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  • 251.
    Hakobyan, Shoghik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rzhepishevska, Olena
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Boily, Jean-François
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ramstedt, Madeleine
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Influence of Chelation Strength and Bacterial Uptake of Gallium Salicylidene Acylhydrazide on Biofilm Formation and Virulence by Pseudomonas aeruginosa2016In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 160, p. 24-32Article in journal (Refereed)
    Abstract [en]

    Development of antibiotic resistance in bacteria causes major challenges for our society and has prompted a great need for new and alternative treatment methods for infection. One promising approach is to target bacterial virulence using for example salicylidene acylhydrazides (hydrazones). Hydrazones coordinate metal ions such as Fe(III) and Ga(III) through a five-membered and a six-membered chelation ring. One suggested mode of action is via restricting bacterial Fe uptake. Thus, it was hypothesized that the chelating strength of these substances could be used to predict their biological activity on bacterial cells. This was investigated by comparing Ga chelation strength of two hydrazone complexes, as well as bacterial Ga uptake, biofilm formation, and virulence in the form of production and secretion of a toxin (ExoS) by Pseudomonas aeruginosa. Equilibrium constants for deprotonation and Ga(III) binding of the hydrazone N′-(5-chloro-2-hydroxy-3-methylbenzylidene)-2,4-dihydroxybenzhydrazide (ME0329), with anti-virulence effect against P. aeruginosa, were determined and compared to bacterial siderophores and the previously described Ga(III) 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (Ga-ME0163) and Ga-citrate complexes. In comparison with these two complexes, it was shown that the uptake of Ga(III) was higher from the Ga-ME0329 complex. The results further show that the Ga-ME0329 complex reduced ExoS expression and secretion to a higher extent than Ga-citrate, Ga-ME0163 or the non-coordinated hydrazone. However, the effect against biofilm formation by P. aeruginosa, by the ME0329 complex, was similar to Ga-citrate and lower than what has been reported for Ga-ME0163.

  • 252.
    Hall, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The chloroplast lumen: New insights into thiol redox regulation and functions of lumenal proteins2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In higher plants oxygenic photosynthesis primarily takes place in the chloroplasts of leaves. Within the chloroplasts is an intricate membrane system, the thylakoid membrane, which is the site of light harvesting and photosynthetic electron transport. Enclosed by this membrane is the lumen space, which initially was believed to only contain a few proteins, but now is known to house a distinct set of >50 proteins, many for which there is still no proposed function. The work presented in this thesis is focused on understanding the functions of the proteins in the lumen space. Using proteomic methods, we investigated first the regulation of lumenal proteins by light and secondly by dithiol-disulphide exchange, mediated by the disulphide reductase protein thioredoxin. We furthermore performed structural and functional studies of the lumenal pentapeptide repeat proteins and of the PsbP-domain protein PPD6. When studying the diurnal expression pattern of the lumen proteins, using difference gel electrophoresis, we observed an increased abundance of fifteen lumen protein in light-adapted Arabidopsis thaliana plants. Among these proteins were subunits of the oxygen evolving complex, plastocyanin and proteins of unknown function. In our analysis of putative lumenal targets of thioredoxin, we identified nineteen proteins, constituting more than 40 % of the lumen proteins observable by our methods. A subset of these putative target proteins were selected for further studies, including structure determination by x-ray crystallography. The crystal structure of the pentapeptide repeat protein TL15 was solved to 1.3 Å resolution and further biochemical characterization suggested that it may function as a novel type of redox regulated molecular chaperone in the lumen. PPD6, a member of the PsbP-family of proteins, which is unique in that it possesses a conserved disulphide bond not found in any other PsbP-family protein, was also expressed, purified and crystallized. A preliminary x-ray analysis suggests that PPD6 exists as a dimer in the crystalline state and binds zinc ions. The high representation of targets of thioredoxin among the lumen proteins, along with the characterization of the pentapeptide repeat protein family, implies that dithiol-disulphide exchange reactions play an important role in the thylakoid lumen of higher plants, regulating processes such as photoprotection, protein turnover and protein folding.

  • 253.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hasegawa, Yoshiaki
    Yoshimura, Fuminobu
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural and functional characterization of shaft, anchor, and tip proteins of the Mfa1 fimbria from the periodontal pathogen Porphyromonas gingivalis2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 1793Article in journal (Refereed)
    Abstract [en]

    Very little is known about how fimbriae of Bacteroidetes bacteria are assembled. To shed more light on this process, we solved the crystal structures of the shaft protein Mfa1, the regulatory protein Mfa2, and the tip protein Mfa3 from the periodontal pathogen Porphyromonas gingivalis. Together these build up part of the Mfa1 fimbria and represent three of the five proteins, Mfa1-5, encoded by the mfa1 gene cluster. Mfa1, Mfa2 and Mfa3 have the same overall fold i.e., two β-sandwich domains. Upon polymerization, the first β-strand of the shaft or tip protein is removed by indigenous proteases. Although the resulting void is expected to be filled by a donor-strand from another fimbrial protein, the mechanism by which it does so is still not established. In contrast, the first β-strand in Mfa2, the anchoring protein, is firmly attached by a disulphide bond and is not cleaved. Based on the structural information, we created multiple mutations in P. gingivalis and analysed their effect on fimbrial polymerization and assembly in vivo. Collectively, these data suggest an important role for the C-terminal tail of Mfa1, but not of Mfa3, affecting both polymerization and maturation of downstream fimbrial proteins.

  • 254.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    von Sydow, Lotta
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Storm, Patrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kieselbach, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schröder, Wolfgang
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The lumenal pentapeptide repeat proteins TL15 and TL20.3 are novel chaperone-like proteins in the chloroplast lumen of higher plantsManuscript (preprint) (Other academic)
    Abstract [en]

    In the thylakoid lumen of Arabidopsis thaliana, three pentapeptide repeat family proteins of unknown function are localized. Pentapeptide repeat proteins (PRP) are comprised of at least eight tandem repeats of five amino acids of the consensus sequence A(D/N)LXX, which fold into a quadrilateral beta helix structure. Here we have solved the crystal structure of the mature form of the lumenal PRP protein TL15 to 1.3 Å resolution. TL15 is comprised of a main pentapeptide domain, consisting of a total of 19 pentapeptide repeats which form five turns of a beta helix, and a C-terminal alpha helix domain consisting of two alpha helices. The alpha helices form a ‘cap’ at the C-terminal end of the beta helix and are connected by a disulphide bond between the conserved cysteine residues C122 and C142. Furthermore we show that the lumenal PRPs TL15 and TL20.3 can assist in refolding of a chemically denatured substrate in vitro, indicating foldase chaperone activity. The three lumenal PRPs have been previously identified as targets of thioredoxin, and interestingly we observed a greatly increased chaperone activity of TL15 and TL20.3 after reduction of their disulphide bonds. Our results provide the high resolution crystal structure of the TL15 protein and our analysis of chaperone activity suggests that TL15 and TL20.3 may constitute a novel type of redox-regulated molecular chaperones in the chloroplast lumen of higher plants.

