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  • 1.
    Aksoy, N. H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Aksaray Univ, Dept Biochem, Aksaray, Turkey..
    Mannervik, B.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Inhibitory effects of ethacrynic acid on glutathione S-transferase A1-1 from Callithrix jacchus2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 348-348Article in journal (Other academic)
  • 2. Andersen, Gorm
    et al.
    Andersen, Birgit
    Dobritzsch, Doreen
    Karolinska Institutet.
    Schnackerz, Klaus D
    Piskur, Jure
    A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 7, p. 1804-1017Article in journal (Refereed)
    Abstract [en]

    In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.

  • 3.
    Andersson, Mattias K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 10, p. 2255-2267Article in journal (Refereed)
    Abstract [en]

    Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

  • 4.
    Bardales, José R.
    et al.
    Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Villamarín, J. Antonio
    Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
    Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 18, p. 4479-4489Article in journal (Refereed)
    Abstract [en]

    Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.

  • 5.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Käthe
    Salminen, Tiina
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 10, p. 2387-2398Article in journal (Refereed)
    Abstract [en]

    We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π–π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

  • 6.
    Bromée, Torun
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Sjödin, Paula
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Boswell, Tim
    Larsson, Tomas A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Salaneck, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Zoorob, Rima
    Mohell, Nina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Larhammar, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Neuropeptide Y-family receptors Y6 and Y7 in chicken: Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 9, p. 2048-2063Article in journal (Refereed)
    Abstract [en]

    The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a Kd of 0.80 ± 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a Kd of 0.14 ± 0.01 nm (mean ± SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.

  • 7.
    Chen, Yang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Koripella, Ravi Kiran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 18, p. 3789-3803Article in journal (Refereed)
    Abstract [en]

    Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 A resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the individual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 A relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46-56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694-699]. The mutations can be classified as affecting FA binding, EF-G-ribosome interactions, EF-G conformation, and EF-G stability.

  • 8. Claesson, Magnus
    et al.
    Siitonen, Vilja
    Dobritzsch, Doreen
    Karolinska Institutet.
    Metsä-Ketelä, Mikko
    Schneider, Gunter
    Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 17, p. 3251-3263Article in journal (Refereed)
    Abstract [en]

    The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.

  • 9.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 32007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 22, p. 5979-5986Article in journal (Refereed)
    Abstract [en]

    This study of the full-length bifunctional nonstructural protein 3 from hepatitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, purified and characterized. The substitutions resulted in indistinguishable Km values and slightly lower kcat values compared to the wild-type. The Ki values for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10-fold. The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization. Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds.

  • 10.
    Davey, Norman E.
    et al.
    Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
    Seo, Moon-Hyeong
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Yadav, Vikash Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Jeon, Jouhyun
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Nim, Satra
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Krystkowiak, Izabella
    Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Dong, Debbie
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Markova, Natalia
    Malvern Instruments Nord AB, Solna, Sweden..
    Kim, Philip M.
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada.;Univ Toronto, Dept Mol Genet, Toronto, ON M5S 1A1, Canada.;Univ Toronto, Dept Comp Sci, Toronto, ON M5S 1A1, Canada..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 3, p. 485-498Article in journal (Refereed)
    Abstract [en]

    The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.

  • 11.
    Gallant, Caroline J.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    A compendium on single-cell analysis for the curious2019In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 286, no 8, p. 1442-1444Article in journal (Other academic)
  • 12. Ge, Changrong
    et al.
    Gómez-Llobregat, Jordi
    Skwark, Marcin J
    Ruysschaert, Jean-Marie
    Wieslander, Ake
    Lindén, Martin
    Membrane remodeling capacity of a vesicle-inducing glycosyltransferase2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 16, p. 3667-3684Article in journal (Refereed)
    Abstract [en]

    Intracellular vesicles are abundant in eukaryotic cells but absent in the Gram-negative bacterium Escherichia coli. However, strong overexpression of a monotopic glycolipid-synthesizing enzyme, monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii (alMGS), leads to massive formation of vesicles in the cytoplasm of E. coli. More importantly, alMGS provides a model system for the regulation of membrane properties by membrane-bound enzymes, which is critical for maintaining cellular integrity. Both phenomena depend on how alMGS binds to cell membranes, which is not well understood. Here, we carry out a comprehensive investigation of the membrane binding of alMGS by combining bioinformatics methods with extensive biochemical studies, structural modeling and molecular dynamics simulations. We find that alMGS binds to the membrane in a fairly upright manner, mainly by residues in the N-terminal domain, and in a way that induces local enrichment of anionic lipids and a local curvature deformation. Furthermore, several alMGS variants resulting from substitution of residues in the membrane anchoring segment are still able to generate vesicles, regardless of enzymatic activity. These results clarify earlier theories about the driving forces for vesicle formation, and shed new light on the membrane binding properties and enzymatic mechanism of alMGS and related monotopic GT-B fold glycosyltransferases.

  • 13.
    Giampietro, C.
    et al.
    FIRC Inst Mol Oncol, IFOM, Milan, Italy..
    Disanza, A.
    FIRC Inst Mol Oncol, IFOM, Milan, Italy..
    Scita, G.
    FIRC Inst Mol Oncol, IFOM, Milan, Italy..
    Dejana, Elisabetta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. FIRC Inst Mol Oncol, IFOM, Milan, Italy..
    VE-cadherin modulates YAP intracellular localization and signalling2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, p. 48-48Article in journal (Refereed)
  • 14.
    Hamnevik, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Maurer, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ntuku, Siphosethu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed)
    Abstract [en]

    Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes.

