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  • 101. Kiesler, E
    et al.
    Hase, M
    Brodin, D
    Visa, N
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs2005In: The journal of cell Biology, Vol. 168, no 7, p. 1013-1025Article in journal (Refereed)
  • 102. Kiesler, E
    et al.
    Miralles, F
    Östlund, A-K
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    Visa, N
    Department of Molecular Biology and Functional Genomics.
    The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal2003In: Journal of Cell Science, Vol. 116, p. 3949-3956Article in journal (Refereed)
  • 103. Kiesler, E
    et al.
    Visa, N
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Intranuclear Pre-mRNA Trafficking in an Insect Model System2004In: Progress in Molecular and Subcellular BiologyArticle in journal (Other academic)
  • 104.
    Kiesler, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells.

    Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences.

    Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1.

    Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

  • 105.
    Kiesler, Eva
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hase, Manuela
    Visa, Neus
    Hrp59, an hnRNP M-like protein in Chironomus and Drosophila, binds to exonic splicing enhancers in a subset of pre-mRNAs2004In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 168, no 7, p. 1031-1025Article in journal (Refereed)
    Abstract [en]

    Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59-RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

  • 106.
    Kiesler, Eva
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Miralles, Franscesc
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    HEL/UAP56 Binds Cotranscriptionally to the Balbiani Ring Pre-mRNA in an Intron-Independent Manner and Accompanies the BR mRNP to the Nuclear Pore2002In: Current Biology, ISSN 0960-9822, Vol. 12, no 10, p. 859-62Article in journal (Refereed)
    Abstract [en]

    The splicing factor UAP56/HEL/Sub2p is essential for mRNA export [1], [2], [3] and [4]. It has been proposed [1] and [2] that UAP56/HEL/Sub2p interacts with the pre-mRNA during splicing and recruits the export factor Aly/REF/Yra1 (reviewed in [5]) to the spliced mRNA. However, UAP56/HEL/Sub2p also participates in the transport of intronless mRNAs, and thus its role in export is not necessarily coupled to splicing [2], [3] and [4]. Here, we characterize the HEL protein of Chironomus tentans and we analyze in situ the interaction of HEL with a natural export substrate, the Balbiani ring pre-messenger ribonucleoprotein (BR pre-mRNP, reviewed in [6]). Using immunoelectron microscopy, we show that HEL binds to the BR pre-mRNP cotranscriptionally and that incorporation of HEL into the pre-mRNP is independent of the location of introns along the BR pre-mRNA. We also show that HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation to the cytoplasm. Aly/REF is also released from the BR mRNP during translocation but after dissociation of HEL. In summary, we have shown that binding of HEL to the BR pre-mRNA occurs independently of splicing, and we have established the point in the export pathway at which HEL and Aly/REF interact with the mRNP.

  • 107.
    Kiesler, Eva
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Miralles, Franscesc
    Visa, Neus
    HEL/UAP56 Binds to Hrp65 in Chironomus tentans and is Present in Nucleoplasmic Fibers that Interact Post-Transcriptionally with mRNP ParticlesManuscript (Other academic)
  • 108.
    Kiesler, Eva
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Miralles, Franscesc
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal2003In: Journal of Cell Science, ISSN 1477-9137, Vol. 116, no 19, p. 3949-3956Article in journal (Refereed)
    Abstract [en]

    Hrp65, an evolutionary conserved RNA-binding protein from the midge Chironomus tentans, has a conserved DBHS (Drosophila behavior, human splicing) domain that is also present in several mammalian proteins. In a yeast two-hybrid screening we found that Hrp65 can interact with itself. Here we confirm the Hrp65 self-interaction by in vitro pull-down experiments and map the sequences responsible for the interaction to a region that we refer to as the protein-binding domain located within the DBHS domain. We also show that the protein-binding domains of Drosophila NonA and human PSF, two other proteins with conserved DBHS domains, bind to Hrp65 in the yeast two-hybrid system. These observations indicate that the protein-binding domain can mediate homodimerization of Hrp65 as well as heterodimerization between different DBHS-containing proteins. Moreover, analyses of recombinant Hrp65 by gel-filtration chromatography show that Hrp65 can not only dimerize but also oligomerize into complexes of at least three to six molecules. Furthermore, we have analyzed the functional significance of the Hrp65 self-interaction in cotransfection assays, and our results suggest that the interaction between different Hrp65 isoforms is crucial for their intracellular localization.

