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  • 1.
    Alenius, Mattias
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University.
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 42, p. 26083-26086Article in journal (Refereed)
    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.

  • 2. Aleshkov, S B
    et al.
    Fa, M
    Karolin, J
    Strandberg, L
    Johansson, L B
    Wilczynska, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    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.1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 35, p. 21231-8Article in journal (Refereed)
    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.

  • 3. Andres Valderrama, J
    et al.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Carmona, Manuel
    Diaz, Eduardo
    AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp CIB2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 4, p. 1892-1904Article in journal (Refereed)
    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.

  • 4. Balciunas, Darius
    et al.
    Hallberg, Magnus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ronne, Hans
    Functional interactions within yeast mediator and evidence of differential subunit modifications2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 6, p. 3831-3839Article in journal (Refereed)
    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.

  • 5. Baldock, PA
    et al.
    Allison, SJ
    Lundberg, Pernilla
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Cell Biology.
    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.2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 26, p. 19092-19102Article in journal (Refereed)
    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.

  • 6. Bergö, Martin
    et al.
    Wu, Gengshu
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Ruge, Toralph
    Olivecrona, Thomas
    Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, p. 11927-11932Article in journal (Refereed)
  • 7. Bernardo, Lisandro
    et al.
    Johansson, Linda U M
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    σ54-promoter discrimination and regulation by ppGpp and DksA2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 2, p. 828-838Article in journal (Refereed)
    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.

  • 8. 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.2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 4, p. 2887-95Article in journal (Refereed)
    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.

  • 9.
    Botelho, Hugo M.
    et al.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    Leal, Sonia S.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    Cardoso, Isabel
    Molecular Neurobiology Unit, Instituto de Biologia Molecular e Celular, Porto, Portugal.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Fritz, Günter
    Department of Neuropathology, University of Freiburg, Germany.
    Gomes, Claudio M.
    Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
    S100A6 Amyloid Fibril formation is Calcium-modulated and enhances Superoxide Dismutase-1 (SOD1) aggregation2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 50, p. 42233-42242Article in journal (Refereed)
    Abstract [en]

    S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel beta-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metalmodulated aggregation propensity may be a key aspect in their physiology and function.

  • 10.
    Brattsand, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
    Egelrud, Torbjörn
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
    Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 42, p. 30033-40Article in journal (Refereed)
    Abstract [en]

    A new human 33-kDa serine protease was purified from human epidermis, and its cDNA was cloned from a keratinocyte library, from mRNA from a human keratinocyte line (HaCat) and from mRNA from human skin. Polyclonal antibodies specific for the new protein detected three groups of proteins in partially purified extracts of cornified eptihelium of human plantar skin. The three components are proposed to correspond to proenzyme, active enzyme, and proteolytically modified active enzyme. After N-deglycosylation, there was a decrease in apparent molecular mass of all detected components. Expression of the cloned cDNA in a eukaryotic virus-derived system yielded a recombinant protein that could be converted to an active protease by treatment with trypsin. Polymerase chain reaction analyses of cDNA from a number of human tissues showed high expression of the new enzyme in the skin and low expression in brain, placenta, and kidney. Homology searches yielded the highest score for porcine enamel matrix protease (55% amino acid sequence homology). High scores were also obtained for human and mouse neuropsin and for human stratum corneum chymotryptic enzyme. The function of this new protease, tentatively named stratum corneum tryptic enzyme, may be related to stratum corneum turnover and desquamation in the epidermis.

  • 11.
    Brännström, Kristoffer
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Segerman, Bo
    Gullberg, Martin
    Functional dissection of GTP hydrolysis and exchange within the ternary complex of tubulin heterodimers and Op18/stathmin family members2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 19, p. 16651-16657Article in journal (Refereed)
  • 12.
    Byström, Roberth
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersen, Peter M
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oliveberg, Mikael
    Stockholms universitet.
    SOD1 mutations targeting surface hydrogen bonds promote ALS without reducing apo-state stability2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 25, p. 19544-52Article in journal (Refereed)
    Abstract [en]

    In good accord with the protein-aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apo-SOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apo SOD1 is the elimination or introduction of a single charge, i.e., D76V/Y, D101N and N139D/K. The thermodynamic stability and folding behaviour of these mutants are indistinguishable from the wildtype control. Moreover, D101N is an outlier in the plot of stability loss vs. patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.

  • 13. Bárcena-Uribarri, Iván
    et al.
    Thein, Marcus
    Barbot, Mariam
    Sans-Serramitjana, Eulalia
    Bonde, Mari
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mentele, Reinhard
    Lottspeich, Friedrich
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 27, p. 18614-18624Article in journal (Refereed)
    Abstract [en]

    P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 M KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to +/-100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

  • 14.
    Cameron, Sarina R.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nandi, Soumyadeep
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kahn, Tatyana G.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Barrasa, Juan I.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Stenberg, Per
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Division of Chemical, Biological, Radioactive and Nuclear (CBRN) Security and Defence, FOI–Swedish Defence Research Agency, 906 21 Umeå Sweden.
    Schwartz, Yuri B.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    PTE, a novel module to target Polycomb Repressive Complex 1 to the human cyclin D2 (CCND2) oncogene2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 37, p. 14342-14358Article in journal (Refereed)
    Abstract [en]

