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  • 151. Coutard, B.
    et al.
    Gorbalenya, A. E.
    Snijder, E. J.
    Leontovich, A. M.
    Poupon, A.
    De lamballerie, X.
    Charrel, R.
    Gould, E. A.
    Gunther, S.
    Norder, H.
    Klempa, B.
    Bourhy, H.
    Rohayem, J.
    L'hermite, E.
    Nordlund, P.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Stuart, D. I.
    Owens, R. J.
    Grimes, J. M.
    Tucker, P. A.
    Bolognesi, M.
    Mattevi, A.
    Coll, M.
    Jones, T. A.
    Aqvist, J.
    Unge, T.
    Hilgenfeld, R.
    Bricogne, G.
    Neyts, J.
    La Colla, P.
    Puerstinger, G.
    Gonzalez, J. P.
    Leroy, E.
    Cambillau, C.
    Romette, J. L.
    Canard, B.
    The VIZIER project: Preparedness against pathogenic RNA viruses2008Ingår i: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 78, nr 1, s. 37-46Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

  • 152.
    Covarrubias, Adrian Suarez
    et al.
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Högbom, Martin
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Bergfors, Terese
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Carroll, Paul
    Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London.
    Mannerstedt, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Oscarson, Stefan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Parish, Tanya
    Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London.
    Jones, T Alwyn
    Department of Cell and Molecular Biology, Uppsala University, Sweden.
    Mowbray, Sherry L
    Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
    Structural, biochemical, and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis.2008Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 381, nr 3, s. 622-33Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C(gamma) in the formation of the alphabeta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth; such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.

  • 153.
    Crona, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Deoxyribonucleic acid (DNA) chains in which our genetic blueprint is stored are built from four DNA precursors by DNA polymerases. The enzyme ribonucleotide reductase (RNR) provides the only de novo synthesis pathway of deoxyribonucleotides from ribonucleotides and is essential for nearly all organisms. All four ribonucleotides are substrates for RNR and key to this flexibility is a sophisticated allosteric regulation. Nucleotide effectors (ATP, dATP, dTTP or dGTP) binding to the allosteric specificity site determines substrate specificity for the active site. When present at high concentrations, dATP binds to the allosteric overall activity site and inhibits activity by an unknown mechanism. Three approaches, RNR activity measurements, subunit interaction studies and quaternary structure studies were applied to four different class I RNRs to address the allosteric overall regulation. We found that allosteric overall inhibition was closely linked to formation of tight and large RNR protein complexes; α4β4 complex for the Escherichia coli class Ia RNR and α6β2 for the Dictyostelium discoideum class Ia RNR with functional allosteric inhibitions. The Aeh1 phage class Ia RNR with a non-functional dATP inhibition showed weak remnant inhibition features, while the Bacillus anthracis class Ib RNR without the allosteric overall regulation domain lacked these features. In addition, we presented the first biochemical characterization of a mechanism to restore protein function after gene fragmentation, we showed that the B. anthracis class Ib RNR was most active when reconstituted with manganese and in the presence of a physiological redoxin protein and we found that the class Ia RNR is the principal RNR in D. discoideum, although the coexisting class II RNR could partly compensate class I RNR inhibition during axenic growth. Finally, our improved method for studying RNR interactions has potential for RNR inhibitor screening.

  • 154.
    Crona, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Avesson, Lotta
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala .
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Hinas, Andrea
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala .
    Klose, Ralph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Söderbom, Fredrik
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala .
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    The two classes of ribonucleotide reductase in the social amoeba Dictystelium discoideumManuskript (preprint) (Övrigt vetenskapligt)
  • 155.
    Crona, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Avesson, Lotta
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Lundin, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hinas, Andrea
    Klose, Ralph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Söderbom, Fredrik
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 12, s. 8198-8208Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class IIRNRis most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B-12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

  • 156.
    Crona, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Furrer, Ernst
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Torrents, Eduard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Edgell, David R.
    Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario,.
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses2010Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 23, nr 8, s. 633-641Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors

  • 157.
    Crona, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Moffatt, Connor
    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
    Edgell, David R.
    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
    Friedrich, Nancy C.
    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
    Hofer, Anders
    Department of Medical Biochemistry and Biophysics, Umeå University.
    Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element2011Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, nr 4, s. 1381-1389Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)2 large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

  • 158.
    Crona, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Torrents, Eduard
    Cellular Biotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain.
    Hofer, Anders
    Department of Medical Biochemistry & Biophysics, Umeå University.
    Furrer, Ernst
    Tomter, Ane
    Department of Molecular Biosciences, University of Oslo, Norway.
    Rohr, Åsmund
    Andersson, Kristoffer
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    NrdH-redoxin mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from Bacillus anthracisManuskript (preprint) (Övrigt vetenskapligt)
  • 159. Cuviello, Flavia
    et al.
    Tellgren-Roth, Åsa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lara, Patricia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ruud Selin, Frida
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Monné, Magnus
    Bisaccia, Faustino
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ostuni, Angela
    Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)2015Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, nr 24, s. 3921-3928Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout-Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.

  • 160.
    Cymer, Florian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hedman, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ismail, Nurzian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants2015Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 16, s. 10208-10215Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.

