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  • 1. Bergh, F T
    et al.
    Flinn, Elisabeth M
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Svaren, J
    Wright, Anthony P
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Horz, W
    Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 12, p. 9035-9042Article in journal (Refereed)
    Abstract [en]

    Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR, Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

  • 2. Brooks, Andrew J
    et al.
    Johansson, Magnus
    James Cook University.
    John, Anna V
    Xu, Yibin
    Jans, David A
    Vasudevan, Subhash G
    The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 39, p. 36399-36407Article in journal (Refereed)
    Abstract [en]

    Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.

  • 3. Cunnea, P M
    et al.
    Miranda-Vizuete, A
    Bertoli, G
    Simmen, T
    Damdimopoulos, A E
    Hermann, Stefan
    Södertörn University, Avdelning Naturvetenskap.
    Leinonen, S
    Huikko, M P
    Gustafsson, J A
    Sitia, R
    Spyrou, G
    ERdJ5, an endoplasmic reticulum (ER)-resident protein containing DnaJ and thioredoxin domains, is expressed in secretory cells or following ER stress2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 2, p. 1059-1066Article in journal (Refereed)
  • 4. Deadman, Mary E.
    et al.
    Lundström, Susanna L.
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences.
    Moxon, E. Richard
    Hood, Derek W.
    Specific amino acids of the glycosyltransferase LpsA direct the addition of glucose or galactose to the terminal inner core heptose of Haemophilus influenzae lipopolysaccharide via alternative linkages2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 40, p. 29455-29467Article in journal (Refereed)
    Abstract [en]

    Lipopolysaccharide is the major glycolipid of the cell wall of the bacterium Haemophilus influenzae, a Gram-negative commensal and pathogen of humans. Lipopolysaccharide is both a virulence determinant and a target for host immune responses. Glycosyltransferases have high donor and acceptor substrate specificities that are generally limited to catalysis of one unique glycosidic linkage. The H. influenzae glycosyltransferase LpsA is responsible for the addition of a hexose to the distal heptose of the inner core of the lipopolysaccharide molecule and belongs to the glycosyltransferase family 25. The hexose added can be either glucose or galactose and linkage to the heptose can be either beta 1-2 or beta 1-3. Each H. influenzae strain uniquely produces only one of the four possible combinations of linked sugar in its lipopolysaccharide. We show that, in any given strain, a specific allelic variant of LpsA directs the anomeric linkage and the added hexose, glucose, or galactose. Site-directed mutagenesis of a single key amino acid at position 151 changed the hexose added in vivo from glucose to galactose or vice versa. By constructing chimeric lpsA gene sequences, it was shown that the 3' end of the gene directs the anomeric linkage (beta 1-2 or beta 1-3) of the added hexose. The lpsA gene is the first known example where interstrain variation in lipopolysaccharide core structure is directed by the specific sequence of a genetic locus encoding enzymes directing one of four alternative possible sugar additions from the inner core.

  • 5.
    Elgan, Tobias H.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Berndt, Kurt D.
    Södertörn University, School of Life Sciences. Karolinska Insitute.
    Quantifying Escherichia coli Glutaredoxin-3 Substrate Specificity Using Ligand-induced Stability2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 47, p. 32839-32847Article in journal (Refereed)
    Abstract [en]

    Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N- and C-terminal of glutathione, van der Waals interactions from the unique gamma-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.

  • 6.
    Ferreira, Monica E
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Hermann, Stefan
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Prochasson, P
    Stowers Institute for Medical Research, Kansas City, USA.
    Workman, J L
    Stowers Institute for Medical Research, Kansas City, USA.
    Berndt, Kurt D
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Athony P H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Mechanism of transcription factor recruitment by acidic activators2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 23, p. 21779-21784Article in journal (Refereed)
    Abstract [en]

    Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.

  • 7. Filling, C
    et al.
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intitute.
    Benach, J
    Knapp, S
    Prozorovski, T
    Nordling, E
    Ladenstein, R
    Jörnvall, H
    Oppermann, U
    Critical residues for structure and catalysis in short-chain dehydrogenases/reductases2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 28, p. 25677-25684Article in journal (Refereed)
    Abstract [en]

    Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wildtype enzyme at 1.2-Angstrom resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.

