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  • 1.
    Abelein, Axel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kaspersen, Jørn Døvling
    Nielsen, Søren Bang
    Jensen, Grethe Vestergaard
    Christiansen, Gunna
    Pedersen, Jan Skov
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Otzen, Daniel E.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates2013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 32, s. 23518-23528Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.

  • 2.
    Aboulaich, Nabila
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Vener, Alexander V
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Strålfors, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes2006Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 17, s. 11446-11449Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

  • 3.
    Adlerz, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holback, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Multhaup, Gerd
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    IGF-1-induced Processing of the Amyloid Precursor Protein Family Is Mediated by Different Signaling Pathways2007Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 14, s. 10203-10209Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.

  • 4.
    Alenius, Mattias
    et al.
    Umeå University.
    Bohm, S
    Umeå University.
    Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection1997Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, nr 42, s. 26083-26086Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin Ca-type domains followed by two fibronectin type III domains, Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane, These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II, In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections, The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.

  • 5.
    Alenius, Mattias
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå University.
    Bohm, Staffan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection1997Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, nr 42, s. 26083-26086Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe here the cloning of mouse complementary DNAs encoding a novel protein, Rb-8 neural cell adhesion molecule (RNCAM), with a predicted extracellular region of five immunoglobulin Ca-type domains followed by two fibronectin type III domains, Alternative splicing is likely to generate two RNCAM isoforms, which are differently attached to the cell membrane, These structural features and overall sequence identity identify this protein as a novel member of a cell adhesion molecule subgroup together with vertebrate neural cell adhesion molecule, Aplysia cell adhesion molecule, and Drosophila fasciclin II, In insects, fasciclin II is present on a restricted subset of embryonic central nervous system axons where it controls selective axon fasciculation. Intriguingly, RNCAM likewise is expressed in subsets of olfactory and vomeronasal neurons with topographically defined axonal projections, The spatial expression RNCAM corresponds precisely to that of certain odorant receptor expression zones of the olfactory epithelium. These expression patterns thus render RNCAM the first described cell adhesion molecule with a potential regulatory role in formation of selective axonal projections important for olfactory sensory information coding.

  • 6. Aleshkov, S B
    et al.
    Fa, M
    Karolin, J
    Strandberg, L
    Johansson, L B
    Wilczynska, M
    Ny, Tor
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.1996Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, nr 35, s. 21231-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

  • 7.
    Amagasaki, Kenichi
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Kaneto, Hideaki
    Osaka University Graduate School of Medicine, Department of Internal Medicine and Therapeutics (A8), 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
    Heldin, Carl-Henrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Lennartsson, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    c-Jun N-terminal kinase is necessary for platelet-derived growth factor-mediated chemotaxis in primary fibroblasts2006Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 31, s. 22173-22179Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.

  • 8.
    Ammoun, Sylwia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Lindholm, Dan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Wootz, Hanna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Åkerman, Karl E. O.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Kukkonen, Jyrki P.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    G-protein-coupled OX1 Orexin/hcrtr-1 Hypocretin Receptors Induce Caspase-dependent and -independent Cell Death through p38 Mitogen-/Stress-activated Protein Kinase2006Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, s. 834-842Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have investigated the signaling of OX1 receptors to cell death using Chinese hamster ovary cells as a model system. OX1 receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca2+ elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.

  • 9.
    Anandapadamanaban, Madhanagopal
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska fakulteten.
    Kyriakidis, Nikolaos C.
    Karolinska Univ Hosp, Sweden; UDLA, Ecuador.
    Csizmok, Veronika
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Wallenhammar, Amélie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Espinosa, Alexander C.
    Karolinska Univ Hosp, Sweden.
    Ahlner, Alexandra
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Round, Adam R.
    Grenoble Outstn, France; European XFEL GmbH, Germany.
    Trewhella, Jill
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten. Univ Sydney, Australia.
    Moche, Martin
    Karolinska Inst, Sweden.
    Wahren-Herlenius, Marie
    Karolinska Univ Hosp, Sweden.
    Sunnerhagen, Maria
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E22019Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 294, nr 30, s. 11404-11419Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-angstrom crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called linchpin residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.