    Polycomb group proteins are essential epigenetic repressors. They form multiple protein complexes of which two kinds, PRC1 and PRC2, are indispensable for repression. Although much is known about their biochemical properties, how mammalian PRC1 and PRC2 are targeted to specific genes is poorly understood. Here, we establish the cyclin D2 (CCND2) oncogene as a simple model to address this question. We provide the evidence that the targeting of PRC1 to CCND2 involves a dedicated PRC1-targeting element (PTE). The PTE appears to act in concert with an adjacent cytosine-phosphate-guanine (CpG) island to arrange for the robust binding of PRC1 and PRC2 to repressed CCND2. Our findings pave the way to identify sequence-specific DNA-binding proteins implicated in the targeting of mammalian PRC1 complexes and provide novel link between polycomb repression and cancer.

  • 15.
    Cava, Felipe
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Zafra, Olga
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Magalon, Axel
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
    Blasco, Francis
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 44, p. 45369-45378Article in journal (Refereed)
    Abstract [en]

    A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.

  • 16. Chai, Qian
    et al.
    Singh, Bhupender
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Peisker, Kristin
    Metzendorf, Nicole
    Ge, Xueliang
    Dasgupta, Santanu
    Sanyal, Suparna
    Organization of ribosomes and nucleoids in Escherichia coli cells during growth and in quiescence.2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 16, p. 11342-52Article in journal (Refereed)
    Abstract [en]

    We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

  • 17.
    Chilkova, Olga
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Jonsson, Bengt-Harald
    Johansson, Erik
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 16, p. 14082-14086Article in journal (Refereed)
  • 18.
    Conaway, H. Herschel
    et al.
    Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas.
    Pirhayati, Amir
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Persson, Emma
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Pettersson, Ulrika
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Pharmacology.
    Svensson, Olle
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Lindholm, Catharina
    Center for Bone and Arthritis Research at the Institute for Medicine, Sahlgrenska Academy at the University of Gothenburg.
    Henning, Petra
    Center for Bone and Arthritis Research at the Institute for Medicine, Sahlgrenska Academy at the University of Gothenburg.
    Tuckermann, Jan
    Tissue-specific Hormone Action, Leibniz Institute for Age Research, Fritz Lipmann Institute, D-07745 Jena, Germany.
    Lerner, Ulf H.
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Retinoids Stimulate Periosteal Bone Resorption by Enhancing the Protein RANKL: a Response Inhibited by Monomeric Glucocorticoid Receptor2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, p. 31425-31436Article in journal (Refereed)
    Abstract [en]

    Increased vitamin A (retinol) intake has been suggested to increase bone fragility. In the present study, we investigated effects of retinoids on bone resorption in cultured neonatal mouse calvarial bones and their interaction with glucocorticoids (GC). All-trans-retinoic acid (ATRA), retinol, retinalaldehyde, and 9-cis-retinoic acid stimulated release of (45)Ca from calvarial bones. The resorptive effect of ATRA was characterized by mRNA expression of genes associated with osteoclast differentiation, enhanced osteoclast number, and bone matrix degradation. In addition, the RANKL/OPG ratio was increased by ATRA, release of (45)Ca stimulated by ATRA was blocked by exogenous OPG, and mRNA expression of genes associated with bone formation was decreased by ATRA. All retinoid acid receptors (RAR alpha/beta/gamma) were expressed in calvarial bones. Agonists with affinity to all receptor subtypes or specifically to RAR alpha enhanced the release of (45)Ca and mRNA expression of Rankl, whereas agonists with affinity to RAR beta/gamma or RAR gamma had no effects. Stimulation of Rankl mRNA by ATRA was competitively inhibited by the RAR alpha antagonist GR110. Exposure of calvarial bones to GC inhibited the stimulatory effects of ATRA on 45Ca release and Rankl mRNA and protein expression. This inhibitory effect was reversed by the glucocorticoid receptor (GR) antagonist RU 486. Increased Rankl mRNA stimulated by ATRA was also blocked by GC in calvarial bones from mice with a GR mutation that blocks dimerization (GR(dim) mice). The data suggest that ATRA enhances periosteal bone resorption by increasing the RANKL/OPG ratio via RAR alpha receptors, a response that can be inhibited by monomeric GR.

  • 19.
    Costa, Tiago R D
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edqvist, Petra J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Åhlund, Monika K
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, p. 25269-25284Article in journal (Refereed)
    Abstract [en]

    YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.