  • 255.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The HhoA protease from Synechocystis sp. PCC 6803: novel insights into structure and activity regulation2017In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 198, no 3, p. 147-153Article in journal (Refereed)
    Abstract [en]

    Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.

  • 256. 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.

  • 257.
    Hammarström, Sten
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Is there a role for CEA in innate immunity in the colon?2001In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 9, no 3, p. 119-25Article in journal (Refereed)
    Abstract [en]

    Carcinoembryonic antigen (CEA) is a well known tumor marker associated with the progression of colorectal tumors. The CEA family of glycoproteins has been fully characterized and the function of some of its members is now beginning to be understood. Here, we advance the hypothesis that, rather than functioning in cell adhesion as has been suggested previously, CEA plays a role in protecting the colonic mucosa from microbial invasion. This hypothesis is based on new microscopic, molecular, phylogenetic and microbiological evidence.

  • 258. Hammer, Neal D
    et al.
    McGuffie, Bryan A
    Zhou, Yizhou
    Badtke, Matthew P
    Reineke, Ashley A
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gestwicki, Jason E
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chapman, Matthew R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 422, no 3, p. 376-389Article in journal (Refereed)
    Abstract [en]

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

  • 259.
    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.

  • 260.
    Harju, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bergman, Anders
    Olsson, Mats
    Roos, Anna
    Haglund, Peter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Determination of atropisomeric and planar polychlorinated biphenyls, their enantiomeric fractions and tissue distribution in grey seals using comprehensive 2D gas chromatography2003In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1019, no 1-2, p. 127-142Article in journal (Refereed)
    Abstract [en]

    High prevalence of uterine occlusions and sterility is found among Baltic ringed and grey seal. Polychlorinated biphenyls (CBs) are suspected to be the main cause. The CB concentrations are higher in affected than in healthy animals, but the natural variation is considerable. Thus, it might be possible to assess the health status of seals by CB analysis. The ratios of chiral compounds (enantiomeric fractions (EFs)) such as atropisomeric CBs are of particular interest, since these may reflect differences in metabolic rates. An analytical procedure was developed and used to determine the levels of atropisomeric CBs, planar-CBs (WHO-PCBs) and total CBs in seals of different health status. Comprehensive 2D gas chromatography (GC×GC) was used to separate the target analytes from other CBs and interferences and a micro electron-capture detector (μECD) was used for detection. EFs of the atropisomeric CBs were difficult to determine as the levels were low and the interferences many. Two column combinations had to be used to avoid biased results—both had a chiral column as first-dimension column. The second-dimension column was coated with either a high-polarity cyanopropyl or a liquid crystal phase. EFs were determined for five atropisomeric CBs, i.e. CBs 91, 95, 132, 149 and 174. The results were verified by GC×GC–time-of-flight mass spectrometry (TOF-MS). Some atropisomeric CBs had EFs that deviated strongly from the racemic-mixture value. The deviations were larger in liver than blubber, which indicates enantioselective metabolism. However, there was no selective passage of the studied atropisomeric CBs across placenta and no selective blood–brain barrier. Similarly, no correlation between EFs and health status was observed, although there was a correlation between the total CB levels and health status.

  • 261. Hartmann, Laura
    et al.
    Pedrotti, Lorenzo
    Weiste, Christoph
    Fekete, Agnes
    Schierstaedt, Jasper
    Göttler, Jasmin
    Kempa, Stefan
    Krischke, Markus
    Dietrich, Katrin
    Mueller, Martin J
    Vicente-Carbajosa, Jesus
    Hanson, Johannes
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Molecular Plant Physiology, Utrecht University, The Netherlands .
    Dröge-Laser, Wolfgang
    Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots2015In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 27, no 8, p. 2244-2260Article in journal (Refereed)
    Abstract [en]

    Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity.

  • 262. Hasegawa, Y.
    et al.
    Iijima, Y.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nagano, K.
    Yoshida, Y.
    Lamont, RJ.
    Kikuchi, T.
    Mitani, A.
    Yoshimura, F.
    Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis2016In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 95, no 11, p. 1291-1297Article in journal (Refereed)
    Abstract [en]

    Fimbriae are protein-based filamentous appendages that protrude from the bacterial cell surface and facilitate host adhesion. Two types of fimbriae, FimA and Mfa1, of the periodontal pathogen Porphyromonas gingivalis are responsible for adherence to other bacteria and to host cells in the oral cavity. Both fimbrial forms are composed of 5 proteins, but there is limited information about their polymerization mechanisms. Here, the authors evaluated the function of Mfa5, one of the Mfa1 fimbrial accessory proteins. Using mfa5 gene disruption and complementation studies, the authors revealed that Mfa5 affects the incorporation of other accessory proteins, Mfa3 and Mfa4, into fibers and the expression of fimbriae on the cell surface. Mfa5 is predicted to have a C-terminal domain (CTD) that uses the type IX secretion system (T9SS), which is limited to this organism and related Bacteroidetes species, for translocation across the outer membrane. To determine the relationship between the putative Mfa5 CTD and the T9SS, mutants were constructed with in-frame deletion of the CTD and deletion of porU, a C-terminal signal peptidase linked to T9SS-mediated secretion. The ∆CTD-expressing strain presented a similar phenotype to the mfa5 disruption mutant with reduced expression of fimbriae lacking all accessory proteins. The ∆porU mutants and the ∆CTD-expressing strain showed intracellular accumulation of Mfa5. These results indicate that Mfa5 function requires T9SS-mediated translocation across the outer membrane, which is dependent on the CTD, and subsequent incorporation into fibers. These findings suggest the presence of a novel polymerization mechanism of the P. gingivalis fimbriae.