  • 15.
    Hasse, Dirk
    et al.
    University of Rostock, Germany .
    Mikkat, Stefan
    Hagemann, Martin
    Bauwe, Hermann
    Alternative splicing produces an H-protein with better substrate properties for the P-protein of glycine decarboxylase.2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 23Article in journal (Refereed)
    Abstract [en]

    Several thousand plant genes are known to produce multiple transcripts, but the precise function of most of the alternatively encoded proteins is not known. Alternative splicing has been reported for the H-protein subunit of glycine decarboxylase in the genus Flaveria. H-protein has no catalytic activity itself but is a substrate of the three enzymatically active subunits, P-, T- and L-protein. In C(4) species of Flaveria, two H-proteins originate from single genes in an organ-dependent manner. Here, we report on differences between the two alternative H-protein variants with respect to their interaction with the glycine-decarboxylating subunit, P-protein. Steady-state kinetic analyses of the alternative Flaveria H-proteins and artificially produced 'alternative' Arabidopsis H-proteins, using either pea mitochondrial matrix extracts or recombinant cyanobacterial P-protein, consistently demonstrate that the alternative insertion of two alanine residues at the N-terminus of the H-protein elevates the activity of P-protein by 20%in vitro, and could promote glycine decarboxylase activity in vivo.

  • 16.
    Henningsson, Frida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hergeth, Sonja
    SLU.
    Cortelius, Robert
    SLU.
    Åbrink, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pejler, Gunnar
    SLU.
    A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 21, p. 4901-4912Article in journal (Refereed)
    Abstract [en]

    In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.

  • 17.
    Härdin, Hanna M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Mathematics.
    Zagaris, Antonios
    Willms, Allan R.
    Westerhoff, Hans V.
    Clusters of reaction rates and concentrations in protein networks such as the phosphotransferase system2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 2, p. 531-548Article, review/survey (Refereed)
    Abstract [en]

    To understand the functioning of living cells, it is often helpful or even necessary to exploit inherent timescale disparities and focus on long-term dynamic behaviour. In the present study, we explore this type of behaviour for the biochemical network of the phosphotransferase system. We show that, during the slow phase that follows a fast initial transient, the network reaction rates are partitioned into clusters corresponding to connected parts of the reaction network. Rates within any of these clusters assume essentially the same value: differences within each cluster are vastly smaller than that from one cluster to another. This rate clustering induces an analogous clustering of the reactive compounds: only the molecular concentrations on the interface between these clusters are produced and consumed at substantially different rates and hence change considerably during the slow phase. The remaining concentrations essentially assume their steady-state values already by the end of the transient phase. Further, we find that this clustering phenomenon occurs for a large number of parameter values and also for models with different topologies; to each of these models, there corresponds a particular network partitioning. Our results show that, in spite of its complexity, the phosphotransferase system tends to behave in a rather simple (yet versatile) way. The persistence of clustering for the perturbed models we examined suggests that it is likely to be encountered in various environmental conditions, as well as in other signal transduction pathways with network structures similar to that of the phosphotransferase system.

  • 18.
    Ignatovica, Vita
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Megnis, Kaspars
    Lapins, Maris
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Schiöth, Helgi B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Klovins, Janis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Identification and analysis of functionally important amino acids in human purinergic 12 receptor using a Saccharomyces cerevisiae expression system2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 1, p. 180-191Article in journal (Refereed)
    Abstract [en]

    The purinergic 12 receptor (P2Y12) is a major drug target for anticoagulant therapies, but little is known about the regions involved in ligand binding and activation of this receptor. We generated four randomized P2Y12 libraries and investigated their ligand binding characteristics. P2Y12 was expressed in a Saccharomyces cerevisiae model system. Four libraries were generated with randomized amino acids at positions 181, 256, 265 and 280. Mutant variants were screened for functional activity in yeast using the natural P2Y12 ligand ADP. Activation results were investigated using quantitative structure-activity relationship (QSAR) models and ligand-receptor docking. We screened four positions in P2Y12 for functional activity by substitution with amino acids with diverse physiochemical properties. This analysis revealed that positions E181, R256 and R265 alter the functional activity of P2Y12 in a specific manner. QSAR models for E181 and R256 mutant libraries strongly supported the experimental data. All substitutions of amino acid K280 were completely inactive, highlighting the crucial role of this residue in P2Y12 function. Ligand-receptor docking revealed that K280 is likely to be a key element in the ligand-binding pocket of P2Y12. The results of this study demonstrate that positions 181, 256, 265 and 280 of P2Y12 are important for the functional integrity of the receptor. Moreover, K280 appears to be a crucial feature of the P2Y12 ligand-binding pocket. These results are important for rational design of novel antiplatelet agents.

  • 19.
    Ingvarsson, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl-tRNA synthetase2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 19, p. 3947-3962Article in journal (Refereed)
    Abstract [en]

    Two structures of monomeric methionyl-tRNA synthetase, from Mycobacterium smegmatis, in complex with the ligands methionine/adenosine and methionine, were analyzed by X-ray crystallography at 2.3 Å and at 2.8 Å, respectively. The structures demonstrated the flexibility of the multidomain enzyme. A new conformation of the structure was identified in which the connective peptide domain bound more closely to the catalytic domain than described previously. The KMSKS(301-305) loop in our structures was in an open and inactive conformation that differed from previous structures by a rotation of the loop of about 90° around hinges located at Asn297 and Val310. The binding of adenosine to the methionyl-tRNA synthetase methionine complex caused a shift in the KMSKS domain that brought it closer to the catalytic domain. The potential use of the adenosine-binding site for inhibitor binding was evaluated and a potential binding site for a specific allosteric inhibitor was identified.