  • 10. Andersen, Jan Terje
    et al.
    Pehrson, Rikard
    Tolmachev, Vladimir
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
    Daba, Muluneh Bekele
    Abrahmsén, Lars
    Ekblad, Caroline
    Extending Half-life by Indirect Targeting of the Neonatal Fc Receptor (FcRn) Using a Minimal Albumin Binding Domain2011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 7, s. 5234-5241Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The therapeutic and diagnostic efficiency of engineered small proteins, peptides, and chemical drug candidates is hampered by short in vivo serum half-life. Thus, strategies to tailor their biodistribution and serum persistence are highly needed. An attractive approach is to take advantage of the exceptionally long circulation half-life of serum albumin or IgG, which is attributed to a pH-dependent interaction with the neonatal Fc receptor (FcRn) rescuing these proteins from intracellular degradation. Here, we present molecular evidence that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn. We show that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent FcRn-albumin binding kinetics. The same result was obtained in the presence of IgG. An in vivo study performed in rat confirmed that the clinically relevant human epidermal growth factor 2 (HER2)-targeting Affibody molecule fused to ABD has a similar half-life and biodistribution profile as serum albumin. The proof-of-concept described may be broadly applicable to extend the in vivo half-life of short lived biological or chemical drugs ultimately resulting in enhanced therapeutic or diagnostic efficiency, a more favorable dosing regimen, and improved patient compliance.

  • 11.
    Andersson, M.
    et al.
    Karolinska Institute, Stockholm, Sweden.
    Holmgren, A.
    Karolinska Institute, Stockholm, Sweden.
    Spyrou, Giannis
    Karolinska Institute, Stockholm, Sweden and Karolinska Institute, Novum, Huddinge, Sweden.
    NK-lysin, a disulfide-containing effector peptide of T-lymphocytes, is reduced and inactivated by human thioredoxin reductase. Implication for a protective mechanism against NK-lysin cytotoxicity1996Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, nr 17, s. 10116-10120Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The cytotoxic and antibacterial polypeptide NK-lysin has a molecular mass of approximately 9 kDa and contains three disulfide bonds. The activity was highly dependent on intact disulfides, because the bactericidal effect on Escherichia coli and the cytolytic effect on human 3B6 lymphocytes was inhibited when NK-lysin was treated with dithiothreitol prior to incubation with the cells. NK-lysin was a direct substrate for human or calf thymus thioredoxin reductase and preincubation of the peptide with mammalian thioredoxin reductase, and NADPH abolished its antibacterial and cytolytic activities. The addition of human thioredoxin further enhanced the inhibitory effect of thioredoxin reductase and NADPH. In contrast, e. coli thioredoxin reductase showed no direct disulfide reductase activity with NK-lysin in agreement with previous data showing large differences in structure and substrate specificity between the mammalian and E. coli enzymes. NK-lysin is the first identified macromolecular disulfide substrate for human thioredoxin reductase apart from human thioredoxin. When 3B6 cells were incubated with NADPH, thioredoxin, and thioredoxin reductase prior to addition of NK-lysin, cytotoxicity was markedly reduced. These data suggest that thioredoxin reductase inactivates NK-lysin and provides a mechanism by which the cytotoxic activity of NK-lysin is regulated.

  • 12. Andres Valderrama, J
    et al.
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Carmona, Manuel
    Diaz, Eduardo
    AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp CIB2014Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, nr 4, s. 1892-1904Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp(60) phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N-6-TGCAT) present at two different locations within the P-N promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp(60) of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other -proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria.

  • 13.
    Andréasson, Claes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för cellbiologi.
    Rampelt, Heike
    Fiaux, Jocelyne
    Druffel-Augustin, Silke
    Bukau, Bernd
    The endoplasmic reticulum Grp170 acts as a nucleotide exchange factor of Hsp70 via a mechanism similar to that of the cytosolic Hsp112010Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 16, s. 12445-53Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.

  • 14.
    Annerén, Cecilia
    et al.
    Harvard University, Department of Molecular and Cellular Biology.
    Cowan, Chad A
    Harvard University, Department of Molecular and Cellular Biology.
    Melton, Douglas A
    Harvard University, Department of Molecular and Cellular Biology.
    The Src family of tyrosine kinases is important for embryonic stem cell self-renewal.2004Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 30, s. 31590-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human embryonic stem (ES) cells. We demonstrate that cYes kinase activity is regulated by leukemia inhibitory factor (LIF) and serum and is down-regulated when cells differentiate. Moreover, selective chemical inhibition of Src family kinases decreases growth and expression of stem cell genes that mark the undifferentiated state, including Oct3/4, alkaline phosphatase, fibroblast growth factor 4, and Nanog. A synergistic effect on differentiation is observed when ES cells are cultured with an Src family inhibitor and low levels of retinoic acid. Src family kinase inhibition does not interfere with LIF-induced JAK/STAT3 (Janus-associated tyrosine kinases/signal transducer and activator of transcription 3) or p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells.