  • 263.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Verma-Gaur, Jiyoti
    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).
    Broad feedback inhibition of pre-B-cell receptor signaling components2013In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 54, no 3-4, p. 247-253Article in journal (Refereed)
    Abstract [en]

    During B lymphocyte development, first immunoglobulin heavy chain gene segments and then immunoglobulin light chain gene segments are rearranged to create antibody diversity. Early in the development, expression of a pre-B-cell receptor (pre-BCR) that has membrane-bound Ig heavy chain protein associated with surrogate light chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces survival and clonal expansion to select cells with good heavy chains, but it also down-regulates transcription of the genes for the SLC proteins and CD19 and limits its own proliferative signaling. Here we have analyzed whether the down-regulation is limited to the SLC proteins and CD19, and we show that the pre-BCR of primary mouse pre-B-cells instead is subject to a broad feedback inhibition of pre-BCR signaling components. Activation of signaling leads to down-regulation of the receptor proteins, many co-receptors and proteins participating in signal pathways from the receptor. Thus the down-regulation of the pre-BCR is much broader than previously assumed. We also show that Ca2+/calmodulin inhibition of the transcription factor E2A is required for the feedback inhibition of the pre-BCR signaling proteins. (C) 2012 Elsevier Ltd. All rights reserved.

  • 264. Havlasová, Jana
    et al.
    Hernychová, Lenka
    Brychta, Martin
    Hubálek, Martin
    Lenco, Jurai
    Larsson, Pär
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Swedish Defence Research Agency, Umeå, Sweden.
    Lundqvist, Margaretha
    Swedish Defence Research Agency, Umeå, Sweden.
    Forsman, Mats
    Swedish Defence Research Agency, Umeå, Sweden.
    Kročová, Zulana
    Stulík, Jiri
    Macela, Aks
    Proteomic analysis of anti-Franciselia tularensis LVS antibody response in murine model of tularemia2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 8, p. 2090-2103Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens.

  • 265.
    He, Feng
    et al.
    University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology.
    Amrani, Nadia
    University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology.
    Johansson, Marcus J O
    University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology.
    Jacobson, Allan
    University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology.
    Chapter 6. Qualitative and quantitative assessment of the activity of the yeast nonsense-mediated mRNA decay pathway.2008In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 449, p. 127-47Article in journal (Refereed)
    Abstract [en]

    The yeast Saccharomyces cerevisiae provides an ideal model system for elucidation of the molecular mechanisms that regulate the nonsense-mediated mRNA decay (NMD) pathway. This chapter describes an array of molecular biological, genetic, and biochemical tools that facilitate the characterization of transcripts that comprise NMD substrates and provide insights into the roles of the upf/nmd proteins in mRNA decay and translation termination. Examples illustrate the use of these methods in wild-type and NMD-deficient cells to monitor the abundance, structure, and half-lives of nonsense-containing mRNAs, the read through of premature termination codons by the ribosome, and the positioning of ribosomes at or near normal and premature termination codons.

  • 266.
    He, Shu-Lan
    et al.
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Tan, Wu-Hong
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Zhang, Zeng-Tie
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Zhang, Feng
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Qu, Cheng-Juan
    Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
    Lei, Yan-Xia
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Zhu, Yan-He
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Yu, Han-Jie
    Department of Biotechnology, Northwest University, Xi'an, China.
    Xiang, You-Zhang
    Shandong Institute for prevention & Treatment of Endemic Disease, Jinan, China.
    Guo, Xiong
    Key Laboratory of Environment and Gene Related Diseases, Xi'an Jiaotong University, Ministry Education, Xi'an, China; Key Laboratory of Trace Elements and Endemic Diseases, Xi'an Jiaotong University, Ministry of Health, Xi'an, China.
    Mitochondrial-related gene expression profiles suggest an important role of PGC-1alpha in the compensatory mechanism of endemic dilated cardiomyopathy.2013In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 17, p. 2604-2616, article id 23954821Article in journal (Refereed)
    Abstract [en]

    Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4 × 44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios ≥ 2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD.

  • 267.
    Hedberg, Christian
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Max Planck Institute of Molecular Physiology, Department of Chemical Biology, Dortmund, Germany.
    Itzen, Aymelt
    Center for Integrated Protein Science Munich, Chemistry Department, Technische Universität München, Garching, Germany.
    Molecular perspectives on protein adenylylation2015In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 10, no 1, p. 12-21Article, review/survey (Refereed)
    Abstract [en]

    In the cell, proteins are frequently modified covalently at specific amino acids with post-translational modifications, leading to a diversification of protein functions and activities. Since the introduction of high-resolution mass spectrometry, new post-translational modifications are constantly being discovered. One particular modification is the adenylylation of mammalian proteins. In adenylylation, adenosine triphosphate (ATP) is utilized to attach an adenosine monophosphate at protein threonine or tyrosine residues via a phosphodiester linkage. Adenylylation is particularly interesting in the context of infections by bacterial pathogens during which mammalian proteins are manipulated through AMP attachment via secreted bacterial factors. In this review, we summarize the role and regulation of enzymatic adenylylation and the mechanisms of catalysis. We also refer to recent methods for the detection of adenylylated proteins by modification-specific antibodies, ATP analogues equipped with chemical handles, and mass spectrometry approaches. Additionally, we review screening approaches for inhibiting adenylylation and briefly discuss related modifications such as phosphocholination and phosphorylation.

  • 268. Heggelund, Julie E.
    et al.
    Haugen, Espen
    Lygren, Birgitte
    Mackenzie, Alasdair
    Holmner, Åsa
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Vasile, Francesca
    Reina, Jose J.
    Bernardi, Anna
    Krengel, Ute
    Both El Tor and classical cholera toxin bind blood group determinants2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 418, no 4, p. 731-735Article in journal (Refereed)
    Abstract [en]

    Cholera is a disease which shows a clear blood group profile, with blood group 0 individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group 0 individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics. (C) 2012 Elsevier Inc. All rights reserved.

