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  • 1.
    Bäckström, Stefan
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Wolf-Watz, Magnus
    Chemistry.
    Xie, X Q
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, Torleif
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Crystallization and preliminary studies of the DNA-binding runt domain of AML1.2001In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 57, no Pt 2, p. 269-71Article in journal (Refereed)
    Abstract [en]

    The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

  • 2.
    Bäckström, Stefan
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Wolf-Watz, Magnus
    Chemistry.
    Grundström, Christine
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, Torleif
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.2002In: J Mol Biol, ISSN 0022-2836, Vol. 322, no 2, p. 259-72Article in journal (Refereed)
    Abstract [en]

    The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

  • 3. Dalhus, Bjørn
    et al.
    Saarinen, Markuu
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Eklund, Pär
    Johansson, Kenth
    Karlsson, Andreas
    Ramaswamy, S
    Bjørk, Alexandra
    Synstad, Bjørnar
    Naterstad, Kristine
    Sirevåg, Reidun
    Eklund, Hans
    Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.2002In: J Mol Biol, ISSN 0022-2836, Vol. 318, no 3, p. 707-21Article in journal (Refereed)
    Abstract [en]

    The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.

  • 4.
    Ekström, Fredrik
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Stier, Gunter
    Eaton, Julian T
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Crystallization and X-ray analysis of a bacterial non-haem iron-containing phenylalanine hydroxylase from the Gram-negative opportunistic pathogen Pseudomonas aeruginosa.2003In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 59, no Pt 7, p. 1310-2Article in journal (Refereed)
    Abstract [en]

    Monooxygenases are frequently involved in the pathways that mediate the pivotal role of microorganisms in recycling carbon from the environment. A structural study of a monooxygenase from Pseudomonas aeruginosa that was identified as a phenylalanine hydroxylase has been initiated. The single-domain monomeric protein harbours a non-haem iron at the active site. The sequence identity to the catalytic domains of tyrosine and tryptophan hydroxylases suggests that the enzyme is not restricted to the substrate phenylalanine alone. Here, the cloning, purification and crystallization of native and SeMet-labelled P. aeruginosa phenylalanine hydroxylase are reported. Crystals grew in space group P6(1), with unit-cell parameters a = b = 210.5, c = 100.7 A, and diffracted to a d spacing of 2.0 A. Crystals of SeMet-labelled protein were used to collect a three-wavelength multiple anomalous dispersion (MAD) data set around the Se K edge.

  • 5.
    Ekström, Fredrik
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Stier, Gunter
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Crystallization of the actin-binding domain of human alpha-actinin: analysis of microcrystals of SeMet-labelled protein2003In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, no Pt 4, p. 724-726Article in journal (Refereed)
    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.

  • 6.
    Eneqvist, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Andersson, Karin
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Olofsson, Anders
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The beta-slip: a novel concept in transthyretin amyloidosis.2000In: Mol Cell, ISSN 1097-2765, Vol. 6, no 5, p. 1207-18Article in journal (Refereed)
    Abstract [en]

    Transthyretin is a tetrameric plasma protein associated with two forms of amyloid disease. The structure of the highly amyloidogenic transthyretin triple mutant TTRG53S/E54D/L55S determined at 2.3 A resolution reveals a novel conformation: the beta-slip. A three-residue shift in beta strand D places Leu-58 at the position normally occupied by Leu-55 now mutated to serine. The beta-slip is best defined in two of the four monomers, where it makes new protein-protein interactions to an area normally involved in complex formation with retinol-binding protein. This interaction creates unique packing arrangements, where two protein helices combine to form a double helix in agreement with fiber diffraction and electron microscopy data. Based on these findings, a novel model for transthyretin amyloid formation is presented.

  • 7.
    Eneqvist, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Lundberg, Erik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Karlsson, Anders
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Santos, Ceci­lia R A
    Power, Deborah M
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 25, p. 26411-6Article in journal (Refereed)
    Abstract [en]

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

  • 8.
    Eneqvist, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Ando, Yukio
    Miyakawa, Taihei
    Katsuragi, Shoichi
    Jass, Jana
    Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Disulfide-bond formation in the transthyretin mutant Y114C prevents amyloid fibril formation in vivo and in vitro.2002In: Biochemistry, ISSN 0006-2960, Vol. 41, no 44, p. 13143-51Article in journal (Refereed)
    Abstract [en]

    The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.