  • 15. Ariza, A.
    et al.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Offen, W.A.
    Roberts, S.M.
    Wilson, K.S.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Davies, G.J.
    Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 442011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 39, s. 33890-33900Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-beta-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

  • 16. Arkhammar, P
    et al.
    Juntti-Berggren, L
    Larsson, O
    Welsh, M
    Nånberg, Eewa
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Sjöholm, A
    Köhler, M
    Berggren, P O
    Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels.1994Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 269, nr 4, s. 2743-2749Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse beta-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When beta-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca(2+)-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released.(ABSTRACT TRUNCATED AT 400 WORDS)

  • 17.
    Assarsson, Maria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, M E
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Persson, B O
    Sahlin, M
    Barra, A L
    Sjöberg, B M
    Nordlund, P
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.2001Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 29, s. 26852-26859Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

  • 18. Azarias, Guillaume
    et al.
    Kruusmagi, Markus
    Connor, Siobhan
    Akkuratov, Evgeny E.
    Liu, Xiao-Li
    Lyons, David
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Broberger, Christian
    Aperia, Anita
    A Specific and Essential Role for Na,K-ATPase alpha 3 in Neurons Co-expressing alpha 1 and alpha 32013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 4, s. 2734-2743Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous alpha 1, and the more selectively expressed alpha 3. Although neurological syndromes are associated with alpha 3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na+ imaging techniques to study the role of alpha 3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of alpha 3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na+ concentration ([Na+](i)) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+](i), similar to what could be expected following suprathreshold neuronal activity, selective inhibition of alpha 3 almost completely abolished the capacity to restore [Na+](i) in soma and dendrite. Recordings of Na, K-ATPase specific current supported the notion that when [Na+](i) is elevated in the neuron, alpha 3 is the predominant isoform responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of alpha 3 function in the central nervous system. The alpha isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na+ binding site. Following activation of protein kinase A, both the alpha 3-dependent current and restoration of dendritic [Na+](i) were significantly attenuated, indicating that alpha 3 is a target for phosphorylation and may participate in short term regulation of neuronal function.

  • 19.
    Bakali, Amin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Herman, Maria Dolores
    Johnson, Kenneth A.
    Kelly, Amélie A.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hallberg, B. M.
    Nordlund, Pär
    Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site2007Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 27, s. 19644-19652Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.

  • 20. Balciunas, Darius
    et al.
    Hallberg, Magnus
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ronne, Hans
    Functional interactions within yeast mediator and evidence of differential subunit modifications2003Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 6, s. 3831-3839Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain. To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains. We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested. lexA-Srb10 is a weak activator that depends on Srb8 and Srb11. lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator. Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms.

  • 21. Baldock, PA
    et al.
    Allison, SJ
    Lundberg, Pernilla
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral cellbiologi.
    Lee, NJ
    Slack, K
    Lin, EJ
    Enriquez, RF
    McDonald, MM
    Zhang, L
    During, MJ
    Little, DG
    Eisman, JA
    Gardiner, EM
    Yulyaningsih, E
    Lin, S
    Sainsbury, A
    Herzog, H
    Novel role of Y1 receptors in the coordinated regulation of bone and energy homeostasis.2007Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 26, s. 19092-19102Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The importance of neuropeptide Y (NPY) and Y2 receptors in the regulation of bone and energy homeostasis has recently been demonstrated. However, the contributions of the other Y receptors are less clear. Here we show that Y1 receptors are expressed on osteoblastic cells. Moreover, bone and adipose tissue mass are elevated in Y1(-/-) mice with a generalized increase in bone formation on cortical and cancellous surfaces. Importantly, the inhibitory effects of NPY on bone marrow stromal cells in vitro are absent in cells derived from Y1(-/-) mice, indicating a direct action of NPY on bone cells via this Y receptor. Interestingly, in contrast to Y2 receptor or germ line Y1 receptor deletion, conditional deletion of hypothalamic Y1 receptors in adult mice did not alter bone homeostasis, food intake, or adiposity. Furthermore, deletion of both Y1 and Y2 receptors did not produce additive effects in bone or adiposity. Thus Y1 receptor pathways act powerfully to inhibit bone production and adiposity by nonhypothalamic pathways, with potentially direct effects on bone tissue through a single pathway with Y2 receptors.