  • 161.
    Cymer, Florian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ismail, Nurzian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Weak pulling forces exerted on N-in-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli2014Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, nr 10, s. 1930-1934Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transmembrane helices (TMHs) in membrane proteins can be orientated with their N-terminus towards the cytoplasm (N-in), or facing the non-cytoplasmic side (N-out). Most membrane proteins are inserted co-translationally into membranes, aided by Sec-type translocons. Since the final orientation of N-in-and N-out-orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion. We measured pulling forces exerted on N-in-orientated TMHs during co-translational insertion into the inner membrane of Escherichia coli. Our results demonstrate that Nin-orientated TMHs experience a weaker pulling force but retain the overall biphasic force profile seen previously for Nout-orientated TMHs (Ismail et al., 2012 [1]).

  • 162.
    Cymer, Florian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    White, Stephen H.
    Mechanisms of Integral Membrane Protein Insertion and Folding2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 5, s. 999-1022Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The biogenesis, folding, and structure of alpha-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons-often referred to as protein-conducting channels-for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. (C) 2014 Elsevier Ltd. All rights reserved.

  • 163.
    da Silva, Diogo V.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordholm, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Madjo, Ursula
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pfeiffer, Annika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daniels, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 1, s. 644-653Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Neuraminidase (NA) is one of the two major influenza surface antigens and the main influenza drug target. Although NA has been well characterized and thought to function as a tetramer, the role of the transmembrane domain (TMD) in promoting proper NA assembly has not been systematically studied. Here, we demonstrate that in the absence of the TMD, NA is synthesized and transported in a predominantly inactive state. Substantial activity was rescued by progressive truncations of the stalk domain, suggesting the TMD contributes to NA maturation by tethering the stalk to the membrane. To analyze how the TMD supports NA assembly, the TMD was examined by itself. The NA TMD formed a homotetramer and efficiently trafficked to the plasma membrane, indicating the TMD and enzymatic head domain drive assembly together through matching oligomeric states. In support of this, an unrelated strong oligomeric TMD rescued almost full NA activity, whereas the weak oligomeric mutant of this TMD restored only half of wild type activity. These data illustrate that a large soluble domain can force assembly with a poorly compatible TMD; however, optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.

  • 164.
    da Silveira Vieira da Silva, Diogo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Influenza neuraminidase assembly: Evolution of domain cooperativity2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    Influensa är en av de mest smittsamma sjukdomarna som drabbar människor och de flesta kan räkna med att bli infekterade många gånger under sin livstid. Influensaviruset attackerar främst luftvägarna, men kan även leda till t.ex. lunginflammation. De enskilda viruspartiklarna (virionerna) kan komma i olika former, men den vanligaste formen som används för att beskriva viruset är den sfäriska. På en virions yta så finns det två olika typer av membranproteiner, som kan liknas med två olika sorters spikar som sticker ut från viruset. Den ena ”spiken” kallas neuraminidas, eller bara kort för NA, och den andra för hemagglutinin (HA). När man har andats in ett influensavirus så kan viruset ta sig till de övre luftvägarna och vidare ner i luftstrupen för att där använda sig av HA för att ta sig in i en cell. Viruset använder sig sedan av cellen för att skapa många nya virioner, som tar sig ut ur cellen för att infektera fler celler. NA är det protein som virionerna använder sig av för att klyva sig loss från modercellen.

    Målet för avhandlingen var att studera NA och beskriva hur proteinet måste vara ihopsatt för att vara aktivt. NA har en uppbyggnad liknande en trädklunga, där fyra stycken identiska träd (med tillhörande rötter, stammar och trädkronor) går ihop och bildar en enda aktiv enhet, en s.k. tetramer. ”Rötterna” hos NA är den transmembrana domänen (TMD), den del av proteinet som sitter fast i influenaviruskroppen. ”Stammen”, eller stjälkdelen av NA, binder samman TMD med den största delen, huvuddomänen som motsvarar ”trädkronan”. Det är just huvuddomänen som är ansvarig för att klyva loss viruspartiklar från en modercell.

    Vi har i våra studier sett att det kan vara väldigt viktigt att TMD-domänerna går ihop i grupper om fyra för att hela NA ska kunna gå ihop i en tetramer och aktivt kunna klyva loss viruspartiklarna. När vi studerade TMD från olika influensavirus så märkte vi att vissa egenskaper hos TMD krävs för att de skulle kunna gå ihop, men också att dessa egenskaper inte fanns hos alla influensavirus. Virusen har evolverat över lång tid och har anpassat sig efter värdorganismerna (inklusive människan) och har hittat olika lösningar på problemet med att behöva bilda en tetramer. När vi gjorde ändringar i en TMD som vanligtvis gick ihop till en tetramer, och därmed förhindrade detta, så noterade vi att huvuddomänens funktion påverkades vilket ledde till att influensaviruset hade svårt att spridas. Vidare så har våra pågående studier på stjälkdelen visat att även denna del kan ha stor betydelse för tetrameriseringen av NA, speciellt i de fall där TM-domänen saknar egenskaper för att gå ihop.

    Avhandlingen tillför inte bara ny och viktig information till influensaforskningen, utan även potentiellt för framställandet av nya influensavacciner/-mediciner.