  • 9.
    Eneqvist, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Structural distribution of mutations associated with familial amyloidotic polyneuropathy in human transthyretin.2001In: Amyloid, ISSN 1350-6129, Vol. 8, no 3, p. 149-68Article in journal (Refereed)
    Abstract [en]

    The human plasma protein transthyretin (TTR) is a highly stable soluble homotetrameric protein. Still, conformational changes in the wild type protein can lead to self-assembly into insoluble amyloid fibrils. In addition, 74 point mutations are known to enhance amyloid formation causing familial amyloidotic polyneuropathy (PAP). Alignment of TTR sequences from twenty different species shows that only six of these mutations occur as natural amino acids in other organisms. In this paper we analyse the distribution of FAP mutations within the three-dimensional structure of TTR. Contradictory to what might be expected from protein stability studies, the mutations are not restricted to structurally rigid parts of the molecule, nor are they concentrated at the monomer interaction sites.

  • 10. Enerly, Espen
    et al.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Lambertsson, Andrew
    Silencing the Drosophila ribosomal protein L14 gene using targeted RNA interference causes distinct somatic anomalies.2003In: Gene, ISSN 0378-1119, Vol. 320, p. 41-8Article in journal (Other academic)
    Abstract [en]

    The Drosophila Minutes are haploinsufficient mutations that are defective in ribosomal protein (rp) production, resulting in short, thin bristles, delayed development and recessive lethality. In a Minute fly, the amount of rp gene messenger RNA (mRNA) is reduced to >or=50% of the normal amount of gene product, and becomes rate limiting for ribosome biogenesis, cell proliferation and growth. Haploinsufficiency increases the vulnerability to complete loss of gene function (homozygous null state) if hit by a second mutation. Because of the homozygous lethality, it has only been possible to study the effects of Minute mutations in heterozygous animals. To be able to study the consequences of a loss-of-function of an rp gene (0%>mRNA<50%) in developing and differentiated cells we used heritable RNA interference (RNAi) in combination with the yeast GAL4/UAS binary system to spatiotemporally knock down the ribosomal protein L14 (RpL14) gene. We show, at the RNA and phenotypic levels, that RNAi efficiently reduces RpL14 gene expression throughout development, causing lethality and distinct and dramatic somatic anomalies in both developing and differentiated cells.

  • 11.
    Gariani, Talal
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Samuelsson, Tore
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY.2006In: J Struct Biol, ISSN 1047-8477, Vol. 153, no 1, p. 85-96Article in journal (Refereed)
    Abstract [en]

    The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG’s GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.

  • 12.
    Gariani, Talal
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Crystallization and preliminary X-ray diffraction studies of the signal recognition particle receptor FtsY from Mycoplasma mycoides.2000In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 56, no Pt 8, p. 1030-2Article in journal (Refereed)
    Abstract [en]

    The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system. All four proteins are part of a unique subfamily of GTPases. Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified. Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials. Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E. coli). A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation. The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit.

  • 13.
    Hainzl, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Interaction of signal-recognition particle 54 GTPase domain and signal-recognition particle RNA in the free signal-recognition particle.2007In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 104, no 38, p. 14911-6Article in journal (Refereed)
    Abstract [en]

    The signal-recognition particle (SRP) is a ubiquitous protein-RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-A resolution of the SRP54-SRP19-SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.

  • 14.
    Hainzl, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Structural insights into SRP RNA: an induced fit mechanism for SRP assembly.2005In: RNA, ISSN 1355-8382, Vol. 11, no 7, p. 1043-50Article in journal (Refereed)
    Abstract [en]

    Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.

  • 15.
    Hainzl, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Structure of the SRP19 RNA complex and implications for signal recognition particle assembly.2002In: Nature, ISSN 0028-0836, Vol. 417, no 6890, p. 767-71Article in journal (Refereed)
    Abstract [en]

    The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.

  • 16.
    Huang, Shenghua
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sjöblom, Björn
    Chemistry.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Jonsson, Bengt-Harald
    Chemistry.
    Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies of a T199P/C206S mutant.2002In: Biochemistry, ISSN 0006-2960, Vol. 41, no 24, p. 7628-35Article in journal (Refereed)
    Abstract [en]

    Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.

  • 17.
    Huang, Shenghua
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Xue, Y
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Chirica, L
    Lindskog, Sven
    Chemistry.
    Jonsson, Bengt-Harald
    Chemistry.
    Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide.1998In: J Mol Biol, ISSN 0022-2836, Vol. 283, no 1, p. 301-10Article in journal (Refereed)
    Abstract [en]

    The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.

  • 18.
    Hörnberg, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Eneqvist, Therese
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    A comparative analysis of 23 structures of the amyloidogenic protein transthyretin.2000In: J Mol Biol, ISSN 0022-2836, Vol. 302, no 3, p. 649-69Article in journal (Refereed)
    Abstract [en]

    Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.