  • 8.
    Flinn, Elizabeth M
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intstitutet.
    Wallberg, A E
    Hermann, Stefan
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Grant, P A
    Workman, J L
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Recruitment of Gen5-containing complexes during c-Myc-dependent gene activation - Structure and function aspects2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 26, p. 23399-23406Article in journal (Refereed)
    Abstract [en]

    The N-terminal domain of c-Myc plays a key role in cellular transformation and is involved in both activation and repression of target genes as well as in modulated proteolysis of c-Myc via the proteasome. Given this functional complexity, it has been difficult to clarify the structures within the N terminus that contribute to these different processes as well as the mechanisms by which they function. We have used a simplified yeast model system to identify the primary determinants within the N terminus for W chromatin remodeling of a promoter, (ii) gene activation from a chromatin template in vivo, and (iii) interaction with highly purified Gcn5 complexes as well as other chromatin-remodeling complexes in vitro. The results identify two regions that contain autonomous chromatin opening and gene activation activity, but both regions are required for efficient interaction with chromatin-remodeling complexes in vitro. The conserved Myc boxes do not play a direct role in gene activation, and Myc box II is not generally required for in vitro interactions with remodeling complexes. The yeast SAGA complex, which is orthologous to the human GCN5-TRRAP complex that interacts with Myc in human cells, plays a role in Myc-mediated chromatin opening at the promoter but may also be involved in later steps of gene activation.

  • 9.
    Hermann, Stefan
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap.
    How transcriptional activators bind target proteins2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 43, p. 40127-40132Article in journal (Refereed)
    Abstract [en]

    The product of the proto-oncogene c-myc influences many cellular processes through the regulation of specific target genes. Through its transactivation domain (TAD), c-Myc protein interacts with several transcription factors, including TATA-binding protein (TBP). We present data that suggest that in contrast to some other transcriptional activators, an extended length of the c-Myc TAD is required for its binding to TBP. Our data also show that this interaction is a multistep process, in which a rapidly forming low affinity complex slowly converts to a more stable form. The initial complex formation results from ionic or polar interactions, whereas the slow conversion to a more stable form is hydrophobic in nature. Based on our results, we suggest two alternative models for activation domain/target protein interactions, which together provide a single universal paradigm for understanding activator-target factor interactions.

  • 10. Hurme, R
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Namork, E
    Rhen, M
    DNA binding exerted by a bacterial gene regulator with an extensive coiled-coil domain1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 29, p. 12626-12631Article in journal (Refereed)
    Abstract [en]

    Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel, sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.

  • 11. Johansson, J
    et al.
    Gudmundsson, G H
    Rottenberg, M E
    Berndt, Kurt D
    Karolinska Institutet.
    Agerberth, B
    Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-371998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 6, p. 3718-3724Article in journal (Refereed)
    Abstract [en]

    The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO3-, SO42-, or CF3CO2- causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl- is less efficient, A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer, The extent of alpha-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL 37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered, In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 mu M, and at 13-25 mu M the peptide is cytotoxic against several eukaryotic cells, In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

  • 12. Juntti-Berggren, L
    et al.
    Webb, D L
    Arkhammar, P O G
    Schultz, V
    Schweda, Elke K H
    Södertörn University, Avdelning Naturvetenskap.
    Tornheim, K
    Berggren, P O
    Dihydroxyacetone-induced oscillations in cytoplasmic free Ca2+ and the ATP/ADP ratio in pancreatic beta-cells at substimulatory glucose2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 42, p. 40710-40716Article in journal (Refereed)
    Abstract [en]