  • 22. Bambou, Jean-Christophe
    et al.
    Giraud, Antoine
    Menard, Sandrine
    Begue, Bernadette
    Rakotobe, Sabine
    Heyman, Martine
    Taddei, François
    Cerf-Bensussan, Nadine
    Gaboriau-Routhiau, Valérie
    In vitro and ex vivo activation of the TLR5 signaling pathway in intestinal epithelial cells by a commensal Escherichia coli strain.2004Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 41, s. 42984-92Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The capacity of non-pathogenic enteric bacteria to induce a pro-inflammatory response is under debate in terms of its effect on the symbiosis between the mammalian host and its commensal gut microflora. Activation of NF-kappaB and induction of interleukin-8 (IL-8) and CCL-20 by the commensal Escherichia coli strain MG1655 were first studied in vitro in the human intestinal epithelial cell (IECs) lines HT29-19A and Caco-2, transfected or not with plasmids encoding dominant negative Toll-like receptor (TLR) 5 and myeloid differentiation factor-88 (MyD88) adaptor protein. The response of enterocytes in situ was then assessed using murine ileal biopsies mounted in Ussing chambers. Commensal E. coli induced NF-kappaB DNA binding, NF-kappaB transcriptional activity, CCL-20 expression, and IL-8 secretion in the human IEC lines. E. coli MG1655 flagellin was necessary and sufficient to trigger this pro-inflammatory pathway via its interaction with TLR5 and the subsequent recruitment of the adaptor protein MyD88. Following epithelial cell polarization, signaling could be induced by live E. coli and flagellin on the apical side of HT29-19A. The in vivo relevance of our findings was confirmed, because immunohistochemical staining of murine ileum demonstrated expression of TLR5 in the apical part of enterocytes in situ. Furthermore, flagellin added on the mucosal side of murine ileal biopsies mounted in Ussing chambers induced a basolateral production of KC, a functional murine homolog of human IL-8. These findings provide strong evidence that flagellin released by flagellated commensal bacteria in the intestinal lumen can induce a pro-inflammatory response in enterocytes in vivo.

  • 23.
    Barkefors, Irmeli
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Fredlund Fuchs, Peder
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Heldin, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bergström, Tobias
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Forsberg-Nilsson, Karin
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kreuger, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells2011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 27, s. 24189-24199Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.

  • 24.
    Barkefors, Irmeli
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Le Jan, Sébastien
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Jakobsson, Lars
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Hejll, Eduar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Carlson, Gustav
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Johansson, Henrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Jarvius, Jonas
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Park, Jeong Won
    Li Jeon, Noo
    Kreuger, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis2008Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, nr 20, s. 13905-13912Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 microm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.

  • 25. Barnes, Brian R
    et al.
    Marklund, Stefan
    Steiler, Tatiana L
    Walter, Mark
    Hjälm, Göran
    Amarger, Valerie
    Mahlapuu, Margit
    Leng, Ying
    Johansson, Carina
    Galuska, Dana
    Lindgren, Kerstin
    Åbrink, Magnus
    Stapleton, David
    Zierath, Juleen R
    Andersson, Leif
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle2004Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 37, s. 38441-38447Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.

  • 26. Bart, Genevieve
    et al.
    Vico, Nuria Ortega
    Hassinen, Antti
    Pujol, Francois M.
    Deen, Ashik Jawahar
    Ruusala, Aino
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Tammi, Raija H.
    Squire, Anthony
    Heldin, Paraskevi
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kellokumpu, Sakari
    Tammi, Markku I.
    Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS32015Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 18, s. 11479-11490Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

  • 27.
    Bauer, M. K. A.
    et al.
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen.
    Vogt, M.
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen.
    Los, Marek Jan
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen, Germany.
    Siegel, J.
    Department of Virology, Albrecht-Ludwigs-University, Freiburg, Germany.
    Wesselborg, Sebastian
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen, Germany.
    Schulze-Osthoff, Klaus
    Department of Internal Medicine I, Medical Clinics, Eberhard-Karls-University, Tübingen.
    Role of reactive oxygen intermediates in activation-induced CD95 (APO-1/Fas) ligand expression1998Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, nr 14, s. 8048-8055Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Activation-induced cell death of T lymphocytes requires the inducible expression of CD95 (APO-1/Fas) ligand, which triggers apoptosis in CD95-bearing target cells by an autocrine or paracrine mechanism. Although execution of the CD95 death pathway is largely independent of reactive oxygen intermediates, activation-induced cell death is blocked by a variety of antioxidants. In the present study, we investigated the involvement of redox processes in the regulation of CD95 ligand (CD95L) expression in Jurkat T cells. We show that various antioxidants potently inhibited the transcriptional activation of CD95L following T cell receptor litigation or stimulation of cells with phorbol ester and ionomycin. Conversely, a prooxidant such as hydrogen peroxide alone was able to increase CD95L expression. As detected by Western blot and cytotoxicity assays, functional expression of CD95L protein was likewise diminished by antioxidants. Inhibition of CD95L expression was associated with a decreased DNA binding activity of nuclear factor (NF)-kappa B, an important redox-controlled transcription factor. Moreover, inhibition of NF-kappa B activity by a transdominant I kappa B mutant attenuated CD95L expression. Our data suggest that, although reactive oxygen intermediates do not act as mediators in the execution phase of CD95-mediated apoptosis, they are involved in the transcriptional regulation of CD95L expression.

