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  • 251. Gurvich, Olga L
    et al.
    Näsvall, S Joakim
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Baranov, Pavel V
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Atkins, John F
    Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, p. 3079-3092Article in journal (Refereed)
    Abstract [en]

    The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5’-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNA(Pro) mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting.

  • 252.
    Gustavsson, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands.

    The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins.

    We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

  • 253.
    Gustavsson, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Armulik, Annika
    Brakebusch, Cord
    Fässler, Reinhard
    Johansson, Staffan
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia2002In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 115, no 13, p. 2669-2678Article in journal (Refereed)
    Abstract [en]

    Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

  • 254.
    Gustavsson, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, Maria
    Myosin X recruits FAK and vinculin to the tip complexes of filopodiaManuscript (Other academic)
  • 255. Gómez-Consarnau, Laura
    et al.
    Akram, Neelam
    Lindell, Kristoffer
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Pedersen, Anders
    Neutze, Richard
    Milton, Debra L
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    González, José M
    Pinhassi, Jarone
    Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation2010In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 8, article id e1000358Article in journal (Refereed)
    Abstract [en]

    Proteorhodopsins are globally abundant photoproteins found in bacteria in the photic zone of the ocean. Although their function as proton pumps with energy-yielding potential has been demonstrated, the ecological role of proteorhodopsins remains largely unexplored. Here, we report the presence and function of proteorhodopsin in a member of the widespread genus Vibrio, uncovered through whole-genome analysis. Phylogenetic analysis suggests that the Vibrio strain AND4 obtained proteorhodopsin through lateral gene transfer, which could have modified the ecology of this marine bacterium. We demonstrate an increased long-term survival of AND4 when starved in seawater exposed to light rather than held in darkness. Furthermore, mutational analysis provides the first direct evidence, to our knowledge, linking the proteorhodopsin gene and its biological function in marine bacteria. Thus, proteorhodopsin phototrophy confers a fitness advantage to marine bacteria, representing a novel mechanism for bacterioplankton to endure frequent periods of resource deprivation at the ocean’s surface.

  • 256. Haglund, Kaisa
    et al.
    Nezis, Ioannis P
    Lemus, Dafne
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wesche, Jörgen
    Liestol, Knut
    Dikic, Ivan
    Palmer, Ruth
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stenmark, Harald
    Cindr interacts with anillin to control cytokinesis in Drosophila melanogaster2010In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 20, no 10, p. 944-950Article in journal (Refereed)
    Abstract [en]

    Cytokinesis, the final step of cell division, conventionally proceeds to cell separation by abscission, or complete cytokinesis [1, 2], but may in certain tissues be incomplete, yielding daughter cells that are interconnected in syncytia by stable intercellular bridges [3]. The mechanisms that determine complete versus incomplete cytokinesis are not known. Here we report a novel in vivo role of the Drosophila CD2AP/CIN85 ortholog Cindr in both complete and incomplete cytokinesis. We also show evidence for the presence of persistent intercellular bridges in the major larval imaginal disc epithelia. During conventional division of both cultured and embryonic cells, Cindr localizes to cleavage furrows, intercellular bridges, and midbodies. Moreover, in cells undergoing incomplete cytokinesis in the female germline and the somatic ovarian follicle cell and larval imaginal disc epithelia, Cindr localizes to arrested cleavage furrows and stable intercellular bridges, respectively. In these structures, Cindr colocalizes with the essential cytokinesis regulator Anillin. We show that Cindr interacts with Anillin and that depletion of either Cindr or Anillin gives rise to binucleate cells and fewer intercellular bridges in vivo. We propose that Cindr and Anillin cooperate to promote intercellular bridge stability during incomplete cytokinesis in Drosophila melanogaster.

