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  • 251.
    Forslund, Kristoffer
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Swedish e-Science Research Center .
    Evolution of Protein Domain Architectures2012Ingår i: Evolutionary Genomics: Statistical and Computational Methods, Vol 2 / [ed] Anisimova, M, Totowa, NJ: Humana Press, 2012, s. 187-216Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    This chapter reviews the current research on how protein domain architectures evolve. We begin by summarizing work on the phylogenetic distribution of proteins, as this directly impacts which domain architectures can be formed in different species. Studies relating domain family size to occurrence have shown that they generally follow power law distributions, both within genomes and larger evolutionary groups. These findings were subsequently extended to multidomain architectures. Genome evolution models that have been suggested to explain the shape of these distributions arc reviewed, as well as evidence for selective pressure to expand certain domain families more than others. Each domain has an intrinsic combinatorial propensity, and the effects of this have been studied using measures of domain versatility or promiscuity. Next, we study the principles of protein domain architecture evolution and how these have been inferred from distributions of extant domain arrangements. Following this, we review inferences of ancestral domain architecture and the conclusions concerning domain architecture evolution mechanisms that can be drawn from these. Finally, we examine whether all known cases of a given domain architecture can be assumed to have a single common origin (monophyly) or have evolved convergently (polyphyly).

  • 252.
    Fotouhi, Asal
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cornella, Nicola
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Ramezani, Mehrafarin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Wojcik, Andrzej
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Haghdoost, Siamak
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB or UVC2015Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 793, nr SI, s. 161-165Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The longer wave parts of UVR can increase the production of reactive oxygen species (ROS) which can oxidize nucleotides in the DNA or in the nucleotide pool leading to mutations. Oxidized bases in the DNA are repaired mainly by the DNA base excision repair system and incorporation of oxidized nucleotides into newly synthesized DNA can be prevented by the enzyme MTH1. Here we hypothesize that the formation of several oxidized base damages (from pool and DNA) in close proximity, would cause a high number of base excision repair events, leading to DNA double strand breaks (DSB) and therefore giving rise to cytogenetic damage. If this hypothesis is true, cells with low levels of MTH1 will show higher cytogenetic damage after the longer wave parts of UVR. We analyzed micronuclei induction (MN) as an endpoint for cytogenetic damage in the human lymphoblastoid cell line, TK6, with a normal and a reduced level of MTH1 exposed to UVR. The results indicate a higher level of micronuclei at all incubation times after exposure to the longer wave parts of UVR. There is no significant difference between wildtype and MTH1-knockdown TK6 cells, indicating that MTH1 has no protective role in UVR-induced cytogenetic damage. This indicates that DSBs induced by UV arise from damage forms by direct interaction of UV or ROS with the DNA rather than through oxidation of dNTP.

  • 253. Fourati, Zaineb
    et al.
    Howard, Rebecca J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Heusser, Stephanie A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Hu, Haidai
    Ruza, Reinis R.
    Sauguet, Ludovic
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Delarue, Marc
    Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels2018Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 23, nr 4, s. 993-1004Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking[1] . The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore, or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.

  • 254. Freimann, Krista
    et al.
    Arukuusk, Piret
    Kurrikoff, Kaido
    Vasconcelos, Luís Daniel Ferreira
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Veiman, Kadi-Liis
    Uusna, Julia
    Margus, Helerin
    Garcia-Sosa, Alfonso T.
    Pooga, Margus
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Optimization of in vivo DNA delivery with NickFect peptide vectors2016Ingår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 241, s. 135-143Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    As the field of gene therapy progresses, an increasingly urgent need has arisen for efficient and non-toxic vectors for the in vivo delivery of nucleic acids. Cell-penetrating peptides (CPP) are very efficient transfection reagents in vitro, however, their application in vivo needs improvement. To enhance in vivo transfection we designed various CPPs based on previous knowledge of internalization studies and physiochemical properties of NickFect (NF) nanoparticles. We show that increment of the helicity of these Transportan10 analogues improves the transfection efficiency. We rationally design by modifying the net charge and the helicity of the CPP a novel amphipathic α-helical peptide NF55 for in vivo application. NF55 condenses DNA into stable nanoparticles that are resistant to protease degradation, promotes endosomal escape, and transfects the majority of cells in a large cell population. We demonstrate that NF55 mediates DNA delivery in vivo with gene induction efficiency that is comparable to commercial transfection reagents. In addition to gene induction in healthy mice, NF55/DNA nanoparticles showed promising tumor transfection in various mouse tumor models, including an intracranial glioblastoma model. The efficiency of NF55 to convey DNA specifically into tumor tissue increased even further after coupling a PEG2000 to the peptide via a disulphide-bond. Furthermore, a solid formulation of NF55/DNA displayed an excellent stability profile without additives or special storage conditions. Together, its high transfection efficacy and stability profile make NF55 an excellent vector for the delivery of DNA in vivo.

  • 255. Freimann, Krista
    et al.
    Kurrikoff, Kaido
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Galanin receptors as a potential target for neurological disease2015Ingår i: Expert opinion on therapeutic targets, ISSN 1472-8222, E-ISSN 1744-7631, Vol. 19, nr 12, s. 1665-1676Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    INTRODUCTION: Galanin is a 29/30 amino acid long neuropeptide that is widely expressed in the brains of many mammals. Galanin exerts its biological activities through three different G protein-coupled receptors, GalR1, GalR2 and GalR3. The widespread distribution of galanin and its receptors in the CNS and the various physiological and pharmacological effects of galanin make the galanin receptors attractive drug targets.