  • 165.
    Dahlin, Paul
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik.
    Analysis of sterol metabolism in the pathogenic oomycetes Saprolegnia parasitica and Phytophthora infestans2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The primary objective of this thesis was to investigate the sterol metabolism of two pathogenic oomycetes, specifically the processes of sterol synthesis and sterol acquisition in the fish pathogen Saprolegnia parasitica (Saprolegniales) and the plant pathogen Phytophthora infestans (Peronosporales). Furthermore, the effects of steroidal glycoalkaloids from Solanaceous plants, on P. infestans, were examined. The improved understanding of these processes should help to identify approaches for the identification of new oomycete inhibitors targeting sterol metabolism in agriculture and aquaculture farming systems, and to guide plant-breeding strategies to defend solanaceous plants against oomycetes.

    For these reasons, the molecular basis of the metabolic pathways of sterol synthesis and/or sterol acquisition was investigated. Sterols are derived from isoprenoids and indispensable in various biological processes. Our biochemical investigation of an oxidosqualene cyclase revealed that sterol synthesis in S. parasitica begins with the formation of lanosterol (Paper I), and a reconstruction of the complete sterol synthesis pathway to the final compound, fucosterol, in S. parasitica was performed using bioinformatics (Paper II). Complementary to this work, the extent to which P. infestans, which is incapable of de novo sterol synthesis, is able to modify exogenously provided sterols was investigated by determining the growth impact of various sterol supplements in the growth media (Paper II). 

    Building on the sterol investigations, the solanaceous sterol derivatives from the glycoalkaloid family were analysed. These compounds contain both a steroidal and a carbohydrate (glycan) moiety. Data obtained by feeding various deuterium-labeled sterols to potato shoots, supported the theory that steroidal glycoalkaloids in Solanum tuberosum are produced from cholesterol (Paper III).  Since these steroidal glycoalkaloids are thought to play a role in plant defense, their physiological effects on P. infestans were investigated (Paper IV). Unexpectedly we found that non-glycosylated steroidal alkaloids had a greater inhibitory effect than steroidal glycoalkaloids.  Steroidal glycoalkaloids derived from other Solanaceous species exhibited different physiological effects on the growth of P. infestans

    This research was conducted on two oomycete species belonging to the Saprolegniales and Peronosporales orders, hence the results presented are likely to be representative of each of these two oomycete orders.

  • 166.
    Dahlroth, Sue-Li
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Expression and structure-function characterisation of herpesviral proteins2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult.

    The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

  • 167.
    Dahlroth, Sue-Li
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lieu, Victoria
    Haas, Juergen
    Nordlund, Paer
    Screening Colonies of Pooled ORFeomes (SCOOP): A rapid and efficient strategy for expression screening ORFeomes in Escherichia coli2009Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 68, nr 2, s. 121-127Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have designed and evaluated a novel strategy for screening large gene collections available as GATEWAY-adapted ORFeomes for soluble recombinant overexpression in Escherichia coli, called ""Screening Colonies of ORFeome Pools"" (SCOOP). From a large gene collection we could, without expensive multi-well based cloning and expression screening, determine which targets were suitable for large-scale expression and purification. Normalized bacterial overnight cultures of an ORF collection of entry clones derived from the Kaposi's sarcoma associated herpesvirus (KSHV) were pooled and used for the isolation of plasmid DNA. The resulting ORF library was subcloned into a prokaryotic expression vector in a single recombination reaction and was subsequently screened with the colony filtration (CoFi) blot for soluble recombinant overexpression in E. coli. ORFs determined to express soluble recombinant proteins were identified by sequencing and analysed by small-scale IMAC and SDS-PAGE. As a reference, we subcloned all ORFs individually using a traditional multi-well based procedure and screened them for soluble expression. Our results show that the two processes have a similar efficiency as 23 and 25 out of 74 assessable clones were identified as soluble expressers using SCOOP and the traditional multi-well procedure, respectively. Because SCOOP minimises costs for cloning and expression screening, it constitutes an interesting alternative for establishing expression of large gene collections. SCOOP also allows affordable screening in alternative vectors, expression strains and physical conditions, which is challenging in large-scale protein production programs. With this strategy in hand success rates for future proteome-wide protein production efforts can be significantly increased. 

  • 168.
    Dallner, Gustav
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Regulation of coenzyme Q biosynthesis2011Ingår i: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 164, s. s12-S12Artikel i tidskrift (Övrigt vetenskapligt)
  • 169.
    Daniel, Chammiran
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Silberberg, Gilad
    Behm, Mikaela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Öhman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Alu elements shape the primate transcriptome by cis-regulation of RNA editing2014Ingår i: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 15, nr 2, artikel-id R28Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: RNA editing by adenosine to inosine deamination is a widespread phenomenon, particularly frequent in the human transcriptome, largely due to the presence of inverted Alu repeats and their ability to form double-stranded structures - a requisite for ADAR editing. While several hundred thousand editing sites have been identified within these primate-specific repeats, the function of Alu-editing has yet to be elucidated. Results: We show that inverted Alu repeats, expressed in the primate brain, can induce site-selective editing in cis on sites located several hundred nucleotides from the Alu elements. Furthermore, a computational analysis, based on available RNA-seq data, finds that site-selective editing occurs significantly closer to edited Alu elements than expected. These targets are poorly edited upon deletion of the editing inducers, as well as in homologous transcripts from organisms lacking Alus. Sequences surrounding sites near edited Alus in UTRs, have been subjected to a lesser extent of evolutionary selection than those far from edited Alus, indicating that their editing generally depends on cis-acting Alus. Interestingly, we find an enrichment of primate-specific editing within encoded sequence or the UTRs of zinc finger-containing transcription factors. Conclusions: We propose a model whereby primate-specific editing is induced by adjacent Alu elements that function as recruitment elements for the ADAR editing enzymes. The enrichment of site-selective editing with potentially functional consequences on the expression of transcription factors indicates that editing contributes more profoundly to the transcriptomic regulation and repertoire in primates than previously thought.