  • 28.
    Belogurov, Georgiy A
    et al.
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Penttinen, Anni
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Huopalahti, Saila
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Baykov, Alexander A
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Lahti, Reijo
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl2002Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, nr 25Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.

  • 29.
    Bengoechea-Alonso, Maria T.
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Ericsson, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    A phosphorylation cascade controls the degradation of active SREBP12009Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, nr 9, s. 5885-5895Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

  • 30.
    Bengtson, Per
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Larson, Cecilia
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Larson, Göran
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene2001Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 34, s. 31575-31582Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLex) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLex and SLex-related epitopes. One individual showed lower levels of SLex expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg110 → Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. TheFUT7 Arg110 is conserved in all previously cloned vertebrate α1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of ≤1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLex and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

  • 31. Bengtsson, E
    et al.
    Aspberg, A
    Heinegård, D
    Sommarin, Y
    Spillmann, Dorothe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    The amino-terminal part of PRELP binds to heparin and heparan sulfate2000Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, nr 52, s. 40695-40702Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    PRELP (proline, arginine-rich end leucine-rich repeat protein) is an extracellular matrix leucine-rich repeat protein. The amino-terminal region of PRELP differs from that of other leucine-rich repeat proteins in containing a high number of proline and arginine residues. The clustered proline and basic residues are conserved in rat, bovine, and human PRELP. Although the function of PRELP is not yet known, the clustered arginine residues suggest a heparan sulfate/heparin-binding capacity. We show here that PRELP indeed binds heparin and heparan sulfate. Truncated PRELP without the amino-terminal region does not bind heparin. The dissociation constant for the interaction of PRELP with heparin was determined by an in solution binding assay and by surface plasmon resonance analysis to be in the range of 10-30 nm. A 6-mer heparin oligosaccharide was the smallest size showing binding to PRELP. The binding increased with increasing length up to an 18-mer and depended on the degree of sulfation of heparin as well as heparan sulfate. Sulfate groups at all positions were shown to be of importance for the binding. Fibroblasts bind PRELP, and this interaction is inhibited with heparin, suggesting a function for PRELP as a linker between the matrix and cell surface proteoglycans.

  • 32. Ben-Saadon, Ronen
    et al.
    Fajerman, Ifat
    Ziv, Tamar
    Hellman, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Schwartz, Alan L
    Ciechanover, Aaron
    The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system: Direct evidence for ubiquitination at the N-terminal residue2004Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 40, s. 41414-41421Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.

  • 33. Berg, Stefan
    et al.
    Edman, Maria
    Li, Lu
    Wikström, Malin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes: Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea2001Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 25, s. 22056-22063Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Synthesis of the nonbilayer-prone α-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the                     lipid surface charge density in the membrane ofAcholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive                     bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX7E catalytic motif of the CAZy family 4 of α-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from                      Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity                     for these proteins. However, alMGS and  spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol                     and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and                     spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis                     of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface                     binding and activity is proposed for this regulatory enzyme.

  • 34. Bergh, F T
    et al.
    Flinn, Elisabeth M
    Södertörns högskola, Avdelning Naturvetenskap. Karolinska Institute.
    Svaren, J
    Wright, Anthony P
    Södertörns högskola, Avdelning Naturvetenskap. Karolinska Institute.
    Horz, W
    Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor2000Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, nr 12, s. 9035-9042Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 35.
    Bergqvist, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sundström, Sara
    Karolinska Institutet.
    Dimberg, Lina Y.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Masucci, Maria G.
    Karolinska Institutet.
    The Hepatitis C Virus Core Protein Modulates T Cell Responses by Inducing Spontaneous and Altering T-cell Receptor-triggered Ca2+ Oscillations2003Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 21, s. 18877-18883Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infection, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Expression of the HCV core (C) protein modulates transcription of the IL-2 promoter in T lymphocytes by activating the nuclear factor of activated T lymphocyte (NFAT) pathway. Here we report on the effect of HCV C on Ca2+ signaling, which is essential for activation of NFAT. Expression of HCV C correlated with increased levels of cytosolic Ca2+ and spontaneous Ca2+ oscillations in transfected Jurkat cells. Triggering of the T-cell receptor induced a prolonged Ca2+ response characterized by vigorous high frequent oscillations in a high proportion of the responding cells. This was associated with decreased sizes and accelerated emptying of the intracellular calcium stores. The effect of HCV C on calcium mobilization was not dependent on phospholipase C-1 (PLC-) activity or increased inositol 1,4,5-trisphosphate (IP3) production and did not require functional IP3 receptors, suggesting that insertion of the viral protein in the endoplasmic reticulum membrane may be sufficient to promote Ca2+ leakage with dramatic downstream consequences on the magnitude and duration of the response. Our data suggest that expression of HCV C in infected T lymphocytes may contribute to the establishment of persistent infections by inducing Ca2+ oscillations that regulate both the efficacy and information content of Ca2+ signals and are ultimately responsible for induction of gene expression and functional differentiation.