    AREAS COVERED: This review provides an overview of the role of galanin and its receptors in the CNS, the involvement of the galaninergic system in various neurological diseases and the development of new galanin receptor-specific ligands.

    EXPERT OPINION: Recent advances and novel approaches in migrating the directions of subtype-selective ligand development and chemical modifications of the peptide backbone highlight the importance of the galanin neurochemical system as a potential target for drug development.

  • 256. Friedlaender, Jonathan S
    et al.
    Hunley, Keith
    University of New Mexico.
    Dunn, Michael
    Radboud University; Max Planck Institute for Psycholinguistics.
    Terrill, Angela
    Radboud University.
    Lindström, Eva
    Stockholms universitet, Humanistiska fakulteten, Institutionen för lingvistik, Avdelningen för allmän språkvetenskap.
    Friedlaender, Françoise
    Linguistics More Robust Than Genetics: (Letter to the editors)2009Övrigt (Refereegranskat)
  • 257.
    Fristedt, Ulrika
    Stockholms universitet.
    Characterization of the structure and function of the Pho84 high-affinity phosphate transporter of Saccharomyces cerevisiae1999Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    All living organisms, from bacteria and yeast to mammalian cells, must respond to a fluctuating extracellular environment. Information contained in such fluctuations is signaled via the cell surface to the interior of the cell, resulting in responses such as altered gene expression and protein activity. Membrane transport proteins play a central role in such signal transduction. These proteins are responsible for the uptake of essential nutrients, ions and metabolites as well.

    Transport of inorganic phosphate across the plasma membrane is the first step in phosphate utilization by the cell. The intracellular level of phosphate in the yeast Saccharomyces cerevisiae is regulated by the PHO network of scattered genes coding for both structural and regulatory units.

    In S. cerevisiae, phosphate uptake across the plasma membrane is mediated by specific proteins proposed to be organized into at least three different transport systems. The PHO84 and PHO89 genes encode two high-affinity phosphate co-transporters. Hydropathy analysis suggests that these proteins are arranged into 12 transmembrane domains. While the derepressible Pho84 protein catalyzes proton-coupled phosphate transport at acidic pH, the Pho89 protein, which is also derepressible, catalyzes sodium-dependent phosphate uptake at alkaline pH.

    Three different model systems have been developed for the characterization of the Pho84 protein, i.e., inside-out plasma membrane vesicles and reconstitution into liposomes alone or with cytochrome c oxidase. The results obtained suggest that the phosphate transport is bidirectional and that it is the direction of the driving-force rather than the orientation of the protein, which determines the direction of transport of inorganic phosphate catalyzed by Pho84p. Each component of the Dp which drives phosphate uptake has been investigated in the co-reconstituted model system and the findings suggest that this transport can be driven by either one of the components. Divalent cations such as Mn2+ and Co2+ stimulate phosphate uptake by proteoliposomes, suggesting that a metal-phosphate complex is most likely the entity being transported.

  • 258.
    Fritzell, Kajsa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The ADAR editing enzymes as therapeutic targets2018Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Editing by deamination of adenosine to inosine (A-to-I) in double-stranded RNA is a common event in the human transcriptome. Altered levels of the essential ADAR1 activity are associated with disease. Children with mutations in the ADAR1 gene suffer from fatal Aicardi-Goutières syndrome (AGS) characterized by aberrant interferon expression. In contrast, ADAR1 overexpression is associated with increased malignancy in several cancers including breast cancer, lung cancer and liver cancer. ADAR1 silencing in breast cancer cell lines leads to a significant increase in apoptosis, suggesting that ADAR1 acts as an anti-apoptotic factor and promotes cancer progression. Yet, suitable high-throughput assays to monitor editing activity in human cells have limited research progress. Here we describe the development of a bioluminescent reporter system that facilitates the rapid and accurate determination of endogenous editing activity. The system is based on the highly sensitive and quantitative Nanoluciferase that is conditionally expressed upon reporter transcript editing. Stably introduced into cancer cell lines, we show that the system can measure elevated endogenous ADAR1 editing activity induced by externally provided interferon as well as knockdown of ADAR1 and ADAR2. In an optimized single-well setup we used the reporter in a stable HeLa cell line to screen a small molecule library of 33 000 compounds for potential inhibitors. This yielded a primary hit rate of 0.9% at 70% inhibition. Thus, we have generated a key tool for high-throughput identification of modifiers of A-to-I editing activity in cancer cells.