  • 109. Klaue, Y
    et al.
    Källman, Annika M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bonin, M
    Nellen, W
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates2003In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 9, no 7, p. 839-846Article in journal (Refereed)
    Abstract [en]

    Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has                     been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.

  • 110. Kolberg, Matthias
    et al.
    Logan, Derek
    Bleifuss, Günther
    Pötsch, Stephan
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lubitz, Wolfgang
    Lassmann, Günter
    Lendzian, Friedhelm
    A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 12, p. 11233-46Article in journal (Other academic)
    Abstract [en]

    The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.

  • 111. Kolberg, Matthis
    et al.
    Bleifuss, Günter
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Protein thiyl directly observed by EPR spectroscopy2002In: Arcives of Biochemistry & Biophysics, Vol. 403, p. 141-144Article in journal (Refereed)
  • 112. Korayem, A M
    et al.
    Fabbri, M
    Takahashi, K
    Scherfer, C
    Lindgren, M
    Schmidt, O
    Ueda, R
    Dushay, M S
    Theopold, U
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A Drosophila salivary glad mucin is also expressed in immune tissues: evidence for a function in coagulation and the entrapment of bacteria2004In: Insect Biochemistry and Molecular Biology, Vol. 34, p. 1297-1304Article in journal (Refereed)
  • 113.
    Korayem, Ahmed M
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lesch, Christine
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Fabbri, Marco
    Lindgren, Malin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Schmidt, Otto
    Dushay, Mitchell S
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Evidence for an immune function of lepidopteran silk proteins2007In: Biochemical and Biophysical Research Communications, ISSN 0006-291X, Vol. 352, p. 317-322Article in journal (Refereed)
  • 114. Kylberg, Karin
    et al.
    Björk, Petra
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Fomproix, Nathalie
    Ivarsson, Birgitta
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Daneholt, Bertil
    Exclusion of mRNPs and ribosomal particels from a thin zone beneath the nuclear envelope revealed upon inhibition of transport2010In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 316, no 6, p. 1028-1038Article in journal (Refereed)
    Abstract [en]

    We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.

  • 115.
    Källman, A.M.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Öhman, M
    RNA editing and alternative splicing: the importance of co-transcriptional coordinationManuscript (Other academic)
  • 116.
    Källman, Annika
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Selective ADAR editing and the coordination with splicing2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Maturation of precursor messenger RNAs (pre-mRNA) in eukaryotes includes processes like capping, 3’ end formation and splicing. Some premRNAs undergo an additional process called RNA editing where a single nucleotide is modified to generate a new nucleotide identity. One such RNA editing event is the hydrolytic deamination of adenosine (A) that results in inosine (I). Because of its base pairing properties, inosine is recognized as guanosine (G) by cellular machineries, like the spliceosome or the ribosome. The enzymes catalyzing A to I editing are the ADARs (Adenosine deaminases that act on RNA). In vivo, these enzymes selectively locate and deaminate individual adenosines within a few substrates, all with long double stranded helixes interrupted by bulges and internal loops. It is not yet fully understood how ADARs discriminate between sites for selective editing and other adenosines in a double stranded context. Edited sites are often located near exon/intron borders where editing depends on intron sequences to form the double stranded structure required for ADAR recognition. Thus editing has to occur prior to splicing. In the work described in this thesis we have investigated the mechanism of selective editing and the possible coordination of editing and splicing.

    We have found that ADAR1 and ADAR2 display different editing specificities for a natural substrate, in vitro. ADAR2, but not ADAR1 perform site selective editing, similar to what occurs in vivo. Further, we have found that the selective editing is not determined by mismatches in the vicinity of the edited site. It is rather the immediate structure surrounding the adenosine that affects editing selectivity by ADAR2 in vitro.

    The coordination of editing and splicing was investigated in vivo. We found that the C-terminal domain (CTD) of the RNA polymerase II is required for efficient ADAR2 editing during ongoing transcription, while splicing of substrates for editing is independent of the CTD.

  • 117.
    Källman, Annika M.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    ADAR2 A to I editing: site selectivity and editing efficiency are separate events2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 16, p. 4874-4881Article in journal (Refereed)
    Abstract [en]

    ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double‐stranded. Site‐selective A→I editing has been detected at specific sites within  a few tructured pre‐mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre‐mRNA of the glutamate receptor subunit B (GluR‐B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double‐stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.                 