  • 20. Crona, Mikael
    et al.
    Torrents, Eduard
    Rohr, Asmund K.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Furrer, Ernst
    Tomter, Ane B.
    Andersson, K. Kristoffer
    Sahlin, Margareta
    Sjoberg, Britt-Marie
    NrdH-Redoxin Protein Mediates High Enzyme Activity in Manganese-reconstituted Ribonucleotide Reductase from Bacillus anthracis2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 38, p. 33053-33060Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

  • 21. Descheemaeker, K A
    et al.
    Wyns, S
    Nelles, L
    Auwerx, J
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Collen, D
    Interaction of AP-1-, AP-2-, and Sp1-like proteins with two distinct sites in the upstream regulatory region of the plasminogen activator inhibitor-1 gene mediates the phorbol 12-myristate 13-acetate response.1992In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 267, no 21, p. 15086-91Article in journal (Refereed)
    Abstract [en]

    Phorbol 12-myristate 13-acetate induces a 3- and 10-fold induction of chloramphenicol acetyltransferase (CAT) activity in HT1080 and HeLa cells, respectively, following transient transfection of a 336-base pair plasminogen activator inhibitor-1 (PAI-1) promoter fragment linked to a CAT reporter gene. Substitution mutations in the regions encompassing nucleotides -78 to -69 (TGGGTGGGGC) or -61 to -54 (TGAGTTCA), but not in the regions -155 to -149 (TGCCTCA) or -84 to -76 (AGTGAGTGG) reduced this induction. Gel electrophoresis of double-stranded -65 to -50 oligonucleotides of the PAI-1 promoter region and nuclear extracts from Hela cells produced a gel shift pattern similar to that obtained with a AP-1 consensus oligomer, and excess unlabeled AP-1 oligomer reverted binding, suggesting that this region of the PAI-1 promoter is an AP-1-like binding site. Gel electrophoresis of double-stranded -82 to -65 oligonucleotides with HeLa nuclear extracts revealed a gel shift pattern of three bands; Sp1 consensus oligomer competed with the binding to two of these bands and AP-2 consensus sequence oligomer with the binding to the third band. The -82 to -65 oligomer also bound to purified AP-2 and Sp1 proteins. Southwestern blotting of HeLa nuclear extracts revealed that the labeled oligomer spanning region -82 to -65 bound to proteins with molecular masses of 52 and 72 kDa. Consensus AP-2 oligonucleotides competed for binding of the labeled -82 to -65 oligonucleotide to the 52-kDa protein, but consensus Sp-1 oligonucleotides did not compete for binding to the 72-kDa compound. The 72-kDa component binding to the -82 to -65 region may represent a new protein involved in transcriptional regulation.

  • 22. Desmarais, Samantha M
    et al.
    Tropini, Carolina
    Miguel, Amanda
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Monds, Russell D
    de Pedro, Miguel A
    Huang, Kerwyn Casey
    High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 52, p. 31090-31100Article in journal (Refereed)
    Abstract [en]

    The bacterial cell wall is a network of glycan strands crosslinked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

  • 23.
    Domkin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 21, p. 18574-8Article in journal (Refereed)
    Abstract [en]

    The ribonucleotide reductase system in Saccharomyces cerevisiae includes four genes (RNR1 and RNR3 encoding the large subunit and RNR2 and RNR4 encoding the small subunit). RNR3 expression, nearly undetectable during normal growth, is strongly induced by DNA damage. Yet an rnr3 null mutant has no obvious phenotype even under DNA damaging conditions, and the contribution of RNR3 to ribonucleotide reduction is not clear. To investigate the role of RNR3 we expressed and characterized the Rnr3 protein. The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. However, a strong synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated in experiments with the catalytically inactive Rnr1-C428A mutant, which increased the endogenous activity of Rnr3 by at least 10-fold. In vivo, the levels of Rnr3 after DNA damage never reached more than one-tenth of the Rnr1 levels. We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme, which may be important when Rnr1 is limiting for dNTP production. In complex with inactive Rnr1-C428A, the activity of Rnr3 is controlled by effector binding to Rnr1-C428A. This result indicates cross-talk between the Rnr1 and Rnr3 polypeptides of the large subunit.

  • 24. Dongre, Mitesh
    et al.
    Singh, Naorem Santa
    Dureja, Chetna
    Peddada, Nagesh
    Solanki, Ashish K
    Ashish, Ganguly
    Raychaudhuri, Saumya
    Evidence on how a conserved glycine in the hinge region of HapR regulates its DNA binding ability: lessons from a natural variant.2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17, p. 15043-9Article in journal (Refereed)
    Abstract [en]

    HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR(V2)) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapR(V2) to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR(V2) (HapR(V2G)), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR(V2) and HapR(V2G) proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

  • 25. Dossena, Silvia
    et al.
    Gandini, Rosaria
    Tamma, Grazia
    Vezzoli, Valeria
    Nofziger, Charity
    Tamplenizza, Margherita
    Salvioni, Elisabetta
    Bernardinelli, Emanuele
    Meyer, Giuliano
    Valenti, Giovanna
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fuerst, Johannes
    Paulmichl, Markus
    The molecular and functional interaction between ICLN and HSPCo38 modulates the regulation of cell volume2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 47, p. 40659-40670Article in journal (Refereed)
    Abstract [en]