  • 257.
    Hagström, Åke
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Azam, Farooq
    Andersson, Agneta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Raassoulzadegan, Fereidoun
    Microbial loop in an oligothropic pelagic marine ecosystem: Possible roles of cyanobacteria and nanoflagellates in the organic fluxes1988In: Marine Ecology Progress Series, ISSN 0171-8630, E-ISSN 1616-1599, Vol. 49, no 1-2, p. 171-178Article in journal (Refereed)
  • 258.
    Hallberg, Bengt
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Crizotinib: latest champion in the cancer wars?2010In: New England Journal of Medicine, ISSN 0028-4793, E-ISSN 1533-4406, Vol. 363, no 18, p. 1760-1762Article in journal (Refereed)
  • 259.
    Hallberg, Bengt
    et al.
    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).
    Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology2013In: Nature Reviews. Cancer, ISSN 1474-175X, E-ISSN 1474-1768, ISSN 1474-1768, Vol. 13, no 10, p. 685-700Article in journal (Refereed)
    Abstract [en]

    The burgeoning field of anaplastic lymphoma kinase (ALK) in cancer encompasses many cancer types, from very rare cancers to the more prevalent non-small-cell lung cancer (NSCLC). The common activation of ALK has led to the use of the ALK tyrosine kinase inhibitor (TKI) crizotinib in a range of patient populations and to the rapid development of second-generation drugs targeting ALK. In this Review, we discuss our current understanding of ALK function in human cancer and the implications for tumour treatment.

  • 260.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Verma-Gaur, Jiyoti
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wallenius, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Grundström, Thomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mechanisms regulating diversification and affinity maturation of antibodies2012In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 30, no Suppl 1, p. S42-S42Article in journal (Other academic)
  • 261.
    Heby, Olle
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, L
    Rentala, M
    Targeting the polyamine biosynthetic enzymes: a promising approach to therapy of African sleeping sickness, Chagas' disease, and leishmaniasis2007In: Amino Acids, Vol. 33, p. 359-366Article, review/survey (Other (popular science, discussion, etc.))
  • 262.
    Hedman, H.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Alenius, Mattias
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lundgren, E.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Defective expression of beta 1-integrins in cells with constitutively active alpha L beta 2-integrins1997In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 232, no 2, p. 270-276Article in journal (Refereed)
    Abstract [en]

    We have investigated a potential relationship between expression of beta 1-integrins and adhesiveness of the beta 2-integrin LFA-1 (alpha L beta 2, CD11a/CD18). By an approach of random mutagenesis and selection we established clones from the human acute lymphatic leukemia cell line HPB-ALL with (i) constitutively active LFA-1 and (ii) with no apparent integrin-beta 1 cell surface expression. Thirty seven of 42 clones selected for activated LFA-1 were found to have lost apparent integrin-beta 1 expression. Conversely, 7 of 21 clones selected for lack of beta 1 expression were found to have activated LFA-1. Since this pointed toward a possible coupling between beta 1 expression and LFA-1 activity, we further analyzed at which level beta 1 expression was blocked. We focused on one clone, HAP4, with activated LFA-I and no detectable beta 1 cell surface expression and found, surprisingly, that it expressed wild-type levels of beta 1 mRNA and, in Western blots of whole cell lysates, apparently normal levels of beta 1 protein. However, in addition to beta 1 of the expected molecular weight, HAP4 expressed a unique 48-kDa band recognized by the polyclonal anti-beta 1 antiserum. Immunoprecipitation experiments revealed that the epitope recognized by the anti-beta 1 antibody 4B4 was hidden or lost. The alpha 4-chain was found in its precursor form but it did not associate with any beta-chain, and it was not processed to its mature form. Instead alpha 4-chains were eventually degraded. Taken together this showed that beta 1-chains were produced but not properly processed in HAP4. From this we propose that HAP4 is deficient in a gene product required both for proper beta 1 folding and for repression of LFA-1 adhesiveness.

  • 263. Hemann, Michael T
    et al.
    Bric, Anka
    Teruya-Feldstein, Julie
    Herbst, Andreas
    Nilsson, Jonas A
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Cordon-Cardo, Carlos
    Cleveland, John L
    Tansey, William P
    Lowe, Scott W
    Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants.2005In: Nature, ISSN 1476-4687, Vol. 436, no 7052, p. 807-11Article in journal (Refereed)
    Abstract [en]

    The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitt's lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis.

  • 264.
    Henriksson, M L
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Telepnev, Maxim
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras2000In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 347, no 1, p. 217-222Article in journal (Refereed)
  • 265.
    Henriksson, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Peden, Alex
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stefansson, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Palmer, Ruth
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aitken, Alastair
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, no 20, p. 4921-4929Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.

  • 266.
    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 Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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.