  • 118.
    Larsson Birgander, Pernilla
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The influence of nucleotides on ribonucleotide reductase assambly in class I ribonucleotide reductase from Escherichia coli2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The components of DNA, the deoxyribonucleotides, are produced from the components of RNA, the ribonucleotides. One single substitution is needed to convert a ribonucleotide into a deoxyribonucleotide i.e. a replacement of a hydroxyl group with a hydrogen atom. The reaction is catalysed by ribonucleotide reductase, an enzyme that is present in all living organisms. At first this conversion may seem trivial but in reality it is a difficult chemical reaction requiring much energy. In ribonucleotide reductase this energy is provided by an amino acid radical that upon each catalytic turnover is transferred from its stable position in the R2 protein to the active site of protein R1. The need for deoxyribonucleotides in the cell varies, therefore the activity of ribonucleotide reductase must be regulated. A complex allosteric regulation controls both the level of enzymatic activity and the substrate specificity to make sure that the deoxyribonucleotides are produced in correct amounts. In this work, we have shown that the reason why enzymatic activity is turned off when dATP binds is due to formation of a constrained R1: R2 interaction and we have proposed that a conserved hydrogen bond is important in this mechanism. We evaluated the effect of nucleotides on the R1: R2 interaction further using the surface plasmon resonance technique and found that allosteric effectors and substrates as well as the presence of thioredoxin considerably enhances the interaction.

    The second allosteric site that controls substrate specificity is located at the interaction area of the two polypeptides constituting protein R1. We have identified key residues in the dimerisation process of the two polypeptides and we have established a stabilisation effect of allosteric effectors and substrates on the dimer interaction using a mutant protein. The radical is believed to be transferred between the two proteins that constitute ribonucleotide reductase by a chain of hydrogen bonded amino acid residues. We have developed a new in vivo activity assay in which we have shown the physiological importance of these residues. Another methodological approach in this work was an attempt to turn protein R1 of ribonucleotide reductase into a selenoprotein by genetically substituting one of the active site cysteines for a selenocysteine. In a selenocysteine-substituted protein, this particular residue can be distinguished from the other cysteines which would be advantageous in subsequent biophysical characterisations of thiyl radicals.

  • 119.
    Larsson Birgander, Pernilla
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bug, Stefanie
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gordon, Euan
    Dahlroth, Sue-Li
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kasrayan, Alex
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Westman, MariAnn
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Euan, Gordon
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R12005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 15, p. 14997-15003Article in journal (Refereed)
    Abstract [en]

    Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (α2) and R2 (β2), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

  • 120.
    Larsson Birgander, Pernilla
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Kasrayan, Alex
    Sjöberg, Britt-Marie
    Mutant R1 proteins from Escherichia coli class Ia ribonucleotide reductase with altered responses to dATP inhibition2004In: Journal of Biological chemistry, ISSN 0021-9258, Vol. 279, no 15, p. 14496-14501Article in journal (Refereed)
  • 121.
    Larsson, Karl-Magnus
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, Jessica
    Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Nordlund, Pär
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Logan, Derek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural Basis for Allosteric Substrate Specificity Regulation in Anaerobic Ribonucleotide Reductases2001In: Structure, Vol. 9, p. 739-750Article in journal (Refereed)
  • 122.
    Larsson-Birgander, Pernilla
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Kasrayan, Alex
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Mutant R1 proteins from Escherichia coli class Ia ribonucleotide reductase with altered responses to dATP inhibition.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 15, p. 14496-501Article in journal (Other academic)
    Abstract [en]

    Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.