  • 170.
    Daniel, Chammiran
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Öhman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    RNA editing and its impact on GABAa receptor function2009Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, s. 1399-1403Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A-to-I (adenosine-to-inosine) RNA editing catalysed by the ADARs (adenosine deaminases that act on RNA) is a post-transcriptional event that contributes to protein diversity in metazoans. In mammalian neuronal ion channels, editing alters functionally important amino acids and creates receptor subtypes important for the development of the nervous system. The excitatory AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and kainate glutamate receptors, as well as the inhibitory GABAA [GABA (γ-aminobutyric acid) type A] receptor, are subject to A-to-I RNA editing. Editing affects several features of the receptors, including kinetics, subunit assembly and cell-surface expression. Here, we discuss the regulation of editing during brain maturation and the impact of RNA editing on the expression of different receptor subtypes.

  • 171.
    Danielsson, Daniel
    et al.
    Department of Clinical Science, Intervention and Technology, Division of ENT Diseases, Karolinska Institute, Stockholm.
    Brehwens, Karl
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Halle, Martin
    Department of Molecular Medicine and Surgery, Reconstructive Plastic Surgery, Karolinska Institute and Karolinska University Hospital, Stockholm.
    Marczyk, Michal
    Data Mining Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland.
    Polanska, Joanna
    Data Mining Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland.
    Munck-Wikland, Eva
    Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Karolinska University Hospital and Karolinska Institutet, Stockholm.
    Wojcik, Andrzej
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Haghdoost, Simak
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Reduced oxidative stress response as a risk factor for normal tissue damage after radiotherapy: a study on mandibular osteoradionecrosisManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background

    The use of radiotherapy (RT) to treat cancer involves exposure of normal tissues. Factors that promote the development of normal tissue damage are poorly understood. An increased individual sensitivity to ionizing radiation is a likely candidate, but general phenotypes for late adverse effects of RT are difficult to define. We have found osteoradionecrosis (ORN) in the mandible as a well-defined model phenotype for an in-depth study of clinical and biological risk factors for developing late adverse effects to RT.

    Methods

    A cohort of patients with stage 2/3 ORN following RT for head and neck cancer (HCN) was studied and compared to a closely matched control group. Blood samples from the patients were collected and irradiated in vitro and the capacity to handle radiation-induced oxidative stress was investigated by measuring the level of 8-oxo-dG in serum 60 min post exposure. The patients were also genotyped for eight SNPs in genes involved in the oxidative stress response and previously studied in the context of individual radiosensitivity. Results from these endpoints were analyzed in conjunction with clinical data using multivariate analysis and an ORN risk model was constructed.

     

    Findings

    A significant difference in 8-oxo-dG levels was found between the patient cohorts, indicating a heterogeneous response to oxidative stress induced by the in vitro γ-radiation. The SNP rs1695 in GSTP1 was found to be significantly more frequent in the ORN+ compared to ORN- group. Multivariate analysis of the clinical and biological factors revealed concomitant brachytherapy plus the two biomarkers to be the most significant.

     

    Interpretation: The current study indicates that patient-related factors are a major source of individual variation in normal tissue response to RT. Two of the studied genetic biomarkers are strong factors in the described risk model of ORN.

  • 172. Danielsson, Frida
    et al.
    Skogs, Marie
    Huss, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Rexhepaj, Elton
    O'Hurley, Gillian
    Klevebring, Daniel
    Ponten, Fredrik
    Gad, Annica K. B.
    Uhlen, Mathias
    Lundberg, Emma
    Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model2013Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, nr 17, s. 6853-6858Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.

  • 173.
    Danielsson, Jens
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kurnik, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lang, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric beta-Barrels2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 38, s. 33070-33083Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric beta-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent beta-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a bolt-on domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in beta-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

  • 174.
    Dantoft, Widad
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Role of POU/Oct transcription factors in Drosophila immunity2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In response to infection, Drosophila melanogaster relies on the ability to mount a robust and potent innate immune response, characterized by induction of cellular and humoral processes. While rapid immune induction is of utmost importance, it is equally important to restrict and repress immune gene expression, where and when it is not needed. The aim of my studies was to elucidate the in vivo role of the Oct1/POU2F1 homolog Nubbin (Nub), an evolutionarily conserved transcription factor, in innate immunity.

    The transcription factor Nub-PD, a product of the nub gene, was studied in wild type and mutant (nub1) flies, as well as in transgenic flies that either overexpress or down-regulate Nub-PD. Infection-studies were conducted after both oral and septic infection. Expression analysis of target genes using quantitative mRNA measurements and microarray analysis of wild type and nub1, reveal that Nub-PD acts as a bona fide repressor of NF-κB/Relish-dependent expression of immune defense genes, e.g. antimicrobial peptides. I show that Nub-PD represses transcription in uninfected flies by binding to oct DNA sequence motifs located upstream of a number of immune genes. In the event of infection, repression by Nub-PD is alleviated through rapid signal-dependent degradation, in a proteasome- and Imd-dependent manner, allowing initiation of antimicrobial peptide gene expression in both fat body and intestine. Lastly, we show that nub1 mutants also exhibit a shortened lifespan as well as elevated loads of gut bacteria with altered composition. We suggest that the aberrant immune gene activation, elevated bacterial loads, and altered bacterial composition in nub1 can collectively explain the shortened lifespan.

