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  • 1. Arkblad, Eva L
    et al.
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Pestov, Nikolay B
    Dmitriev, Ruslan I
    Kostina, Maria B
    Stenvall, Jörgen
    Tranberg, Mattias
    Rydström, Jan
    A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress.2005Inngår i: Free Radic Biol Med, ISSN 0891-5849, Vol. 38, nr 11, 1518-25 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 2. Bogomolovas, Julius
    et al.
    Simon, Bernd
    Sattler, Michael
    Stier, Gunter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP). Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
    Screening of fusion partners for high yield expression and purification of bioactive viscotoxins2009Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 64, nr 1, 16-23 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni(2+) affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 11 of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.

  • 3. Bourbon, Henri-Marc
    et al.
    Aguilera, Andres
    Ansari, Aseem Z
    Asturias, Francisco J
    Berk, Arnold J
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Blackwell, T Keith
    Borggrefe, Tilman
    Carey, Michael
    Carlson, Marian
    Conaway, Joan W
    Conaway, Ronald C
    Emmons, Scott W
    Fondell, Joseph D
    Freedman, Leonard P
    Fukasawa, Toshio
    Gustafsson, Claes M
    Han, Min
    He, Xi
    Herman, Paul K
    Hinnebusch, Alan G
    Holmberg, Steen
    Holstege, Frank C
    Jaehning, Judith A
    Kim, Young-Joon
    Kuras, Laurent
    Leutz, Achim
    Lis, John T
    Meisterernest, Michael
    Naar, Anders M
    Nasmyth, Kim
    Parvin, Jeffrey D
    Ptashne, Mark
    Reinberg, Danny
    Ronne, Hans
    Sadowski, Ivan
    Sakurai, Hiroshi
    Sipiczki, Matthias
    Sternberg, Paul W
    Stillman, David J
    Strich, Randy
    Struhl, Kevin
    Svejstrup, Jasper Q
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Winston, Fred
    Roeder, Robert G
    Kornberg, Roger D
    A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II.2004Inngår i: Molecular Cell, ISSN 1097-2765, Vol. 14, nr 5, 553-7 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 4.
    Bäckström, S.
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Sauer, UH.
    The importance of low resolution data: A SAD bromide exampleManuskript (Annet vitenskapelig)
  • 5.
    Bäckström, S.
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Wolf-Watz, M.
    Grundström, C.
    Härd, T.
    Grundström, T.
    The RUNX1 Runt domain at 1.25 Å resolution: a structural switch and specifically bound chloride ions modulate DNA binding.2002Inngår i: J Mol Biol, Vol. 322, nr 2, 259-272 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 6.
    Bäckström, Stefan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    The hematopoietic transcription factor RUNX1: a structural view2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.

    The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.

    The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.

    During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.

    The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution

    synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.

  • 7.
    Edling, Charlotte E.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Pedersen, Malin
    Experimental Clinical Chemistry, Lund University, Malmö University Hospital, Malmö.
    Carlsson, Leif
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Rönnstrand, Lars
    Experimental Clinical Chemistry, Lund University, Malmö University Hospital, Malmö.
    Palmer, Ruth H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Haematopoietic progenitor cells utilise conventional PKC to suppress PKB/Akt activity in response to c-Kit stimulation2007Inngår i: British Journal of Haematology, ISSN 0007-1048, E-ISSN 1365-2141, Vol. 136, nr 2, 260-268 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Receptor tyrosine kinase (RTK) c-Kit signalling is crucial for the proliferation, survival and differentiation of haematopoietic stem cells (HSCs). To further understand the mechanisms underlying these events we explored how the downstream mediators interact. The present study investigated the function of conventional protein kinase Cs (c-PKC) in c-Kit mediated signalling pathways in HSC-like cell lines. This analysis supported earlier findings, that steel factor (SF) activates c-PKC, extracellular signal-regulated kinase (Erk) and protein kinase B (PKB). The present results were consistent with an important role of c-PKC in the positive activation of Erk and for proliferation. Further, it was observed that c-PKC negatively regulated PKB activity upon SF stimulation, indicating that c-PKC acts as a suppressor of c-Kit signalling. Finally, these observations were extended to show that c-PKC mediated the phosphorylation of the endogenous c-Kit receptor on serine 746, resulting in decreased overall tyrosine phosphorylation of c-Kit upon SF stimulation. This report showed that this specific feedback mechanism of c-PKC mediated phosphorylation of the c-Kit receptor has consequences for both proliferation and survival of HSC-like cell lines.