  • 36.
    Bergqvist, Niclas
    et al.
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten.
    Nyman, Elin
    Linköpings universitet, Institutionen för medicinsk teknik, Avdelningen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten. AstraZeneca RandD, Sweden.
    Cedersund, Gunnar
    Linköpings universitet, Institutionen för medicinsk teknik, Avdelningen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten.
    Stenkula, Karin G.
    Lund University, Sweden.
    A systems biology analysis connects insulin receptor signaling with glucose transporter translocation in rat adipocytes2017Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, nr 27, s. 11206-11217Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type 2 diabetes is characterized by insulin resistance, which arises from malfunctions in the intracellular insulin signaling network. Knowledge of the insulin signaling network is fragmented, and because of the complexity of this network, little consensus has emerged for the structure and importance of the different branches of the network. To help overcome this complexity, systems biology mathematical models have been generated for predicting both the activation of the insulin receptor (IR) and the redistribution of glucose transporter 4 (GLUT4) to the plasma membrane. Although the insulin signal transduction between IR and GLUT4 has been thoroughly studied with modeling and time-resolved data in human cells, comparable analyses in cells from commonly used model organisms such as rats and mice are lacking. Here, we combined existing data and models for rat adipocytes with new data collected for the signaling network between IR and GLUT4 to create a model also for their interconnections. To describe all data (amp;gt;140 data points), the model needed three distinct pathways from IR to GLUT4: (i) via protein kinase B (PKB) and Akt substrate of 160 kDa (AS160), (ii) via an AS160-independent pathway from PKB, and (iii) via an additional pathway from IR, e.g. affecting the membrane constitution. The developed combined model could describe data not used for training the model and was used to generate predictions of the relative contributions of the pathways from IR to translocation of GLUT4. The combined model provides a systems-level understanding of insulin signaling in rat adipocytes, which, when combined with corresponding models for human adipocytes, may contribute to model-based drug development for diabetes.

  • 37.
    Bergström, Rosita
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Evolutionsbiologi.
    Savary, Katia
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Morén, Anita
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Guibert, Sylvain
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Evolutionsbiologi.
    Heldin, Carl-Henrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Ohlsson, Rolf
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Evolutionsbiologi.
    Moustakas, Aristidis
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Transforming growth factor β promotes complexes between Smad proteins and the CCCTC-binding factor on the H19 imprinting control region chromatin2010Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 26, s. 19727-19737Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Whether signal transduction pathways regulate epigenetic states in response to environmental cues remains poorly understood. We demonstrate here that Smad3, signaling downstream of transforming growth factor beta, interacts with the zinc finger domain of CCCTC-binding factor (CTCF), a nuclear protein known to act as "the master weaver of the genome." This interaction occurs via the Mad homology 1 domain of Smad3. Although Smad2 and Smad4 fail to interact, an alternatively spliced form of Smad2 lacking exon 3 interacts with CTCF. CTCF does not perturb well established transforming growth factor beta gene responses. However, Smads and CTCF co-localize to the H19 imprinting control region (ICR), which emerges as an insulator in cis and regulator of transcription and replication in trans via direct CTCF binding to the ICR. Smad recruitment to the ICR requires intact CTCF binding to this locus. Smad2/3 binding to the ICR requires Smad4, which potentially provides stability to the complex. Because the CTCF-Smad complex is not essential for the chromatin insulator function of the H19 ICR, we propose that it can play a role in chromatin cross-talk organized by the H19 ICR.

  • 38. Bergö, Martin
    et al.
    Wu, Gengshu
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Ruge, Toralph
    Olivecrona, Thomas
    Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on2002Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, s. 11927-11932Artikkel i tidsskrift (Fagfellevurdert)
  • 39. Bernardo, Lisandro
    et al.
    Johansson, Linda U M
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Skärfstad, Eleonore
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    σ54-promoter discrimination and regulation by ppGpp and DksA2009Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, nr 2, s. 828-838Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The sigma(54)-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of sigma(54)-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of sigma(54)-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of sigma(54)-dependent transcription in combination with a series of sigma(54)-promoters with graded affinities for sigma(54)-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted sigma(54)-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous sigma(54)-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive sigma(54)-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity sigma(54)-promoters that is conferred by the strong DNA binding properties of the P. putida sigma(54)-RNA polymerase holoenzyme.

  • 40.
    Bernert, Berit
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Porsch, Helena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Heldin, Paraskevi
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)2011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 49, s. 42349-42359Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.