  • 20.
    Johansson, Ann-Sofi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Berglind-Dehlin, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Karlsson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Gellerfors, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Physiochemical characterization of the Alzheimers disease related peptides Aβ1-42 Arctic and Aβ1-42wt2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 12, p. 2618-2630Article in journal (Refereed)
    Abstract [en]

    The amyloid beta peptide (A beta) is crucial for the pathogenesis of Alzheimer's disease. Aggregation of monomeric A beta into insoluble amyloid fibrils proceeds through several soluble A beta intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimer's disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimer's disease, and synthetic A beta 1-40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887-893]. To increase our understanding of the mechanisms involved in A beta aggregation, especially A beta monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of A beta 1-42wt and A beta 1-42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both A beta 1-42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes - monomer oligomerization and protofibril fibrillization - were affected differently by changes in the micro-environment and that the Arctic mutation alters the peptide response to such changes.

  • 21.
    Johansson, Ann-Sofi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Garlind, Anita
    Berglind-Dehlin, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Karlsson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Gellerfors, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ekholm-Pettersson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Palmblad, Jan
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Docosahexaenoic acid stabilizes soluble amyloid-β protofibrils and sustains amyloid-β induced neurotoxicity in vitro2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 4, p. 990-1000Article in journal (Refereed)
    Abstract [en]

    Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-β (Aβ) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Aβ, and their effect on Aβ aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Aβ42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-β-induced toxicity in PC12 cells over time, whereas Aβ without docosahexaenoic acid stabilization resulted in reduced toxicity, as Aβ formed fibrils. Arachidic acid had no effect on Aβ aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on Aβ42 harboring the Arctic mutation (AβE22G). Consequently, AβArctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Aβ aggregates and how endogenous lipids can affect Aβ aggregation.

  • 22.
    Kamran, Mohammad
    et al.
    Jawaharlal Nehru Univ, Special Ctr Mol Med, New Delhi 110067, India.
    Dubey, Priyanka
    Jawaharlal Nehru Univ, Special Ctr Mol Med, New Delhi 110067, India.
    Verma, Vijay
    Jawaharlal Nehru Univ, Special Ctr Mol Med, New Delhi 110067, India.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Dhar, Suman K.
    Jawaharlal Nehru Univ, Special Ctr Mol Med, New Delhi 110067, India.
    Helicobacter pylori shows asymmetric and polar cell divisome assembly associated with DNA replisome2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 13, p. 2531-2547Article in journal (Refereed)
    Abstract [en]

    DNA replication and cell division are two fundamental processes in the life cycle of a cell. The majority of prokaryotic cells undergo division by means of binary fission in coordination with replication of the genome. Both processes, but especially their coordination, are poorly understood in Helicobacter pylori. Here, we studied the cell divisome assembly and the subsequent processes of membrane and peptidoglycan synthesis in the bacterium. To our surprise, we found the cell divisome assembly to be polar, which was well-corroborated by the asymmetric membrane and peptidoglycan synthesis at the poles. The divisome components showed its assembly to be synchronous with that of the replisome and the two remained associated throughout the cell cycle, demonstrating a tight coordination among chromosome replication, segregation and cell division in H. pylori. To our knowledge, this is the first report where both DNA replication and cell division along with their possible association have been demonstrated for this pathogenic bacterium.

  • 23.
    Kardassis, Dimitris
    et al.
    Department of Biochemistry, University of Crete Medical School, Heraklion, Greece.
    Murphy, Carol
    Biomedical Research Institute, Foundation for Research & Technology-Hellas, Ioannina, Greece.
    Fotsis, Theodore
    Biomedical Research Institute, Foundation for Research & Technology-Hellas, Ioannina, Greece.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Stournaras, Christos
    Department of Biochemistry, University of Crete Medical School, Heraklion, Greece.
    Control of transforming growth factor beta signal transduction by small GTPases2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 11, p. 2947-2965Article, review/survey (Refereed)
    Abstract [en]

    The integrated roles of small GTPases in executing the transforming growth factor beta (TGFbeta) signaling pathway have attracted increasing attention in recent years. In this review, we summarize recent findings on TGFbeta signaling during receptor endocytosis, Smad trafficking and actin cytoskeleton remodeling, and emphasize the role of small GTPases in these processes. First, we give an overview of the different endocytic routes taken by TGFbeta receptors, their impact on active TGFbeta signaling versus degradation and their regulation by the small GTPases Rab, RalA/Ral-binding protein 1 and Rap2. Second, we focus on the mechanisms and regulation of Smad trafficking in the cytoplasm, through the nuclear pores and into the nucleus, and the contribution of Ran GTPase to these events. Third, we summarize the role of Rho small GTPases in early and late cytoskeleton remodeling in various cell models and diseases, and the positive and negative cross-talk between Rho GTPases and the TGFbeta/Smad pathway. The biological significance of this exciting research field, the perspectives and critical open questions are discussed.

  • 24. Korkmaz, Brice
    et al.
    Jegot, Gwenhael
    Lau, Laurie C.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pitois, Elodie
    Juliano, Luiz
    Walls, Andrew F.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gauthier, Francis
    Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 15, p. 2635-2646Article in journal (Refereed)
    Abstract [en]

    Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.

  • 25.
    Lindberg, Diana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 24, p. 6309-6320Article in journal (Refereed)
    Abstract [en]

    The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre-steady-state measurements on a series of styrene oxide derivatives. A preference for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans-stilbene oxide was established, with E-values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme formation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by 13C-NMR spectroscopy of 18O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans-stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency.

  • 26. Liu, Xiao Li
    et al.
    Kilpeläinen, Pekka
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Sun, Yi
    Wartiovaara, Jorma
    Morgunova, Ekaterina
    Pikkarainen, Timo
    Yan, Kunimasa
    Jonsson, Anders P.
    Tryggvason, Karl
    Characterization of the interactions of the nephrin intracellular domain2005In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, no 1, p. 228-243Article in journal (Refereed)
    Abstract [en]

    Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.