  • 8.
    Ekström, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylase2003Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH).

    To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe.

    The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum.

    The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.

  • 9.
    Ekström, Fredrik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Bäckström, Stefan
    Stier, Gunter
    Flatmark, Torgeir
    Sauer, Uwe H.
    Pseudomonas aeruginosa phenylalanine hydroxylase at 1.6 Å resolution: structural and biochemical characterizationManuskript (Annet vitenskapelig)
  • 10.
    Ekström, Fredrik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Stier, Gunter
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Crystallization of the actin-binding domain of human alpha-actinin: analysis of microcrystals of SeMet-labelled protein2003Inngår i: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, nr Pt 4, 724-726 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.

  • 11.
    Eneqvist, Therese
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Lundberg, Erik
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Karlsson, Anders
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Santos, Ceci­lia R A
    Power, Deborah M
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine.2004Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, nr 25, 26411-6 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

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

  • 12.
    Eneqvist, Therese
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Lundberg, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Nilsson, Lars
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Abagyan, Ruben
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    The transthyretin-related protein family2003Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 270, nr 3, 518-532 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.

  • 13. Gaur, Rahul
    et al.
    Björk, Glenn
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Tuck, Simon
    Umeå centrum för molekylär patogenes (UCMP).
    Varshney, Umesh
    Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block.2007Inngår i: J Biosci, ISSN 0250-5991, Vol. 32, nr 4, 747-54 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Queuosine (Q), a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora. However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C.elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

  • 14.
    Hörnberg, Andreas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Transthyretin from a structural perspective2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation.

    Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation.

    We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils.

    The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.

  • 15.
    Hörnberg, Andreas
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Wikström Hultdin, Ulrika
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    The effect of iodide and chloride on transthyretin structure and stability2005Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 26, 9290-9299 s.Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.

  • 16.
    Johansson, Anna-Mia
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Stenberg, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Pettersson, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Larsson, Jan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation2007Inngår i: PLoS Genet, ISSN 1553-7404, Vol. 3, nr 11, e209- s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional "peak" in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome.

  • 17.
    Larsen, Morten K
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Faergeman, Nils J
    Knudsen, Jens
    MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans.2006Inngår i: Mol Biol Cell, ISSN 1059-1524, Vol. 17, nr 10, 4318-29 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 18.
    Larsson, Jan
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Svensson, Malin J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Stenberg, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Mäkitalo, Maria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Painting of fourth in genus Drosophila suggests autosome-specific gene regulation2004Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, nr 26, 9728-9733 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Painting of fourth (POF) is a chromosome-specific protein in Drosophila and represents the first example of an autosome-specific protein. POF binds to chromosome 4 in Drosophila melanogaster, initiating at the proximal region, followed by a spreading dependent on chromosome 4-specific sequences or structures. Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila, a complex including the male-specific lethal proteins, "paints" the male X chromosome, mediating its hypertranscription, explained to some extent by the acetylation of lysine 16 on histone H4. Here, we show that Pof is essential for viability in both sexes and for female fertility. POF binding to an autosome, the F element, is conserved in genus Drosophila, indicating functional conservation of the autosome specificity. In three of nine studied species, POF binds to the male X chromosome. When bound to the male X, it also colocalizes with the dosage compensation protein male-specific lethal 3, suggesting a relationship to dosage compensation. The chromosome specificity is determined at the species level and not by the amino acid sequence. We argue that POF is involved in a chromosome-specific regulatory function.

  • 19.
    Lorén, Christina
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Investigating the function of the Receptor Tyrosine Kinase ALK during Drosophila melanogaster development2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The Drosophila melanogaster gene Anaplastic Lymphoma Kinase (DAlk) is homologous to mammalian Alk, which is a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans the t(2;5) translocation involving the Alk locus encodes an active form of Alk that is the causative agent in Non-Hodgkin’s Lymphoma (Morris et al., 1994). Alk has also been associated with other cancers such as inflammatory myofibroblastic tumours (IMTs). The physiological function of the Alk RTK has not been described in any system until very recently, and is still not defined in vertebrates. The molecular similarity between Drosophila Alk and mammalian Alk suggested that mutation of Alk in flies may affect similar functional and developmental processes, and thus lead to some understanding of Alk function in vivo.