  • 19.
    Hörnberg, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Eneqvist, Therese
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The β-strand D of transthyretin trapped in two discrete conformations2004In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1700, no 1, p. 93-104Article in journal (Refereed)
    Abstract [en]

    Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.

  • 20.
    Johansson, Anna-Mia
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Bernhardsson, Carolina
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster.2007In: EMBO J, ISSN 0261-4189, Vol. 26, no 9, p. 2307-2316Article in journal (Refereed)
    Abstract [en]

    Drosophila melanogaster exhibits two expression-regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male-specific lethal complex doubles transcription of genes on the male X-chromosome and the chromosome 4-specific protein Painting of fourth, POF. POF is the first example of an autosome-specific protein and its presence raises the question of the universality of chromosome-specific regulation. Here we show that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo-4th karyotype flies. However, Pof is essential for survival of haplo-4th individuals and expression of chromosome 4 genes in diplo-4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome.

  • 21.
    Johansson, Anna-Mia
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Pettersson, Fredrik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation2007In: PLoS Genet, ISSN 1553-7404, Vol. 3, no 11, p. e209-Article in journal (Refereed)
    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.

  • 22.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Leipuviene, Ramune
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Pollard, Michael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Qian, Qiang
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 3, p. 750-757Article in journal (Refereed)
    Abstract [en]

    The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and BACTERIA: In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.

  • 23.
    Karlsson, Anders
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Structural and functional properties of transthyretin2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information.

    The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.

    The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly.

    We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals.

  • 24.
    Karlsson, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Eneqvist, Therese
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Cys114-linked dimers of transthyretin are compatible with amyloid formation.2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 39, p. 13063-70Article in journal (Refereed)
    Abstract [en]

    The Tyr114Cys substitution in the human plasma protein transthyretin leads to a particularly aggressive form of familial amyloidotic polyneuropathy. In a previous study we demonstrated that ATTR Tyr114Cys forms intermolecular disulfide bonds, which partly impair fibril formation and result in a more amorphous morphology. Apart from the introduced cysteinyl group in position 114, the native sequence contains one cysteine located at position 10. To deduce the role of intermolecular disulfide bridging in fibril formation we generated and characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular cysteine bridge at position 114 enhances the exposure of cysteine 10, thereby facilitating additional intermolecular cysteine assemblies. We also purified a disulfide-linked dimeric form of TTR Cys10Ala/Tyr114Cys, which was recognized by the anti-TTR amyloid-specific monoclonal antibody MAb (39-44). Moreover, this dimeric molecule can form protofibrils indistinguishable from the fibrils formed under reducing conditions, as judged by atomic force microscopy. Assuming that both molecules of the dimer are part of the core of the fibril, the assembly is incompatible with a preserved native or near-native dimeric interphase. Our findings raise the question of whether TTR-amyloid architecture is indeed the result of one highly stringent assembly of structures or if different fibrils may be built from different underlying structures.

  • 25.
    Karlsson, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Heating of proteins as a means of improving crystallization: a successful case study on a highly amyloidogenic triple mutant of human transthyretin.2007In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 63, no Pt 8, p. 695-700Article in journal (Refereed)
    Abstract [en]

    The use of high temperatures in the purification procedures of heat-stable proteins is a well established technique. Recently, rapid pre-heat treatment of protein samples prior to crystallization trials was described as a final polishing step to improve the diffraction properties of crystals [Pusey et al. (2005), Prog. Biophys. Mol. Biol. 88, 359-386]. The present study demonstrates that extended high-temperature incubation (328 K for 48 h) of the highly amyloidogenic transthyretin mutant TTR G53S/E54D/L55S successfully removes heterogeneities and allows the reproducible growth of well diffracting crystals. Heat treatment might be applied as an optimization method to other cases in which the protein/biomolecule fails to form diffracting crystals.

  • 26.
    Larsson, Jan
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Meller, Victoria H
    Dosage compensation, the origin and the afterlife of sex chromosomes.2006In: Chromosome Res, ISSN 0967-3849, Vol. 14, no 4, p. 417-31Article in journal (Other academic)
    Abstract [en]

    Over the past 100 years Drosophila has been developed into an outstanding model system for the study of evolutionary processes. A fascinating aspect of evolution is the differentiation of sex chromosomes. Organisms with highly differentiated sex chromosomes, such as the mammalian X and Y, must compensate for the imbalance in gene dosage that this creates. The need to adjust the expression of sex-linked genes is a potent force driving the rise of regulatory mechanisms that act on an entire chromosome. This review will contrast the process of dosage compensation in Drosophila with the divergent strategies adopted by other model organisms. While the machinery of sex chromosome compensation is different in each instance, all share the ability to direct chromatin modifications to an entire chromosome. This review will also explore the idea that chromosome-targeting systems are sometimes adapted for other purposes. This appears the likely source of a chromosome-wide targeting system displayed by the Drosophila fourth chromosome

  • 27.
    Lundberg, Erik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Transthyretin and the transthyretin-related protein: A structural study2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transthyretin (TTR) is one of several proteins involved in amyloid disease in humans. Unknown conformational changes of the native state of TTR result in aggregation of TTR molecules into amyloid fibrils, which accumulate in extracellular tissues. This may result in different clinical symptoms, e.g. polyneuropathy or cardiomyopathy, depending on their site of accumulation.