    In conclusion, this work reveals a novel function of Nub-PD as a negative regulator of immune and stress response genes in vivo. Furthermore, my work sheds light on the importance of Nub-PD in host survival and in supporting a balanced composition of the gut microbiota.

  • 175.
    Daryanavard, Seyed
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Jeppsson-Dadoun, Amin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Andersson, Lars I.
    Hashemi, Mahdi
    Colmsjö, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Abdel-Rehim, Mohamed
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Molecularly imprinted polymer in microextraction by packed sorbent for the simultaneous determination of local anesthetics: lidocaine, ropivacaine, mepivacaine and bupivacaine in plasma and urine samples2013Ingår i: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 27, nr 11, s. 1418-1488Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This study presents the use of molecularly imprinted polymer (MIP) as packing material for microextraction by packed syringe (MEPS) to achieve higher extraction selectivity. Pentycaine was used as template for MIP. Development and validation of the determination of lidocaine, ropivacaine, mepivacaine and bupivacaine in human plasma and urine samples utilizing MIP-MEPS and liquid chromatography–tandem mass spectrometry (LC-MS/MS) were carried out. The MEPS MIP-cartridge could be used for 100 extractions before it was discarded. The extraction recovery ranged from 60 to 80%. The correlation coefficients values were >0.999 for all assays using lidocaine, ropivacaine, mepivacaine and bupivacaine in the calibration range 5–2000 nmol/L. The accuracy of the studied compounds, given as a percentage variation from the nominal concentration values, ranged from -4.9 to 8.4% using plasma and urine samples. The between-batch precision, given as the relative standard deviation, at three different concentrations (quality control samples) was ranged from −4.7 to 14.0% and from 1.8 to 12.7% in plasma and urine, respectively. The lower limit of quantification and limit of detection of the studied substances were 5.0 and 1.0 nm, respectively

  • 176. Dash-Wagh, Suvarna
    et al.
    Jacob, Stefan
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Fridberger, Anders
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ulfendahl, Mats
    Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect62012Ingår i: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 1, s. e61-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.

  • 177.
    Dawitz, Hannah
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Mitochondria fulfill a plethora of functions, including harboring metabolic pathways and converting energy stored in metabolites into ATP, the common energy source of the cell. This last function is performed by the oxidative phosphorylation system, consisting of the respiratory chain and the ATP synthase. Electrons are channeled through the complexes of the respiratory chain, while protons are translocated across the inner mitochondrial membrane. This process establishes an electrochemical gradient, which is used by the ATP synthase to generate ATP. The subunits of two of the respiratory chain complexes, the bc1 complex and the cytochrome c oxidase, are encoded by two genetic origins, the nuclear and the mitochondrial genome. Therefore, the assembly of these complexes needs to be coordinated and highly regulated.

    Several proteins are involved in the biogenesis of the bc1 complex. Amongst these proteins, the Cbp3-Cbp6 complex was shown to regulate translation and assembly of the bc1 complex subunit cytochrome b. In this work, we established a homology model of yeast Cbp3. Using a site-specific crosslink approach, we identified binding sites of Cbp3 to its obligate binding partner Cbp6 and its client, cytochrome b, enabling a deeper insight in the molecular mechanisms of bc1 complex biogenesis. 

    The bc1 complex and the cytochrome c oxidase form macromolecular structures, called supercomplexes. The detailed assembly mechanisms and functions of these structures remain to be solved. Two proteins, Rcf1 and Rcf2, were identified associating with supercomplexes in the yeast Saccharomyces cerevisiae. Our studies demonstrate that, while Rcf1 has a minor effect on supercomplex assembly, its main function is to modulate cytochrome c oxidase activity. We show that cytochrome c oxidase is present in three structurally different populations. Rcf1 is needed to maintain the dominant population in a functionally active state. In absence of Rcf1, the abundance of a population with an altered active site is increased. We propose that Rcf1 is needed, especially under a high work load of the respiratory chain, to maintain the function of cytochrome c oxidase.

    This thesis aims to unravel molecular mechanisms of proteins involved in biogenesis and functionality of respiratory chain complexes to enable a deeper understanding. Dysfunctional respiratory chain complexes lead to severe disease, emphasizing the importance of this work.

  • 178.
    Dawitz, Hannah
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schäfer, Jacob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schaart, Judith Maria
    Magits, Wout
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rcf1 modulates cytochrome c oxidase activity especially under energy-demanding conditionsManuskript (preprint) (Övrigt vetenskapligt)
  • 179.
    de Klerk, Nele
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Maudsdotter, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Gebreegziabher, Hanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Saroj, Sunil D.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Eriksson, Beatrice
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Eriksson, Olaspers Sara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Roos, Stefan
    Linden, Sara
    Sjölinder, Hong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jonsson, Ann-Beth
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression2016Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 84, nr 5, s. 1526-1535Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori. In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA. Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity.