    Identifying functional partners for protein/protein interactions can be a difficult challenge. We proposed the use of the operon structure of the Caenorhabditis elegans genome as a 'New Gene-Finding Tool' (Eichmueller et al, JBC, 2004, 279:7136) that could be functionally translated to the human system. Here we show the validity of this approach by studying the predicted functional interaction between ICln and HSPC038. In Caenorhabditis elegans, the gene encoding for the ICln homolog (icln-1) is embedded in an operon with two other genes, Nx (the human homolog of Nx is HSPC038) and Ny. ICln is a highly conserved, ubiquitously expressed multifunctional protein that plays a critical role in the regulatory volume decrease after cell swelling. Following hypotonic stress, ICln translocates from the cytosol to the plasma membrane, where it has been proposed to participate in the activation of the swelling induced chloride current (IClswell). Here we show that the interaction between human ICln and HSPC038 plays a role in volume regulation after cell swelling and that HSPC038 acts as an escort, directing ICln to the cell membrane after cell swelling and facilitating the activation of IClswell. Assessment of the NMR structure of HSPC038 showed the presence of a zinc finger motif. Moreover, NMR and additional biochemical techniques enabled us to identify the putative ICln/HSPC038 interacting sites, thereby explaining the functional interaction of both proteins on a molecular level.

  • 26. Dries, Daniel R.
    et al.
    Zhu, Yi
    Brooks, Mieu M.
    Forero, Diego A.
    Adachi, Megumi
    Cenik, Basar
    West, James M.
    Han, Yu-Hong
    Yu, Cong
    Arbella, Jennifer
    Nordin, Annelie
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Psychiatry.
    Adolfsson, Rolf
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Psychiatry.
    Del-Favero, Jurgen
    Lu, Q. Richard
    Callaerts, Patrick
    Birnbaum, Shari G.
    Yu, Gang
    Loss of Nicastrin from Oligodendrocytes Results in Hypomyelination and Schizophrenia with Compulsive Behavior2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 22, p. 11647-11656Article in journal (Refereed)
    Abstract [en]

    The biological underpinnings and the pathological lesions of psychiatric disorders are centuries-old questions that have yet to be understood. Recent studies suggest that schizophrenia and related disorders likely have their origins in perturbed neurodevelopment and can result from a large number of common genetic variants or multiple, individually rare genetic alterations. It is thus conceivable that key neurodevelopmental pathways underline the various genetic changes and the still unknown pathological lesions in schizophrenia. Here, we report that mice defective of the nicastrin subunit of gamma-secretase in oligodendrocytes have hypomyelination in the central nervous system. These mice have altered dopamine signaling and display profound abnormal phenotypes reminiscent of schizophrenia. In addition, we identify an association of the nicastrin gene with a human schizophrenia cohort. These observations implicate gamma-secretase and its mediated neurodevelopmental pathways in schizophrenia and provide support for the "myelination hypothesis" of the disease. Moreover, by showing that schizophrenia and obsessive-compulsive symptoms could be modeled in animals wherein a single genetic factor is altered, our work provides a biological basis that schizophrenia with obsessive-compulsive disorder is a distinct subtype of schizophrenia.

  • 27.
    Drobni, Mirva
    et al.
    Umeå University, Faculty of Medicine, Odontology.
    Olsson, Ing-Marie
    Umeå University, Faculty of Science and Technology, Chemistry.
    Eriksson, Christer
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Odontology, Cariology.
    Multivariate design and evaluation of a set of RGRPQ-derived innate immunity peptides2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 22, p. 15164-15171Article in journal (Refereed)
    Abstract [en]

    Oral commensal Streptococcus gordonii proteolytically cleave the salivary PRP-1 polypeptide into an RGRPQ innate peptide. The Arg and Gln termini are crucial for RGRPQ-mediated ammonia production and proliferation by S. gordonii SK12 and adhesion inhibition and desorption by Actinomyces naeslundii T14V, respectively. Here we have applied (i) a multivariate approach using RGRPQ-related peptides varied at amino acids 2, 3, and 4 simultaneously and (ii) size and N- and C-terminal modifications of RGRPQ to generate structure activity information. While the N-terminal arginine motif mediated ammonia production independent of peptide size, other responses required more or less full-length peptide motifs. The motifs for adhesion inhibition and desorption were the same. The adhesion and proliferation motifs required similarly a hydrophobic/low polarity amino acid 4 but differentially a hydrophilic or hydrophobic character of amino acids 2/3, respectively; polar peptides with small/hydrophilic and hydrophilic amino acids 2 and 3, respectively, had high adhesion inhibition/desorption activity, and lipophilic peptides with large/hydrophobic amino acids 2 and 3 had high proliferation activity. Accordingly, while RIWWQ had increased proliferation but abolished adhesion/desorption activity, peptides designed with hydrophilic amino acids 2 and 3 were predicted to behave in the opposite way. Moreover, a RGRPQ mimetic for all three responses should mimic small hydrophilic, large nitrogen-containing, and hydrophobic/low polarity amino acids 2, 3, and 4, respectively. Peptides fulfilling these criteria were 1-1.6-fold improved in all three responses. Thus, both mimetics and peptides with differential proliferation and adhesion activities may be generated for evaluation in biofilm models.