  • 267. Hillier, Charles
    et al.
    Pardo, Mercedes
    Yu, Lu
    Bushell, Ellen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Sanderson, Theo
    Metcalf, Tom
    Herd, Colin
    Anar, Burcu
    Rayner, Julian C.
    Billker, Oliver
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Choudhary, Jyoti S.
    Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality2019In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 28, no 6, p. 1635-1647Article in journal (Refereed)
    Abstract [en]

    Malaria represents a major global health issue, and the identification of new intervention targets remains an urgent priority. This search is hampered by more than one-third of the genes of malaria-causing Plasmodium parasites being uncharacterized. We report a large-scale protein interaction network in Plasmodium schizonts, generated by combining blue native-polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. This integrative approach, spanning 3 species, identifies > 20,000 putative protein interactions, organized into 600 protein clusters. We validate selected interactions, assigning functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between AP2-domain transcription factors and epigenetic regulation. Our interactome represents a high-confidence map of the native organization of core cellular processes in Plasmodium parasites. The network reveals putative functions for uncharacterized proteins, provides mechanistic and structural insight, and uncovers potential alternative therapeutic targets.

  • 268.
    Hjalmarsson, Karin J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli1983In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 258, no 2, p. 1343-1351Article in journal (Refereed)
    Abstract [en]

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

  • 269.
    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.

  • 270.
    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.

  • 271.
    Holmqvist, Per-Henrik
    et al.
    The Wenner-Gren Institute, Developmental Biology, Stockholm University, Stockholm.
    Boija, Ann
    The Wenner-Gren Institute, Developmental Biology, Stockholm University, Stockholm.
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Crona, Filip
    The Wenner-Gren Institute, Developmental Biology, Stockholm University, Stockholm.
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mannervik, Mattias
    The Wenner-Gren Institute, Developmental Biology, Stockholm University, Stockholm.
    Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos2012In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 8, no 6, p. e1002769-Article in journal (Refereed)
    Abstract [en]

    CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP-seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation.

  • 272.
    Holmström, Anna
    et al.
    Swedish Defence Research Agency, Division of CBRN Defence and Security, SE-901 82 Umeå, Sweden.
    Olsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cherepanov, Peter
    Maier, Elke
    Nordfelth, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pettersson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Benz, Roland
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Forsberg, Åke
    LcrV is a channel size-determining component of the Yop effector translocon of Yersinia2001In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 39, no 3, p. 620-632Article in journal (Refereed)
    Abstract [en]

    Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact‐dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127–195 of LcrV and to amino acids 106–173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size‐determining structural component of the Yop translocon.

  • 273.
    Holmström, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Petterson, Jonas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Håkansson, Sebastian
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tafazoli, Farideh
    Department of Medical Microbiology, Linköping University, S-581 85 Linköping, Sweden.
    Fällman, Maria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Magnusson, Karl-Eric
    Department of Medical Microbiology, Linköping University, S-581 85 Linköping, Sweden.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Department of Microbiology, National Defence Research Establishment, S-901 82 Umeå, Sweden..
    YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane.1997In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 24, no 1, p. 73-91Article in journal (Refereed)
    Abstract [en]

    Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

  • 274. Howick, Virginia M.
    et al.
    Russell, Andrew J. C.
    Andrews, Tallulah
    Heaton, Haynes
    Reid, Adam J.
    Natarajan, Kedar
    Butungi, Hellen
    Metcalf, Tom
    Verzier, Lisa H.
    Rayner, Julian C.
    Berriman, Matthew
    Herren, Jeremy K.
    Billker, Oliver
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, UK.
    Hemberg, Martin
    Talman, Arthur M.
    Lawniczak, Mara K. N.
    The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle2019In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 365, no 6455, article id eaaw2619Article in journal (Refereed)
    Abstract [en]

    Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites.

  • 275.
    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.

  • 276.
    Huang, Bo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J.O.
    Byström, Anders S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An early step in wobble uridine tRNA modification requires the Elongator complex2005In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 4, p. 424-436Article in journal (Refereed)
    Abstract [en]

    Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1–ELP6) genes or the three killer toxin insensitivity (KTI11–KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1–ELP6 and KTI11–KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm5U, ncm5U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1–elp6 and kti11–kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1–ELP6 or KTI11KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.

  • 277. Huang, Bo
    et al.
    Lu, Jian
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae.2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 10, p. 2183-2194Article in journal (Refereed)
    Abstract [en]

    We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.

  • 278.
    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.

  • 279.
    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.