    Glucose stimulation of pancreatic beta-cells causes oscillatory influx of Ca2+, leading to pulsatile insulin secretion. We have proposed that this is due to oscillations of glycolysis and the ATP/ADP ratio, which modulate the activity of ATP-sensitive K+ channels. We show here that dihydroxyacetone, a secretagogue that feeds into glycolysis below the putative oscillator phosphofructokinase, could cause a single initial peak in cytoplasmic free Ca2+ ([Ca2+](i)) but did not by itself cause repeated oscillations in [Ca2+](i) in mouse pancreatic beta-cells. However, in the presence of a substimulatory concentration of glucose (4 mM), dihydroxyacetone induced [Ca2+](i) oscillations. Furthermore, these oscillations correlated with oscillations in the ATP/ADP ratio, as seen previously with glucose stimulation. Insulin secretion in response to dihydroxyacetone was transient in the absence of glucose but was considerably enhanced and somewhat prolonged in the presence of a substimulatory concentration of glucose, in accordance with the enhanced [Ca2+](i) response. These results are consistent with the hypothesized role of phosphofructokinase as the generator of the oscillations. Dihydroxyacetone may affect phosphofructokinase by raising the free concentration of fructose 1,6-bisphosphate to a critical level at which it activates the enzyme autocatalytically, thereby inducing the pulses of phosphofructokinase activity that cause the metabolic oscillations.

  • 13. Karlsson, C.
    et al.
    Korayem, A. M.
    Scherfer, C.
    Loseva, O.
    Dushay, Mitchell S.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Theopold, U.
    Proteomic analysis of the Drosophila larval hemolymph clot2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 50, p. 52033-52041Article in journal (Refereed)
    Abstract [en]

    Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.

  • 14. Khorosjutina, Olga
    et al.
    Wanrooij, Paulina H.
    Walfridsson, Julian
    Södertörn University, School of Life Sciences, Molecular biology.
    Szilagyi, Zsolt
    Zhu, Xuefeng
    Baraznenok, Vera
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology.
    Gustafsson, Claes M.
    A Chromatin-remodeling Protein Is a Component of Fission Yeast Mediator2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 39, p. 29729-29737Article in journal (Refereed)
    Abstract [en]

    The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cells. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We here demonstrate that Med15 exists in a protein complex together with Hrp1, a CHD1 ATP-dependent chromatin-remodeling protein. The Med15-Hrp1 subcomplex is not a component of the core Mediator complex but can interact with the L-Mediator conformation. Deletion of med15(+) and hrp1(+) causes very similar effects on global steady-state levels of mRNA, and genome-wide analyses demonstrate that Med15 associates with a distinct subset of Hrp1-bound gene promoters. Our findings therefore indicate that Mediator may directly influence histone density at regulated promoters.

  • 15.
    Le Rouzic, E
    et al.
    Université Paris 5, Paris, France.
    Mousnier, A
    Université Paris VI and Université Paris VII, Paris, France.
    Rustum, Cecilia
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Stutz, F
    Université Paris 5, Paris, France.
    Hallberg, Einar
    Stockholm University.
    Dargemont, C
    Université Paris VI and Université Paris VII, Paris, France.
    Benichou, S
    Université Paris 5, Paris, France.
    Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG12002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 47, p. 45091-45098Article in journal (Refereed)
    Abstract [en]

    The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

  • 16. Lind, U
    et al.
    Greenidge, P
    Gillner, M
    Koehler, K F
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Carlstedt-Duke, J
    Functional probing of the human glucocorticoid receptor steroid-interacting surface by site-directed mutagenesis - Gln-642 plays an important role in steroid recognition and binding2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 25, p. 19041-19049Article in journal (Refereed)
    Abstract [en]

    To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17 alpha-OH group. This effect was alleviated by the presence of a 16 alpha-CH3 group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid, Met-EGO and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point.

  • 17. Lind, U.
    et al.
    Greenidge, P.
    Gustafsson, J. -A
    Wright, Anthony P. H.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Instiute.
    Carlstedt-Duke, J.
    Valine 571 functions as a regional organizer in programming the glucocorticoid receptor for differential binding of glucocorticoids and mineralocorticoids1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 26, p. 18515-18523Article in journal (Refereed)
    Abstract [en]