  • 41. Birkenstock, Timo
    et al.
    Liebeke, Manuel
    Winstel, Volker
    Krismer, Bernhard
    Gekeler, Cordula
    Niemiec, Maria J
    Bisswanger, Hans
    Lalk, Michael
    Peschel, Andreas
    Exometabolome analysis identifies pyruvate dehydrogenase as a target for the antibiotic triphenylbismuthdichloride in multiresistant bacterial pathogens.2012Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 4, s. 2887-95Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The desperate need for new therapeutics against notoriously antibiotic-resistant bacteria has led to a quest for novel antibacterial target structures and compounds. Moreover, defining targets and modes of action of new antimicrobial compounds remains a major challenge with standard technologies. Here we characterize the antibacterial properties of triphenylbismuthdichloride (TPBC), which has recently been successfully used against device-associated infections. We demonstrate that TPBC has potent antimicrobial activity against many bacterial pathogens. Using an exometabolome profiling approach, a unique TPBC-mediated change in the metabolites of Staphylococcus aureus was identified, indicating that TPBC blocks bacterial pyruvate catabolism. Enzymatic studies showed that TPBC is a highly efficient, uncompetitive inhibitor of the bacterial pyruvate dehydrogenase complex. Our study demonstrates that metabolomics approaches can offer new avenues for studying the modes of action of antimicrobial compounds, and it indicates that inhibition of the bacterial pyruvate dehydrogenase complex may represent a promising strategy for combating multidrug-resistant bacteria.

  • 42. Bjorkhem, I
    et al.
    Araya, Zufan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Rudling, M
    Angelin, B
    Einarsson, C
    Wikvall, Kjell
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver: no coordinate regulation of CYP7A1 and CYP27A1.2002Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, nr 30, s. 26804-26807Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    It has been reported that there is a coordinate regulation of sterol 27-hydroxylase (CYP27A1) and cholesterol 7_-hydroxylase (CYP7A1) in rats. Thus, the levels of the mRNA corresponding to these two enzymes were found to change in the same direction in rat liver and in isolated rat hepatocytes. In contrast, other groups have not seen such regulation of CYP27A1 in rabbit liver or in rat liver when using an activity assay. In the present work, the effect of bile acid treatment on human CYP27A1/luciferase reporter activity was studied in a transient transfection assay in human liver-derived HepG2 cells. Neither the endogenous 27-hydroxylase activity nor the CYP27A1/luciferase reporter activity were down-regulated by treatment of HepG2 cells with chenodeoxycholic acid or taurochenodeoxycholic acid. We also measured CYP27A1 mRNA and CYP7A1 mRNA in liver of humans subjected to treatment with chenodeoxycholic acid, ursodeoxycholic acid, hydroxymethylglutaryl (HMG)-CoA reductase inhibitor and a combination of HMG-CoA reductase inhibitor and cholestyramine. There was a 60-fold variation in the levels of CYP7A1 mRNA but only a 5-fold variation in the levels of CYP27A1 mRNA. There was no correlation between the two mRNA species. It is concluded that, in humans, there is little or no coordinate regulation of CYP7A1 and CYP27A1 at the transcriptional level, and that CYP27A1 is not subject to a negative feedback control by bile acids. The results underline that marked species differences may exist in mechanisms for control of synthesis of bile acids and cholesterol homeostasis.

  • 43.
    Björkelid, Christofer
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Bergfors, Terese
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Raichurkar, Anand Kumar V.
    AstraZeneca India Private Limited.
    Mukherjee, Kakoli
    AstraZeneca India Private Limited.
    Malolanarasimhan, Krishnan
    AstraZeneca India Private Limited.
    Bandodkar, Balachandra
    AstraZeneca India Private Limited.
    Jones, T. Alwyn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase2013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 25, s. 18260-18270Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

  • 44.
    Blaukat, Andree
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Pizard, Anne
    Breit, Andreas
    Wernstedt, Christer
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Alhenc-Gelas, François
    Müller-Esterl, Werner
    Dikic, Ivan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function2001Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 44, s. 40431-40440Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.

  • 45. Boban, Mirta
    et al.
    Ljungdahl, Per O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Foisner, Roland
    Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation2015Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 4, s. 2489-2495Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to epsilon-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.

  • 46. Bocedi, Alessio
    et al.
    Fabrini, Raffaele
    Lo Bello, Mario
    Caccuri, Anna Maria
    Federici, Giorgio
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cornish-Bowden, Athel
    Ricci, Giorgio
    Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function2016Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, nr 52, s. 26739-26749Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of n(H) are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.