  • 27.
    Lord, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Englund, Hillevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Söderberg, Linda
    Tucker, Stina
    Clausen, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Gordon, Marcia
    Morgan, Dave
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ekholm Pettersson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nilsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 4, p. 995-1006Article in journal (Refereed)
    Abstract [en]

    Oligomeric assemblies of amyloid-β (Aβ) are suggested to be central in the pathogenesis of Alzheimer's disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long-term potentiation. The tg-ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg-ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg-ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg-ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg-ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg-ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age-dependent manner in tg-ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.

  • 28.
    Misra, Suniti
    et al.
    Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
    Heldin, Paraskevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hascall, Vincent C.
    Department of Biomedical Engineering ⁄ ND20, Cleveland Clinic, Cleveland, OH, USA.
    Karamanos, Nikos K.
    Department of Chemistry, Laboratory of Biochemistry, University of Patras, Greece.
    Skandalis, Spyros S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Markwald, Roger R.
    Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
    Ghatak, Shibnath
    Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
    Hyaluronan-CD44 interactions as potential targets for cancer therapy2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 9, p. 1429-1443Article, review/survey (Refereed)
    Abstract [en]

    It is becoming increasingly clear that signals generated in tumor microenvironments are crucial to tumor cell behavior, such as survival, progression and metastasis. The establishment of these malignant behaviors requires that tumor cells acquire novel adhesion and migration properties to detach from their original sites and to localize to distant organs. CD44, an adhesion/homing molecule, is a major receptor for the glycosaminoglycan hyaluronan, which is one of the major components of the tumor extracellular matrix. CD44, a multistructural and multifunctional molecule, detects changes in extracellular matrix components, and thus is well positioned to provide appropriate responses to changes in the microenvironment, i.e. engagement in cell-cell and cell-extracellular matrix interactions, cell trafficking, lymph node homing and the presentation of growth factors/cytokines/chemokines to co-ordinate signaling events that enable the cell responses that change in the tissue environment. The potential involvement of CD44 variants (CD44v), especially CD44v4-v7 and CD44v6-v9, in tumor progression has been confirmed for many tumor types in numerous clinical studies. The downregulation of the standard CD44 isoform (CD44s) in colon cancer is postulated to result in increased tumorigenicity. CD44v-specific functions could be caused by their higher binding affinity than CD44s for hyaluronan. Alternatively, CD44v-specific functions could be caused by differences in associating molecules, which may bind selectively to the CD44v exon. This minireview summarizes how the interaction between hyaluronan and CD44v can serve as a potential target for cancer therapy, in particular how silencing CD44v can target multiple metastatic tumors.

  • 29.
    Motallebipour, Mehdi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ameur, Adam
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Koch, Christoph
    Wellcome Trust Sanger Institute.
    Dunham, Ian
    Wellcome Trust Sanger Institute.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Novel genes in cell cycle control and lipid metabolism with dynamically regulated binding sites for sterol regulatory element-binding protein 1 and RNA polymerase II in HepG2 cells detected by chromatin immunoprecipitation with microarray detection2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 7, p. 1878-1890Article in journal (Refereed)
    Abstract [en]

    Sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2) are important regulators of genes involved in cholesterol and fatty acid metabolism, but have also been implicated in the regulation of the cell cycle and have been associated with the pathogenesis of type 2 diabetes, atherosclerosis and obesity, among others. In this study, we aimed to characterize the binding sites of SREBP-1 and RNA polymerase II through chromatin immunoprecipitation and microarray analysis in 1% of the human genome, as defined by the Encyclopaedia of DNA Elements consortium, in a hepatocellular carcinoma cell line (HepG2). Our data identified novel binding sites for SREBP-1 in genes directly or indirectly involved in cholesterol metabolism, e.g. apolipoprotein C-III (APOC3). The most interesting biological findings were the binding sites for SREBP-1 in genes for host cell factor C1 (HCFC1), involved in cell cycle regulation, and for filamin A (FLNA). For RNA polymerase II, we found binding sites at classical promoters, but also in intergenic and intragenic regions. Furthermore, we found evidence of sterol-regulated binding of SREBP-1 and RNA polymerase II to HCFC1 and FLNA. From the results of this work, we infer that SREBP-1 may be involved in processes other than lipid metabolism.

  • 30.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Transforming growth factor beta and bone morphogenetic protein actions in cancer progression2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 35-35Article in journal (Other academic)
  • 31. Nitharwal, Ram G.
    et al.
    Verma, Vijay
    Subbarao, Naidu
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Choudhury, Nirupam R.
    Dhar, Suman K.
    DNA binding activity of Helicobacter pylori DnaB helicase: the role of the N-terminal domain in modulating DNA binding activities2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 2, p. 234-250Article in journal (Refereed)
    Abstract [en]

    Replicative helicases are major motor proteins essential for chromosomal DNA replication in prokaryotes. Usually hexameric in solution, their DNA binding property must have different roles at stages ranging from the loading onto a branched structure at initiation from the origin to the highly processive translocation during elongation. Here, we have analysed the DNA binding activity of Helicobacter pylori (Hp) replicative helicase, DnaB. The results indicate that while the C-terminal region is important for its DNA binding activity, the N-terminus appears to dampen the proteins affinity for DNA. The masking activity of the N-terminus does not require ATP or hexamerization of HpDnaB and can be overcome by deleting the N-terminus. It can also be neutralized by engaging the N-terminus in an interaction with a partner like the C-terminus of DnaG primase. The inhibitory effect of the N-terminus on DNA binding activity is consistent with the 3D homology model of HpDnaB. Electron microscopy of the HpDnaBssDNA complex showed that HpDnaB preferentially bound at the ends of linear ssDNA and translocated along the DNA in the presence of ATP. These results provide an insight into the stimulatory and inhibitory effects of different regions of HpDnaB on DNA binding activities that may be central to the loading and translocation functions of DnaB helicases.