  • 269. Heller, K
    et al.
    Ochtrop, Philipp
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Albers, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Itzen, A
    Enzymatic phosphocholination as a tool for protein labeling2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 12-12Article in journal (Other academic)
    Abstract [en]

    Posttranslational modification (PTM) of proteins is a versatile cellular process to regulate the activities of proteins. The high regioselectivity and catalysis rate of posttranslationally modifying enzymes utilizing high-energy precursors can potentially be exploited to equip proteins or peptide sequences with a label of choice site selectively and efficiently. We and others have recently described and analyzed a new reversible PTM called phosphocholination in which a phosphocholine group is transferred from a cytidine diphosphate choline (CDP-choline) to a serine residue of the small GTPase Rab1 [1–3]. The enzymes AnkX and Lem3 catalyze the modification and the corresponding demodification reactions, respectively. Interestingly, we could demonstrate that the modifying enzyme AnkX only requires a short amino acid sequence for substrate recognition. Therefore, we envision AnkX as a tool for the site directed labeling of target proteins. Here we report on the progress of developing a novel reversible protein labeling strategy based on the enzymes AnkX and Lem3 and on derivatives of CDP-choline. We demonstrate the optimization of AnkX and Lem3 enzyme activities and the identification of optimal and minimal peptide target sequences. Results indicate that indeed arbitrary proteins of interest can be functionalized with phosphocholine derivatives. In summary, this work yields first insights into the development of a CDP-choline based fully reversible protein labeling strategy.

  • 270.
    Heller, Katharina
    et al.
    Center for Integrated Protein Science Munich, Technische Universität München, Department Chemistry, Lichtenbergstrasse 4, 85748 Garching, Germany.
    Ochtrop, Philipp
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Albers, Michael F.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Zauner, Florian B.
    Center for Integrated Protein Science Munich, Technische Universität München, Department Chemistry, Lichtenbergstrasse 4, 85748 Garching, Germany.
    Itzen, Aymelt
    Center for Integrated Protein Science Munich, Technische Universität München, Department Chemistry, Lichtenbergstrasse 4, 85748 Garching, Germany.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Max Planck Institute of Molecular Physiology, Department of Chemical Biology, Dortmund, Germany.
    Covalent Protein Labeling by Enzymatic Phosphocholination2015In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 35, p. 10327-10330Article in journal (Refereed)
    Abstract [en]

    We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.

  • 271.
    Helminen, Heikki
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Hyttinen, Mika
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Arokoski, Jari
    Department of Physical and Rehabilitation Medicine, Kuopio University Hospital, Kuopio, Finland.
    Lapveteläinen, Tuomo
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Jurvelin, Jukka
    Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, Kuopio, Finland.
    Kiviranta, Ilkka
    Department of Surgery, Jyväskylä Central Hospital, Jyväskylä, Finland.
    Tammi, Markku
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life. A hypothesis.2000In: Journal of Bone and Mineral Metabolism, ISSN 0914-8779, E-ISSN 1435-5604, Vol. 18, no 5, p. 245-257, article id 10959613Article, review/survey (Refereed)
    Abstract
  • 272.
    Helminen, Heikki
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Säämänen, Anna-Marja
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Jurvelin, Jukka
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Kiviranta, Ilkka
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Parkkinen, Jyrki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Tammi, Markku
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Kuormituksen vaikutus nivelrustoon [The effects of loading on articular cartilage].1992In: Duodecim, ISSN 0012-7183, E-ISSN 2242-3281, Vol. 108, no 12, p. 1097-1107, article id 1366060Article, review/survey (Refereed)
    Abstract [fi]

    Nivelen kuormitus on tärkeimpiä nivelruston aineenvaihduntaan ja rakenteeseen vaikuttavia fysiologisia tekijöitä. Kohtuullinen rytminen kuormitus lisää nuoren ihmisen nivelruston proteoglykaanipitoisuutta. Tämän vaikutuksesta rusto jäykistyy ja kasvaa paksuutta. Hyvin voimakas kuormitus ei aiheuta tällaista positiivista vastetta. Toisaalta nivelkuormituksen puuttuminen pienentää ruston proteoglykaanipitoisuutta ja heikentää kimmo-ominaisuuksia. Nämä surkastumismuutokset ovat suurimmaksi osaksi–elleivät kokonaan–korjautuvia. Kohtuullisella nivelkuormituksella voidaan siis ylläpitää ja parantaa nivelruston ominaisuuksia. Pitkäaikaisen liikkumattomuuden jälkeen nivelrusto on heikompi kuin normaalisti ja voi vaurioitua niveltä voimakkaasti kuormitettaessa. Siksi nivelen kuormitusta pitää lisätä toipumisvaiheessa vähitellen.

  • 273.
    Henriksson, Maria L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sundin, Charlotta
    Umeå University, Faculty of Medicine, Molecular Biology.
    Jansson, Anna L.
    Forsberg, Åke
    Palmer, Ruth H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, no 3, p. 617-28Article in journal (Refereed)
    Abstract [en]

    Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

  • 274.
    Hermann, Stefan
    et al.
    Department of Biosciences, Karolinska institute, Novum, Huddinge, Sweden.
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Onions, Jacqueline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hughes, Kate
    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).
    Calcium regulation of basic helix-loop-helix transcription factors1998In: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 23, no 2-3, p. 135-142Article in journal (Refereed)
    Abstract [en]

    The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.

  • 275. Hermansen, Russell A.
    et al.
    Hvidsten, Torgeir R.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway.
    Sandve, Simen Rod
    Liberles, David A.
    Extracting functional trends from whole genome duplication events using comparative genomics2016In: Biological Procedures Online, ISSN 1480-9222, E-ISSN 1480-9222, Vol. 18, article id 11Article, review/survey (Refereed)
    Abstract [en]

    Background: The number of species with completed genomes, including those with evidence for recent whole genome duplication events has exploded. The recently sequenced Atlantic salmon genome has been through two rounds of whole genome duplication since the divergence of teleost fish from the lineage that led to amniotes. This quadrupoling of the number of potential genes has led to complex patterns of retention and loss among gene families. Results: Methods have been developed to characterize the interplay of duplicate gene retention processes across both whole genome duplication events and additional smaller scale duplication events. Further, gene expression divergence data has become available as well for Atlantic salmon and the closely related, pre-whole genome duplication pike and methods to describe expression divergence are also presented. These methods for the characterization of duplicate gene retention and gene expression divergence that have been applied to salmon are described. Conclusions: With the growth in available genomic and functional data, the opportunities to extract functional inference from large scale duplicates using comparative methods have expanded dramatically. Recently developed methods that further this inference for duplicated genes have been described.