    By employing an EMS mutagenesis screen we were able to obtain loss-of-function mutants in the Drosophila DAlk gene. Eleven independent DAlk mutants were identified and characterized. DAlk is normally expressed in the developing gut and in the CNS. DAlk mutant animals have a lethal phenotype and die at late embryonic stages or as 1st instar larva. In DAlk mutant embryos there is a complete failure in the development of the midgut whereas the CNS appears normal. The midgut consists of visceral musculature that is syncytial and is formed by fusion of multiple myoblasts. This is a dynamic process where two types of myoblasts, i.e. fusion-competent-myoblasts and founder-cells that function as seeds for muscle formation, fuse. In DAlk homozygous embryos there is no founder cell specification, which explains the failure of midgut formation in these embryos.

    Recently a novel secreted molecule Jelly Belly (Jeb) was identified. Jeb is expressed in the tissue neighbouring the DAlk expressing cells of the developing visceral mesoderm. Jeb mutant embryos show a phenotype that is similar to that of DAlk mutant embryos. We have been able to show that Jeb is the ligand for DAlk in the developing visceral mesoderm and that Jeb binding stimulates a DAlk driven ERK signaling pathway. This leads to the expression of Dumbfounded (duf)/kin of Irregular chiasm-C (kirre), a founder-cell specific immunoglobulin that has an important role in myoblast aggregation and fusion.

    The functional Drosophila midgut is made up of the visceral muscle that encircles the endodermal tube. This tube formation includes migration of cells originating in the anterior and posterior parts of the embryo, first along the anterior-posterior axis using the visceral mesoderm as a template, then dorsally and ventrally. In DAlk mutant embryos there is no visceral muscle fusion and both the visceral mesoderm and the endoderm fail to undergo dorsal-ventral migration.

  • 20.
    Lorén, Christina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Englund, Camilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Grabbe, Caroline
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Hunter, Tony
    Palmer, Ruth H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    A crucial role for the Anaplastic lymphoma kinase receptor tyrosine kinase in gut development in Drosophila melanogaster2003Inngår i: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 4, nr 8, 781-786 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, which encodes a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans, the t(2;5) translocation, which involves the ALK locus, produces an active form of ALK, which is the causative agent in non-Hodgkin's lymphoma. The physiological function of the Alk RTK, however, is unknown. In this paper, we describe loss-of-function mutants in the Drosophila Alk gene that cause a complete failure of the development of the gut. We propose that the main function of Drosophila Alk during early embryogenesis is in visceral mesoderm development.

  • 21.
    Lorén, Christina
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Palmer, RH
    Visceral muscle fusion is essential for the dorsal-ventral migration of the endoderm in Drosophila.Manuskript (Annet vitenskapelig)
  • 22.
    Lorén, Christina
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Scully, A
    Grabbe, C
    Umeå universitet, Medicinsk fakultet, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Edeen, PT
    Thomas, J
    McKeown, M
    Hunter, T
    Palmer, RH
    Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo.2001Inngår i: Genes to Cells, ISSN 1356-9597, Vol. 6, nr 6, 531-544 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 23. Mijovilovich, Ana
    et al.
    Ekström, Fredrik
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Meyer-Klaucke, Wolfram
    Sauer, Uwe H.
    A close look at the catalytic centre of phenylalanine hydroxylase from the opportunistic pathogen Pseudomonas aeruginosa.Manuskript (Annet vitenskapelig)
  • 24. Morgado, Isabel
    et al.
    Melo, Eduardo P
    Lundberg, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Estrela, Nídia L
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Power, Deborah M
    Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal2008Inngår i: Molecular and Cellular Endocrinology, ISSN 0303-7207, E-ISSN 1872-8057, Vol. 295, nr 1-2, 48-58 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

  • 25. Nilsson, L
    et al.
    Li, X
    Tiensuu, T
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Auty, R
    Greenwald, I
    Tuck, S
    Caenorhabditis elegans lin-25: cellular focus, protein expression and requirement for sur-2 during induction of vulval fates1998Inngår i: Development, Vol. 125, nr 23, 4809-4819 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 26. Nilsson, L
    et al.
    Tiensuu, T
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Tuck, S
    Caenorhabditis elegans lin-25: a study of its role in multiple cell fate specification events involving Ras and the identification and characterization of evolutionary conserved domains2000Inngår i: Genetics, Vol. 156, nr 3, 1083-1096 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 27.
    Nilsson, Lars
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Conradt, Barbara
    Ruaud, Anne-Françoise
    Chen, Carlos Chih-Hsiung
    Hatzold, Julia
    Bessereau, Jean-Louis
    Grant, Barth
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Caenorhabditis elegans num-1 negatively regulates endocytic recycling2008Inngår i: Genetics, Vol. 179, 375-387 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in C. elegans, endocytic recycling is inhibited by NUM-1A, the nematode Numb homologue. NUM-1A::GFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling both in C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.