  • 32.
    Ottesen, A. H.
    et al.
    Oslo Univ Hosp, Inst Expt Med Res, Oslo, Norway.;Univ Oslo, Oslo, Norway.;Akershus Univ Hosp, Div Med, Lorenskog, Norway.;Ctr Heart Failure Res, Oslo, Norway..
    Carlson, C. R.
    Oslo Univ Hosp, Inst Expt Med Res, Oslo, Norway.;Univ Oslo, Oslo, Norway..
    Laver, D. R.
    Univ Newcastle, Sch Biomed Sci & Pharm, Callaghan, NSW, Australia.;Hunter Med Res Inst, Callaghan, NSW, Australia..
    Myhre, P. L.
    Univ Oslo, Oslo, Norway.;Akershus Univ Hosp, Div Med, Lorenskog, Norway.;Ctr Heart Failure Res, Oslo, Norway..
    Dalhus, B.
    Oslo Univ Hosp, Dept Microbiol, Oslo, Norway.;Oslo Univ Hosp, Dept Med Biochem, Oslo, Norway.;Univ Oslo, Rikshosp, Oslo, Norway..
    Lunde, P. K.
    Oslo Univ Hosp, Inst Expt Med Res, Oslo, Norway.;Univ Oslo, Oslo, Norway..
    Lunde, M.
    Oslo Univ Hosp, Inst Expt Med Res, Oslo, Norway.;Univ Oslo, Oslo, Norway..
    Hoff, J. E.
    Univ Oslo, Oslo, Norway.;Akershus Univ Hosp, Div Med, Lorenskog, Norway.;Ctr Heart Failure Res, Oslo, Norway..
    Godang, K.
    Univ Oslo, Oslo, Norway.;Akershus Univ Hosp, Div Med, Lorenskog, Norway.;Ctr Heart Failure Res, Oslo, Norway..
    Stridsberg, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemical endocrinology.
    Omland, T.
    Univ Oslo, Oslo, Norway.;Akershus Univ Hosp, Div Med, Lorenskog, Norway.;Ctr Heart Failure Res, Oslo, Norway..
    Christensen, G.
    Oslo Univ Hosp, Inst Expt Med Res, Oslo, Norway.;Univ Oslo, Oslo, Norway..
    Rosjo, H.
    Univ Oslo, Oslo, Norway.;Akershus Univ Hosp, Div Med, Lorenskog, Norway.;Ctr Heart Failure Res, Oslo, Norway..
    Louch, W. E.
    Oslo Univ Hosp, Inst Expt Med Res, Oslo, Norway.;Univ Oslo, Oslo, Norway..
    Secretoneurin attenuates Ca2+-dependent arrhythmogenesis2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no SI, p. 146-147, article id P.1.5-081Article in journal (Other academic)
  • 33.
    Parmryd, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Önfelt, Björn
    Consequences of Membrane Topography2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 12, p. 2775-2784Article, review/survey (Refereed)
    Abstract [en]

    The surface of mammalian cells is neither smooth nor flat and cells have several times more plasma membrane than the minimum area required to accommodate their shape. We discuss the biological function of this apparent excess membrane that allows the cells to migrate, undergo shape changes and probably plays a role in signal transduction. Methods for studying membrane folding and topography; atomic force microscopy, scanning ion conductance microscopy, fluorescence polarisation microscopy and linear dichroism, are described and evaluated. Membrane folding and topography is frequently ignored when interpreting microscopy data. This has resulted in several misconceptions for instance regarding colocalisation, membrane organisation and molecular clustering. We suggest simple ways to avoid these pitfalls and invoke Occam's razor - that simple explanations are preferable to complex ones. Topography, i. e. deviations from a smooth surface, should always be ruled out as the cause of anomalous data before other explanations are presented.

  • 34.
    Pettersson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Holmberg, Lisa
    Axelson, Magnus
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    CYP7B1-mediated metabolism of dehydroepiandrosterone and 5alpha-androstane-3beta,17beta-diol--potential role(s) for estrogen signaling2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 8, p. 1778-1789Article in journal (Refereed)
    Abstract [en]

    CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3 beta-Adiol may play an important role for estrogen receptor beta-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor beta activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3 beta-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3 beta-Adiol (a CYP7B1 substrate) and 7 alpha-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor beta activation. The data obtained indicated that 3 beta-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor beta activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3 beta-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the K-cat/K-m values were in the same order of magnitude. A high dehydroepiandrosterone/3 beta-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3 beta-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3 beta-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3 beta-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor beta signaling.

  • 35.
    Philipson, Ola
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lord, Anna
    Gumucio, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nilsson, Lars N. G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Animal models of amyloid-β-related pathologies in Alzheimer’s disease2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 6, p. 1389-1409Article, review/survey (Refereed)
    Abstract [en]

    In the early 1990s, breakthrough discoveries on the genetics of Alzheimer's disease led to the identification of missense mutations in the amyloid-beta precursor protein gene. Research findings quickly followed, giving insights into molecular pathogenesis and possibilities for the development of new types of animal models. The complete toolbox of transgenic techniques, including pronuclear oocyte injection and homologous recombination, has been applied in the Alzheimer's disease field, to produce overexpressors, knockouts, knockins and regulatable transgenics. Transgenic models have dramatically advanced our understanding of pathogenic mechanisms and allowed therapeutic approaches to be tested. Following a brief introduction to Alzheimer's disease, various nontransgenic and transgenic animal models are described in terms of their values and limitations with respect to pathogenic, therapeutic and functional understandings of the human disease.