    The glucocorticoid receptor (GR) interacts specifically with glucocorticoids, whereas its closest relative, the mineralocorticoid receptor (MR), interacts with both glucocorticoids and mineralocorticoids, such as aldosterone. To investigate the mechanism underlying the glucocorticoid/mineralocorticoid specificity of the GR, we used a yeast model system to screen for GR ligand-binding domain mutants, substituted with MR residues in the segment 565-574, that can be efficiently activated by aldosterone. In all such increased activity mutants, valine 571 was replaced by methionine, even though most mutants also contained substitutions of other residues with their MR counterparts. Further analysis in yeast and COS-7 cells has revealed that the identity of residue 571 determines the behavior of other MR substituted residues in the 565-574 segment. Generally, MR substitutions in this region are only consistent with aldosterone binding if residue 571 is also replaced with methionine (MR conformation). If residue 571 is valine (GR conformation), most other MR substitution mutants drastically reduce interaction with both mineralocorticoid and glucocorticoid hormones. Based on these functional data, we hypothesize that residue 571 functions as a regional organizer involved in discriminating between glucocorticoid and mineralocorticoid hormones. We have used a molecular model of the GR ligand-binding domain in an attempt to interpret our functional data in structural terms.

  • 18.
    Schubert, Maria
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Petersson, Ulrika A
    Södertörn University, Avdelning Naturvetenskap. Stockholm University.
    Haas, B J
    Institute for Genomic Research, Rockville, USA.
    Funk, C
    Stockholm University.
    Schröder, Wolfgang P
    Södertörn University, Avdelning Naturvetenskap.
    Kieselbach, T
    Karolinska Institute.
    Proteome map of the chloroplast lumen of Arabidopsis thaliana2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 10, p. 8354-8365Article in journal (Refereed)
    Abstract [en]

    The thylakoid membrane of the chloroplast is the center of oxygenic photosynthesis. To better understand the function of the luminal compartment within the thylakoid network, we have carried out a systematic characterization of the luminal thylakoid proteins from the model organism Arabidopsis thaliana. Our data show that the thylakoid lumen has its own specific proteome, of which 36 proteins were identified. Besides a large group of peptidyl-prolyl cis-trans isomerases and pro. teases, a family of novel PsbP domain proteins was found. An analysis of the luminal signal peptides showed that 19 of 36 luminal precursors were marked by a twin-arginine motif for import via the Tat pathway. To compare the model organism Arabidopsis with another typical higher plant, we investigated the proteome from the thylakoid lumen of spinach and found that the luminal proteins from both plants corresponded well. As a complement to our experimental investigation, we made a theoretical prediction of the luminal proteins from the whole Arabidopsis genome and estimated that the thylakoid lumen of the chloroplast contains similar to80 proteins.

  • 19. Shi, L X
    et al.
    Lorkovic, Z J
    Oelmuller, R
    Schröder, Wolfgang P
    The low molecular mass PsbW protein is involved in the stabilization of the dimeric photosystem II complex in Arabidopsis thaliana2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 48, p. 37945-37950Article in journal (Refereed)
    Abstract [en]

    Arabidopsis thaliana plants have been transformed with an antisense gene to the psbW of photosystem II (PSII). Eight transgenic lines containing low levels of psbW mRNA have been obtained. Transgenic seedlings with low contents of PsbW protein (more than 96% reduced) were selected by Western blotting and used for photosynthetic functional studies. There were no distinct differences in phenotype between the antisense and wild type plants during vegetative period under normal growth light intensities. However, a sucrose gradient separation of briefly solubilized thylakoid membranes revealed that no dimeric PSII supracomplex could be detected in the transgenic plants lacking the PsbW protein. Furthermore, analysis of isolated thylakoids demonstrated that the oxygen-evolving rate in antisense plants decreased by 50% compared with the wild type, This was found to be due to up to 40% of D1 and D2 reaction center proteins of PSII disappearing in the transgenic plants. The absence of the PsbW protein also altered the contents of other PSII proteins to differing extents. These results show that in the absence of the PsbW protein, the stability of the dimeric PSII is diminished and consequently the total number of PSII complexes is greatly reduced. Thus the nuclear encoded PsbW protein may play a crucial role in the biogenesis and regulation of the photosynthetic apparatus.