  • 123. Lassman, G
    et al.
    Kolberg, M
    Bleifuss, G
    Gräslund, A
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, B-M
    Department of Molecular Biology and Functional Genomics.
    Lubitz, W
    Protein thiyl radicals in disordered systems: A comparative EPR study at low temperature2003In: Phys. Chem. Chem. Phys., Vol. 5, p. 2442-2453Article in journal (Refereed)
  • 124. Laurencikiene, J
    et al.
    Källman, A M
    Fong, N
    Bentley, D L
    Öhman, M
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    RNA editing and alternative splicing: the importance of co-transcriptional coordination2006In: EMBO Reports, Vol. 7, no 3, p. 303-307Article in journal (Other academic)
  • 125.
    Lesch, Christine
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Goto, Akira
    Lindgren, Malin
    Bidla, Gawa
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dushay, Mitchell S.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A role for Hemolectin in coagulation and immunity in Drosophila melanogaster2007In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 31, no 12, p. 1255-1263Article in journal (Refereed)
    Abstract [en]

    Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(−) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

  • 126. Lesch, Christine
    et al.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Methods to study hemolymph clotting in insects2008In: Insect Immunology, Academic Press , 2008Chapter in book (Other academic)
  • 127. Li, D
    et al.
    Korayem, A M
    Zhao, Z
    Schmidt, O
    Theopold, U
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Scherfer, C
    Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoxidase activating cascade2002In: Insect Biochemistry & Molecular Biology, Vol. 32, p. 919-928Article in journal (Refereed)
  • 128. Li, D
    et al.
    Roberts, H
    Schneider, MV
    Theopold, U
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Schmidt, O
    Genetic analysis of two distinct reproductive strategies in sexual and asexual flield populations of an endoparasitic wasp, Venturia canescens2003In: Heredity, Vol. 90, p. 291-297Article in journal (Refereed)
  • 129. Li, D
    et al.
    Theopold, U
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Schmidt, O
    Posstible function of two insect phospholipid-hydroperoxide glutathione peroxidases2003In: Journal of insect Physiology, Vol. 49, p. 1-9Article in journal (Refereed)
  • 130.
    Lindgren, Malin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Molecular and functional characterization of the insect hemolymph clot2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the Drosophila hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as Galleria mellonella. The Drosophila clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in Drosophila coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, in vivo when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as Galleria mellonella and Ephestia kuehniella, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.

  • 131. Lindgren, Malin
    et al.
    Riazi, Raha
    Lesch, Christine
    Wilhelmsson, Christine
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Dushay, Mitchell S
    Fondue and transglutaminase in the Drosophila larval clot2008In: Journal of insect physiology, ISSN 0022-1910, E-ISSN 1879-1611, Vol. 54, p. 586-592Article in journal (Refereed)
  • 132. Lindmark, Hans
    et al.
    Johansson, Karin C
    Stöven, Svenja
    Hultmark, Dan
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderhäll, Kenneth
    Enteric bacteria Counteract Lipopolysaccharide Induction of Antimicrobial Peptide Genes2001In: The Journal of Immunology, Vol. 167, p. 6920-6923Article in journal (Refereed)
  • 133. Logan, Derek
    et al.
    Mulliez, Etienne
    Larsson, Karl-Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bodevin, Sabrina
    Department of Molecular Biology and Functional Genomics.
    Atta, Mohamed
    Garnaud, Pierre E
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Fontecave, Marc
    A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase.2003In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 100, no 7, p. 3826-31Article in journal (Other academic)
    Abstract [en]

    A Zn(Cys)(4) center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4. The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Mutant enzymes of the homologous RNR from Escherichia coli, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical. The possible roles of the metal center are discussed in relationship to the currently proposed reaction mechanism for generation of the glycyl radical in class III RNRs.