  • 36. Rydengård, Victoria
    et al.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mörgelin, Matthias
    Schmidtchen, Artur
    Histidine-rich glycoprotein exerts antibacterial activity2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 2, p. 377-389Article in journal (Refereed)
    Abstract [en]

    Histidine-rich glycoprotein (HRGP), an abundant heparin-binding protein found in plasma and thrombocytes, exerts antibacterial effects against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fluorescence studies and electron microscopy to assess membrane permeation showed that HRGP induces lysis of E. faecalisbacteria in the presence of Zn2+ or at low pH. Heparin blocked binding of the protein to E. faecalis and abolished antibacterial activity. Furthermore, truncated HRGP, devoid of the heparin-binding and histidine-rich domain, was not antibacterial. It has previously been shown that peptides containing consensus heparin-binding sequences (Cardin and Weintraub motifs) are antibacterial. Thus, the peptide (GHHPH)4, derived from the histidine-rich region of HRGP and containing such a heparin-binding motif, was antibacterial for E. faecalis in the presence of Zn2+ or at low pH. The results show a previously undisclosed antibacterial activity of HRGP and suggest that the histidine-rich and heparin-binding domain of HRGP mediates the antibacterial activity of the protein.

  • 37. Salazar, Vivian A.
    et al.
    Rubin, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Moussaoui, Mohammed
    Pulido, David
    Victoria Nogues, Maria
    Venge, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Boix, Ester
    Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 24, p. 5432-5446Article in journal (Refereed)
    Abstract [en]

    Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post-translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichiacoli cell cultures and compared with the recombinant non-glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self-aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post-translational modifications to the antimicrobial role of ECP in host defense.

  • 38.
    Schibich, D.
    et al.
    Univ Heidelberg ZMBH, Ctr Mol Biol, Heidelberg, Germany.;German Canc Res Ctr, Heidelberg, Germany..
    Gloge, F.
    Univ Heidelberg ZMBH, Ctr Mol Biol, Heidelberg, Germany.;German Canc Res Ctr, Heidelberg, Germany..
    Pohner, I.
    Heidelberg Inst Theoret Studies, Heidelberg, Germany..
    Björkholm, Patrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Stockholm Univ, Dept Biochem & Biophys, Ctr Biomembrane Res, Stockholm, Sweden.;Stockholm Univ, Sci Life Lab, Solna, Sweden..
    Wade, R. C.
    Univ Heidelberg ZMBH, Ctr Mol Biol, Heidelberg, Germany.;Heidelberg Inst Theoret Studies, Heidelberg, Germany.;Heidelberg Univ, Interdisciplinary Ctr Sci Comp IWR, Heidelberg, Germany..
    von Heijne, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Stockholm Univ, Sci Life Lab, Solna, Sweden..
    Bukau, B.
    Univ Heidelberg ZMBH, Ctr Mol Biol, Heidelberg, Germany.;German Canc Res Ctr, Heidelberg, Germany..
    Kramer, G.
    Univ Heidelberg ZMBH, Ctr Mol Biol, Heidelberg, Germany.;German Canc Res Ctr, Heidelberg, Germany..
    Genome-wide ribosome profiling of bacterial SRP interaction with nascent polypeptides2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, p. 39-39Article in journal (Refereed)
  • 39. Sharma, Atul
    et al.
    Nitharwal, G
    Singh, Bhupender
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Dar, Ashraf
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Dhar, Suman K.
    Helicobacter pylori single-stranded DNA binding protein - functional characterization and modulation of H-pylori DnaB helicase activity2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, p. 519-531Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpDnaB proteins were co-localized in sharp, distinct foci in exponentially growing H. pylori cells, whereas both were spread over large areas in its dormant coccoid form, suggesting the absence of active replication forks in the latter. These results confirm the multiple roles of SSB during DNA replication and provide evidence for altered replicative metabolism in the spiral and coccoid forms that may be central to the bacterial physiology and pathogenesis.

  • 40.
    Sharma, Atul
    et al.
    Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
    Nitharwal, Ram G.
    Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
    Singh, Bhupender
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Dar, Ashraf
    Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Dhar, Suman K.
    Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
    Helicobacter pylori single-stranded DNA bindingprotein – functional characterization and modulationof H. pylori DnaB helicase activity2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, p. 519-531Article in journal (Refereed)
  • 41.
    Sooriyaarachchi, Sanjeewani
    et al.
    Department of Molecular Biology, SLU.
    Ubhayasekera, Wimal
    Department of Molecular Biology, SLU.
    Boos, Winfried
    Department of Biology, University of Konstanz, Germany.
    Mowbray, Sherry
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    X-ray structure of glucose/galactose receptor from Salmonella typhimurium in complex with the physiological ligand, (2R)-glyceryl-β-d- galactopyranoside2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 7, p. 2116-2124Article in journal (Refereed)
    Abstract [en]

    Periplasmic binding proteins are abundant in bacteria by virtue of their essential roles as high-affinity receptors in ABC transport systems and chemotaxis. One of the best studied of these receptors is the so-called glucose/galactose-binding protein (GBP). In the present paper, we report the x-ray structure of the Salmonella typhimurium protein bound to the physiologically relevant ligand, (2R)-glyceryl-b-D-galactopyranoside (GGal), solved by molecular replacement, and refined to 1.87 Å resolution with R and R-free values of 17% and 22%. The structure identifies three amino acid residues that are diagnostic of GGal binding (Thr110, Asp154 and Gln261), as opposed to binding to the monosaccharides, glucose and galactose. These three residues are conserved in essentially all available GBP sequences, indicating that the binding of GGal is the rule rather than the exception for receptors of this type. The role of GGal in bacterial biology is discussed. Further, comparison of the available structures provides the most complete description of the conformational changes of GBP to date. The structures follow a smooth and continuous path from the most closed structure (that bound to GGal) to the most open one (an apo structure).