  • 276.
    Hernández-Prieto, Miguel Angel
    Umeå University, Faculty of Science and Technology, Chemistry.
    The Small Cab-like Proteins in the cyanobacterium Synechocystis sp. PCC 68032009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The Small Cab-like Proteins (SCPs) in the cyanobacterium Synechocystis sp. PCC 6803 accumulate in cells grown under different stress conditions. Genes coding for SCPs have been found in all sequenced organisms performing oxygenic photosynthesis and even in the genomes of cyanophages. Deletion of multiple scp genes in Synechocystis resulted in mutants with severely impaired growth and altered pigment content. These findings indicate the importance of SCPs in photosynthesis; however, their specific function is not well understood. SCPs share a chlorophyll-binding motif with the plant light harvesting complex, suggesting that they bind chlorophyll. Here I describe my findings, which unambiguously show that SCPs are able to bind chlorophyll in vitro. Although they affect both the stoichiometric ratio of Photosystem I to II and chlorophyll stability, they do not seem to be directly involved in non-photochemical quenching. I was able to reveal the location of the SCPs within the cyanobacterial cell: in stressed cells they attach to Photosystem II in the thylakoid membrane. Furthermore, I revealed the presence of another light-harvesting like (Lil)/SCP protein in Synechocystis sp. PCC 6803. The gene, slr1544, codifying for this newly characterised LilA protein, co-transcribes together with scpD and also appears to bind to Photosystem II during stress.

  • 277.
    Hernández-Prieto, Miguel Angel
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Tibiletti, Tania
    Umeå University, Faculty of Science and Technology, Chemistry.
    Kirilovsky, Diana
    2Unité de Recherche Associée 2096, Centre National de la Recherche Scientifique, Service de Bioénergétique, 91191 Gif sur Yvette, France.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Chemistry.
    Imbalance in tetrapyrrole metabolites caused by deletion of scp genes in the cyanobacterium Synechocystis sp. PCC 6803 results in dramatic cellular effectsManuscript (preprint) (Other academic)
    Abstract [en]

    Light-harvsting like (Lil) proteins contain a chlorophyll-binding domain similar to the chlorophyll a/b binding antenna proteins of plants. The five small Cab-like proteins (SCPs) of the cyanobacterium Synechocystis sp. PCC 6803 belong to the Lil family. They have been shown to stabilize chlorophyll and to play a role in the tetrapyrrole biosynthesis pathway (Xu, H., Vavilin, D., Funk, C. and Vermaas, W. (2004) J. Biol. Chem. 279, 27971-27979). Here we show that deletion of the five scp genes in a PSI-less mutant results in a decrease of Photosystem II amount in the thylakoid membrane. This reduction of Photosystem II leads to increased cell volume, disorganized thylakoid membranes in the cell, and metabolic imbalance. We conclude that the lack of SCPs alters the assembly/repair of Photosystem II, causing an imbalance in the energetic level of the mutant cells.

  • 278.
    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.

  • 279.
    Hoengenaert, Lennart
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The role of microtubule binding proteins and post-translational modifications of tubulin during plant vessel formation in Arabidopsis2015Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In vascular plants, xylem tracheary elements (TEs) form the conducting tubes responsible for the hydro-mineral sap distribution between roots and leaves. Besides their role in sap transport, they also strengthen plant stems allowing upward growth of plants on land. TEs enable plants to resist mechanical constrains by laying down a thick lateral patterned secondary cell wall. The deposition of secondary cell wall material in TEs is strictly controlled by the underlying network of cortical microtubules. Such a microtubular network is a highly dynamic structure that organizes the content of the cell by anchoring or moving cellular components. The dynamic instability of the microtubules is controlled by three main factors. First, the amount of tubulin; the basic building blocks of microtubules, which are necessary for the microtubular assembly or disassembly. Second, microtubule associated proteins (MAPs), proteins capable of binding microtubules, which control microtubule stability and organization. Third, the cytoplasmic energy in GTP levels which regulate the energy dependent assembly of microtubules.

    In this research project we try to decipher if secondary cell wall patterning in TEs is achieved to a process of local microtubule stabilization or destabilization. To accomplish this, we investigated variations in tubulin quantities during TE differentiation in Arabidopsis cell cultures. Our data shows that fluctuation in tubulin levels during TE differentiation are not correlated to the process of secondary cell wall formation. In addition, we investigated how the microtubular state changes between undifferentiating and differentiating cell cultures. Using a new protocol we were able to show that differentiating TEs are enriched with stable microtubules. Since the increase in stability is not correlated to increasing tubulin amounts, other factors such as TE-specific MAPs and post-translational modifications (PTMs) of tubulin, have been investigated.

    Because such PTMs could act as molecular beacons, modifying the properties and behaviour of microtubules or its interacting MAPs, we investigated changes in tubulin-tyrosination during TE differentiation. Our experiments show a positive correlation between tubulin-tyrosination, secondary cell wall formation and the amount of available tubulin. This suggest that the tyrosination of tubulin might act as a signal for microtubular stabilization.

    Furthermore we evaluated the effect of xylem-specific MAPs on TE patterning in planta. Using tDNA mutants of MAP70-5 and MIDD1, respectively stabilizing and destabilizing MAPs, we were able to show that TE secondary cell wall patterning is mainly determined by microtubular stabilization rather than destabilization.

  • 280.
    Hofer, Anders
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Crona, Mikael
    Logan, Derek T
    Sjöberg, Britt-Marie
    DNA building blocks: keeping control of manufacture2012In: Critical reviews in biochemistry and molecular biology, ISSN 1040-9238, E-ISSN 1549-7798, Vol. 47, no 1, p. 50-63Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α(6)β(2) complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli. Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

  • 281. Hoffmann, Andreas
    et al.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Institute of Physics, Martin Luther University Halle-Wittenberg, Halle, Germany.
    Oberwinkler, Tanja
    Siedler, Frank
    Socorro Cortina, Niña
    Balbach, Jochen
    Oesterhelt, Dieter
    Novel sulfated phosphoglycolipids from Natronomonas moolapensis2015In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 191, p. 8-15Article in journal (Refereed)
    Abstract [en]

    Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol. However, by NMR spectroscopy and enzymatic assays the existence of two isomeric molecules with different hexoses (1-(6-sulfo-D-glcp/galf-beta 1,2-glycero)phospho-2,3-diphytanylglycerol) could be shown. The structural origin from phosphatidylglycerol distinguishes these glycolipids within Archaea, because all other characterized haloarchaeal glycolipids consist of diphytanylglycerol directly linked to an oligoglycosyl moiety. Now the door is open to investigate the physical and functional consequences of these architectural differences of the head groups.