  • 134. Loseva, O
    et al.
    Engström, Y
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Analysis of Signal-dependent Changes in the Proteome of Drosophila Blood Cells During an Immune Response2004In: Molecular and Cellular Proteomics, Vol. 3, no 8, p. 796-808Article in journal (Refereed)
  • 135.
    Lundgren, Josefin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Studies of metazoan proteasome function and regulation2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Biological processes depend upon the structural and functional quality of the molecules that comprise living organisms. The integrity of molecules such as DNA, RNA, proteins, carbohydrates and lipids is crucial and the precise three-dimensional shape and the detailed chemistry of these molecules orchestrate the biochemical processes vital for life. Within a cell, each protein must be present at a specific concentration during certain specific conditions. To maintain cellular homeostasis and the ability to respond to the environment the proteome is in a dynamic state of synthesis and degradation. In eukaryotic cells the ubiquitin-proteasome pathway is the principal mechanism for regulated protein turnover in both the cytoplasm and the nucleus. The 20S proteasome is a cylindrical multi-subunit protease. Proteasomes play an essential role in the targeted and timely ordered degradation of key regulatory proteins and their inhibitors. The 26S proteasome is a 2.500 kDa complex composed of the 20S proteasome sandwiched between two 19S regulators. This is the enzymatic complex responsible for ATP-dependent ubiquitin mediated protein degradation. A polyubiquitin chain attached to a protein serves as a general recognition signal for destruction via the 26S proteasome. It is known that the 19S regulator confers ubiquitin recognition and substrate unfolding to the 20S proteasome, however, the specific functions for many of the different subunits within the 19S complex are not known. We have used RNA interference to study the S13/Rpn11 and S5a/Rpn10 subunits of Drosophila melanogatser proteasomes. We have produced stable cell lines with the human S13 gene under inducible promoters that was used to rescue the knockdown phenotype after RNA interference. The rescue was successful in demonstrating that the human protein is a functional homologue to the Drosophila protein. We call the technique RNAi+c (RNA interference + complementation). This procedure enabled us to also test different mutants of the human S13 protein for their ability to function in the proteasome. Using RNA interference to a Drosophila proteasome subunit in combination with complementation with a corresponding human protein we have been able to study residues important for the deubiquitinating activity of this subunit (Paper I). Interestingly, upon a decrease of either S13 or S5a we see an induction in the levels of active 20S proteasomes. Increase in the levels of the non-targeted 19S subunit can be detected when RNAi treatment is carried out on either S13 or S5a. We have used RNA interference and proteasomal inhibition together with whole genome microarray analysis to reveal a co-regulated network of proteasome genes. This network likely contributes to an overall regulatory system that maintains proper proteasome levels in the cell. Initial studies of the mechanism of transcriptional co-regulation of proteins involved in the 26S proteasome pathway were also performed (Paper II). Finally, the biological function of the proteasome regulator PA28g/REGg is not known. We have studied this regulator in Drosophila using RNA interference and promoter mapping (Paper III).

  • 136.
    Lundgren, Josefin
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Masson, Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Mirzaei, Zahra
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Identification and characterization of a Drosophila proteasome regulatory network2005In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 25, no 11, p. 4662-4675Article in journal (Refereed)
    Abstract [en]

    Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5' untranslated regions (UTRs). Transfer of the 5' UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.

  • 137.
    Lundgren, Josefin
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Masson, Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Realini, Claudio
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Use of RNA interference and Complementation to study the function of the Drosophila and Human 26S proteasome Subunit S132003In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 15, p. 5320-5330Article in journal (Refereed)
  • 138.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    RNRdb, a curated database of the universal enzyme familyribonucleotide reductase, reveals a high level of misannotation insequences deposited to Genbank2009In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 10, p. 589-Article in journal (Refereed)
    Abstract [en]

    Background: Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway fordeoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Whileribonucleotide reduction has a single evolutionary origin, significant differences between RNRsnevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation.These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependenceand cobalamin dependence), and form the basis for the classification of RNRs into three classes.Description: In RNRdb (Ribonucleotide Reductase database), we have collated and curated allknown RNR protein sequences with the aim of providing a resource for exploration of RNRdiversity and distribution. By comparing expert manual annotations with annotations stored inGenbank, we find that significant inaccuracies exist in larger databases. To our surprise, only 23%of protein sequences included in RNRdb are correctly annotated across the key attributes of class,role and function, with 17% being incorrectly annotated across all three categories. This illustratesthe utility of specialist databases for applications where a high degree of annotation accuracy maybe important. The database houses information on annotation, distribution and diversity of RNRs,and links to solved RNR structures, and can be searched through a BLAST interface. RNRdb isaccessible through a public web interface at http://rnrdb.molbio.su.se.Conclusion: RNRdb is a specialist database that provides a reliable annotation and classificationresource for RNR proteins, as well as a tool to explore distribution patterns of RNR classes. Therecent expansion in available genome sequence data have provided us with a picture of RNRdistribution that is more complex than believed only a few years ago; our database indicates thatRNRs of all three classes are found across all three cellular domains. Moreover, we find a numberof organisms that encode all three classes.

  • 139.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The evolution of ribonucleotide reductases2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyses the transformation of RNA building blocks, ribonucleotides, to DNA building blocks, deoxyribonucleotides. This is the only extant reaction pathway for de novo synthesis of DNA building blocks and the enzyme is thus necessary for life. RNR is found in all but a few organisms.