  • 259.
    Fängström, Britta
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Athanassiadis, Ioannis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Odsjö, Tjelvar
    Department of Contaminant Research, Swedish Museum of Natural History, Stockholm, Sweden.
    Norén, Koidu
    Bergman, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK), Avdelningen för miljökemi.
    Temporal trends of polybrominated diphenyl ethers and hexabromocyclododecane in milk from Stockholm mothers, 1980-2004.2008Ingår i: Molecular Nutrition & Food Research, ISSN 1613-4125, E-ISSN 1613-4133, Vol. 52, s. 187-193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Environmental and human exposures to brominated flame retardants (BFR) have been of emerging concern since some BFR are persistent and bioaccumulative compounds. Among those, polybrominated diphenyl ethers (PBDE) have frequently been reported in low to high ng/g concentrations in human blood around the world while hexabromocyclododecane (HBCDD) only occasionally has been reported and then in the low ppb concentrations in human blood. The present study concerns PBDE congener and HBCDD concentrations in human milk from Stockholm from 1980 to 2004. HBCDD concentrations has increased four to five times since 1980 until 2002 but seems to have stabilized at this concentration in the last years (2003/04). Similarly, BDE-153 has continued to increase at least to 2001, after which it has stabilized in the mother's milk. Other PBDE congeners with four to five bromine substituents peaked 5 years earlier (1995) and are all decreasing. DecaBDE (BDE-209) is not a suitable biomarker for time trend studies according to the present results, showing no changes over time. This is likely due to its short apparent half-life in humans and poor transfer from blood to milk.

  • 260. Gadalla, Salah-Eldin
    et al.
    Öjemalm, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lara Vasquez, Patricia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ericsson, Christer
    Zhao, Jian
    Nister, Monica
    EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells2013Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 439, nr 2, s. 203-208Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell marker and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

  • 261.
    Galian-Barrueco, Carmen
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Björkholm, Patrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bulleid, Neil
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 20, s. 16399-16409Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is marginal: less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.

  • 262. Galmozzi, Andrea
    et al.
    Sonne, Si B.
    Altshuler-Keylin, Svetlana
    Hasegawa, Yutaka
    Shinoda, Kosaku
    Luijten, Ineke H. N.
    University of California, USA.
    Won Chang, Jae
    Sharp, Louis Z.
    Cravatt, Benjamin F.
    Saez, Enrique
    Kajimura, Shingo
    ThermoMouse: An In Vivo Model to Identify Modulators of UCP1 Expression in Brown Adipose Tissue2014Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 9, nr 5, s. 1584-1593Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis.

  • 263.
    Gao, Qiuju
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Meteorologiska institutionen (MISU).
    Nilsson, Ulrika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Ilag, Leopold L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Leck, Caroline
    Stockholms universitet, Naturvetenskapliga fakulteten, Meteorologiska institutionen (MISU).
    Monosaccharide compositional analysis of marine polysaccharides by hydrophilic interaction liquid chromatography-tandem mass spectrometry2011Ingår i: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 399, nr 7, s. 2517-2529Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A simple and sensitive method was developed using hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry for determination of monosaccharides liberated from marine polysaccharides by acidic hydrolysis. Optimal separation of diastereomeric monosaccharides including hexoses, pentoses, and deoxyhexoses was achieved using an aminopropyl bonded column with mobile phase containing ternary solvents (acetonitrile/methanol/water) in conjunction with MS/MS in SRM mode. Mechanisms for fragmentation of deprotonated monosaccharides with regard to cross-ring cleavage were proposed. Matrix effects from coeluting interferences were observed and isotopic-labeled internal standard was used to compensate for the signal suppression. The method demonstrated excellent instrumental limits of detection (LOD), ranging from 0.7 to 4.2 pg. Method LODs range from 0.9 to 5.1 nM. The proposed method was applied to the analysis of polysaccharides in seawater collected from the open leads of the central Arctic Ocean in the summer of 2008.

  • 264. Garcia-Bartolome, Alberto
    et al.
    Penas, Ana
    Marin-Buera, Lorena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Hospital Universitario, Madrid, Spain.
    Lobo-Jarne, Teresa
    Perez-Perez, Rafael
    Moran, Maria
    Arenas, Joaquin
    Martin, Miguel A.
    Ugalde, Cristina
    Respiratory chain enzyme deficiency induces mitochondrial location of actin-binding gelsolin to modulate the oligomerization of VDAC complexes and cell survival2017Ingår i: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 26, nr 13, s. 2493-2506Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used two-dimensional difference gel electrophoresis analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patients' fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes.

  • 265.
    Gatsinzi, Tom
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Different strategies to inhibit NF-κB in order to sensitize Stypehuman neuroblastoma cells to TRAIL-induced apoptosisManuskript (preprint) (Övrigt vetenskapligt)
  • 266.
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Property-controlling Enzymes at the Membrane Interface2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Monotopic proteins represent a specialized group of membrane proteins in that they are engaged in biochemical events taking place at the membrane interface. In particular, the monotopic lipid-synthesizing enzymes are able to synthesize amphiphilic lipid products by catalyzing two biochemically distinct molecules (substrates) at the membrane interface. Thus, from an evolutionary point of view, anchoring into the membrane interface enables monotopic enzymes to confer sensitivity to a changing environment by regulating their activities in the lipid biosynthetic pathways in order to maintain a certain membrane homeostasis. We are focused on a plant lipid-synthesizing enzyme DGD2 involved in phosphate shortage stress, and analyzed the potentially important lipid anchoring segments of it, by a set of biochemical and biophysical approaches. A mechanism was proposed to explain how DGD2 adjusts its activity to maintain a proper membrane. In addition, a multivariate-based bioinformatics approach was used to predict the lipid-binding segments for GT-B fold monotopic enzymes. In contrast, a soluble protein Myr1 from yeast, implicated in vesicular traffic, was also proposed to be a membrane stress sensor as it is able to exert different binding properties to stressed membranes, which is probably due to the presence of strongly plus-charged clusters in the protein. Moreover, a bacterial monotopic enzyme MGS was found to be able to induce massive amounts of intracellular vesicles in Escherichia coli cells. The mechanisms involve several steps: binding, bilayer lateral expansion, stimulation of lipid synthesis, and membrane bending. Proteolytic and mutant studies indicate that plus-charged residues and the scaffold-like structure of MGS are crucial for the vesiculation process. Hence, a number of features are involved governing the behaviour of monotopic membrane proteins at the lipid bilayer interface.