  • 28. Dukuzumuremyi, Jean-Marie
    et al.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Åkerström, Bo
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Schesser, Kurt
    The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 45, p. 35281-35290Article in journal (Refereed)
    Abstract [en]

    The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

  • 29. Edman, Maria
    et al.
    Berg, Stefan
    Storm, Patrik
    Wikström, Malin
    Vikström, Susanne
    Öhman, Anders
    Wieslander, Ake
    Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 10Article in journal (Refereed)
    Abstract [en]

    In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC ) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC ), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

  • 30.
    Eneqvist, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Lundberg, Erik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Karlsson, Anders
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Santos, Ceci­lia R A
    Power, Deborah M
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 25, p. 26411-6Article in journal (Refereed)
    Abstract [en]

    Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).

  • 31.
    Faraz, Mahmood
    et al.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Herdenberg, Carl
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Holmlund, Camilla
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Henriksson, Roger
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Hedman, Håkan
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 9, p. 3421-3435Article in journal (Refereed)
    Abstract [en]

    Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor and a negative regulator of several receptor tyrosine kinases. The molecular mechanisms by which LRIG1 mediates its tumor suppressor effects and regulates receptor tyrosine kinases remain incompletely understood. Here, we performed a yeast two-hybrid screen to identify novel LRIG1-interacting proteins and mined data from the BioPlex (biophysical interactions of ORFeome-based complexes) protein interaction data repository. The putative LRIG1 interactors identified in the screen were functionally evaluated using a triple co-transfection system in which HEK293 cells were co-transfected with platelet-derived growth factor receptor α, LRIG1, and shRNAs against the identified LRIG1 interactors. The effects of the shRNAs on the ability of LRIG1 to down-regulate platelet-derived growth factor receptor α expression were evaluated. On the basis of these results, we present an LRIG1 protein interaction network with many newly identified components. The network contains the apparently functionally important LRIG1-interacting proteins RAB4A, PON2, GAL3ST1, ZBTB16, LRIG2, CNPY3, HLA-DRA, GML, CNPY4, LRRC40, and LRIG3, together with GLRX3, PTPRK, and other proteins. In silico analyses of The Cancer Genome Atlas data sets revealed consistent correlations between the expression of the transcripts encoding LRIG1 and its interactors ZBTB16 and PTPRK and inverse correlations between the transcripts encoding LRIG1 and GLRX3. We further studied the LRIG1 function–promoting paraoxonase PON2 and found that it co-localized with LRIG1 in LRIG1-transfected cells. The proposed LRIG1 protein interaction network will provide leads for future studies aiming to understand the molecular functions of LRIG1 and the regulation of growth factor signaling.

  • 32. Feng, P
    et al.
    Ohlsson, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The structure of the TATA-less rat tissue-type plasminogen activator gene. Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.1990In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 265, no 4, p. 2022-7Article in journal (Refereed)
    Abstract [en]

    The genomic region carrying the rat tissue-type plasminogen activator (tPA) gene including its 5'-flanking sequence has been isolated and characterized by restriction enzyme analysis, Southern blotting, and DNA sequencing of all coding parts and the promoter region. The gene is approximately 25 kilobase pairs in size and comprises 14 exons separated by 13 introns. All the exon/intron boundaries agree with the GT-AG rule. The organization of the rat tPA gene is very similar to its human counterpart, and the location of the introns in the protein structure is identical to the human tPA gene. To characterize the promoter region, the transcription initiation site was identified by S1 nuclease protection experiments. A DNA fragment carrying 621 nucleotides of the 5'-flanking sequence was found to confer basal promoter activity and hormone responsiveness to a reporter gene construct in primary cultures of rat granulosa cells. Analysis of the rat tPA promoter sequence and a comparison with the human and mouse counterparts reveal several species-specific differences: the rat and mouse tPA promoters lack typical TATA and CAAT sequences found in the human tPA gene. Furthermore, the rat tPA promoter contains a consensus cAMP-responsive element shown to be required for cAMP responsiveness in eucaryotic genes. At the same position as the cAMP-responsive element in the rat gene, the mouse and human tPA genes have a 12-O-tetradecanoylphorbol-13-acetate-responsive element known to mediate activation by phorbol esters. The differences in the promoter sequences of the rat, mouse, and human tPA genes may have implications for the regulation of the tPA gene in different species.

  • 33.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA Polymerase epsilon Catalytic Core and Holoenzyme Have Comparable Catalytic Rates2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3825-3835Article in journal (Refereed)
    Abstract [en]

    The holoenzyme of yeast DNApolymerase ε (Pol ε) consists of four subunits– Pol2, Dpb2, Dpb3, and Dpb4. A proteasesensitivesite results in a N-terminalproteolytic fragment of Pol2, called Pol2core,that consists of the catalytic core of Pol ε andretains both polymerase and exonucleaseactivities. Pre-steady-state kinetics showedthat the exonuclease rates on single-stranded,double-stranded, and mismatched DNA werecomparable between Pol ε and Pol2core. Singleturnover pre-steady-state kinetics alsoshowed that the kpol of Pol ε and Pol2core werecomparable when pre-loading the polymeraseonto the primer-template before adding Mg2+and dTTP. However, a global fit of the dataover six sequential nucleotide incorporationsrevealed that the overall polymerization rateand processivity was higher for Pol ε than forPol2core. The largest difference was observedwhen challenged for the formation of aternary complex and incorporation of thefirst nucleotide. Pol ε needed less than asecond to incorporate a nucleotide, butseveral seconds passed before Pol2coreincorporated detectable levels of the firstnucleotide. We conclude that the accessorysubunits and the C-terminus of Pol2 do notinfluence the catalytic rate of Pol ε butfacilitate the loading and incorporation of thefirst nucleotide by Pol ε.