  • 180.
    de Klerk, Nele
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Saroj, Sunil D.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Wassing, Gabriela M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Maudsdotter, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jonsson, Ann-Beth
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The Host Cell Transcription Factor EGR1 Is Induced by Bacteria through the EGFR-ERK1/2 Pathway2017Ingår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 7, artikel-id 16Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The essential first step in bacterial colonization is adhesion to the host epithelial cells. The early host-responses post-bacterial adhesions are still poorly understood. Early growth response 1 (EGR1) is an early response transcriptional regulator that can be rapidly induced by various environmental stimuli. Several bacteria can induce EGR1 expression in host cells, but the involved bacterial characteristics and the underlying molecular mechanisms of this response are largely unknown. Here, we show that EGR1 can be induced in host epithelial cells by different species of bacteria independent of the adherence level, Gram-staining type and pathogenicity. However, bacterial viability and contact with host cells is necessary, indicating that an active interaction between bacteria and the host is important. Furthermore, the strongest response is observed in cells originating from the natural site of the infection, suggesting that the EGR1 induction is cell type specific. Finally, we show that EGFRERK1/2 and beta 1-integrin signaling are the main pathways used for bacteria-mediated EGR1 upregulation. In conclusion, the increase of EGR1 expression in epithelial cells is a common stress induced, cell type specific response upon host-bacteria interaction that is mediated by EGFRERK1/2 and beta 1-integrin signaling.

  • 181.
    de Marothy, Tuuli Minttu Virkki
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Marginally hydrophobic transmembrane α-helices shaping membrane protein folding2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Most membrane proteins are inserted into the membrane co-translationally utilizing the translocon, which allows a sufficiently long and hydrophobic stretch of amino acids to partition into the membrane. However, X-ray structures of membrane proteins have revealed that some transmembrane helices (TMHs) are surprisingly hydrophilic. These marginally hydrophobic transmembrane helices (mTMH) are not recognized as TMHs by the translocon in the absence of local sequence context.

    We have studied three native mTMHs, which were previously shown to depend on a subsequent TMH for membrane insertion. Their recognition was not due to specific interactions. Instead, the presence of basic amino acids in their cytoplasmic loop allowed membrane insertion of one of them. In the other two, basic residues are not sufficient unless followed by another, hydrophobic TMH. Post-insertional repositioning are another way to bring hydrophilic residues into the membrane. We show how four long TMHs with hydrophilic residues seen in X-ray structures, are initially inserted as much shorter membrane-embedded segments. Tilting is thus induced after membrane-insertion, probably through tertiary packing interactions within the protein.

    Aquaporin 1 illustrates how a mTMH can shape membrane protein folding and how repositioning can be important in post-insertional folding. It initially adopts a four-helical intermediate, where mTMH2 and TMH4 are not inserted into the membrane. Consequently, TMH3 is inserted in an inverted orientation. The final conformation with six TMHs is formed by TMH2 and 4 entering the membrane and TMH3 rotating 180°. Based on experimental and computational results, we propose a mechanism for the initial step in the folding of AQP1: A shift of TMH3 out from membrane core allows the preceding regions to enter the membrane, which provides flexibility for TMH3 to re-insert in its correct orientation.

  • 182.
    de Maré, Fredrick
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Diiron-carboxylate proteins: structural studies of rubrerythrin and protein R2 of ribonucleotide reductase1996Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 183.
    Dehvari, Nodi
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    Mahmud, Tapan
    Persson, Johanna
    Bengtsson, Tore
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för fysiologi.
    Graff, Caroline
    Winblad, Bengt
    Ronnback, Annica
    Behbahani, Homira
    Amyloid precursor protein accumulates in aggresomes in response to proteasome inhibitor2012Ingår i: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 60, nr 5, s. 533-542Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aggresomes are cytoplasmic inclusions which are localized at the microtubule organizing center (MTOC) as a result of induced proteasome inhibition, stress or over-expression of certain proteins. Aggresomes are linked to the pathogenesis of many neurodegenerative diseases. Here we studied whether amyloid precursor protein (APP), a type-I transmembrane glycoprotein, is localized in aggresomes after exposure to stress condition. Using confocal microscopy we found that APP is located in aggresomes and co-localized with vimentin, gamma-tubulin, 20S and ubiquitin at the MTOC in response to proteasome dysfunction. An interaction between vimentin and APP was found after proteasome inhibition suggesting that APP is an additional protein constituent of aggresomes. Suppression of the proteasome system in APP-HEK293 cells overexpressing APP or transfected with APP Swedish mutation caused an accumulation of stable, detergent-insoluble forms of APP containing poly-ubiquitinated proteins. In addition, brain homogenates from transgenic mice expressing human APP with the Arctic mutation demonstrated an interaction between APP and the aggresomal-marker vimentin. These data suggest that malfunctioning of the proteasome system caused by mutation or overexpression of pathological or non-pathological proteins may lead to the accumulation of stable aggresomes, perhaps contributing to the neurodegeneration.

  • 184. Devesse, Laurence
    et al.
    Smirnova, Irina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lönneborg, Rosa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kapp, Ulrike
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Leonard, Gordon A.
    Dian, Cyril
    Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators2011Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 81, nr 2, s. 354-367Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Activation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apo- forms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.