  • 267.
    Ge, Changrong
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Georgiev, Alexander
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öhman, Anders
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kelly, Amélie A.
    Tryptophan residues promote membrane association for a plant lipid glycosyltransferase involved in phosphate stress2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 8, s. 6669-6684Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

  • 268.
    Ge, Changrong
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University Libre Brussels, Belgium; Karolinska Institutet, Sweden.
    Gómez Llobregat, Jordi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Skwark, Marcin J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Aalto University, Finland.
    Ruysschaert, Jean-Marie
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindén, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane remodeling capacity of a vesicle-inducing glycosyltransferase2014Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, nr 16, s. 3667-3684Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intracellular vesicles are abundant in eukaryotic cells but absent in the Gram-negative bacterium Escherichia coli. However, strong overexpression of a monotopic glycolipid-synthesizing enzyme, monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii (alMGS), leads to massive formation of vesicles in the cytoplasm of E. coli. More importantly, alMGS provides a model system for the regulation of membrane properties by membrane-bound enzymes, which is critical for maintaining cellular integrity. Both phenomena depend on how alMGS binds to cell membranes, which is not well understood. Here, we carry out a comprehensive investigation of the membrane binding of alMGS by combining bioinformatics methods with extensive biochemical studies, structural modeling and molecular dynamics simulations. We find that alMGS binds to the membrane in a fairly upright manner, mainly by residues in the N-terminal domain, and in a way that induces local enrichment of anionic lipids and a local curvature deformation. Furthermore, several alMGS variants resulting from substitution of residues in the membrane anchoring segment are still able to generate vesicles, regardless of enzymatic activity. These results clarify earlier theories about the driving forces for vesicle formation, and shed new light on the membrane binding properties and enzymatic mechanism of alMGS and related monotopic GT-B fold glycosyltransferases.

  • 269.
    Ge, Changrong
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Raussens, Vincent
    Ruysschaert, Jean-Marie
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Modulation of Escherichia coli Cell Membrane by a Monotopic Lipid Glycosyltransferase - an Exploration of Potential MechanismsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Intracellular vesicles are abundant in eukaryotic cells but are rare in Gram-negative bacterium Escherichia coli. Strongly overexpression of a monotopic glycolipid-synthesizing enzyme could induce massive formation of “foreign” vesicles in the cytoplasm. Here we investigate how this membrane-associated enzyme is able to bend and deform the plasma membrane. Limited proteolysis combined with ESI-MS suggested interface binding is mediated through both its two Rossmann fold topological domains. Detailed subcellular localization and liposome binding assay indicates different interface anchoring regions in the protein, and anionic lipid seems to influence the binding properties of the anchoring segments. Genetic engineering of a known membrane-bound segment to explore its vesiculation potentials led to the identification of important catalytic residues (regions). Flow cytometry and infrared spectroscopy were also performed on bacterial cells to get more insight into the cellular morphology and internal complexity. The linking region between two domains was demonstrated to be crucial for both catalytic function and vesiculation capacity of the enzyme. Based on our findings, we propose, that scaffold-like structural feature of this enzyme is most likey one of key elements contributing to vesiculation.        

  • 270.
    Ge, Changrong
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana2014Ingår i: Molecular Plant, ISSN 1674-2052, Vol. 7, nr 1, s. 121-136Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide (TP) directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis (MVDA) with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed. dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for the chloroplast import.

  • 271.
    Georgiev, Alexander
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Interpretable Numerical Descriptors of Amino Acid Space2009Ingår i: Journal of Computational Biology, ISSN 1066-5277, E-ISSN 1557-8666, Vol. 16, nr 5, s. 703-723Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Informative numerical representations of amino acid residues are essential for successful in silico modeling or establishing the structure-activity relationships of proteins. A straightforward approach is adopted here for representing more than 500 amino acid indices from the AAindex database by a set of uncorrelated scales, satisfying the VARIMAX criterion. Different measures are considered in order to demonstrate the improved interpretability of the current scales as compared to previously published ones. Performance is also addressed in a classification problem of G-protein coupled receptors, and is found to be similar or higher than the performance achieved by six other scale sets. Finally, a unique correspondence between numerical indices and mutation matrices is derived and discussed in light of the evolutionary conservation of amino acid properties. Conclusions from this study highlight the discord between ease of interpretation of amino acid scales and their relevance to protein structure conservation, as well as general considerations for designing custom scale sets.

  • 272.
    Georgiev, Alexander
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Stress and the Role of GYF Domain Proteins2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from Saccharomyces cerevisiae, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner.

    Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants.

    We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.