  • 34. Gangabadage, Chinthaka Saneth
    et al.
    Zdunek, Janusz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tessari, Marco
    Nilsson, Solveig
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Wijmenga, Sybren Sipke
    Structure and dynamics of human apolipoprotein CIII2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 25, p. 17416-17427Article in journal (Refereed)
    Abstract [en]

    Human apolipoprotein CIII (apoCIII) is a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins. ApoCIII inhibits lipoprotein lipase as well as binding of lipoproteins to cell surface heparan sulfate proteoglycans and receptors. High levels of apoCIII are often correlated with elevated levels of blood lipids (hypertriglyceridemia). Here, we report the three-dimensional NMR structure and dynamics of human apo-CIII in complex with SDS micelles, mimicking its natural lipid-bound state. Thanks to residual dipolar coupling data, the first detailed view is obtained of the structure and dynamics of an intact apolipoprotein in its lipid-bound state. ApoCIII wraps around the micelle surface as a necklace of six approximately 10-residue amphipathic helices, which are curved and connected via semiflexible hinges. Three positively charged (Lys) residues line the polar faces of helices 1 and 2. Interestingly, their three-dimensional conformation is similar to that of the low density lipoprotein receptor binding motifs of apoE/B and the receptor-associated protein. At the C-terminal side of apoCIII, an array of negatively charged residues lines the polar faces of helices 4 and 5 and the adjacent flexible loop. Sequence comparison shows that this asymmetric charge distribution along the solvent-exposed face of apoCIII as well as other structural features are conserved among mammals. This structure provides a template for exploration of molecular mechanisms by which human apoCIII inhibits lipoprotein lipase and receptor binding.

  • 35. Ge, Changrong
    et al.
    Georgiev, Alexander
    Öhman, Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Wieslander, Åke
    Kelly, Amelie A.
    Tryptophan Residues Promote Membrane Association for a Plant Lipid Glycosyltransferase Involved in Phosphate Stress2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 8, p. 6669-6684Article in journal (Refereed)
    Abstract [en]

    Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

  • 36.
    Golovleva, Irina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Bhattacharya, Sanjoy
    Wu, Zhiping
    Shaw, Natacha
    Yang, Yanwu
    Andrabi, Khurshid
    West, Karen A
    Burstedt, Marie
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Forsman, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Holmgren, Gösta
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Sandgren, Ola
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Noy, Noa
    Quin, Jun
    Crabb, John W
    Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 14, p. 12397-12402Article in journal (Refereed)
    Abstract [en]

    Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.

  • 37. Gratz, Nina
    et al.
    Siller, Maria
    Schaljo, Barbara
    Pirzada, Zaid A
    Gattermeier, Irene
    Vojtek, Ivo
    Kirschning, Carsten J
    Wagner, Hermann
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kovarik, Pavel
    Akira, Shizuo
    Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR92008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 29, p. 19879-19887Article in journal (Refereed)
    Abstract [en]

    Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.

  • 38. Griese, Julia J
    et al.
    Kositzki, Ramona
    Schrapers, Peer
    Branca, Rui M M
    Nordström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lehtiö, Janne
    Haumann, Michael
    Högbom, Martin
    Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 42, p. 25254-25272Article in journal (Refereed)
    Abstract [en]

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn-II and Fe-II in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R.M., Lehtio , J., Graslund, A., Lubitz, W., Siegbahn, P. E., and Hogbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 1718917194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.

  • 39.
    Guittet, Olivier
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Håkansson, Pelle
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Voevodskaya, Nina
    Fridd, Susan
    Gräslund, Astrid
    Arakawa, Hirofumi
    Nakamura, Yusuke
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 44, p. 40647-40651Article in journal (Refereed)
  • 40. Gullberg, M
    et al.
    Noreus, K
    Brattsand, G
    Friedrich, B
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes.1990In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 265, no 29Article in journal (Refereed)
    Abstract [en]

    Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells.

  • 41.
    Gustavsson, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yuan, Ming
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Temporal dissection of beta1-integrin signaling indicates a role for p130Cas-Crk in filopodia formation.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 22, p. 22893-22901Article in journal (Refereed)
    Abstract [en]

    Invasin-promoted spreading of beta1-integrin-deficient cells, transfected with the beta1A- or beta1B-integrin splice variants, were used to dissect early beta1-integrin signaling events. The beta1B isoform, which has a different membrane-distal part of the cytoplasmic tail from beta1A, is defective in signaling and function. When plated on surfaces coated with the high affinity ligand invasin, beta1B-integrin-expressing cells spread by forming filopodia with distinct adhesive phosphotyrosine complexes at the tips, without signs of lamellipodia. This suggested that the beta1B-integrin mediated a partial signaling sufficient for formation of filopodia but insufficient for lamellipodia formation. When screening for proteins present in the distal filopodial phosphotyrosine complexes of beta1B cells, p130Cas and the filopodia proteins vasodilator-stimulated phosphoprotein and talin were found, whereas the typical focal complex proteins focal adhesion kinase, paxillin, and vinculin were not. Invasin-promoted adhesion induced complex formation of p130Cas and the adapter Crk. Moreover, Crk together with Dock180 were present at the filopodial tips of beta1B-integrin-expressing cells, and there was a prominent Rac1 activation. Expression of dominant negative variants of p130Cas or CrkII blocked beta1B-integrin-mediated filopodia formation, indicating that this signaling scaffold is central in this process.