    Our long-term goal is to identify structural changes associated with amyloid formation. For this work, structural characterization of TTR from other species than human may provide valuable information.

    The three-dimensional X-ray crystallographic structure of TTR from sea bream (Sparus aurata) was determined at 1.75 Å resolution. Human and sea bream TTR were found to be structurally very similar. However, interesting differences were present in the area at and around -strand D, which in fish forms an extended loop region. Interestingly, this area is believed to dissociate from the structure prior to amyloid formation, to allow -strands A and B to participate in polymerization.

    During evolution, TTR from different species have developed differences in preference to their natural ligands, the thyroid hormones 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4). While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution. A significantly wider entrance and narrower thyroid hormone binding channel provide a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.

    In a separate work, we identified a novel protein family with structural similarity to TTR, which we named the transthyretin-related protein (TRP) family. To attain information about this protein family, we cloned, expressed, purified and characterized TRP from Escherichia coli (EcTRP). Furthermore, we solved the structure of EcTRP to 1.65 Å resolution. As predicted, EcTRP and human TTR are structurally very similar. Interesting structural differences are found in the area corresponding to the thyroid hormone binding site in TTR, which due to its amino acid conservation within the TRP family we identified as a putative ligand-binding site in TRPs. The function of the TRP is not known, however, recent studies suggest that it might be involved in purine catabolism.

    It has been shown that partial acid denaturation of human TTR results in amyloid-fibril formation. Interestingly, we have shown that sea bream TTR also forms amyloid-like fibrils in vitro, even though it shares only 52% sequence identity to human TTR. Corresponding studies on EcTRP did not generate amyloid-like fibrils. EcTRP has 30% sequence identity to human TTR. The fact that two of the proteins form amyloid fibrils and one does not means that they can serve as a model system for the study of amyloid formation. Further studies on these three proteins are currently performed to attain more information about the mechanism of amyloid formation.

  • 28.
    Lundberg, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Bäckström, Stefan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The transthyretin-related protein: structural investigation of a novel protein family2006In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 155, no 3, p. 445-457Article in journal (Refereed)
    Abstract [en]

    The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.

  • 29.
    Lundberg, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Sauer-Eriksson, A. Elisabeth
    Stability and amyloid formation of transthyretin and the transthyretin-related proteinManuscript (preprint) (Other academic)
  • 30.
    Olofsson, Anders
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Borowik, Tomasz
    Gröbner, Gerhard
    Faculty of Science and Technology, Chemistry.
    Sauer-Eriksson, Elisabeth
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Negatively Charged Phospholipid Membranes Induce Amyloid Formation of Medin via an alpha-Helical Intermediate2007In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 374, no 1, p. 186-94Article in journal (Refereed)
    Abstract [en]

    Medin, a recently discovered 5.5 kDa peptide, is associated with amyloid deposits in the medial layer of human arteries and the prevalence is nearly 100% within individuals above 50 years. Presently, not much is known about its biochemical and biophysical properties or its pathway from soluble peptide to insoluble amyloid. Here we have characterized the behavior of medin in the presence of lipid membranes, using circular dichroism, isothermal titration calorimetry, differential scanning calorimetry, size exclusion chromatography, and atomic force microscopy (AFM). Medin was shown to exist as a monomer in solution with a predominantly random-coil structure. It binds lipid vesicles that have either a neutral or a negative surface potential. Upon association to membranes containing acidic lipids, it undergoes an electrostatically driven conformational change towards a mainly α-helical state. Prolonged incubation converts medin from an α-helical structure into an amyloid β-sheet fibrillar state as confirmed by AFM. Based on these findings, we propose a mechanism of medin-amyloid formation where medin electrostatically associates in its monomeric form to biological interfaces displaying a negative potential. This process both increases the local peptide concentration and induces an aggregation-prone α-helical fold.

  • 31.
    Olofsson, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Ippel, Johannes H
    Wijmenga, Sybren S
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ohman, Anders
    Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 7, p. 5699-707Article in journal (Refereed)
    Abstract [en]

    The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.