  • 28.
    Nyström-Friberg, Josefin
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Lundstedt, Staffan
    Tuck, Simon
    S. Caenorhabditis elegans RSKS-1, a homologue of p70 S6 kinase, functions in sensory neurons to modulate fat metabolism and entry into dauer.Artikkel i tidsskrift (Fagfellevurdert)
  • 29.
    Nyström-Friberg, Josefin
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Nilsson, Lars
    Tuck, Simon
    A new technique for genetic mosaic analysis in the nematode Caenorhabditis elegansManuskript (Annet vitenskapelig)
  • 30.
    Olofsson, Anders
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Amyloid fibril dynamics revealed by combined hydrogen/deuterium exchange and nuclear magnetic resonance2009Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 385, nr 2, 374-376 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A general method to explore the dynamic nature of amyloid fibrils is described, combining hydrogen/deuterium exchange and nuclear magnetic resonance spectroscopy to determine the exchange rates of individual amide protons within an amyloid fibril. Our method was applied to fibrils formed by the amyloid-beta(1-40) peptide, the major protein component of amyloid plaques in Alzheimer's disease. The fastest exchange rates were detected among the first 14 residues of the peptide, a stretch known to be poorly structured within the fibril. Considerably slower exchange rates were observed in the remainder of the peptide within the beta-strand-turn-beta-strand motif that constitutes the fibrillar core.

  • 31. Rocheleau, Christian E
    et al.
    Rönnlund, Agneta
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Sundaram, Meera V
    Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling.2005Inngår i: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 102, nr 33, 11757-62 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 32. Schumacher, B
    et al.
    Schertel, C
    Wittenburg, N
    Tuck, S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Mitani, S
    Gartner, A
    Conradt, B
    Shaham, S
    C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage.2005Inngår i: Cell Death Differ, ISSN 1350-9047, Vol. 12, nr 2, 153-61 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 33.
    Shen, Zhai-Zhong
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Nyström-Friberg, Josefin
    Padgett, Richard W.
    Sitaram, Raviprakash T.
    Tillberg, Karin
    Leroi, Armand
    Tuck, Simon
    Identification of new loci involved in the regulation of body size in C. elegansManuskript (Annet vitenskapelig)
  • 34.
    Tiensuu, Teresa
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Cell fate specification by Ras-mediated cell signalling in C. elegans2003Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.