    There are three classes of RNR, all evolutionarily related. The classification is built on differences in generation of the radical that is central to the reaction. As a consequence, RNR classes have different operational constraints. Class I requires oxygen, while class III is poisoned by oxygen. Class II is independent of oxygen, but dependent on vitamin B12 and, hence, cobalt. This makes RNR interesting from an evolutionary as well as environmental point of view.

    RNR must have evolved before the transition from RNA encoded genomes to DNA encoded. The extant radical-based reaction is likely similar to the ancestral reaction, which entails that the ancestral enzyme was a protein and not an RNR.

    My results are consistent with both class II and III being present in the last universal common ancestor. Class I RNR evolved later, presumably once oxygen levels had risen. From commonalities in extant RNRs we can reconstruct their last common ancestor as an enzyme that 1) used a transient cysteine-radical in the reaction, 2) reduced all four ribonucleotides, 3) regulated which nucleotide was reduced after binding an effector nucleotide in the dimer interface of the enzyme and 4) had activity regulation through binding of a nucleotide to another part of the enzyme.

    The presence of a specific RNR class is likely to influence the environmental range of organisms, which makes horizontal transfer of RNR particularly interesting. Horizontal transfer of RNR genes is widespread, both between closely related organisms and between domains. For instance, all three classes are present in eukaryotes, but likely all three are results of horizontal transfer.

  • 140.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gribaldo, Simonetta
    Unite Biologie Moléculaire du Gène chez les Extremophiles (BMGE), Departement de Microbiologie, Institut Pasteur, Paris, France.
    Torrents, Eduard
    Institute for Bioengineering of Catalonia (IBEC), Scientific Park of Barcelona, Barcelona, Spain.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ribonucleotide reduction: horizontal transfer of a required function spans all three domains2010In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 10, no 383Article in journal (Other academic)
    Abstract [en]

    Background Ribonucleotide reduction is the only de novo pathway for synthesis ofdeoxyribonucleotides, the building blocks of DNA. The reaction is catalysed byribonucleotide reductases (RNRs), an ancient enzyme family comprised of threeclasses. Each class has distinct operational constraints, and are broadly distributedacross organisms from all three domains, though few class I RNRs have beenidentified in archaeal genomes, and classes II and III likewise appear rare acrosseukaryotes. In this study, we examine whether this distribution is best explained bypresence of all three classes in the Last Universal Common Ancestor (LUCA), or byhorizontal gene transfer (HGT) of RNR genes. We also examine to what extentenvironmental factors may have impacted the distribution of RNR classes.

    Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possesseda class I RNR, but that the eukaryotic class I enzymes are not directly descended fromclass I RNRs in archaea. Instead, our results indicate that archaeal class I RNR geneshave been independently transferred from bacteria on two occasions. While LECApossessed a class I RNR, our trees indicate that this is ultimately bacterial in origin.We also find convincing evidence that eukaryotic class I RNR has been transferred tothe bacteroidetes, providing a stunning example of HGT from eukaryotes back tobacteria. Based on our phylogenies and available genetic and genomic evidence, classII and III RNRs in eukaryotes also appear to have been transferred from bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of archaeaand eukaryotes appears, through a process of elimination, to have been a dimeric classII RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT.

    Conclusions Horizontal gene transfer has clearly played an important role in the evolution of theRNR repertoire of organisms from all three domains of life. Our results clearly showthat class I RNRs have spread to archaea and eukaryotes via transfers from thebacterial domain, indicating that class I likely evolved in the bacteria. We find noclear evolutionary trace placing either class II or III RNRs in the LUCA, despite thefact that ribonucleotide reduction is an essential cellular reaction and was pivotal tothe transition from RNA to DNA genomes. Instead, a general pattern emerges whereenvironmental and enzyme operational constraints, especially the presence or absenceof oxygen, coupled with horizontal transmission are major determinants of the RNR repertoire of genomes.