  • 32.
    Olofsson, Anders
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Lindhagen-Persson, Malin
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Amide solvent protection analysis demonstrates that amyloid-beta(1-40) and amyloid-beta(1-42) form different fibrillar structures under identical conditions.2007In: Biochem J, ISSN 1470-8728, Vol. 404, no 1, p. 63-70Article in journal (Refereed)
    Abstract [en]

    AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39–43 residue long Ab (amyloid-b)-peptide. The most abundant species, Ab(1–40) and Ab(1–42), are both present within senile plaques, but Ab(1–42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Ab-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Ab(1–40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Ab(1–42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Ab(1–40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two b-strands connected with a bend. This protection pattern partly resembles the pattern found in Ab(1–42) fibrils, but the Ab(1–40) fibrils display a significantly increased protection for the N-terminal residues Phe4–His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37–Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.

  • 33.
    Olofsson, Anders
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The solvent protection of alzheimer amyloid-beta-(1-42) fibrils as determined by solution NMR spectroscopy.2006In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 281, no 1, p. 477-83Article in journal (Refereed)
    Abstract [en]

    Alzheimer disease is a neurodegenerative disorder that is tightly linked to the self-assembly and amyloid formation of the 39-43-residue-long amyloid-beta (Abeta) peptide. Considerable evidence suggests a correlation between Alzheimer disease development and the longer variants of the peptide, Abeta-(1-42/43). Currently, a molecular understanding for this behavior is lacking. In the present study, we have investigated the hydrogen/deuterium exchange of Abeta-(1-42) fibrils under physiological conditions, using solution NMR spectroscopy. The obtained residue-specific and quantitative map of the solvent protection within the Abeta-(1-42) fibril shows that there are two protected core regions, Glu11-Gly25 and Lys28-Ala42, and that the residues in between, Ser26 and Asn27, as well as those in the N terminus, Asp1-Tyr10, are solvent-accessible. This result reveals considerable discrepancies when compared with a previous investigation on Abeta-(1-40) fibrils and suggests that the additional residues in Abeta-(1-42), Ile41 and Ala42, significantly increase the solvent protection and stability of the C-terminal region Lys28-Ala42. Consequently, our findings provide a molecular explanation for the increased amyloidogenicity and toxicity of Abeta-(1-42) compared with shorter Abeta variants found in vivo.

  • 34.
    Petzold, Katja
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Öhman, Anders
    Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Backman, Lars
    Chemistry.
    Folding of the αΙΙ-spectrin SH3 domain under physiological salt conditions2008In: Archives of Biochemistry and Biophysics, Vol. 474, no 1, p. 39-47Article in journal (Refereed)
    Abstract [en]

    The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of II-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the II-spectrin SH3 domain.

  • 35. Qi, Dai
    et al.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Mannervik, Mattias
    Drosophila Ada2b is required for viability and normal histone H3 acetylation.2004In: Mol Cell Biol, ISSN 0270-7306, Vol. 24, no 18, p. 8080-9Article in journal (Refereed)
    Abstract [en]

    Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent

  • 36.
    Sauer-Eriksson, Elisabeth
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Hainzl, Tobias
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    S-domain assembly of the signal recognition particle.2003In: Curr Opin Struct Biol, ISSN 0959-440X, Vol. 13, no 1, p. 64-70Article, review/survey (Other (popular science, discussion, etc.))
    Abstract [en]

    The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.

  • 37. Stabell, Marianne
    et al.
    Eskeland, Ragnhild
    Bjørkmo, Mona
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Aalen, Reidunn B
    Imhof, Axel
    Lambertsson, Andrew
    The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development.2006In: Nucleic Acids Res, ISSN 1362-4962, Vol. 34, no 16, p. 4609-21Article in journal (Refereed)
    Abstract [en]

    Mammalian G9a is a histone H3 Lys-9 (H3-K9) methyltransferase localized in euchromatin and acts as a co-regulator for specific transcription factors. G9a is required for proper development in mammals as g9a-/g9a- mice show growth retardation and early lethality. Here we describe the cloning, the biochemical and genetical analyses of the Drosophila homolog dG9a. We show that dG9a shares the structural organization of mammalian G9a, and that it is a multi-catalytic histone methyltransferase with specificity not only for lysines 9 and 27 on H3 but also for H4. Surprisingly, it is not the H4-K20 residue that is the target for this methylation. Spatiotemporal expression analyses reveal that dG9a is abundantly expressed in the gonads of both sexes, with no detectable expression in gonadectomized adults. In addition we find a low but clearly observable level of dG9a transcript in developing embryos, larvae and pupae. Genetic and RNAi experiments reveal that dG9a is involved in ecdysone regulatory pathways.