  • 282.
    Hofsten, Jonas von
    Umeå University, Faculty of Medicine, Molecular Biology.
    Developmental and reproductive regulation of NR5A genes in teleosts2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In mammals sex chromosomes direct and initiate the development of male and female gonads and subsequently secondary sex characteristics. In most vertebrates each individual is pre-destined to either become male or female. The process by which this genetic decision is carried out takes place during the embryonic development and involves a wide range of genes. The fushi tarazu factor-1 (FTZ-F1) is a nuclear receptor and transcription factor, which in mammals has proven to be essential for gonad development and directs the differentiation of testicular Sertoli cells. A mammalian FTZ-F1 homologue subtype, steroidogenic factor-1 (SF-1), is a member of the nuclear receptor 5A1 (NR5A1) group and regulate several enzymes involved in steroid hormone synthesis. It also regulates the expression of the gonadotropin releasing hormone receptor GnRHr and the β-subunit of the luteinizing hormone (LH), indicating that it functions at all levels of the reproductive axis. Another mammalian FTZ-F1 subtype, NR5A2, is in contrast to SF-1, not linked to steroidogenesis or sex determination. Rather, NR5A2 is involved in cholesterol metabolism and bile acid synthesis in liver. Hormones and environmental factors such as temperature and pH can influence teleost development and reproductive traits, rendering them vulnerable to pollutants and climate changes. Very little is known about teleost FTZ-F1 expression, regulation and function. In this thesis, expression patterns of four zebrafish FTZ-F1 genes (ff1a, b, c and d) and two Arctic char genes (acFF1α and β) were studied during development, displaying complex embryonic expression patterns. Ff1a expression was in part congruent with expression of both mammalian NR5A1 and NR5A2 genes but also displayed novel expression domains. The complexity of the expression pattern of ff1a led to the conclusion that the gene may be involved in several developmental processes, including gonad development, which also was indicated by its transcriptional regulation via Sox9a. Two ff1a homologues were also cloned in Arctic char and were shown to be involved in the reproductive cycle, as the expression displayed seasonal cyclicity and preceded that of the down stream steroidogenic genes StAR and CYP11A. High levels were correlated to elevated plasma levels of 11-ketotestosterone (11KT) in males and 17β-estradiol (E2) in females respectively. Treatment with 11KT did not affect FTZ-F1 expression directly but was indicated to alter expression of CYP11A and 3β-hydroxysteroid dehydrogenase. E2 treatment was indicated to down-regulate the expression of testicular FTZ-F1, which may contribute to the feminising effect previously observed in E2 treated salmonids. Ff1d is a novel FTZ-F1 gene, expressed in pituitary and interrenal cells during development, suggesting steroidogenic functions. In adult testis and ovary ff1d was co-expressed with anti-Mullerian hormone (AMH), a gene connected to sex determination in mammals and previously not characterised in teleost fish. The co-expression between ff1d and AMH was found in Sertoli and granulosa cells, which is congruent with the co-expression of mammalian SF-1 and AMH. This suggests that ff1d and AMH may have similar functions in teleost sex differentiation and reproduction, as their mammalian homologues. In conclusion, this study present data that connects members of the teleost FTZ-F1 family to reproduction, cholesterol metabolism and sex determination and differentiation.

  • 283.
    Hogg, Matthew
    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.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ganai, Rais Ahmad
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundström, Else-Britt
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural basis for processive DNA synthesis by yeast DNA polymerase ε2014In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 21, no 1, p. 49-56Article in journal (Refereed)
    Abstract [en]

    DNA polymerase ε (Pol ε) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ε (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ε has the highest fidelity among B-family polymerases despite the absence of an extended b-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ε to encircle the nascent doublestranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes

  • 284.
    Holmfeldt, Linda
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    On the role of small regulatory molecules in the interplay between σ54- and σ70-dependent transcription2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Signal responsive transcriptional control in bacteria is mediated through both specific and global regulatory circuits to attune promoter output to prevailing conditions. Divergent transcription of a regulatory gene and a cognate promoter under its control provides an opportunity for interplay between transcription dependent on RNA polymerases utilizing various σ-factors, each of which programs the holoenzyme to recognize different classes of promoters. The work presented in this thesis analyses the consequences and mechanisms behind interplay between σ54- and σ70-dependent transcription within the dmp-system of Pseudomonas sp. CF600. The dmp-system confers the ability to grow at the expense of (methyl)phenols and is controlled by two promoters that drive non-overlapping divergent transcription from a common intergenic region: i) the σ54-Po promoter, which controls an operon encoding a suit of specialized catabolic enzymes, and ii) the σ70-Pr promoter, which controls production of the aromatic sensor DmpR - a mechano-activator whose transcription-promoting activity is obligatory for activity of the σ54-Po promoter.

    The σ54-Po promoter and its dependence on two non-classical transcriptional regulators - the alarmone ppGpp and its co-factor DksA that directly target RNA polymerase - are the focus of the first part of the thesis. These studies utilized ppGpp and DksA deficient strains, mutant RNA polymerases that bypass the need for ppGpp and DksA, reconstituted in vitro transcription systems, and a series of DmpR-regulated hybrid σ54-promoters with different affinities for σ54-RNA polymerase, together with analysis of protein levels of key transcriptional components. Collectively with previous work, these studies provide the experimental support for a robust but purely passive mechanism for ppGpp and DksA global regulation of σ54-transcription, which is likely to also be pertinent for transcription mediated via any alternative σ-factor (Papers I-III). The second part of the thesis focuses on additional roles of ppGpp and DksA through their direct and indirect effects on the activity of the σ70-Pr promoter. These studies unexpectedly revealed that the σ70-Pr promoter is regulated by a novel mechanism in which σ54-RNA polymerase occupancy and activity at the σ54-Po promoter stimulates σ70-Pr output. Evidence is presented that ppGpp and DksA, through DmpR levels, control a feed forward loop to reinforce silence of the σ54-Po promoter under high energy conditions with robust transcription from σ54-Po when the catabolic enzymes are needed. The interplay outlined above effectively places a σ70-dependent promoter under dual control of two forms of RNA polymerases, and also makes it subservient to regulatory signals that elicit activity of σ54-RNA polymerase. The possibility that such dual sensitivity may be a prevalent, but previously unappreciated, mechanism by which bacteria integrate diverse and/or conflicting signals to gain appropriate transcriptional control is discussed.

  • 285.
    Holmfeldt, Per
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Regulation of tubulin heterodimer partitioning during interphase and mitosis2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The microtubule cytoskeleton, which consists of dynamic polymers of alpha/beta tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubule-regulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products.

    Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKII and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome.

    In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyper-polymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules.

    Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability.