  • 42.
    Sreedharan, Smitha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Sällman Almén, Markus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Carlini, Valeria P
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Haitina, Tatjana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Stephansson, Olga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Sommer, Wolfgang H
    Department of Psychopharmacology, Central institute of Mental Health, Mannheim, Germany.
    Heilig, Marcus
    Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA.
    de Barioglio, Susan R
    Departamento de Farmacología, Universidad Nacional de Córdoba, Argentina.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Schiöth, Helgi B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 24, p. 4881-4894Article in journal (Refereed)
    Abstract [en]

    The Rhodopsin family of G protein-coupled receptors (GPCRs) includes the phylogenetic α-group consisting of about 100 human members. The α-group is the only group of GPCRs that has many receptors for biogenic amines which are major drug targets. Several members of this group are orphan receptors and their functions are elusive. In this study we present a detailed phylogenetic and anatomical characterization of the Gpr153 receptor and also attempted to study its functional role. We identified the homologue of GPR153 in the elephant shark genome and phylogenetic and synteny analyses revealed that Gpr162 originated from Gpr153, through a duplication event before the radiation of the amphibian lineage. Quantitative real time PCR study reveals wide spread expression of GPR153 in the CNS and all the peripheral tissues investigated. Detailed in situ hybridization on mouse brain showed specifically high expression in the thalamus, cerebellum and the arcuate nucleus. The antisense oligodeoxynucleotide knockdown of GPR153 caused a slight reduction in food intake and the elevated plus maze test showed significant reduction in the percentage of time spent in the centre square, which points towards a probable role in decision making. This report provides the first detailed characterization of the evolution, expression and as well as primary functional properties of the GPR153 gene.

  • 43.
    Stasyk, Taras
    et al.
    Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
    Lutsik-Kordovsky, Maxim
    Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Antonyuk, Volodymyr
    Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
    Klyuchivska, Olga
    Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
    Souchelnytskyi, Serhiy
    Karolinska Biomics Center, Karolinska Institutet, Stockholm, Sweden.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Stoika, Rostyslav
    Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
    A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 5, p. 1260-1269Article in journal (Refereed)
    Abstract [en]

    A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21-32]. The physico-chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl-Asp(OMe)-fluoromethylketone] did not stop toxophallin-induced DNA fragmentation. Thus, toxophallin uses a caspase-independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR-based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l-amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l-amino acid oxidase.

  • 44. Stern, Ana Laura
    et al.
    Naworyta, Agata
    Cazzulo, Juan J.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structures of type B ribose 5-phosphate isomerase from Trypanosoma cruzi shed light on the determinants of sugar specificity in the structural family2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 5, p. 793-808Article in journal (Refereed)
    Abstract [en]

    Ribose-5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key activity of the pentose phosphate pathway. Two unrelated types of sequence/structure possess this activity: type A Rpi (present in most organisms) and type B Rpi (RpiB) (in some bacteria and parasitic protozoa). In the present study, we report enzyme kinetics and crystallographic studies of the RpiB from the human pathogen, Trypanosoma cruzi. Structures of the wild-type and a Cys69Ala mutant enzyme, alone or bound to phosphate, D-ribose 5-phosphate, or the inhibitors 4-phospho-D-erythronohydroxamic acid and D-allose 6-phosphate, highlight features of the active site, and show that small conformational changes are linked to binding. Kinetic studies confirm that, similar to the RpiB from Mycobacterium tuberculosis, the T. cruzi enzyme can isomerize D-ribose 5-phosphate effectively, but not the 6-carbon sugar D-allose 6-phosphate; instead, this sugar acts as an inhibitor of both enzymes. The behaviour is distinct from that of the more closely related (to T. cruzi RpiB) Escherichia coli enzyme, which can isomerize both types of sugars. The hypothesis that differences in a phosphate-binding loop near the active site were linked to the differences in specificity was tested by construction of a mutant T. cruzi enzyme with a sequence in this loop more similar to that of E. coli RpiB; this mutant enzyme gained the ability to act on the 6-carbon sugar. The combined information allows us to distinguish the two types of specificity patterns in other available sequences. The results obtained in the present study provide insights into the action of RpiB enzymes generally, and also comprise a firm basis for future work in drug design.

  • 45. Suder, Piotr
    et al.
    Bierczynska-Krzysik, Anna
    Kraj, Agnieszka
    Brostedt, Peter
    Mak, Pawel
    Stawikowski, Maciej
    Rolka, Krzysztof
    Nyberg, Fred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fries, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Silberring, Jerzy
    Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 22, p. 5113-5120Article in journal (Refereed)
    Abstract [en]

    Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K-M value of the convertase was found to be 9 mu M, and the K-i value of the inhibitor was 1.7 nM. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

  • 46.
    Sutton, Vivien R.
    et al.
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia..
    Brennan, Amelia J.
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia..
    Ellis, Sarah
    Peter MacCallum Canc Ctr, Microscopy & Histol, East Melbourne, Vic, Australia..
    Danne, Jill
    Peter MacCallum Canc Ctr, Microscopy & Histol, East Melbourne, Vic, Australia..
    Thia, Kevin
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia..
    Jenkins, Misty R.
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia..
    Voskoboinik, Ilia
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia..
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden..
    Johnstone, Ricky W.
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic 3010, Australia..
    Andrews, Daniel M.
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia..
    Trapani, Joseph A.
    Peter MacCallum Canc Ctr, Canc Cell Death Killer Cell Biol Labs, East Melbourne, Vic, Australia.;Univ Melbourne, Sir Peter MacCallum Dept Oncol, Melbourne, Vic 3010, Australia..
    Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 5, p. 947-961Article in journal (Refereed)
    Abstract [en]

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+)CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.