  • 38. Stabell, Marianne
    et al.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Aalen, Reidunn B
    Lambertsson, Andrew
    Drosophila dSet2 functions in H3-K36 methylation and is required for development.2007In: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 359, no 3, p. 784-9Article in journal (Refereed)
    Abstract [en]

    Lysine methylation has important functions in biological processes that range from heterochromatin formation to transcription regulation. Here, we demonstrate that Drosophila dSet2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of dSet2 lack dSet2 expression and H3-K36 methylation, indicating that dSet2 is the sole enzyme responsible for this modification in Drosophila melanogaster. dSet2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdysone-controlled genetic program. A transheterozygous EcR null mutation/dSet2 RNAi combination produces a complete (balloon) separation of the wing surfaces, revealing a genetic interaction between EcR and dSet2. Using immunoprecipitation, we demonstrate that dSet2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII).

  • 39.
    Stenberg, Per
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Pettersson, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Saura, Anja O
    Department of Genetics, University of Helsinki, Helsinki, Finland.
    Berglund, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sequence signature analysis of chromosome identity in three Drosophila species2005In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 6, no 158, p. 1-17Article in journal (Refereed)
    Abstract [en]

    Background: All eukaryotic organisms need to distinguish each of their chromosomes. A few protein complexes have been described that recognise entire, specific chromosomes, for instance dosage compensation complexes and the recently discovered autosome-specific Painting of Fourth (POF) protein in Drosophila. However, no sequences have been found that are chromosome-specific and distributed over the entire length of the respective chromosome. Here, we present a new, unbiased, exhaustive computational method that was used to probe three Drosophila genomes for chromosome-specific sequences.

    Results: By combining genome annotations and cytological data with multivariate statistics related to three Drosophila genomes we found sequence signatures that distinguish Muller's F-elements ( chromosome 4 in D. melanogaster) from all other chromosomes in Drosophila that are not attributable to differences in nucleotide composition, simple sequence repeats or repeated elements. Based on these signatures we identified complex motifs that are strongly overrepresented in the F-elements and found indications that the D. melanogaster motif may be involved in POF-binding to the F-element. In addition, the X-chromosomes of D. melanogaster and D. yakuba can be distinguished from the other chromosomes, albeit to a lesser extent. Surprisingly, the conservation of the F-element sequence signatures extends not only between species separated by approximately 55 Myr, but also linearly along the sequenced part of the F-elements.

    Conclusion: Our results suggest that chromosome-distinguishing features are not exclusive to the sex chromosomes, but are also present on at least one autosome ( the F-element) in Drosophila.

  • 40.
    Svensson, Malin J.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Thioredoxins and gene regulation in the Drosophila germline2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The process of spermatogenesis is in many organisms one of the most dramatic cellular transformations - a normal round diploid cell is ultimately transformed into a needle shaped haploid cell with tightly packaged cell machinery. In Drosophila melanogaster this process involves several characteristic stages, one of these being the primary spermato-cyte stage, which is the stage prior to meiosis. This stage is characterized by a loose chromatin structure in the nucleus and exceptionally high rates of transcription and translation to produce essentially all the mRNAs and proteins that are needed later during spermatid formation. Two proteins that are expressed in high levels in primary spermatocytes are ThioredoxinT (TrxT) and Painting of fourth (POF).

    Thioredoxins are small thiol proteins that reduce disulfides in other proteins, a mechanism that is utilized in many different contexts. In this thesis I show that the TrxT gene encodes a testis-specific thioredoxin that specifically associates to Y-chromosome loops in primary spermatocytes. TrxT is located right next to deadhead (dhd), a gene that encodes a female-specific thioredoxin that specifically locates to nuclei in the ovaries. A third thioredoxin in Drosophila is the ubiquitously expressed Thioredoxin-2 (Trx-2). I have found that flies lacking Trx-2 are viable but have shorter life spans than wild type flies, while over-expression of Trx-2 mediates an increased resistance to oxidative stress. Interestingly, a lack of all three thioredoxins does not result in lethality, contrary to what could be expected. All three thioredoxins are conserved among Drosophilids and the striking genomic organization of TrxT and dhd is generally conserved.

    The gene name Painting of fourth originates from the finding that POF stains the 4th chromosome of Drosophila in a banded pattern on salivary gland chromosomes. I show in this thesis that POF binding to the equivalent of the 4th chromosome is conserved in genus Drosophila and that POF co-localizes with both a protein and a histone modification associated with dosage compensation in species where POF also binds the male X. POF is expressed ubiquitously in both males and females, but at very high levels in male testes. I show that POF is present in nuclei of primary spermatocytes, but also in nuclei of maturing spermatids and that a lack of POF in the male germline causes a global down-regulation of chromosome 4 genes. These results combined suggest a function of POF in the first known case of chromosome-wide gene regulation of an autosome.