  • 280. Hudson, Debra L
    et al.
    Layton, Abigail N
    Field, Terry R
    Bowen, Alison J
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Elofsson, Mikael
    Chemistry.
    Stevens, Mark P
    Galyov, Edouard E
    Inhibition of Type III Secretion in Salmonella enterica Serovar Typhimurium by Small-Molecule Inhibitors2007In: Antimicrobial Agents and Chemotherapy, Vol. 51, no 7, p. 2631-5Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) are conserved in many pathogenic gram-negative bacteria. Small molecules that specifically target T3SS in Yersinia and Chlamydia spp. have recently been identified. Here we show that two such compounds inhibit Salmonella T3SS-1, preventing secretion of T3SS-1 effectors, invasion of cultured epithelial cells, and enteritis in vivo.

  • 281.
    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.

  • 282.
    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.

  • 283.
    Hägglund, Emil
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Real-time analysis methods for species identification and genome assembly with MinION-sequencing2016Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
  • 284.
    Hägglund, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Berghard, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Strotmann, J
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Retinoic acid receptor-dependent survival of olfactory sensory neurons in postnatal and adult mice.2006In: Journal of Neuroscience, ISSN 0270-6474, E-ISSN 1529-2401, Vol. 26, no 12, p. 3281-3291Article in journal (Refereed)
  • 285.
    Härtlova, Anetta
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Erttmann, Saskia F.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Raffi, Faizal A. M.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schmalz, Anja M.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Resch, Ulrike
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Anugula, Sharath
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lienenklaus, Stefan
    Nilsson, Lisa M.
    Kroeger, Andrea
    Nilsson, Jonas A.
    Ek, Torben
    Weiss, Siegfried
    Gekara, Nelson O.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    DNA Damage Primes the Type I Interferon System via the Cytosolic DNA Sensor STING to Promote Anti-Microbial Innate Immunity2015In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 42, no 2, p. 332-343Article in journal (Refereed)
    Abstract [en]

    Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.

  • 286.
    Härtlova, Anetta
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Link, Marek
    Balounova, Jana
    Benesova, Martina
    Resch, Ulrike
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Straskova, Adela
    Sobol, Margarita
    Philimonenko, Anatoly
    Hozak, Pavel
    Krocova, Zuzana
    Gekara, Nelson
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Filipp, Dominik
    Stulik, Jiri
    Quantitative proteomics analysis of macrophage-derived lipid rafts reveals induction of autophagy pathway at the early time of Francisella tularensis LVS infection2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 2, p. 796-804Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early, stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.

  • 287.
    Håglin, Sofia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Vitamin A regulated neuronal regeneration and homeostasis2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The olfactory epithelium is a dynamic tissue maintained by continuous neurogenesis throughout life. Upon injury, neurons and other olfactory cell types are regenerated through proliferation of horizontal stem cells. Some genes that regulate vitamin A metabolism are spatially expressed in the olfactory epithelium. Retinoic acid is a vitamin A derivate, a key regulator of proliferation and stem cell activity. Retinoic acid is generated and inactivated by enzymes with opposing expression patterns which create local variations in retinoic acid levels in the olfactory epithelium. The overall aim of this thesis is to elucidate functional relationships between retinoic acid metabolism and the regulation of temporal and spatial features of normal tissue homeostasis and regeneration of neurons within the olfactory epithelium.

    I have studied the association between the activity-dependent retinoic acid inactivating enzyme CYP26B1 and neurogenesis.  During doubled stimulation by odorants and air flow the level of CYP26B1 was further induced in olfactory sensory neurons and proliferation of progenitor/stem cells was increased. In the absence of stimuli, CYP26B1 expression was reduced and proliferation decreased. Stimuli-independent transgenic over-expression of CYP26B1 resul-ted in increased proliferation, which was compared to acute intranasal admini-stration of retinoic acid that reduced the number of proliferating cells.

    The region of the olfactory epithelium with low CYP26B1 and high levels of retinoic acid synthesizing enzymes had the greatest level of proliferation and regenerated efficiently after chemical induced injury. Furthermore, neurons in this region differentiated surprisingly fast. In the region with high CYP26B1 and low levels of retinoic acid synthesizing enzymes the proliferation rate was low and the regeneration after injury was incomplete. Together these results indicate that retinoic acid within the olfactory epithelial stem cell niche regulates local differences in functional neuronal diversity, neurogenesis, and generative capacity of olfactory epithelial progenitor/stem cells.