  • 47. Theocharis, Achilleas D.
    et al.
    Skandalis, Spyridon S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Tzanakakis, George N.
    Karamanos, Nikos K.
    Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 19, p. 3904-3923Article, review/survey (Refereed)
    Abstract [en]

    The expression of proteoglycans (PGs), essential macromolecules of the tumor microenvironment, is markedly altered during malignant transformation and tumor progression. Synthesis of stromal PGs is affected by factors secreted by cancer cells and the unique tumor-modified extracellular matrix may either facilitate or counteract the growth of solid tumors. The emerging theme is that this dual activity has intrinsic tissue specificity. Matrix-accumulated PGs, such as versican, perlecan and small leucine-rich PGs, affect cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Furthermore, expression of cell-surface-associated PGs, such as syndecans and glypicans, is also modulated in both tumor and stromal cells. Cell-surface-associated PGs bind various factors that are involved in cell signaling, thereby affecting cell proliferation, adhesion and motility. An important mechanism of action is offered by a proteolytic processing of cell-surface PGs known as ectodomain shedding of syndecans; this facilitates cancer and endothelial cell motility, protects matrix proteases and provides a chemotactic gradient of mitogens. However, syndecans on stromal cells may be important for stromal cell/cancer cell interplay and may promote stromal cell proliferation, migration and angiogenesis. Finally, abnormal PG expression in cancer and stromal cells may serve as a biomarker for tumor progression and patient survival. Enhanced understanding of the regulation of PG metabolism and the involvement of PGs in cancer may offer a novel approach to cancer therapy by targeting the tumor microenvironment. In this minireview, the implication of PGs in cancer development and progression, as well as their pharmacological targeting in malignancy, are presented and discussed.

  • 48. Ubhayasekera, Wimal
    et al.
    Tang, Ce Mun
    Ho, Sharon W. T.
    Berglund, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Chye, Mee-Len
    Mowbray, Sherry L.
    Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 14, p. 3695-3703Article in journal (Refereed)
    Abstract [en]

    Brassica juncea chitinase is an endo-acting, pathogenesis-related protein that is classified into glycoside hydrolase family 19, with highest homology (50–60%) in its catalytic domain to class I plant chitinases. Here we report X-ray structures of the chitinase catalytic domain from wild-type (apo, as well as with chloride ions bound) and a Glu234Ala mutant enzyme, solved by molecular replacement and refined at 1.53, 1.8 and 1.7 Å resolution, respectively. Confirming our earlier mutagenesis studies, the active-site residues are identified as Glu212 and Glu234. Glu212 is believed to be the catalytic acid in the reaction, whereas Glu234 is thought to have a dual role, both activating a water molecule in its attack on the anomeric carbon, and stabilizing the charged intermediate. The molecules in the various structures differ significantly in the conformation of a number of loops that border the active-site cleft. The differences suggest an opening and closing of the enzyme during the catalytic cycle. Chitin is expected to dock first near Glu212, which will protonate it. Conformational changes then bring Glu234 closer, allowing it to assist in the following steps. These observations provide important insights into catalysis in family 19 chitinases.

  • 49.
    Westermark, Gunilla T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Localized amyloids important in diseases outside the brain: lessons from the islets of Langerhans and the thoracic aorta2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 20, p. 3918-3929Article, review/survey (Refereed)
    Abstract [en]

    It has long been understood that amyloids can be lethal in systemic diseases. More recently, it has been accepted that local cerebral aggregation of the small peptide A beta is involved in the pathogenesis of Alzheimer's disease. Protein aggregation, with the generation of small amyloid deposits in specific organs, also occurs outside the central nervous system and often is associated with increased cell death. In this review, we discuss two lesser known but common localized amyloid fibril-forming proteins: the polypeptide hormone islet amyloid polypeptide (IAPP) and the lactadherin-derived peptide medin. IAPP aggregates and induces the depletion of islet beta-cells in type 2 diabetes and in islets transplanted into type 1 diabetic subjects. Initial amyloid deposition occurs intracellularly and parts of this amyloid consist of proIAPP. Medin derived from lactadherin expressed by smooth muscle cells aggregates into amyloid in certain arteries, particularly the thoracic aortic media layer, and may have a role in the generation of the potentially lethal conditions of thoracic aortic aneurysm and dissection.

  • 50.
    Xiong, Anqi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kundu, Soumi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Forsberg-Nilsson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Heparan sulfate in the regulation of neural differentiation and glioma development2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 22, p. 4993-5008Article, review/survey (Refereed)
    Abstract [en]

    Heparan sulfate proteoglycans (HSPGs) are the main components of the extracellular matrix, where they interact with a large number of physiologically important macromolecules. The sulfation pattern of heparan sulfate (HS) chains determines the interaction potential of the proteoglycans. Enzymes of the biosynthetic and degradation pathways for HS chains are thus important regulators in processes ranging from embryonic development to tissue homeostasis, but also for tumor development. Formation of the nervous system is also critically dependent on intact HSPGs, and several studies have outlined the role of HS in neural induction from embryonic stem cells. High-grade glioma is the most common malignant primary brain tumor among adults, and the outcome is poor. Neural stem cells and glioma stem cells have several common traits, such as sustained proliferation and a highly efficient migratory capacity in the brain. There are also similarities between the neurogenic niche where adult neural stem cells reside, and the tumorigenic niche. These include interactions with the extracellular matrix, and many of the matrix components are deregulated in glioma, e.g. HSPGs and enzymes implementing the biosynthesis and modification of HS. In this article, we will present how HS-regulated pathways are involved in neural differentiation, and discuss their impact on brain development. We will also review and critically discuss the important role of structural modifications of HS in glioma growth and invasion. We propose that targeting invasive mechanisms of glioma cells through modulation of HS structure and HS-mediated pathways may be an attractive alternative to other therapeutic attempts, which so far have only marginally increased survival for glioma patients.

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