenny
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Fallah, Mahsa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rakhimova, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Shevtsova, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lahmann, Martina
    Castaldo, Gaetano
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Coppens, Fanny
    Lo, Alvin W.
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Solnick, Jay V.
    Vandenbussche, Guy
    Oscarson, Stefan
    Hammarström, Lennart
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berg, Douglas E.
    Muyldermans, Serge
    Borén, Thomas
    Remaut, Han
    Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori2016Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 19, nr 1, s. 55-66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Helicobacter pylori adhesin BabA binds mucosal ABO/Le b blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

  • 30.
    Nilsson, Lina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Larsson, Andreas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Swedish Defence Research Agency, CBRN Defence and Security.
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Carlsson, Marcus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kristoffer, Brännström
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity2016Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, nr 4, artikel-id e0153112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment.

  • 31.
    Nilsson, Lina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Golchin, Solmaz A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pettersson, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Transthyretin Interferes with Aβ Amyloid Formation by Redirecting Oligomeric Nuclei into Non-Amyloid Aggregates2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 17, s. 2722-2733Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The pathological Aβ aggregates associated with Alzheimer's disease follow a nucleation-dependent path of formation. A nucleus represents an oligomeric assembly of Aβ peptides that acts as a template for subsequent incorporation of monomers to form a fibrillar structure. Nuclei can form de novo or via surface-catalyzed secondary nucleation, and the combined rates of elongation and nucleation control the overall rate of fibril formation. Transthyretin (TTR) obstructs Aβ fibril formation in favor of alternative non-fibrillar assemblies, but the mechanism behind this activity is not fully understood. This study shows that TTR does not significantly disturb fibril elongation; rather, it effectively interferes with the formation of oligomeric nuclei. We demonstrate that this interference can be modulated by altering the relative contribution of elongation and nucleation, and we show how TTR's effects can range from being essentially ineffective to almost complete inhibition of fibril formation without changing the concentration of TTR or monomeric Aβ.

  • 32. Ouberai, Myriam
    et al.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vestling, Monika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Dumy, Pascal
    Chierici, Sabine
    Garcia, Julian
    Clicked tacrine conjugates as acetylcholinesterase and β-amyloid directed compounds2011Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, nr 4, s. 1140-1147Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The multifaceted nature of Alzheimer's disease (AD) has led to the development of multi-targeted compounds based on the classical AD drug, tacrine, first known to inhibit the acetylcholine-degrading enzyme acetylcholinesterase (AChE). In the present work, we explore the potentiality of multimers of tacrine in this field. The synthesis using the so-called "click chemistry" and the in vitro study of the conjugates are described. Two or four copies of the tacrine molecule are "clicked" on a constrained cyclopeptide template proven to be a convenient tool for multimeric presentation. The multimers significantly inhibit self-induced amyloid fibril formation from Aβ(40) at low inhibitor to Aβ molar ratios at which the tacrine monomer is fully inactive (Thioflavin T assays and AFM observation). Moreover, they have the capacity to bind to Aβ(40) fibrils (SPR assays) while retaining the AChE inhibitory activity of the parent tacrine.

  • 33. Shaikhali, Jehad
    et al.
    Davoine, Celine
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Rouhier, Nicolas
    Bygdell, Joakim
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Wingsle, Gunnar
    Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein2015Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 468, nr 3, s. 385-400Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combinationwith specific TFs as GeBPL.

  • 34.
    Singh, Pardeep
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bartens, Christian
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pyridine-Fused 2-Pyridones via Povarov and A3 Reactions: Rapid Generation of Highly Functionalized Tricyclic Heterocycles Capable of Amyloid Fibril Binding2019Ingår i: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 84, nr 7, s. 3887-3903Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We here describe the use of three-component reactions to synthesize tricyclic pyridine ring-fused 2-pyridones. The developed protocols have a wide substrate scope and allow for the installation of diverse chemical functionalities on the tricyclic central fragment. Several of these pyridine-fused rigid polyheterocycles are shown to bind to Aβ and α-synuclein fibrils, which are associated with neurodegenerative diseases.

  • 35. Szalewska-Palasz, Agnieszka
    et al.
    Johansson, Linda U M
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bernardo, Lisandro M D
    Skärfstad, Eleonore
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Stec, Ewa
    Brännström, Kristoffer
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54.2007Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 25, s. 18046-56Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain sigma70-dependent promoters. A number of promoters that are dependent on alternative sigma-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived sigma54-dependent Po promoter and several other sigma54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro sigma54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the beta- and beta'-subunits of core RNA polymerase that allow high levels of transcription from the sigma54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with sigma-factors to favor sigma54-holoenzyme formation over that with sigma70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent sigma70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of sigma54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent sigma70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative sigma-factors is controlled to meet cellular requirements.

  • 36.
    Wallgren, Marcus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lidman, Martin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pedersen, Anders
    Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Karlsson, B Göran
    Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Reconstitution of the anti-apoptotic bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic bax protein2013Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 4, artikel-id e61452Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

  • 37.
    Zhang, Jin
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, Patrik L.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin2016Ingår i: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 50, nr 21, s. 11984-11993Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 38.
    Zhang, Jin
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Mikael J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Andersson, Patrik L.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals2018Ingår i: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 52, nr 20, s. 11865-11874Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 39. Zhou, Yizhou
    et al.
    Smith, Daniel
    Leong, Bryan J
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Chapman, Matthew R
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Promiscuous cross-seeding between bacterial amyloids promotes interspecies biofilms2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 42, s. 35092-35103Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 40.
    Åberg, Anna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gideonsson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Arnqvist, Anna
    The Helicobacter pylori sialic acid binding adhesin SabA is regulated via a network of two-component systemsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

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

  • 41.
    Åberg, Anna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gideonsson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vallström, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Carina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rakhimova, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Engstrand, Lars
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism2014Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, nr 7, artikel-id e1004234Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 42.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Weise, Christoph
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structural topology and activation of an initial adenylate kinase-substrate complex2013Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 6, s. 1055-1061Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

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