  • 286.
    Holmgren, Marie
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Trametes versicolor as biodegrader and biocatalyst when using lignocellulose for ethanol production2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Energy consumption has increased rapidly during the last century due to population growth and greater industrialization. Lignocellulosic-based biofuels are being developed as alternatives to fossil fuels. For many years the question of how 5-carbon sugars in biomass are utilized in nature has been a vexed one. A patent by Sellstedt and Holmgren (2005) showed increased ethanol production, compared with fermentation using only Saccharomyces cerevisiae, through the use of a fungal mix from a degraded wood sample found in a forest.

    The aim of the work presented in this thesis was to find a fungus that naturally utilizes 5-carbon sugars and metabolizes these sugars to ethanol; elucidate the capacity of this fungus to utilize 5-carbon and 6-carbon sugars simultaneously; assess the ability of the fungus to metabolize the inhibitors produced when lignocellulose is used; and find out whether this fungus could be used for biological pretreatment of lignocellulose to replace industrially produced enzymes.

    The results showed that the fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungi present, by using PCR-amplification and sequencing of DNA, as Chalara parvispora, Xylaria sp and Trametes hirsutaTrametes versicolor. In a reconstitution study, the fungi so identified were shown to produce an amount of ethanol equal to that of the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose.

    T. versicolor could be grown in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and with xylose alone. After 354 h of culture we found very strong correlations between ethanol fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) in the cultures. In a medium containing a 1:1 glucose/xylose ratio, the efficiency of fermentation of total sugars into ethanol was 80 %.

    A variety of inhibitors are formed during pretreatment procedures; they include, for example, phenolics, levulinic acid, HMF and furfural. These inhibitors were used in this study in order to reveal their effects on the growth of cells as well as on sugar utilization, enzyme activities and ethanol production by the white-rot fungus T. versicolor. The inhibitors had a positive effect on fresh weight, the largest increase being observed with the inhibitor furfural. T. versicolor metabolized all the inhibitors during 15 days of experimentation.

    It is known that fungi can degrade cellulose, hemicellulose and lignin through a series of enzymatic reactions. Is it possible to eliminate chemical pretreatment and instead use a biological pretreatment? If T. versicolor could serve as both a biodegrader and a biocatalyst it would lead to reductions in the costs of ethanol production and lower costs for pretreatments for other renewable fuels too. Experiments with different pretreatments applied to Salix viminalis and Populus tremula were conducted with and without fungi, as well as with enzymes, to evaluate whether T. versicolor was suitable as a biodegrader. The results showed that T. versicolor was able to degrade lignocellulose to glucose, and thus is suitable as a biodegrader and in addition has xylanase and beta-glucosidase enzymes that are related to similar enzymes in other fungi.

  • 287.
    Holmgren, Marie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Illanidis, D
    Sellstedt, Anita
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Trametes versicolor involved in biodegradation and biocatalyzation of lignocellulose in ethanol fermentationManuscript (preprint) (Other academic)
  • 288.
    Holmgren, Marie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Sellstedt, Anita
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Identification of white-rot and soft-rot fungi increasing ethanol production from spent sulfite liquor in co-culture with Saccharomyces cerevisiae2008In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 105, no 1, p. 134-140Article in journal (Refereed)
    Abstract [en]

    Aim: To identify fungi that are capable of increasing ethanol production from lignocellulose in spent sulfite liquor.

    Methods and Results: In a batch fermentation study, the fungal mix could produce 24·61 g l−1 ethanol using spent sulfite liquor as substrate. The fungal mix grew well on glucose, xylose, hemicellulose and cellulose. In addition, we were able to identify the fungal mix by use of PCR-amplification of DNA and sequencing, and they were identified as Chalara parvispora and Trametes hirsuta/T. versicolor. In a reconstitution study, the identified fungi were shown to produce equal amount of ethanol as the fungal mix. We were also able to show that C. parvispora could produce ethanol from xylose.

    Conclusion: The present study has shown that ethanol production from biomass can be increased by use of C. parvispora and T. versicolor when compared with fermentation using only S. cerevisiae.

    Significance and Impact of the Study: The study shows that refining biomass by ethanol production from spent sulfite liquor, a lignocellulose material, can be increased by adding C. parvispora and T. versicolor, and it is thus of great potential economical impact.

  • 289.
    Holmner, Åsa
    et al.
    Department of Chemistry and Bioscience, Chalmers University of Technology, PO box 462, SE-40530 Göteborg, Sweden; Department of Chemistry, University of Oslo, PO box 1033 Blindern, NO-0315 Oslo, Norway.
    Askarieh, Glareh
    Ökvist, Mats
    Krengel, Ute
    Blood group antigen recognition by Escherichia coli heat-labile enterotoxin2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 371, no 3, p. 754-764Article in journal (Refereed)
    Abstract [en]

    In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 angstrom crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAc alpha 3(Fuc alpha-2)Gal beta 4(Fuc alpha-3)Glc beta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed.

  • 290.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordvall, Lina-Maria
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Golla, Krishna Prasad
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Modulation of α-synuclein fibrillization by ring-fused 2-pyridones: templation and inhibition involve oligomers with different structure2013In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 532, no 2, p. 84-90Article in journal (Refereed)
    Abstract [en]

    In a recent study we discovered that a ring-fused 2-pyridone compound triggered fibrillization of a key protein in Parkinson's disease, α-synuclein. To reveal how variations in compound structure affect protein aggregation, we now prepared a number of strategic analogs and tested their effects on α-synuclein amyloid fiber formation in vitro. We find that, in contrast to the earlier templating effect, some analogs inhibit α-synuclein fibrillization. For both templating and inhibiting compounds, the key species formed in the reactions are α-synuclein oligomers that contain compound. Despite similar macroscopic appearance, the templating and inhibiting oligomers are distinctly different in secondary structure content. When the inhibitory oligomers are added in seed amounts, they inhibit fresh α-synuclein aggregation reactions. Our study demonstrates that small chemical changes to the same central fragment can result in opposite effects on protein aggregation.

  • 291. Hu, Yunping
    et al.
    Jin, Taiyi
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Environmental Medicine.
    Zhou, Tong
    Pang, Bing
    Wang, Yunfei
    Effects of zinc on gene expressions induced by cadmium in prostate and testes of rats2004In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 17, no 5, p. 571-572Article in journal (Refereed)
  • 292.
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps.

    The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position.

    Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell.

    The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNALys [mcm5s2UUU] and tRNAGln[mcm5s2UUG] can efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes.