  • 273.
    Georgiev, Alexander
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Basic clusters and amphipathic helices contribute to interactions of Myr1/Syh1 with membrane phospholipidsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The ability to associate transiently with membrane bilayers is an important property of many protein regulators of membrane trafficking, lipid transfer proteins, or signaling modules. Membrane association is also a property of Myr1/Syh1, a soluble GYF domain protein from Saccharomyces cerevisiae, previously reported to rescue the temperature sensitive growth of ypt6 and ric1 null strains. Here, we further demonstrate that MYR1 also rescued the vacuole fragmentation phenotype of the ypt6 and ric1 mutants. The mechanism behind these genetic interactions is likely linked to the capacity of the Myr1/Syh1 protein to associate with phospholipid membranes. In order to elucidate further the nature of the interactions with vesicular traffic, we studied protein-protein and protein-phospholipid association of isolated domains from Myr1/Syh1. Using a two-hybrid assay, we confirmed the capacity of Myr1/Syh1 to self-associate in vivo. We measured in vitro the affinity of recombinant Myr1/Syh1 domains fused to GFP for liposomes reconstituted from synthetic and natural yeast lipids by sedimentation techniques. The herewith established affinities of Myr1/Syh1 to specific lipids, combined with evidence for its interactions with membrane traffic and protein synthesis, provide support for a possible function of Myr1/Syh1 as a regulator sensing membrane composition along the vesicular pathways.

  • 274.
    Gestin, Maxime
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Uptake signalling of PepFect 142019Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Cell-penetrating peptides are able to bind and carry various therapeutic agents including oligonucleotides into cells for a therapeutic effect. The aim of the cell-penetrating peptide research field is to produce a simple, safe and potent delivery platform for intracellular therapy and more especially for gene therapy. 

    More than twenty five years after their discovery, numerous sequences of cell penetrating peptides have been designed based on natural substances, chimeric strategy or entirely synthetic products. The precise interactions leading to the uptake of cell-penetrating peptides is as of today still not entirely clear. Global mechanisms of direct penetration and endocytosis are proposed, but little is known about actual molecular interactions building the signalling pathway of cell-penetrating peptides.

    In this thesis, with the help of the cell-penetrating peptide PepFect 14, we study the signalling of the uptake of cell-penetrating peptides either by transcriptome analysis or ligand interfering. We demonstrate the involvement of autophagy in the uptake of both PepFect 14 and the complex formed by PepFect 14 and oligonucleotides. We also present the use of a high throughput assay aimed at identifying new signalling pathways affected by the delivery of oligonucleotides using PepFect 14.

  • 275. Ghalebani, Leila
    et al.
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    pH dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid β peptideManuskript (preprint) (Övrigt vetenskapligt)
  • 276.
    Ghalebani, Leila
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-beta peptide2012Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 421, nr 3, s. 554-560Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-beta (A beta) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with N-15- and C-13,N-15-labeled A beta(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to AD may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the AD peptide under these conditions.

  • 277.
    Giardina, Federica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen. Los Alamos National Laboratory, United States of America .
    Romero-Severson, Ethan Obie
    Albert, Jan
    Britton, Tom
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen.
    Leitner, Thomas
    Inference of Transmission Network Structure from HIV Phylogenetic Trees2017Ingår i: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 13, nr 1, artikel-id e1005316Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Phylogenetic inference is an attractive means to reconstruct transmission histories and epidemics. However, there is not a perfect correspondence between transmission history and virus phylogeny. Both node height and topological differences may occur, depending on the interaction between within-host evolutionary dynamics and between-host transmission patterns. To investigate these interactions, we added a within-host evolutionary model in epidemiological simulations and examined if the resulting phylogeny could recover different types of contact networks. To further improve realism, we also introduced patient-specific differences in infectivity across disease stages, and on the epidemic level we considered incomplete sampling and the age of the epidemic. Second, we implemented an inference method based on approximate Bayesian computation (ABC) to discriminate among three well-studied network models and jointly estimate both network parameters and key epidemiological quantities such as the infection rate. Our ABC framework used both topological and distance- based tree statistics for comparison between simulated and observed trees. Overall, our simulations showed that a virus time-scaled phylogeny (genealogy) may be substantially different from the between-host transmission tree. This has important implications for the interpretation of what a phylogeny reveals about the underlying epidemic contact network. In particular, we found that while the within-host evolutionary process obscures the transmission tree, the diversification process and infectivity dynamics also add discriminatory power to differentiate between different types of contact networks. We also found that the possibility to differentiate contact networks depends on how far an epidemic has progressed, where distance-based tree statistics have more power early in an epidemic. Finally, we applied our ABC inference on two different outbreaks from the Swedish HIV-1 epidemic.

  • 278.
    Glaser, Elzbieta
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Alikhani, Nyosha
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The organellar peptidasome, PreP: a journey from Arabidopsis to Alzheimer's disease2010Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, nr 6-7, s. 1076-1080Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1 A demonstrated a unique totally enclosed large cavity of 10000 A3 that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-beta peptide (Abeta) associated with Alzheimer's disease (AD). Accumulation of Abeta has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Abeta. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Abeta-degrading activity in the human brain mitochondria in relation to AD.