    My research has revealed that ageing as well as constitutive transgenic over-expression of CYP26B1 activated dormant horizontal basal stem cells in the olfactory epithelium in an injury like manner. Continuous stem cell activation by constitutive CYP26B1 expression, repeated injuries or old age results in the appearance of epithelial patches devoid of normal olfactory epithelial cells, containing metaplastic respiratory cells. The respiratory patches either contained ciliated cells or a previously unidentified columnar secretory cell type.  Moreover, we investigated whether increased proliferation of stem cells affected their regenerative potential over time. Repeated injury-repair cycles maximized the number of stem cell division, which decreased their potential to regenerate olfactory epithelial cells. Together these results indicate a premature exhaustion of the stem cell niche upon reduced levels of retinoic acid, repeated injury induced regeneration, and ageing.

  • 288.
    Håglin, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Berghard, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Decreased retinoic acid bioavailability activates dormant olfactory epithelial stem cells and causes age-related respiratory metaplasiaManuscript (preprint) (Other academic)
  • 289.
    Håglin, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Berghard, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Effects of sensory deprivation is mediated by activity-dependent CYP26B1 expression in olfactory sensory neuronsManuscript (preprint) (Other academic)
  • 290.
    Höglund, Andreas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation of DNA damage responses by the Myc oncogene: implications for future anti-cancer therapies2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Myc is a transcription factor frequently found deregulated in human cancer. Cells with deregulated expression of Myc carry a selective advantage against its neighbours due to the fact that Myc-mediated transcription governs crucial cellular events such as proliferation and growth. In addition, Myc has been implicated in several other aspects of tumour biology like cellular immortality, the formation of new blood vessels and the colonization of distant tissues through the process of metastasis. Therapy aimed at disrupting essential pathways regulated by Myc is important because of the many different types of cancers that depend on continued signalling along these pathways.  This thesis describes new treatment opportunities for cancers with a high Myc signature. In Paper Ι, we describe a new role for the DNA methyltransferase inhibitor Decitabine in the treatment of Myc transformed tumours cells. We show that the therapeutic potential of Decitabine in the treatment of Burkitt Lymphoma relies not only on its ability to cause reactivation of silenced genes such as pro-apoptotic PUMA, but also on the DNA damage that this drug induces. In vivo, Decitabine delays disease progression of transplanted lymphoma cells. In Paper ΙΙ, we identify the DNA damage checkpoint kinase Chk1 as a therapeutic target in Myc overexpressing cancers. We show that targeting Chk1 with shRNA or with a novel small molecule inhibitor cause a delay in disease progression of transplanted lymphoma cells in vivo. In Paper ΙΙΙ, the Chk1-related kinase Chk2 is evaluated as a therapeutic target in Myc overexpressing cancers. Myc overexpressing cells are not dependent on Chk2 but we show that Chk2 abrogation using shRNA causes polyploidization and protection against DNA damage. However, Chk2-targeted therapy elicits a synergistic lethal response in combination with inhibition of the DNA repair associated protein PARP. In conclusion, this thesis shows the potential of targeting the DNA damage machinery and the functional hubs important for maintenance of genomic stability in tumours with a deregulated expression of Myc.

  • 291.
    Höglund, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nilsson, Lisa M.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Muralidharan, Somsundar Veppil
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hasvold, Lisa A.
    Merta, Philip
    Rudelius, Martina
    Nikolova, Viktoriya
    Keller, Ulrich
    Nilsson, Jonas A.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Therapeutic implications for the induced levels of Chk1 in Myc- expressing cancer cells2011In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 17, no 22, p. 7067-7079Article in journal (Refereed)
    Abstract [en]

    Purpose: The transcription factor c-Myc (or "Myc") is a master regulator of pathways driving cell growth and proliferation. MYC is deregulated in many human cancers, making its downstream target genes attractive candidates for drug development. We report the unexpected finding that B-cell lymphomas from mice and patients exhibit a striking correlation between high levels of Myc and checkpoint kinase 1 (Chk1). Experimental Design: By in vitro cell biology studies as well as preclinical studies using a genetically engineered mouse model, we evaluated the role of Chk1 in Myc-overexpressing cells. Results: We show that Myc indirectly induces Chek1 transcript and protein expression, independently of DNA damage response proteins such as ATM and p53. Importantly, we show that inhibition of Chk1, by either RNA interference or a novel highly selective small molecule inhibitor, results in caspase-dependent apoptosis that affects Myc-overexpressing cells in both in vitro and in vivo mouse models of B-cell lymphoma. Conclusion: Our data suggest that Chk1 inhibitors should be further evaluated as potential drugs against Myc-driven malignancies such as certain B-cell lymphoma/leukemia, neuroblastoma, and some breast and lung cancers. Clin Cancer Res; 17(22); 7067-79. (C) 2011 AACR.