  • 42. Hamidi, Anahita
    et al.
    von Bulow, Verena
    Hamidi, Rosita
    Winssinger, Nicolas
    Barluenga, Sofia
    Heldin, Carl-Henrik
    Landström, Marene
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Polyubiquitination of Transforming Growth Factor beta (TGF beta)-associated Kinase 1 Mediates Nuclear Factor-kappa B Activation in Response to Different Inflammatory Stimuli2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 1, p. 123-133Article in journal (Refereed)
    Abstract [en]

    The transcription factor nuclear factor kappa B (NF-kappa B) plays a central role in regulating inflammation in response to several external signals. The TGF beta-associated kinase 1 (TAK1) is an upstream regulator of NF-kappa B signaling. In TGF beta-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-kappa B activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1 beta. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-alpha and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGF beta, which in all cases resulted in NF-kappa B activation. Expression of a K34R-mutant TAK1 led to a reduced NF-kappa B activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-alpha-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-kappa B signaling downstream of several receptors.

  • 43.
    Han, Guangye
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mamedov, Fikret
    Styring, Stenbjörn
    Misses during Water Oxidation in Photosystem II Are S State-dependent2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 16, p. 13422-13429Article in journal (Refereed)
    Abstract [en]

    The period of four oscillation of the S state intermediates of the water oxidizing complex in Photosystem II (PSII) is commonly analyzed by the Kok parameters. The important miss factor determines the efficiency for each S transition. Commonly, an equal miss factor has been used in the analysis. We have used EPR signals which probe all S states in the same sample during S cycle advancement. This allows, for the first time, to measure directly the miss parameter for each S state transition. Experiments were performed in PSII membrane preparations from spinach in the presence of electron acceptor at 1 degrees C and 20 degrees C. The data show that the miss parameter is different in different transitions and shows different temperature dependence. We found no misses at 1 degrees C and 10% misses at 20 degrees C during the S-1 -> S-2 transition. The highest miss factor was found in the S-2 -> S-3 transition which decreased from 23% to 16% with increasing temperature. For the S-3 -> S-0 transition the miss parameter was found to be 7% at 1 degrees C and decreased to 3% at 20 degrees C. For the S-0 -> S-1 transition the miss parameter was found to be approximately 10% at both temperatures. The contribution from the acceptor side in the form of recombination reactions as well as from the donor side of PSII to the uneven misses is discussed. It is suggested that the different transition efficiency in each S transition partly reflects the chemistry at the CaMn4O5 cluster. That consequently contributes to the uneven misses during S cycle turnover in PSII.

  • 44. Hansson, L
    et al.
    Bläckberg, L
    Edlund, M
    Lundberg, L
    Strömqvist, M
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Recombinant human milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats.1993In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 268, no 35, p. 26692-8Article in journal (Refereed)
    Abstract [en]

    Human milk bile salt-stimulated lipase ensures efficient utilization of triacylglycerol by breast-fed infants. Cloning and sequencing of cDNA have revealed that the peptide chain consists of 722 amino acid residues showing only little homology to typical lipases. The sequence is identical to that of pancreatic carboxylic-ester hydrolase. The COOH-terminal part contains 16 proline-rich repeats of 11 residues with O-linked carbohydrate. The only N-linked sugar chain is situated close to the active-site serine. Using C127 cells and a bovine papilloma virus vector, high and stable expression of full-length lipase and of several variants, obtained by site-directed mutagenesis, was achieved. The produced proteins were purified and further characterized. Variants lacking all, or all but two, repeats were active with similar specific activity and the same bile salt dependence as the native milk enzyme. Changing the asparagine necessary for N-glycosylation gave the same principal results. Active recombinant full-length lipase was also produced in a bacterial system. We conclude that neither glycosylation (N- or O-linked) nor the proline-rich repeats are essential for catalytic activity or bile salt activation of human milk bile salt-stimulated lipase.

  • 45.
    Hedengren-Olcott, Marika
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Olcott, Michael C
    Mooney, Duane T
    Ekengren, Sophia
    Geller, Bruce L
    Taylor, Barbara J
    Differential activation of the NF-kappaB-like factors Relish and Dif in Drosophila melanogaster by fungi and Gram-positive bacteria.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 20, p. 21121-7Article in journal (Refereed)
    Abstract [en]

    The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G(+)) bacteria activate the Toll/Dif pathway and that Gram-negative (G(-)) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G(+) and G(-) bacteria. In Relish mutants, the Cecropin A gene was induced by the G(+) bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G(+) or G(-) bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G(+) bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G(+) bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G(+) bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.