  • 185.
    Dircksen, Heinrich
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för funktionell zoomorfologi.
    Neupert, Susanne
    Predel, Reinhard
    Verleyen, Peter
    Huybrechts, Jurgen
    Strauss, Johannes
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för funktionell zoomorfologi.
    Hauser, Frank
    Stafflinger, Elisabeth
    Schneider, Martina
    Pauwels, Kevin
    Schoofs, Liliane
    Grimmelikhuijzen, Cornelis J. P.
    Genomics, transcriptomics and peptidomics of Daphnia pulex neuropeptides and protein hormones2011Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, nr 10, s. 4478-4504Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, a first crustacean allatotropin, Ala7-CCAP, one CCHamide, Arg7-corazonin, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin- and one each of three IGF-related peptides, two alternative splice forms of short and long ion transport peptide (ITP), one each of two N-terminally elongated ITPs, myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met4-proctolin, one novel short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, three tachykinins. Two genes encode orcokinins, three genes different allatostatins-C. Paired gene clusters occur for two novel eclosion hormones; bursicons alpha, beta; glycoproteins GPA2, GPB5; and two of the allatostatin-C genes. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.

  • 186.
    Domingo Prim, Judit
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The exosome and the maintenance of genome integrity2016Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The RNA exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome plays a role in DNA repair. We have shown that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DNA double-strand breaks (DSBs) in Drosophila S2 cells and human HeLa cells exposed to either ionizing radiation or I-PpoI endonuclease cleavage. DIS3, the other catalytic subunit of the nuclear exosome, is also recruited to DSBs, whereas the exosome core subunit EXOSC7 is not. Depletion of different exosome subunits does not interfere with the phosphorylation of the histone variants H2Av (Drosophila) or H2AX (humans), but depletion of RRP6/EXOSC10 impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A–V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway in animal cells. Taken together, our results suggest that a 3’-5’ ribonucleolytic activity is required for efficient DNA repair. 

  • 187.
    Dou, Dan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    da Silva, Diogo V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordholm, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wang, Hao
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daniels, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion2014Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 25, nr 21, s. 3363-3374Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N-out-C-in HA and M2 TMDs but not the N-in-C-out TMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (Delta G(app) > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once similar to 70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by similar to 100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

  • 188.
    Dou, Dan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hernández-Neuta, Iván
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Wang, Hao
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Östbye, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Qian, Xiaoyan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Thiele, Swantje
    Resa-Infante, Patricia
    Mounogou Kouassi, Nancy
    Sender, Vicky
    Hentrich, Karina
    Mellroth, Peter
    Henriques-Normark, Birgitta
    Gabriel, Gülsah
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Daniels, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method2017Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 20, nr 1, s. 251-263Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

  • 189.
    Dourado, Daniel F. A. R.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 45, s. 8069-8078Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

  • 190.
    Dowaidar, Moataz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Chimeric gene delivery vectors: Design, synthesis, and mechanisms from transcriptomics analysis2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    Genterapi med hjälp av av oligonukleotider (ON) har en enorm potential för behandling av olika genetiska sjukdomar. För att ha terapeutisk effekt måste dock oligonukleotiderna nå in i cellen och detta försvåras på grund av deras negativa laddningar och snabba nedbrytning. Cellpenetrerande peptider (CPP), är korta katjoniska peptider, som kan användas för att förbättra det cellulära upptaget (transfektionen) av oligonukleotider. I denna avhandling undersöks nya strategier för hur CPP tillsammans med magnetiska nanopartiklar, såsom MNP och Fe3O4, eller grafenoxid (GO) nanopartiklar, kan möjliggöra effektivare transfektion av ON.  Vidare studeras även de möjliga cellulära signalvägar som reglerar CPP-medierat upptag.

    En så kallad ”fragment quantitative structure-activity relationship” (FQSAR) modell  användes för att förutsäga nya effektiva CPP för leverans av plasmider (ringformade DNA-molekyler med omkring 5000 nukleotidbaspar). De bäst prediktade peptiderna visade en signifikant ökad transfektionsförmåga jämfört med den tidigare använda peptiden PeptFect 14 (PF14). De nya peptiderna PF220, PF221, PF222, PF223 och PF224 som identifierades med FQSAR kunde dessutom bilda självmonterande komplex med MNP eller GO nanopartiklar. I cellulära försök uppvisade dessa nya hybridvektorer (CPP/MNP och CPP/GO) en klart förbättrad transfektionsförmåga av såväl plasmider, som splitsningskorrigerande oligonukleotider (SCO) och små interfererande RNA (siRNA), jämfört med PF14-nanopartikel hybridvektorer, såväl som den kommersiella lipidbaserade transfektionsvektorn Lipofectamine™ 2000. Den höga transfektionseffektiviteten hos dessa nya hybridvektorer beror troligen på deras låga cellulära toxicitet och en möjlig synergistisk effekt vid kombinationen av CPP och MNP/GO nanopartiklar. Förmågan hos en CPP/MNP hybridvektor att levera plasmider in vivo undersöktes också och transfektion av celler i såväl lunga och mjälte i behandlade djur kunde påvisas. Dessa nya hybridvektorer utgör således en ny lovande strategi för leverans av ON vid genterapi.

    För att kartlägga de signalvägar som kontrollerar upptaget av CPP-baserade vektorer analyserades  genuttrycket hos celler som transfekterats med PF14 eller PF14-ON, med hjälp av  RNA-sekvensering och qPCR-analys. Resultaten påvisade att en ökning i uttrycket av flera autofagirelaterade gener sker tidigt vid transfektionen. Konfokal- och transmissionselektronmikroskopi demonstrerade vidare en ökad initiering av autofagi och samlokalisering av ON med autofagosomer. Detta visar att CPP-medierad transfektion aktiverar signalvägar som stryr autofagi och öppnar nya möjligheter att använda autofagimodifierare för att förbättra genterapi.