  • 141.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The functional diversity and evolutionary relationships of ferritin-like proteinsManuscript (preprint) (Other academic)
    Abstract [en]

    BackgroundThe ferritin-like proteins are evolutionarily related, as evidenced by the topology oftheir signature metal-binding four-helix bundle. They perform diverse functions suchas iron/oxygen detoxification, iron storage, DNA protection and substrate oxidation.Though evolutionarily related, sequence similarity between families and often withinfamilies is low. To analyse the evolutionary relationships between individual familiesand their functional roles systematically, we turned to 3D structural alignment andphylogenetic methods.ResultsOur phylogenetic network recovers all characterised functional groups of ferritin-likeproteins and suggests evolutionary relationships between them. The evolutionaryhypotheses that are suggested by the phylogeny are tested against availableindependent evidence such as dimerisation geometries, qualitative comparison ofstructures and sequence based partial phylogenies. Generally our hypotheses stand upagainst the evidence, but in a few cases verification have to await further data.ConclusionsTwo large evolutionary groups of ferritin-like proteins are identified from structuralphylogeny: ferritins, bacterioferritins and Dps proteins on one hand, and substrateoxidising proteins, bacterial multicomponent monooxygenases, fatty acid desaturasesand class I ribonucleotide reductase radical generating subunits, on the other. Themethod we present provides a robust way of evolutionarily classifying a functionallydiverse group of distantly related proteins, as well as examining the possible functionsof poorly-characterised proteins.

  • 142.
    Lundkvist, Pär
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Jupiter, Sara
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Segerstolpe, Åsa
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Osheim, Yvonne N
    Beyer, Ann L
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex2009In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 29, no 21, p. 5763-5774Article in journal (Refereed)
    Abstract [en]

    In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A2 cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A0 to A2. Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

  • 143. Lööf, Torsten G.
    et al.
    Morgelin, Matthias
    Johansson, Linda
    Oehmcke, Sonja
    Olin, Anders I.
    Dickneite, Gerhard
    Norrby-Teglund, Anna
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Herwald, Heiko
    Coagulation, an ancestral serine protease cascade, exerts a novel function in early immune defense2011In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 118, no 9, p. 2589-2598Article in journal (Refereed)
    Abstract [en]

    Phylogenetically conserved serine protease cascades play an important role in invertebrate and vertebrate immunity. The mammalian coagulation system can be traced back some 400 million years and shares homology with ancestral serine proteinase cascades that are involved in, for example, Toll receptor signaling in insects and release of antimicrobial peptides during hemolymph clotting. In the present study, we show that the induction of coagulation by bacteria leads to immobilization and killing of Streptococcus pyogenes bacteria inside the clot. The entrapment is mediated via cross-linking of bacteria to fibrin fibers by the action of coagulation factor XIII (fXIII), an evolutionarily conserved transglutaminase. In a streptococcal skin infection model, fXIII(-/-) mice developed severe signs of patho-logic inflammation at the local site of infection, and fXIII treatment of wild-type animals dampened bacterial dissemination during early infection. Bacterial killing and cross-linking to fibrin networks was also detected in tissue biopsies from patients with streptococcal necrotizing fasciitis, supporting the concept that coagulation is part of the early innate immune system.

  • 144. Lööf, Torsten G.
    et al.
    Schmidt, Otto
    Herwald, Heiko
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Coagulation Systems of Invertebrates and Vertebrates and Their Roles in Innate Immunity: The Same Side of Two Coins?2011In: Journal of Innate Immunity, ISSN 1662-811X, Vol. 3, no 1, p. 34-40Article, review/survey (Refereed)
    Abstract [en]

    Bacterial infections represent a serious health care problem, and all multicellular organisms have developed defense mechanisms to eliminate pathogens that enter the host via different paths including wounds. Many invertebrates have an open circulatory system, and effective coagulation systems are in place to ensure fast and efficient closure of wounds. It was proposed early on that coagulation systems in invertebrates play a major role not only in sealing wounds but also in preventing systemic infections. More recent evidence suggests that vertebrates, too, rely on clotting as an immune effector mechanism. Here we discuss the evolution of clotting systems against the background of their versatile function in innate immunity. Copyright (C) 2010 S. Karger AG, Basel