  • 293.
    Huang, Shenghua
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hainzl, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Forsman, Cecilia
    Orphan Biovitrum AB, Umeå, Sweden.
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Structural studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa: Inhibitor complexes with Anions and Acetazolamide2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12, p. e28458-Article in journal (Refereed)
    Abstract [en]

    The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

  • 294.
    Huch, Susanne
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Spatial control of mRNA stability in yeast2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The degradation of mRNA is an important modulator of gene expression and the ultimate fate of messenger mRNA. Important steps in the degradation of mRNA include initial shortening of its poly(A) tail followed by the subsequent removal of the m7G cap. These two processes are linked temporally as well as spatially. In addition to physical interactions between proteins involved in these two processes, deadenylation and decapping enzymes and accessory factors are found in P bodies. P bodies are aggregates of protein and mRNA that are induced upon stress in all eukaryotes examined. In this thesis, I examine the spatial localization of decapping factors and explore the role of P bodies in mRNA turnover in the yeast Saccharomyces cerevisiae.  

    This thesis is based on three underlying principles. First, mRNA decapping factors are membrane associated. More so, we show that decapping factors can be co-localized with the endoplasmic reticulum and Golgi apparatus. Second, although P bodies were proposed as sites of mRNA decay, we found that they stabilize mRNA. We examined the role of P bodies in mRNA turnover using a mutant defective in their assembly, edc3∆ lsm4∆C.  This strain is mutated in two decapping activators.  It combines a deletion of the gene encoding the Edc3 protein and lacks the prion-like domain of Lsm4. Using the edc3∆ lsm4∆C mutant, we demonstrate that mRNA stability is significantly reduced in the absence of P bodies for longer-lived mRNA. The effect of mRNA destabilization was due to increased deadenylation and decapping dependence. Finally, the decapping factor usually found in the cytoplasm, but accumulates in the nucleus in the P body deficient strain (edc3∆ lsm4∆C). This implies a possible role in modulating transcription.

    A model for the functioning of P bodies that is consistent with our work is that P bodies serve a role as a cytoplasmic sink for degradation factors. By regulating the access of the cytosol to proteins involved in mRNA turnover, P bodies can modulate mRNA stability. This suggests a role for P bodies under stress and their potential importance in stress adaptation.

  • 295.
    Huch, Susanne
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gommlich, Jessie
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Muppavarapu, Mridula
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Beckham, Carla
    Nissan, Tracy
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Membrane-association of mRNA decapping factors is independent of stress in budding yeast2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 25477Article in journal (Refereed)
    Abstract [en]

    Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane otation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. 

  • 296.
    Huch, Susanne
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Müller, Maren
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Muppavarapu, Mridula
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gommlich, Jessie
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Balagopal, Vidya
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nissan, Tracy
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae2016In: Biology Open, ISSN 2046-6390, Vol. 5, no 10, p. 1388-1399Article in journal (Refereed)
    Abstract [en]

    The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs or alternatively may imply a role for P bodies in mRNA stabilization.

  • 297.
    Huch, Susanne
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nissan, Tracy
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 44395Article in journal (Refereed)
    Abstract [en]

    Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.

  • 298.
    Huch, Susanne
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nissan, Tracy
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Interrelations between translation and general mRNA degradation in yeast2014In: Wiley Interdisciplinary Reviews: RNA, ISSN 1757-7012, Vol. 5, no 6, p. 747-763Article, review/survey (Refereed)
    Abstract [en]

    Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system.

  • 299.
    Hugosson, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Deciphering the Alk signaling pathway in Drosophila2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In Drosophila melanogaster the visceral mesoderm (VM) develops during embryogenesis in a process where myoblasts become specified to generate two distinct cell types, the founder cells (FCs) and the fusion competent myoblasts (FCMs) that consequently fuses. The cell specification is dependent on cell signaling mediated by the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb), how this further sets up different identity programs that drive myoblasts to differentiate into FCs and FCMs is still not well understood.

    We have analysed whether the Midkine (MDK)/Pleiotrophin (PTN) homologues in Drosophila, Miple1 and Miple2 activate the Alk RTK in vivo. Earlier results from cell culture experiments suggested that vertebrate MDK/PTN is capable of activating ALK, findings that have become controversial with other studies showing contradictory results. We wanted to use Drosophila that have conserved homologues of both MDK/PTN and ALK, to address the question in vivo. We analysed the contribution of Miple in Alk dependent developmental processes such as visceral mesoderm (VM) specification during embryogenesis and in body size regulation of adult flies. Specification of VM as well as body size are not effected by loss of Miple proteins, and over expression of Miple proteins do not effect VM specification or body size. All together we conclude that there is no evidence that Miple1 or Miple2 can activate Alk in vivo. We found that loss of Miple protein effect the median lifespan of the fly which is reduced, interestingly the over expression of Miple proteins can promote an increased median life span in Drosophila.

    We have also analysed how Alk RTK signaling regulates the Gli-like transcription factor Lame duck (Lmd) in vivo on a post-translational level. It has already been reported that Lmd plays an essential role in specification of FCMs in the somatic mesoderm during embryogenesis. We detect Lmd protein exclusively in FCMs of VM in control embryos, but in Alk mutants Lmd protein is present in all cells of VM and opposite to this when Alk is activated in all cells in VM by over expression of Jeb this results in total loss of Lmd protein. This suggests that Alk signaling is regulating Lmd, and we additionally show that Lmd persist in FCMs in mutants where VM is specified but where myoblast fusion do not occur, supporting that Alk activity in FCs is regulating the downregulation of Lmd in FCMs upon fusion.

    Finally we have characterised the Rap1GEF C3G in vivo in Drosophila. In cell culture systems, the GTPase Rap1 has been identified to mediate Alk signaling and that this is regulated by the GEF C3G and interestingly the Drosophila C3G is expressed in the FCs of VM. We generated deletion mutants of C3G which exhibit semi-lethality and reduced life span, but no defects in visceral mesoderm development during embryogenesis. Instead we detected distinct phenotypes in somatic muscles of 3rd instar mutant larvae, with detachment and mistargeting of muscles, which effect localisation of integrins. We suggest that Drosophila C3G regulates Rap1 via inside out signaling of integrins which in turn effects cell adhesion in vivo in Drosophila larval muscles.

  • 300.
    Hugosson, Fredrik
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sjögren, Camilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Birve, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hedlund, Ludmilla
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, Therese
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Palmer, Ruth H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 11, p. e112250-Article in journal (Refereed)
    Abstract [en]

    Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

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