  • 46. Heidler, Juliana
    et al.
    Al-Furoukh, Natalie
    Department of Cardiac Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.
    Kukat, Christian
    Salwig, Isabelle
    Ingelmann, Marie-Elisabeth
    Seibel, Peter
    Krüger, Marcus
    Holtz, Jürgen
    Wittig, Ilka
    Braun, Thomas
    Szibor, Marten
    Nitric oxide-associated protein 1 (NOA1) is necessary for oxygen-dependent regulation of mitochondrial respiratory complexes.2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 37Article in journal (Refereed)
    Abstract [en]

    In eukaryotic cells, maintenance of cellular ATP stores depends mainly on mitochondrial oxidative phosphorylation (OXPHOS), which in turn requires sufficient cellular oxygenation. The crucial role of proper oxygenation for cellular viability is reflected by involvement of several mechanisms, which sense hypoxia and regulate activities of respiratory complexes according to available oxygen concentrations. Here, we focus on mouse nitric oxide-associated protein 1 (mNOA1), which has been identified as an important component of the machinery that adjusts OXPHOS activity to oxygen concentrations. mNOA1 is an evolutionary conserved GTP-binding protein that is involved in the regulation of mitochondrial protein translation and respiration. We found that mNOA1 is located mostly in the mitochondrial matrix from where it interacts with several high molecular mass complexes, most notably with the complex IV of the respiratory chain and the prohibitin complex. Knock-down of mNOA1 impaired enzyme activity I+III, resulting in oxidative stress and eventually cell death. mNOA1 is transcriptionally regulated in an oxygen-sensitive manner. We propose that oxygen-dependent regulation of mNOA1 is instrumental to adjusting OXPHOS activity to oxygen availability, thereby controlling mitochondrial metabolism.

  • 47.
    Hjalmarsson, Karin J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli1983In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 258, no 2, p. 1343-1351Article in journal (Refereed)
    Abstract [en]

    The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

  • 48.
    Ho, Oanh
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rogne, Per
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Login, Fréderic
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity SwitchProtein in the Yersinia Type III Secretion System2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 8, p. 3299-3311Article, review/survey (Refereed)
    Abstract [en]

    Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia the switch to secretion of effector proteins is induced first after that intimate contact between the bacterium and its eukaryotic targetcell has been established and the T3SS proteins YscP and YscU are playing a central role in thisprocess. Here we identify the molecular details of the YscP binding site on YscU by means o fnuclear magnetic resonance (NMR) spectroscopy. The binding interface is centeredon the C-terminal domain of YscU. Disruptingthe YscU/YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the bindingof YscP to the slowly self-cleaving YscU variantP264A conferred significant protection againstauto-proteolysis. The YscP mediated inhibition of YscU auto-proteolysis suggest that the cleavage event may act as a timing switch in the regulationof early vs. late T3SS substrates. We also show that YscUC binds to the inner-rod protein YscI with a Kd of 3.8 μM and with one-to-one stoichiometry. The significant similarity between different members of the YscU, YscP, YscI families suggests that the protein-protein interactions discussed in this study are alsorelevant for other T3SS-containing Gram-negative bacteria.

  • 49. Hughes, K
    et al.
    Edin, Sofia
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Antonsson, A
    Grundström, T
    Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IkappaB kinase.2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 38, p. 36008-36013Article in journal (Refereed)
    Abstract [en]

    Numerous fundamental biological processes involve the NFkappaB family of transcription factors. The mechanisms by which this family of proteins is regulated are therefore of widespread importance. In most cells, NFkappaB is bound to inhibitory IkappaB proteins and sequestered in the cytoplasm. NFkappaB-inducing signals result in activation of a large multisubunit kinase complex, IKK, which phosphorylates IkappaB. IkappaB is subsequently degraded, releasing NFkappaB, which translocates to the nucleus. We previously reported that inhibitors of the calcium-binding protein calmodulin (CaM) prevent phorbol ester-induced phosphorylation of IkappaB. Here we show that KN93, an inhibitor of CaM-dependent kinases (CaMKs), also inhibits the phosphorylation of IkappaB. The effect of both CaM and CaMK inhibitors on IkappaB phosphorylation is due to the inhibition of the activity of CaMK II because neither drug has any effect when a derivative of CaMK II that is insensitive to these inhibitors is expressed. When CaMK II is inhibited, phorbol ester is no longer able to activate IKK, placing CaMK II in the signaling pathway that leads to IKK activation. CaM and CaMK inhibitors also block T cell receptor/CD3-induced activation but have no effect on the ability of the cytokine tumor necrosis factor alpha or the phosphatase inhibitor calyculin A to induce degradation of IkappaB. Finally we show that expression of a constitutively active CaMK II results in the activation of NFkappaB. The results identify CaMK II as a mediator of IKK activation specifically in response to T cell receptor/CD3 and phorbol ester stimulation.

  • 50.
    Hughes, Kate
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edin, Sofia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Antonsson, Åsa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Calmodulin-dependent Kinase II Mediates T Cell Receptor/CD3- and Phorbol Ester-induced Activation of IκB Kinase2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 38, p. 36008-36013Article in journal (Refereed)
123 1 - 50 of 141
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