  • 279.
    Glaser, Elzbieta
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kmiec, Beata
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Teixeira, Pedro Filipe
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mitochondrial and Chloroplastic Targeting Peptides Peptidase, PreP2013Ingår i: Handbook of proteolytic enzymes, vols 1 and 2, 3rd edition / [ed] Rawlings, ND; Salvesen, GS, AMSTERDAM: ELSEVIER SCIENCE BV , 2013, 3rd, s. 1426-1430Kapitel i bok, del av antologi (Refereegranskat)
  • 280. Goel, Suchi
    et al.
    Palmkvist, Mia
    Moll, Kirsten
    Joannin, Nicolas
    Lara, Patricia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Akhouri, Reetesh R.
    Moradi, Nasim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öjemalm, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Westman, Mattias
    Angeletti, Davide
    Kjellin, Hanna
    Lehtio, Janne
    Blixt, Ola
    Ideström, Lars
    Gahmberg, Carl G.
    Storry, Jill R.
    Hult, Annika K.
    Olsson, Martin L.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wahlgren, Mats
    RIFINs are adhesins implicated in severe Plasmodium falciparum malaria2015Ingår i: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 21, nr 4, s. 314-317Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), bind to RBCs-preferentially of blood group A-to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

  • 281.
    Gonska, Nathalie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic.

    Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction.

    The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved.

  • 282.
    Gonska, Nathalie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Young, David R.
    Yuki, Riki
    Okamoto, Takuya
    Antonyuk, Svetlana
    Hasnain, S. Samar
    Muramoto, Kazumasa
    Shiro, Yoshitsugu
    Tosha, Takehiko
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzymeManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Bacterial nitric oxide reductases (NORs) catalyse the reduction of two NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the host. Although NORs are members of the heme-copper oxidase superfamily, and thus relatives of proton-pumping O2-reducing enzymes, the best studied NORs, cNORs (cytochrome c dependent), were found to be non-electrogenic.

    Here, we focus on another type of NOR, qNOR (quinol-dependent). qNOR from Neisseria meningitidis, a human pathogen, was expressed in Escherichia coli and purified as a stable and highly active NO reductase. Spectroscopic and metal analysis of the purified qNOR showed properties largely similar to those in cNORs. Furthermore, the liposome-reconstituted qNOR showed respiratory control ratios consistently above 2, indicative of an electrogenic reaction. We also exchanged residues in a putative proton pathway leading from the cytoplasm to the active site, but there were no significant effects on either turnover rates or electrogenicity. However, the exchange of a glutamate close to the active site (E-498) yielded drastic effects on turnover. We thus suggest that the N. meningitidis qNOR uses cytoplasmic protons, but that the pathway is rather wide and redundant, narrowing around the glutamate-498.

  • 283.
    Gorokhova, Elena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM). Stockholms universitet, Naturvetenskapliga fakulteten, Systemekologiska institutionen.
    Mattsson, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Systemekologiska institutionen.
    Sundström, Annica M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Systemekologiska institutionen.
    A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy2012Ingår i: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 89, nr 3, s. 216-221Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by similar to 25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80 degrees C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies.

  • 284. Grandoch, Maria
    et al.
    Floegel, Ulrich
    Virtue, Sam
    Maier, Julia K.
    Jelenik, Tomas
    Kohlmorgen, Christina
    Feldmann, Kathrin
    Ostendorf, Yanina
    Castaneda, Tamara R.
    Zhou, Zhou
    Yamaguchi, Yu
    Nascimento, Emmani B. M.
    Sunkari, Vivekananda G.
    Goy, Christine
    Kinzig, Martina
    Soergel, Fritz
    Bollyky, Paul L.
    Schrauwen, Patrick
    Al-Hasani, Hadi
    Roden, Michael
    Keipert, Susanne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. German Center for Diabetes Research (DZD), Germany; Helmholtz Zentrum München, Germany.
    Vidal-Puig, Antonio
    Jastroch, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. German Center for Diabetes Research (DZD), Germany; Helmholtz Zentrum München, Germany.
    Haendeler, Judith
    Fischer, Jens W.
    4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue2019Ingår i: Nature Metabolism, E-ISSN 2522-5812, Vol. 1, nr 5, s. 546-559Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Therapeutic increase in brown adipose tissue (BAT) thermogenesis is of great interest, as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, that is synthesized by HA synthases (HAS1, HAS2, and HAS3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here we show that inhibition of HA synthesis by 4-MU or genetic deletion of Has2 and Has3 improves the thermogenic capacity of BAT, reduces body-weight gain, and improves glucose homeostasis independently of adrenergic stimulation in mice on a diabetogenic diet. In this context, we validated a novel magnetic resonce T2 mapping approach for in vivo visualization of BAT activation. Inhibition of HA synthesis increases glycolysis, BAT respiration, and uncoupling protein 1 (UCP1) expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved, prescription-free drug, could be repurposed to treat obesity and diabetes.

  • 285.
    Granholm, Viktor
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kall, Lukas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Quality assessments of peptide-spectrum matches in shotgun proteomics2011Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, nr 6, s. 1086-1093Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The peptide identification process in shotgun proteomics is most frequently solved with search engines. Such search engines assign scores that reflect similarity between the measured fragmentation spectrum and the theoretical spectra of the peptides of a given database. However, the scores from most search engines do not have a direct statistical interpretation. To understand and make use of the significance of peptide identifications, one must thus be familiar with some statistical concepts. Here, we discuss different statistical scores used to show the confidence of an identification and a set of methods to estimate these scores. We also describe the variance of statistical scores and imperfections of scoring functions of peptide-spectrum matches.