  • 292.
    Höglund, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nilsson, Lisa M.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Plym Forshell, Linus
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Maclean, Kirsteen H.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nilsson, Jonas A.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Myc sensitizes p53-deficient cancer cells to the DNA-damaging effects of the DNA methyltransferase inhibitor decitabine2009In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 113, no 18, p. 4281-4288Article in journal (Refereed)
    Abstract [en]

    Decitabine (also referred to as 5-aza-2'-deoxycytidine) is a drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of myelodysplastic syndrome (MDS). The mechanism of action is believed to be the blocking of DNA methylation and thereby reactivating silenced genes involved in harnessing MDS. When analyzing reactivation of genes involved in Burkitt lymphoma (BL), we discovered that decitabine also sensitizes tumor cells by inducing DNA damage. This sensitization is grossly augmented by the MYC oncogene, which is overexpressed in BL, and occurs in cells lacking a functional p53 tumor suppressor pathway. In p53-deficient BL cells and p53(-/-) mouse embryo fibroblasts, Myc overrides a transient G2-block exerted by decitabine via activation of Chk1. This triggers aneuploidy and cell death that correlates with, but can occur in the absence of, Epstein-Barr virus (EBV) reactivation, caspase activation, and/or expression of the BH3-only protein Puma. In vivo modeling of Myc-induced lymphoma suggests that decitabine constitutes a potential new drug against lymphoma that would selectively sensitize tumor cells but spare normal tissue.

  • 293.
    Höglund, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Strömvall, Kerstin
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Plym Forshell, Linus
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nilsson, Jonas A
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chk2 deficiency in Myc overexpressing lymphoma cells elicits a synergistic lethal response in combination with PARP inhibition.2011In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 10, no 20, p. 3598-3607Article in journal (Refereed)
    Abstract [en]

    Myc is a transcription factor frequently found deregulated in human cancer. The Myc- mediated cellular transformation process is associated with fast proliferative cells and inherent genomic instability, giving rise to malignant, invasive neoplasms with poor prognosis for survival. Transcription-independent functions of Myc include stimulation of replication. Excessive Myc expression stimulates a replication-associated DNA damage response that signal via the phosphoinositide 3-kinase (PI3K) related protein kinases (PIKKs) ATM and ATR. These in turn activate the DNA damage transducers Chk1 and Chk2. Here, we show that Myc can stimulate Chek2 transcript indirectly in vitro, as well as in B cells of !-Myc transgenic mice or in the intestine of ApcMin mice. However, Chk2 is dispensable for Myc’s ability to transform cells in vitro and for the survival of established lymphoma cells from !-Myc transgenic mice. Chk2 deficiency induces polyploidy and slow growth but the cells are viable and protected against DNA damage. However, inhibition of both Chk1/Chk2 with AZD7762 induces cell death and significantly delays disease progression of transplanted lymphoma cells in vivo. DNA damage recruits PARP family members to sites of DNA breaks that in turn facilitate the induction of DNA repair. Strikingly, combining Chk2 and PARP inhibition elicits a synergistic lethal response in the context of Myc overexpression. Our data indicates that only certain types of chemotherapy would give rise to a synergistic lethal response in combination with specific Chk2 inhibitors, which will be important if Chk2 inhibitors enter the clinic.