  • 20. Sjöling, Sara
    et al.
    Eriksson, AnnaCarin
    Glaser, Elzbieta
    A helical element in the C-terminal domain of the N. plumbaginifolia F1 beta presequence is important for recognition by the mitochondrial processing peptidase1994In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 269, no 51, p. 32059-32062Article in journal (Refereed)
    Abstract [en]

    The mitochondrial processing peptidase (MPP) in lower eucaryots and mammals is a matrix enzyme, whereas MPP in plants constitutes an integral part of the bc1 complex of the respiratory chain. The isolated spinach leaf bc1 complex catalyzes cleavage of the precursor of Nicotiana plumbaginifolia F1 beta subunit of the ATP synthase, resulting in a production of mature protein and a presequence that consists of 54 amino acids. A synthetic peptide derived from the C-terminal part of the presequence, containing 17 amino acids with a helical structural element, p-F1 beta(38-54), was an efficient inhibitor of the processing, whereas a peptide derived from the N-terminal part of the presequence, p-F1 beta(1-18), was much less effective. ATIII, a helical peptide derived from antithrombin III, was not recognized by MPP. Synthetic peptides corresponding to 4, 6, and 11 amino acids of the C terminus of the presequence, p-F1 beta(51-54), p-F1 beta(49-54), and p-F1 beta(44-54) were almost completely inert. Competition studies show that MPP recognizes the C-terminal domain of the presequence rather than the N-terminal domain. Furthermore, the alpha-helical element of the C-terminal domain is shown to be required for the recognition event.

  • 21.
    Strömblad, Staffan
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Fotedar, A
    Brickner, H
    Theesfeld, C
    de Diaz, E A
    Friedlander, M
    Cheresh, D A
    Loss of p53 compensates for alpha(v)-integrin function in retinal neovascularization2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 16, p. 13371-13374Article in journal (Refereed)
    Abstract [en]

    alpha(v)-Integrin antagonists block neovascularization in various species, whereas 20% of alpha(v)-integrin null mice are born with many normal looking blood vessels. Given that blockade of alpha(v)-integrins during angiogenesis induces p53 activity, we utilized p53 null mice to elucidate whether loss of p53 can compensate for av-integrin function in neovascularization of the retina. Murine retinal vascularization was inhibited by systemic administration of an alpha(v)-integrin antagonist. In contrast, mice lacking p53 were refractory to this treatment, indicating that neovascularization in normal mice depends on alpha(v)-integrin-mediated suppression of p53. Blockade of alpha(v)-integrins during neovascularization resulted in an induction of p21(CIP1) in wild type and, surprisingly, in p53 null retinas, indicating that alpha(v)-integrin ligation regulates p21(CIP1) levels in a p53-independent manner. In conclusion, we demonstrate for the first time an in vivo intracellular mechanism for compensation of integrin function and that p53 and alpha(v)-integrins act in concert during retinal neovascularization.

  • 22. Tanudji, Marcel
    et al.
    Sjöling, Sara
    Glaser, Elzbieta
    Whelan, James
    Signals Required for the Import and Processing of the Alternative Oxidase into Mitochondria1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, p. 1286-1293Article in journal (Refereed)
    Abstract [en]

    The critical residues involved in targeting and processing of the soybean alternative oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean mitochondria indicated that positive residues throughout the length of the presequence were important for import, not just those in the predicted region of amphiphilicity. The position of the positive residues in the C-terminal end of the presequence was also important for import. Processing assays of the various constructs with purified spinach mitochondrial processing peptidase showed that all the −2-position mutants had a drastic effect on processing. In contrast to the import assay, the position of the positive residue could be changed for processing. Deletion mutants confirmed the site-directed mutagenesis data in that an amphiphilic α-helix was not the only determinant of mitochondrial import in this homologous plant system. Import of these constructs into rat liver mitochondria indicated that the degree of inhibition differed and that the predicted region of amphiphilic α-helix was more important with rat liver mitochondria. Processing with a rat liver matrix fraction showed little inhibition. These results are discussed with respect to targeting specificity in plant cells and highlight the need to carry out homologous studies and define the targeting requirements to plant mitochondria.