  • 41.
    Svensson, Malin J
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Chen, J Don
    Pirrotta, Vincenzo
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The ThioredoxinT and deadhead gene pair encode testis- and ovary-specific thioredoxins in Drosophila melanogaster.2003In: Chromosoma, ISSN 0009-5915, Vol. 112, no 3, p. 133-43Article in journal (Other academic)
    Abstract [en]

    So far, two thioredoxin proteins, DHD and Trx-2, have been biochemically characterized in Drosophila melanogaster. Here, with the cloning and characterization of TrxT we describe an additional thioredoxin with testis-specific expression. TrxT and dhd are arranged as a gene pair, transcribed in opposite directions and sharing a 471 bp regulatory region. We show that this regulatory region is sufficient for correct expression of the two genes. This gene pair makes a good model for unraveling how closely spaced promoters are differentially regulated by a short common control region. Both TrxT and DHD proteins are localized within the nuclei in testes and ovaries, respectively. Use of a transgenic construct expressing TrxT fused to Enhanced Yellow Fluorescent Protein reveals a clear association of TrxT with the Y chromosome lampbrush loops ks-1 and kl-5 in primary spermatocytes. The association is lost in the absence of the Y chromosome. Our results suggest that nuclear thioredoxins may have regulatory functions in the germline.

  • 42.
    Svensson, Malin J
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Thioredoxin-2 affects lifespan and oxidative stress in Drosophila2007In: Hereditas, ISSN 0018-0661, E-ISSN 1601-5223, Vol. 144, no 1, p. 25-32Article in journal (Other academic)
    Abstract [en]

    Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have

    several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotidereductase.

    In Drosophila melanogaster there are three ‘‘classical’’ thioredoxins with the conserved active site: deadhead,

    ThioredoxinT and Thioredoxin-2. Here, we report the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene.

    Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the

    organism’s oxidative stress protection system. We found that the mutants have a shorter lifespan than wild-type flies, and

    thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple

    copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant

    functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental

    stress.

  • 43.
    Svensson, Malin J.
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Stenberg, Per
    Johansson, Anna-Mia
    Larsson, Jan
    Painting of fourth in Drosophila spermatogenesisManuscript (Other academic)
  • 44.
    Svensson, Malin J.
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Stenberg, Per
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Organization and regulation of sex-specific thioredoxin encoding genes in genus DrosophilaManuscript (Other academic)
  • 45.
    Svensson, Malin J
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Organization and regulation of sex-specific thioredoxin encoding genes in the genus Drosophila.2007In: Dev Genes Evol, ISSN 0949-944X, Vol. 217, no 9, p. 639-50Article in journal (Refereed)
    Abstract [en]

    Thioredoxins are small thiol proteins that have a

    conserved active site sequence, WCGPC, and reduce

    disulfide bonds in various proteins using the two active site

    cysteines, a reaction that oxidizes thioredoxin and renders it

    inactive. Thioredoxin reductase returns thioredoxin to its

    reduced, active form in a reaction that converts NADPH to

    NADP+. The biological functions of thioredoxins vary

    widely; they have roles in oxidative stress protection, act as

    electron donors for ribonucleotide reductase, and form

    structural components of enzymes. To date, three thioredoxin

    genes have been characterized in Drosophila melanogaster:

    the generally expressed Thioredoxin-2 (Trx-2) and the two

    sex-specific genes ThioredoxinT (TrxT) and deadhead

    (dhd). The male-specific TrxT and the female-specific dhd

    are located as a gene pair, transcribed in opposite directions,

    with only 470 bp between their transcription start points. We

    show in this study that all three D. melanogaster thioredoxins

    are conserved in 11 other Drosophilid species, which are

    believed to have diverged up to 40 Ma ago and that Trx-2 is

    conserved all the way to Tribolium castaneum. We have

    found that the intriguing gene organization and regulation of

    TrxT and dhd is remarkably well conserved and identified

    potential conserved regulatory sequences. In addition, we

    show that the 50–70 C terminal amino acids of TrxT constitute

    a hyper-variable domain, which could play a role in

    sexual conflict and male–female co-evolution.

  • 46.
    Tångrot, Jeanette
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Department of Computing Science.
    The use of structural information to improve biological sequence similarity searches2003Licentiate thesis, monograph (Other academic)
    Abstract [en]

    Bioinformatics is a fast-developing field that make use of computational methods to analyse and structure biological data. An important branch of bioinformatics is structure and function prediction of proteins. To determine the structure of a protein is a crucial part in the characterisation of the molecule. The structure can also give clues about how the protein functions in the cell.