  • 294.
    Hörnberg, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gussing, Fredrik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Berghard, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bohm, Staffan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Retinoic acid selectively inhibits death of basal vomeronasal neurons during late stage of neural circuit formation2009In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 110, no 4, p. 1263-1275Article in journal (Refereed)
    Abstract [en]

    In mouse, sexual, aggressive, and social behaviors are influenced by G protein-coupled vomeronasal receptor signaling in two distinct subsets of vomeronasal sensory neurons (VSNs): apical and basal VSNs. In addition, G protein-signaling by these receptors inhibits developmental death of VSNs. We show that cells of the vomeronasal nerve express the retinoic acid (RA) synthesizing enzyme retinal dehydrogenase 2. Analyses of transgenic mice with VSNs expressing a dominant-negative RA receptor indicate that basal VSNs differ from apical VSNs with regard to a transient wave of RA-regulated and caspase 3-mediated cell death during the first postnatal week. Analyses of G-protein subunit deficient mice indicate that RA and vomeronasal receptor signaling combine to regulate postnatal expression of Kirrel-2 (Kin of IRRE-like), a cell adhesion molecule regulating neural activity-dependent formation of precise axonal projections in the main olfactory system. Collectively, the results indicate a novel connection between pre-synaptic RA receptor signaling and neural activity-dependent events that together regulate neuronal survival and maintenance of synaptic contacts.

  • 295.
    Ingebrand, Nora
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Proteasome Activator PA28αβ and Oxidative Stress in Early Mouse Embryogenesis2016Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
  • 296.
    Isaksson, Elin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fish scale keratocytes constitute a sensitive model system for YopE mediated phenotypes in Yersinia pseudotuberculosis infections2010Manuscript (preprint) (Other academic)
    Abstract [en]

    The bacterial pathogen Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to deliver virulence associated effectors directly into the host cell. Four of the translocated effectors affect the actin cytoskeleton of the cell, demonstrating the importance of inducing actin rearrangements for the establishment of a successful Yersinia infection. To further examine the role of the effectors in actin targeting, we have established a novel model system using fish scale keratocytes. Fish scale keratocytes are rapidly migrating cells present on the surface of teleost skin. The keratocytes are part of the quick wound repair mechanism of fish and are also able to internalize bacteria. The cell consists of a large extended lamellipodium, a two-dimensional actin network regulated by RhoGTPases.                                         By using live cell microscopy, we identified that wild-type Y. pseudotuberculosis caused a cytotoxic effect towards the keratocytes already within 10 minutes of infection. In contrast, a bacterial strain lacking the T3SS was rapidly internalized by the keratocytes. Further, YopE was found to be the sole effector responsible for this cytotoxic effect. YopE inactivates multiple small RhoGTPases via its GAP activity (GTPase activating protein) to induce cytotoxicity in a wide variety of cell types, including HeLa cells. Several domains of YopE are important for proper GAP function. When a number of earlier isolated point mutants of YopE were examined, none of the mutants could induce cytotoxicity towards keratocytes. Thus, this finding is in sharp contrast to earlier observations in HeLa cells, where all but one mutation caused cytotoxicity. In conclusion, the keratocytes appear to be more sensitive towards YopE mediated effects than HeLa cells and would therefore constitute a relevant model system for Yersinia infections for further studies.

  • 297.
    Isaksson, Elin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The multifunctional GAP protein YopE of Yersinia is involved in effector translocation control and virulence2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The Gram-negative bacterium Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to establish infections. The T3SS translocates a diverse set of effector proteins directly into the host cells. The coordinate action of the translocated effectors blocks the innate immune system of the host and ensures extracellular proliferation of the bacterium. YopE is an essential effector that disrupts the actin cytoskeleton of infected host cells. This cytotoxicity is caused by the inactivation of RhoGTPases by the GTPase Activating Protein (GAP) activity of YopE. YopE was demonstrated to inactivate the RhoGTPases Rac1 and RhoA in vivo. However, Rac1 and RhoA inactivation was not a prerequisite for cytotoxicity or virulence. Thus, YopE must have additional targets during infection. Surprisingly, avirulent yopE mutants had lost the control of Yop expression in the presence of target cells and they all overtranslocated effectors. It appeared as if translocated YopE was able to control Yop expression and effector translocation via a feedback inhibition mechanism. This feedback inhibition was dependent on functional GAP activity. Translocation control could also be mediated by exogenous GAP activity, suggesting that effector translocation control might be a general property of all bacterial GAP proteins. Besides YopE, the regulatory protein YopK was also found to be involved in the effector translocation control process. Clearly, as demonstrated in virulence, the roles for YopE and YopK are intimately related.                       Further, YopE possesses a membrane localization domain (MLD) required for proper localization. A yopE∆MLD mutant had lost the feedback inhibition of YopE expression and was avirulent. Hence, the effector translocation control of YopE requires both proper localization as well as functional GAP activity.                                           In addition, fish keratocytes were established as a novel model system for Y. pseudotuberculosis infections. YopE was found to be the sole effector responsible for cytotoxicity towards the keratocytes. Further, induction of cytotoxicity required fully native YopE protein which indicated that the keratocytes would be useful as a sensitive model system for further studies of YopE mediated phenotypes.