  • 191.
    Dowaidar, Moataz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation2017Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Cell-penetrating peptide-based transfection systems (PBTS) are a promising group of drug delivery vectors. Cell-penetrating peptides (CPPs) are short cationic peptides that are able of transporting cell non-permeant cargos into different cell types. Some CPPs can be used to form non-covalent complexes with oligonucleotides for gene delivery applications. For the potential use of CPPs as drug delivery tools, it is important to understand the mechanism of uptake. Here, a fragment quantitative structure–activity relationships (FQSAR) model is generated to predict novel peptides based on approved alpha helical conformers and assisted model construction with energy refinement molecular mechanics simulations of former peptides. The modeled peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were efficient for plasmid transfection with significant enhancement compared to the former group of peptides. Our results confirm that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity. Additionally, using RNA sequencing, we demonstrated the involvement of autophagy pathways in PBTS uptake.

  • 192.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abdelhamid, Hani Nasser
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK). Assuit University, Egypt.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Zou, Xiaodong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Chitosan enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles–cell-penetrating peptide2018Ingår i: Journal of biomaterials applications, ISSN 0885-3282, E-ISSN 1530-8022, Vol. 33, nr 3, s. 392-401Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.

  • 193.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gestin, Maxime
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cerrato, Carmine Pasquale
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Margus, Helerin
    Kivistik, Paula Ann
    Pooga, Margus
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Role of autophagy in PepFect14 transfection2017Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed with qPCR analysis. The gene regulations were then related to the biological process by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection of splice correction oligonucleotide complexed with Pepfect 14 confirming the induction of autophagy process by the uptake of complexes. Finally, colocalization of nucleic acid cargo and autophagosomes, as well as the autophagosome production induced by the treatment, have been shown by confocal microscopy and transmission electron microscopy. We conclude that autophagy is an important response process triggered by the cellular uptake of CPP-based transfection system. This conclusion opens a possibility to use autophagy modifiers in future gene therapy.

  • 194.
    Dowaidar, Moataz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Regberg, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dobchev, Dimitar A.
    Lehto, Tõnis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Karelson, Mati
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems2017Ingår i: International Journal of Peptide Research and Therapeutics, E-ISSN 1573-3904, Vol. 23, nr 1, s. 91-100Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR), a statistical method based on regression data analysis. Here, a fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.

  • 195.
    Drew, David
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    GFP as a tool to monitor membrane protein topology and overexpression in Escherichia coli2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

  • 196.
    Eberle, Andrea B.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik. Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Böhm, Stefanie
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Farrants, Ann-Kristin Östlund
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation2012Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 426, nr 2, s. 147-152Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.

  • 197.
    Edgren, Tomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Electron transport to nitrogenase in Rhodospirillum rubrum2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Biological nitrogen fixation is a key step in the global nitrogen cycle. In this process, dinitrogen in the air is converted to biologically accessible ammonia, which is further assimilated in to the biosphere. Nitrogenase, the enzyme system responsible for dinitrogen reduction, is only found in prokaryotic organisms and biological nitrogen fixation is an energy-demanding process, requiring both ATP and low potential reducing equivalents. In the free-living purple non-sulfur anoxygenic phototroph Rhodospirillum rubrum, efficient electron transfer to nitrogenase is dependent on active electron transport in the chromatophore membrane.

    I have shown that reducing equivalents for nitrogen fixation is generated through the action of the proteins encoded by the fixABCX genes. The membrane associated protein complex encoded by these genes reduces a soluble ferredoxin, which in turn acts as the direct electron donor to nitrogenase in this organism. The heterodimeric flavoprotein FixAB has NADH dehydrogenase activity indicating that the reducing equivalents for nitrogen fixation are derived from the general metabolism of the cell. The membrane associated FixC protein is believed to drive the energetically unfavorable reduction of ferredoxin N using energy derived from the electron transfer processes in the chromatophore membrane in some unknown manner. The membrane associated protein complex encoded by fixABCX most likely constitutes the unknown link between photosynthesis and nitrogen fixation in R. rubrum.

  • 198. Edman, Maria
    et al.
    Berg, Stefan
    Storm, Patrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wikström, Malin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vikström, Susanne
    Öhman, Anders
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae2003Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 10, s. 8420-8428Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyl-diacylglycerol. (MGlcDAG) synthase (aIMGS) (EC 2.4.1.157) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the aIDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of aIDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

  • 199. Edvardsson, Anna
    et al.
    Shapiguzov, Alexey
    Petersson, Ulrika A.
    Stockholms universitet.
    Schroder, Wolfgang P.
    Vener, Alexander V.
    Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-22007Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 33, s. 9432-9442Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.

  • 200. Ehrlich, Kersti
    et al.
    Viirlaid, Säde
    Mahlapuu, Riina
    Saar, Külliki
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kullisaar, Tiiu
    Zilmer, Mihkel
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Soomets, Ursel
    Design, synthesis and properties of novel powerful antioxidants, glutathione analogues2007Ingår i: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 41, nr 7, s. 779-787Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC50 of GSH: 1231.0 +/- 311.8 mu M; EC50 of UPF peptides: from 0.03 to 35 mu M) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC50: 11.4 +/- 1.0 mu M). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 mu M concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.

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