    Since the experimental determination of a protein structure can be both difficult and time-consuming, and in some cases is impossible using current techniques, it is desirable to be able to predict the structure. If two protein sequences are very similar, it is known that they share the same structure. However, there are many proteins that share the same fold, but have no clear sequence similarity. To find these relationships, and be able to predict the structure of these proteins, so called “protein fold recognition methods” have been developed.

    In this thesis, the field of bioinformatics is briefly surveyed, and two fold recognition methods are presented. Both methods use hidden Markov models (HMMs) to find related proteins, and they both exploit the fact that structure is more conserved than sequence, but in two different ways.

    The first paper introduces the reader to the field of molecular biology, and also describes some common tools used for protein sequence comparison. HMMs in general are described in detail, as well as some methods for the construction of multiple structure superposition. Since 3D structure is more conserved than sequence, it is expected that a multiple sequence alignment based on a ultiple

    structure superposition, is more biologically correct than an lignment based on sequence information, especially for proteins with low sequence identities.

    Our structure anchored HMMs (saHMMs), which are presented in the paper, are constructed from multiple sequence alignments that are based on structural superposition. The paper also describes the selection of representatives for each protein family, that were used for the construction of the saHMMs. In this selection, no protein in a given family have a sequence identity higher than a certain threshold to any other protein in the same family. The threshold is

    defined as the border to the so-called twilight zone. The saHMMs are shown to be able to find the family relationships for almost 90% of the test cases, even when the saHMMs are based on two proteins only.

    The second paper describes the secondary structure HMMs (ssHMMs). These HMMs are based on an ordinary multiple sequence alignment, as well as on the secondary structures of the proteins. When a query sequence is compared to the ssHMM, a predicted secondary structure is used, and the score based on the sequence is increased or decreased depending on the match of the secondary

    structures. A rigorous benchmark is also presented, and used to compare automatically generated HMMs with ordinary sequence search methods. The results show that the ordinary sequence search methods tested perform about as well as automatic HMMs built from multiple alignments. The ssHMMs, however, are better at detecting the correct fold of a protein than all the other methods

    tested.

  • 47.
    Tångrot, Jeanette
    et al.
    Umeå University, Faculty of Science and Technology, Department of Computing Science. Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Kågström, Bo
    Umeå University, Faculty of Science and Technology, Department of Computing Science. Umeå University, Faculty of Science and Technology, High Performance Computing Center North (HPC2N).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Accurate Domain Identification with Structure-Anchored Hidden Markov Models, saHMMs2009In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, no 2, p. 343-352Article in journal (Refereed)
    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/.

  • 48.
    Tångrot, Jeanette
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Department of Computing Science.
    Wang, Lixiao
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Kågström, Bo
    Umeå University, Faculty of Science and Technology, Department of Computing Science. Umeå University, Faculty of Science and Technology, High Performance Compting Center North (HPC2N).
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    FISH-Family identification of sequence homologues using structure anchored hidden Markov models2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no Web Server issue, p. W10-W14Article in journal (Refereed)
    Abstract [en]

    The FISH server is highly accurate in identifying the family membership of domains in a query protein sequence, even in the case of very low sequence identities to known homologues. A performance test using SCOP sequences and an E-value cut-off of 0.1 showed that 99.3% of the top hits are to the correct family saHMM. Matches to a query sequence provide the user not only with an annotation of the identified domains and hence a hint to their function, but also with probable 2D and 3D structures, as well as with pairwise and multiple sequence alignments to homologues with low sequence identity. In addition, the FISH server allows users to upload and search their own protein sequence collection or to quarry public protein sequence data bases with individual saHMMs. The FISH server can be accessed at http://babel.ucmp.umu.se/fish/.

  • 49.
    Wang, Lixiao
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    OnD-CRF: predicting order and disorder in proteins using [corrected] conditional random fields2008In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 11, p. 1401-1402Article in journal (Refereed)
    Abstract [en]

    MOTIVATION: Order and Disorder prediction using Conditional Random Fields (OnD-CRF) is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnD-CRF model highest among the fully automatic server group. AVAILABILITY: http://babel.ucmp.umu.se/ond-crf/

  • 50.
    Wolf-Watz, Magnus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bäckström, Stefan
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Härd, Torleif
    Chloride binding by the AML1/Runx1 transcription factor studied by NMR2001In: FEBS Letters, ISSN 0014-5793, Vol. 488, no 1-2, p. 81-4Article in journal (Refereed)
    Abstract [en]

    It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

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