  • 35. Tiensuu, Teresa
    et al.
    Larsen, Morten Krog
    Vernersson, Emma
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    lin-1 has both positive and negative functions in specifying multiple cell fates induced by Ras/MAP kinase signaling in C. elegans.2005Inngår i: Dev Biol, ISSN 0012-1606, Vol. 286, nr 1, 338-51 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 36.
    Tiensuu, Teresa
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Tuck, Simon
    lin-1 functions in multiple cell fate specification events in C. elegans involving Ras/MAP kinase signalingManuskript (Annet vitenskapelig)
  • 37.
    Tiensuu, Teresa
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Tuck, Simon
    lin-25, a gene involved in Ras-mediatedsignalling in C. elegans, associates with the Mediator complexManuskript (Annet vitenskapelig)
  • 38.
    Tångrot, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Kågström, Bo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Högpresterande beräkningscentrum norr (HPC2N).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Accurate Domain Identification with Structure-Anchored Hidden Markov Models, saHMMs2009Inngår i: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, nr 2, 343-352 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The ever increasing speed of DNA sequencing widens the discrepancy between the number of known gene products, and the knowledge of their function and structure. Proper annotation of protein sequences is therefore crucial if the missing information is to be deduced from sequence-based similarity comparisons. These comparisons become exceedingly difficult as the pairwise identities drop to very low values. To improve the accuracy of domain identification, we exploit the fact that the three-dimensional structures of domains are much more conserved than their sequences. Based on structure-anchored multiple sequence alignments of low identity homologues we constructed 850 structure-anchored hidden Markov models (saHMMs), each representing one domain family. Since the saHMMs are highly family specific, they can be used to assign a domain to its correct family and clearly distinguish it from domains belonging to other families, even within the same superfamily. This task is not trivial and becomes particularly difficult if the unknown domain is distantly related to the rest of the domain sequences within the family. In a search with full length protein sequences, harbouring at least one domain as defined by the structural classification of proteins database (SCOP), version 1.71, versus the saHMM database based on SCOP version 1.69, we achieve an accuracy of 99.0%. All of the few hits outside the family fall within the correct superfamily. Compared to Pfam_ls HMMs, the saHMMs obtain about 11% higher coverage. A comparison with BLAST and PSI-BLAST demonstrates that the saHMMs have consistently fewer errors per query at a given coverage. Within our recommended E-value range, the same is true for a comparison with SUPERFAMILY. Furthermore, we are able to annotate 232 proteins with 530 nonoverlapping domains belonging to 102 different domain families among human proteins labelled unknown in the NCBI protein database. Our results demonstrate that the saHMM database represents a versatile and reliable tool for identification of domains in protein sequences. With the aid of saHMMs, homology on the family level can be assigned, even for distantly related sequences. Due to the construction of the saHMMs, the hits they provide are always associated with high quality crystal structures. The saHMM database can be accessed via the FISH server at http://babel.ucmp.umu.se/fish/.

  • 39.
    Tångrot, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Wang, Lixiao
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Kågström, Bo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Högpresterande beräkningscentrum norr (HPC2N).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Design, construction and use of the FISH server2007Inngår i: Applied parallel computing: state of the art in scientific computing, Springer Link , 2007, 647-657 s.Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    At the core of the FISH (Family Identification with Structure anchored Hidden Markov models, saHMMs) server lies the midnight ASTRAL set. It is a collection of protein domains with low mutual sequence identity within homologous families, according to the structural classification of proteins, SCOP. Here, we evaluate two algorithms for creating the midnight ASTRAL set. The algorithm that limits the number of structural comparisons is about an order of magnitude faster than the all-against-all algorithm. We therefore choose the faster algorithm, although it produces slightly fewer domains in the set. We use the midnight ASTRAL set to construct the structure-anchored Hidden Markov Model data base, saHMM-db, where each saHMM represents one family. Sequence searches using saHMMs provide information about protein function, domain organization, the probable 2D and 3D structure, and can lead to the discovery of homologous domains in remotely related sequences.

  • 40.
    Vaitkevicius, Karolis
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lindmark, Barbro
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ou, Gangwei
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Song, Tianyan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Toma, Claudia
    Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
    Iwanaga, Masaaki
    Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
    Zhu, Jun
    Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia.
    Andersson, Agneta
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och geovetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå marina forskningscentrum (UMF).
    Hammarström, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Tuck, Simon
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing2006Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, nr 24, 9280-9285 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.

  • 41.
    Williams, Michael J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Drosophila hemopoiesis and cellular immunity2007Inngår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 178, nr 8, 4711-4716 s.Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    In Drosophila melanogaster larvae, three classes of circulating cellular immune surveillance cells (hemocytes) can be identified: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes are profiessional phagocytes most similar to the mammalian monocyte/macrophage lineage and make up similar to 95% of circulating hemocytes. The other similar to 5% of circulating hemocytes consists of crystal cells, which secrete components necessary for the melanization of invading organisms, as well as for wound repair. A third cell type known as lamellocytes are rarely seen in healthy larvae and are involved in the encapsulation of invading pathogens. There are no obvious mammalian counterparts for crystal cells or lamellocytes, and there is no equivalent to the lymphoid lineage in insects. In this review, I will discuss what is currently known about Drosophila hemopoiesis and the cellular immune response and where possible compare it to vertebrate mechanisms.

  • 42.
    Zettervall, Carl-Johan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Signaling pathways in the activation and proliferation of Drosophila melanogaster blood cells2005Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.

  • 43.
    Zettervall, Carl-Johan
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP).
    Albright, Shannon
    Hultmark, Dan
    Pvf2/Pvr signaling in proliferating and activated Drosophila hemocytes.Manuskript (Annet vitenskapelig)
1 - 43 of 43
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