  • 145.
    Masson, Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Investigating the activation and regulation of the proteasome: an essential proteolytic complex2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The proteasome is a major non-lysosomal proteolytic complex present in eukaryotic cells and has a central role in regulating many protein levels. The complex has been shown to participate in various intracellular pathways including cell cycle regulation or quality control of newly synthesized proteins and many other key pathways. This amazing range of substrates would not be possible without the help of regulators that are able to bind to the 20S proteasome and modulate its activity. Among those, the PA700 or 19S regulator and the PA28 family are the best characterized in higher eukaryotes. The 19S regulatory particle is involved in the recognition of ubiquitinated proteins, targeted for degradation by the proteasome. The PA28 (also termed 11S REG) family is composed of two members the PA28αβ and PA28γ. The function of PA28αβ is related to the adaptive immune response with a proposed contribution in MHC class I peptide presentation whereas the biological role PA28γ remains unknown. The main objectives of the laboratory, and subsequently of this thesis are to use Drosophila melanogaster model system and its advantages to better understand the precise contribution of these different activators in the regulation of the proteasome. Using genomic resources, a unique Drosophila PA28 member was identified, characterized and was shown to be a proteasome regulator with all the properties of PA28γ. Through site-directed mutagenesis we identified a functional nuclear localization signal in the homolog-specific insert region. Study of the promoter region revealed that transcription of Drosophila PA28γ (dPA28γ) gene is under control of DREF, a transcription factor involved in the regulation of genes related to DNA synthesis and cell proliferation. To confirm that dPA28γ has a role in cell cycle progression, the effect of removing dPA28γ from S2 cells was tested using RNA interference. Drosophila cells depleted of dPA28γ showed partial arrest in G1/S cell cycle transition confirming a conserved function between Drosophila and mammalian forms of PA28γ. Finally, characterization of the Dictyostelium regulator, an evolutionarily distant member of the PA28γ, was carried out using fluorogenic degradation assays. We are currently knocking-out the gene in order to determine the biological function of the activator. A second part of my work consisted in the generation of a Drosophila assay used to identify in vivo substrates of the 19S regulator, an assay system that has been originally engineered by Dantuma and coworkers in human cell lines. This was achieved by cloning of GFP behind a series of modified ubiquitins that create substrates degraded through different pathways involving the proteasome pathways. The last project of my thesis concerns the characterization of the mechanism for upregulation of proteasomal gene mRNA after MG132 (proteasome inhibitor) treatment. So far, we found that the 5´-UTR of the genes is responsible for this induction. We are now looking for the precise motif involved in this regulation.

  • 146.
    Masson, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Andersson, Oskar
    Petersen, Ulla-Maja
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Indentification and Characterization of a Drosophila Nuclear Proteasome Regulator2001In: The Journal of Biological Chimistry, Vol. 276, p. 1383-1390Article in journal (Refereed)
  • 147.
    Masson, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Andersson, Oskar
    Petersen, Ulla-Maya
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Identification and characterization of a Drosophila nuclear proteasome activator: a homolog of human 11S REggamma (PA28gamma)2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 2, p. 1383-1390Article in journal (Refereed)
    Abstract [en]

    We report the cloning and characterization of aDrosophila proteasome 11 S REGγ (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGγ and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the DrosophilaREG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. TheDrosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGγ proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that γ class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the α and β REG classes.

  • 148.
    Masson, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lundgren, Josefin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Drosophila Proteasome Regulator REGγ: Transcriptional Activation by DNA Replication-related Factor DREF and Evidence for a Role in Cell Cycle Progression2003In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 327, no 5, p. 1001-1012Article in journal (Refereed)
    Abstract [en]

    The proteasome regulator REG (PA28γ) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGγ in metazoan cells is currently unknown. To understand the role of Drosophila REGγ we have attempted to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGγ transcriptional assays were carried out with mutations in the proposed promoter. Our results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGγ transcription. To confirm that REGγ has a role in cell cycle progression, the effect of removing REGγ from S2 cells was tested using RNA interference. Drosophila cells depleted of REGγ showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGγ is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGγ was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila.

  • 149. Masson, Patrick
    et al.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderbom, Fredrik
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Characterization of a REG/PA28 Proteasome Activator Homolog in Dictyostelium discoideum Indicates that the Ubiquitin- and ATP-Independent REG gamma Proteasome Is an Ancient Nuclear Protease2009In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 8, no 6, p. 844-851Article in journal (Refereed)
    Abstract [en]

    The nuclear proteasome activator REG gamma/PA28 gamma is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REG gamma-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

  • 150.
    Masson, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderbom, Fredrik
    Young, Patrick
    Identification and characterization of a Metazoan Proteasome Regulator 11S REG (PA28) in Dictyostelium DiscoideumManuscript (Other academic)
1234567 101 - 150 of 304
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