  • 286.
    Griese, Julia J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Uppsala University, Sweden.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD2019Ingår i: Acta Crystallographica Section D Structural Biology, ISSN 2059-7983, Vol. D75, s. 764-771Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

  • 287.
    Griese, Julia J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    X ray reduction correlates with soaking accessibility as judged from four non crystallographically related diiron sites2012Ingår i: METALLOMICS, ISSN 1756-5901, Vol. 4, nr 9, s. 894-898Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.

  • 288.
    Griese, Julia J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kositzki, Ramona
    Schrapers, Peer
    Branca, Rui M. M.
    Nordström, Anders
    Lehtiö, Janne
    Haumann, Michael
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor2015Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 42, s. 25254-25272Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.

  • 289. Groves-Chapman, Jessica L.
    et al.
    Runesson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holmes, Philip V.
    Galanin receptor agonists protect against kainic acid-induced excitotoxicity in the rat hippocampus2011Konferensbidrag (Refereegranskat)
    Abstract [en]

    Galanin is a peptide neurotransmitter with neuroprotective actions. Administration of galanin or selective galanin receptor agonists to rats reduces convulsant-induced seizure behavior. However, it remains unclear whether galanin exerts a neuroprotective effect in vivo and which galanin receptor subtype may mediate this effect. The present experiments evaluated the impact of two separate galanin receptor agonists, M1145 and M1154, which are specific for GAL R2 and GAL R1/R2 respectively, on seizure behavior and neuronal excitotoxicity following intracerebroventricular (ICV) administration of kainic acid. Male, Sprague-Dawley rats received ICV infusions of M1145, M1154, or saline prior to an ICV infusion of kainic acid. Seizure behaviors were rated for 30 minutes post-infusion, and rats were euthanized and transcardially perfused 48 hours later. Twenty micron coronal sections of the hippocampal region were obtained and nissl-stained for microscopic cell counting of pyramidal neurons in the CA3 region.Neither galanin receptor agonist reduced the seizure behaviors induced by kainic acid. Cell counting in CA3 revealed that kainic acid treatment alone caused near complete destruction of cells. However, rats pretreated with the galanin receptor agonists showed reduced cell loss. These results provide further evidence of the protective role of galanin in the hippocampus and suggest that the GAL R2 receptor mediates these effects.

  • 290.
    Gruschke, Steffi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Early steps in the biogenesis of the bc1 complex in yeast mitochondria: The role of the Cbp3-Cbp6 complex in cytochrome b synthesis and assembly2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The inner membrane of mitochondria harbors the complexes of the respiratory chain and the ATP synthase, which perform the key metabolic process oxidative phosphorylation. These complexes are composed of subunits from two different genetic origins: the majority of constituents is synthesized on cytosolic ribosomes and imported into mitochondria, but a handful of proteins, which represent core catalytic subunits, are encoded in the organellar DNA and translated on mitochondrial ribosomes. Using yeast as a model organism, I investigated the mitochondrial ribosomal tunnel exit, the region of the ribosome where the nascent chain emerges and that in cytosolic ribosomes serves as a platform to bind biogenesis factors that help the newly synthesized protein to mature. This study provided insights into the structural composition of this important site of mitochondrial ribosomes and revealed the positioning of Cbp3 at the tunnel exit region, a chaperone required specifically for the assembly of the bc1 complex. In my further work I found that Cbp3 structurally and functionally forms a tight complex with Cbp6 and that this complex exhibits fundamental roles in the biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1 complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully synthesized cytochrome b, thereby stabilizing and guiding it further through bc1 complex assembly. The next steps involve the recruitment of the assembly factor Cbp4 to the Cbp3-Cbp6/cytochrome b complex and presumably acquisition of two redox active heme b cofactors. During further assembly Cbp3-Cbp6 is released from cytochrome b, can again bind to the ribosome and activate further rounds of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and assembly allows the complex to act as a regulatory switch to modulate the level of cytochrome b synthesis in response to the bc1 complex assembly process.

  • 291.
    Gruschke, Steffi
    et al.
    Technische Universität Kaiserslautern, Germany.
    Groene, Kerstin
    Heublein, Manfred
    Technische Universität Kaiserslautern, Germany.
    Hoelz, Stefanie
    Israel, Lars
    Imhof, Axel
    Herrmann, Johannes M.
    Ott, Martin
    Proteins at the Polypeptide Tunnel Exit of the Yeast Mitochondrial Ribosome2010Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 25, s. 19022-19028Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.

  • 292.
    Gruschke, Steffi
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Römpler, Katharina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hildenbeutel, Markus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kehrein, Kirsten
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kuehl, Inge
    Bonnefoy, Nathalie
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria2012Ingår i: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 199, nr 1, s. 137-150Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc1 complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc1 complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc1 complex assembly.

  • 293.
    Grāve, Kristīne
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bennett, Matthew D.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Expression of Mycobacterium tuberculosis membrane proteins using folding reporter GFPManuskript (preprint) (Övrigt vetenskapligt)
  • 294.
    Gubanova, Evgenia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Novel functions of SMG-1 in carcinogenesis2013Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Damage to DNA can cause mutations leading to cancer, and the DNA damage response (DDR), leading to transient cell-cycle arrest, DNA repair, senescence and apoptosis, plays a role in preventing tumor formation. At the same time, agents that damage DNA are used as anticancer therapy. The DDR has been extensively studied since the mid-nineties, when the genes of the major checkpoint kinases involved were cloned. Since then, quite a detailed model of the DDR has been worked out. According to the current model, the proximal stress-responsive kinases that are essential for the whole signaling cascade to function properly are ATM and ATR belonging to the family of phosphatidyl-inositol 3-kinase-related kinases (PIKKs). SMG-1 is the latest addition to this family, and accumulating evidence is pointing to its role in genome surveillance.