  • 47. Bodin, Karl
    et al.
    Andersson, Ulla
    Rystedt, Eva
    Ellis, Eva
    Norlin, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi, Biokemi.
    Pikuleva, Irina
    Eggertsen, Gösta
    Björkhem, Ingemar
    Diczfalusy, Ulf
    Metabolism of 4 beta -hydroxycholesterol in humans2002Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, nr 35, s. 31534-31540Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One of the major oxysterols in the human circulation is 4 beta-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4 beta-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7 alpha-, 27-, and 24-hydroxycholesterol to be approximately 0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4 beta-hydroxycholesterol. The apparent half-life of deuterium-labeled 4 beta-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4 beta-Hydroxycholesterol was converted into acidic products at a much slower rate than 7 alpha-hydroxycholesterol in primary human hepatocytes, and 4 beta-hydroxycholesterol was 7 alpha-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 beta-hydroxycholesterol. These results suggest that the high plasma concentration of 4 beta-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7 alpha-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4 beta-hydroxycholesterol as a ligand for the nuclear receptor LXR.

  • 48. Boesler, Carsten
    et al.
    Kruse, Janis
    Söderbom, Fredrik
    Department of Molecular Biology, Uppsala Biomedical Center, Swedish University of Agricultural Sciences, S-75124 Uppsala, Sweden .
    Hammann, Christian
    Sequence and generation of mature ribosomal RNA transcripts in Dictyostelium discoideum2011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 20, s. 17693-17703Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amoeba Dictyostelium discoideum is a well established model organism for studying numerous aspects of cellular and developmental functions. Its ribosomal RNA (rRNA) is encoded in an extrachromosomal palindrome that exists in ∼100 copies in the cell. In this study, we have set out to investigate the sequence of the expressed rRNA. For this, we have ligated the rRNA ends and performed RT-PCR on these circular RNAs. Sequencing revealed that the mature 26 S, 17 S, 5.8 S, and 5 S rRNAs have sizes of 3741, 1871, 162, and 112 nucleotides, respectively. Unlike the published data, all mature rRNAs of the same type uniformly display the same start and end nucleotides in the analyzed AX2 strain. We show the existence of a short lived primary transcript covering the rRNA transcription unit of 17 S, 5.8 S, and 26 S rRNA. Northern blots and RT-PCR reveal that from this primary transcript two precursor molecules of the 17 S and two precursors of the 26 S rRNA are generated. We have also determined the sequences of these precursor molecules, and based on these data, we propose a model for the maturation of the rRNAs in Dictyostelium discoideum that we compare with the processing of the rRNA transcription unit of Saccharomyces cerevisiae.

  • 49.
    Bohm, S.
    et al.
    Center for BioTechnology, NOVUM, Huddinge, Sweden.
    Bakke, M.
    Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden.
    Nilsson, M.
    Center for BioTechnology, NOVUM, Huddinge, Sweden.
    Zanger, U. M.
    Department of Pharmacology, Biocenter, Basel, Switzerland.
    Spyrou, Giannis
    Department of Medical Chemistry I, Karolinska Institute, Stockholm, Sweden.
    Lund, J.
    Center for BioTechnology, NOVUM, Huddinge, Sweden.
    Cooperating nonconsensus cAMP-responsive elements are mediators of adrenocorticotropin-induced VL30 transcription in steroidogenic adrenal cells1993Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 268, nr 6, s. 3952-3963Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pituitary-derived trophic hormones regulate cell-type-specific expression of VL30 retrotransposons in tissues that are engaged in steroidogenesis. We show that adrenocorticotropic hormone and forskolin induced VL30 transcription in the steroidogenic adrenal cell line Y1 and that the transcriptional activation was cell type- and protein kinase A-dependent. Three novel cAMP-responsive elements (CREs), within the VL30 long terminal repeat, were identified and shown to activate transcription synergistically when templates bearing multiple sites were compared with templates bearing a single site. This type of regulation was evident only in forskolin-treated cells, and the response elements were found to be inactive as mediators of constitutive transcription. In vitro binding analyses indicated that a consensus CRE and the nonconsensus VL30 CREs differ with respect to binding affinity and specificity to a number of nuclear factors that were identified to be related to proteins within the CREB, Jun, and C/EBP families of transcription factors. The relatively low affinity and/or a restricted binding specificity of the VL30 CREs made it possible to detect forskolin-induced binding of CREB- and Jun-related proteins to these sequences. We suggest that cAMP-induced transcription, specific for steroidogenic cells, can be mediated by a novel type of nonconsensus CREs and that the mechanism for this type of gene regulation is distinct from that mediated through a consensus CRE. We also report the identification of a novel factor, distinct from previously characterized CRE-binding proteins, that constitutively binds to the identified CREs.

  • 50.
    Borg, Anneli
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Holm, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Shiroyama, Ikue
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Hauryliuk, Vasili
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Pavlov, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Sanyal, Suparna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Ehrenberg, Måns
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome2015Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 6, s. 3440-3454Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

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