    In summary, this thesis work has sought to unravel the multiple functions of translocated YopE. A novel role was elucidated where Yersinia utilizes translocated YopE to control the process of effector translocation into host cells. This regulatory control was connected to virulence in the mouse model of disease. Thus, perhaps YopE should be considered also as a regulatory protein besides being a classical effector.

  • 298.
    Isaksson, Elin L
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aili, Margareta
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Carlsson, Sara E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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).
    The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis.2009In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, no 11, p. 4740-4749Article in journal (Refereed)
    Abstract [en]

    Recent work has shown that a domain of YopE of Yersinia pseudotuberculosis ranging from amino acids 54 to 75 (R. Krall, Y. Zhang, and J. T. Barbieri, J. Biol. Chem. 279:2747-2753, 2004) is required for proper localization of YopE after ectopic expression in eukaryotic cells. This domain, called the membrane localization domain (MLD), has not been extensively studied in Yersinia. Therefore, an in cis MLD deletion mutant of YopE was created in Y. pseudotuberculosis. The mutant was found to secrete and translocate YopE at wild-type levels. However, the mutant was defective in the autoregulation of YopE expression after the infection of HeLa cells. Although the mutant translocated YopE at wild-type levels, it showed a delayed HeLa cell cytotoxicity. This delay was not caused by a change in GTPase activating protein (GAP) activity, since the mutant showed wild-type YopE GAP activity toward Rac1 and RhoA. The MLD mutant displayed a changed intracellular localization pattern of YopE in HeLa cells after infection, and the YopEDeltaMLD protein was found to be dispersed within the whole cell, including the nucleus. In contrast, wild-type YopE was found to localize to the perinuclear region of the cell and was not found in the nucleus. In addition, the yopEDeltaMLD mutant was avirulent. Our results suggest that YopE must target proteins other than RhoA and Rac1 and that the MLD is required for the proper targeting and hence virulence of YopE during infection. Our results raise the question whether YopE is a regulatory protein or a "true" virulence effector protein.

  • 299.
    Ishikawa, Takahiko
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rompikuntal, Pramod Kumar
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lindmark, Barbro
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Milton, Debra L.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wai, Sun Nyunt
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains2009In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 4, no 8, article id e6734Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS.

    METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor.

    CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp.

  • 300.
    Jacobsson, Linn
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Regulation and function of Pax-6 during head and eye development in Drosophila melanogaster2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In Drosophila melanogaster, eyeless and twin of eyeless have important function during eye development. Mutants of both genes give a variety of eye and head phenotypes with the strongest being almost headless meaning that they lack all structures derived from the eye-antennal disc. toy the first eye specification gene expressed in the regulatory network that leads to eye formation and the gene actvates eyeless. What regulates toy is still not clear and has been the focus of this thesis. In Paper I, we analysed part of the upstream region of the toy gene to be able to drive reporter genes with the expected expression pattern in embyo and larval tissues. We found that a 1300-bp region surrounding the toy transcription start is important for correct Toy expression during embryonic and larval development. We also tested for possible redundancy between the toy and ey genes by rescue experiments on some lethal allels in both genes and found that Pax-6 proteins can substitute for each other concerning both head structures and survival. However, rescue is only partial, indicating that the proteins are not fully compatible or that the levels of expression are not sufficiently reproduced by the artificial Gal4-UAS system. Furthermore, we show that inhibition of apoptosis increased survival in strong toy mutants, but did not improve eye phenotypes. In Paper II, we searched for possibly upstream regulators of toy and found that the head gap gene empty spiracles changed the expression pattern of Toy significantly in the embryonic eye-antennal primordium. By clonal analysis and ectopic expressions, we made the conclusion that Ems acts as a repressor of toy during late embryonic development and also at later developmental stages. In Paper III, we investigate presumptive toy enhancer regions within the intron sequences of the gene. Generation and examination of transgenic lines showed that there might be an enhancer region driving toy expression in the embryonic ventral nerve cord within intron 2.

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