  • 23. Vargas, L
    et al.
    Nore, B F
    Berglöf, A
    Heinonen, J E
    Mattsson, P T
    Smith, C I E
    Mohamed, Abdalla J
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Functional interaction of caveolin-1 with Bruton's tyrosine kinase and Bmx2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 11, p. 9351-9357Article in journal (Refereed)
    Abstract [en]

    Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.

  • 24. Wärnmark, A
    et al.
    Wikström, Anja
    KTH.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Gustafsson, J A
    Härd, T
    The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 49, p. 45939-45944Article in journal (Refereed)
    Abstract [en]

    The N-terminal regions of the estrogen receptor ve (ER alpha -N) and beta (ER beta -N) were expressed and purified to homogeneity. Using NAM and circular dichroism spectroscopy, we conclude that both ER alpha -N and ER beta -N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ERa-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ERa-N interacts with TBP, and suggest that structural changes are induced in ERa-N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ER beta -N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ERa-N and ER beta -N. The affinity of the ER alpha -N-TBP interaction was determined to be in the micromolar range (K-D = 10(-6) to 10(-5) m). Our results support models of TBP as a target protein for the N-terminal activation domain of ER alpha. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.

  • 25.
    Wärnmark, Anette
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intsitute.
    Gustafsson, J A
    Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Architectural principles for the structure and function of the glucocorticoid receptor tau 1 core activation domain2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 20, p. 15014-15018Article in journal (Refereed)
    Abstract [en]

    A 58-amino acid region mediates the core transactivation activity of the glucocorticoid receptor tau 1 activation domain. This tau 1 core domain is unstructured in aqueous buffers, but in the presence of trifluoroethanol three Alpha-helical segments are induced. Two of these putative structural modules have been tested in different combinations with regard to transactivation potential in vivo and binding capacity to the coactivators in vitro, The results show that whereas single modules are not transcriptionally active, any combination of two or three modules is sufficient, with trimodular constructs having the highest activity. However, proteins containing one, two, or three segments bind Ada2 and cAMP-response element-binding protein with similar affinity. A single segment is thus able to bind a target factor but cannot transactivate target genes significantly. The results are consistent with models in which activation domains are comprised of short activation modules that allow multiple interactions with coactivators. Our results also suggest that an increased number of modules may not result in correspondingly higher affinity but instead that the concentration of binding sites is increased, which gives rise to a higher association rate. This is consistent with a model where the association rate for activator-target factor interactions rather than the equilibrium constant is the most relevant measure of activator potency.

  • 26.
    Xia, Ling
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Nordman, Tomas
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Olsson, J M
    Damdimopoulos, A
    Björkhem-Bergman, L
    Nalvarte, I
    Eriksson, L C
    Arner, E S J
    Spyrou, Giannis
    Södertörn University, Avdelning Naturvetenskap. Karolinska Instiutet.
    Björnstedt, Mikael
    Karolinska Institutet.
    The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone - A novel mechanism for defense against oxidative stress2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 4, p. 2141-2146Article in journal (Refereed)
  • 27. Åslund, F.
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Holmgren, A.
    Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 49, p. 30780-30786Article in journal (Refereed)
    Abstract [en]

    Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX1X2C). Standard state redox potential (E degrees ') values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E degrees ' resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E degrees ' values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E degrees ' = -233 and -198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E degrees ' values from a number of proteins ranging from -270 mV for E. coli Trx to -124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as -240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

  • 28. Åslund, F
    et al.
    Nordstrand, K
    Berndt, Kurt D
    Karolinska Institutet.
    Nikkola, M
    Bergman, T
    Ponstingl, H
    Jornvall, H
    Otting, G
    Holmgren, A
    Glutaredoxin-3 from Escherichia coli: Amino acid sequence, H-1 and N-15 NMR assignments, and structural analysis1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 12, p. 6736-6745Article in journal (Refereed)
    Abstract [en]

    The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. in addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys(65)) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific H-1 and N-15 nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, (3)J(HN alpha) coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low K-cat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grxl display large functional differences in in vitro protein disulfide oxide-reduction reactions.

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