    In Paper I, we showed that SMG-1 regulates the G1/S checkpoint in response to ionizing radiation (IR) by two mechanisms. In addition to regulating the p53/p21 pathway by phosphorylating p53 and thus regulating its stability and activity, we have demonstrated a novel role for SMG-1 in regulating cell cycle progression and tumor growth via the p53-independent pathway. We identified Cdc25A as a new SMG-1 substrate and found that SMG-1 suppresses CDK2 activity in response to DNA damage, as well as in unperturbed cells.

    Head and neck squamous cell carcinoma (HNSCC) is divided into human papillomavirus (HPV)-positive and HPV-negative subgroups, of which HPV-positive cancers are sensitive to IR treatment and show a more favorable prognosis compared to HPV-negative HNSCCs.  In Paper II, for the first time, we have shown a link between defects in SMG-1 expression and cancer. We demonstrated that HPV-positive HNSCC cancer cell lines and tumors express SMG-1 at lower levels than HPV-negative HNSCCs due to promoter hypermethylation. We concluded that diminished SMG-1 levels may contribute to the enhanced response to radiation therapy exhibited by HPV-positive HNSCCs.  

    Senescence and epithelial-mesenchymal transition (EMT) are both tightly linked to carcinogenesis. Oncogene-induced senescence (OIS) functions as a barrier against tumor progression, while EMT promotes tumor progression and metastasis. In Paper III, we identified previously unknown roles of SMG-1 in these two cellular processes. SMG-1 deficient cells failed to initiate the OIS program induced by activation of Ras. Downregulation of SMG-1 also induced morphologic and molecular changes consistent with EMT. We propose that, by regulating senescence and suppressing EMT, SMG-1 inhibits cancer progression. 

  • 295.
    Gubanova, Evgenia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Science for Life Laboratory; Division of Translational Medicine and Chemical Biology; Department of Medical Biochemistry and Biophysics; Karolinska Institut; Stockholm, Sweden.
    Issaeva, Natalia
    Djureinovic, Tatjana
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    SMG-1 regulates senescence and suppresses epithelial-mesenchymal transitionManuskript (preprint) (Övrigt vetenskapligt)
  • 296.
    Gubanova, Evgenia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Issaeva, Natalia
    Gokturk, Camilla
    Djureinovic, Tatjana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Helleday, Thomas
    SMG-1 suppresses CDK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways2013Ingår i: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 12, nr 24, s. 3770-3780Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.

  • 297.
    Guell, Mireia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Luis, Josep M.
    Sola, Miquel
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N '-dimethylethylenediamine)(2)(2+) complex2009Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 14, nr 2, s. 229-242Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C-H bonds, Stack and coworkers recently reported a synthetic mu-eta(2):eta(2)-peroxodicopper(II)(DBED)(2) complex ( DBED is N,N'-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-mu-oxo-dicopper(III)-phenolate complex, with the O-O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O-O bond cleavage and C-O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O-O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the mu-eta(2):eta(2)-peroxodicopper(II)(DBED)(2) complex by N,N'-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2: 1 Cu(I)O-2-derived species.

  • 298.
    Gustafsson, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural and functional studies of proteins of medical relevance: Protein-ligand complexes in cancer and novel structural folds in bacteria2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    X-ray crystallography is a tool for determining the structures of proteins and protein-ligand complexes. In this thesis the method has been employed to study several proteins of medical relevance.

    Cancer is a terrible disease, severely impacting those affected, as well as their family and friends. Current cancer treatments involve a combination of cytostatic drugs, surgery and radiation treatment. Unfortunately many cytostatic drugs also kill healthy cells, which gives rise to serious side-effects. The discovery of treatments which selectively inhibit proteins essential for cancer cell survival but which are non-essential in normal cells, could reduce such side-effects.

    MTH1 is a protein that degrades oxidised nucleotides, which when incorporated into DNA cause mutations and subsequent cell death. Cancer cells have higher levels of reactive oxygen species, which create oxidised nucleotides.  In Paper I it was discovered that cancer cells are dependent on MTH1 for their survival. Crystal structures of MTH1 in complex with small molecules guided their development into potent MTH1 inhibitors, capable of killing cancer cells. Cells with increased amounts of oxidised nucleotides, or with induced hypoxia, were more susceptible to MTH1 inhibition, as shown in Paper II. In Paper III several MTH1 orthologues from organisms often used in pre-clinical studies were tested for MTH1 inhibition. Leucine 116 of mouse MTH1 was determined to be important for the lower inhibition of the developed inhibitors towards this enzyme. A virtual fragment screening study using commercial chemicals resulted in several potent MTH1 inhibitors, as shown in Paper IV. The crystal structures with the fragments or optimised inhibitors did in most cases agree with the docking pose determined from the virtual screening. In addition to the known function of MTH1 in the degradation of oxidised nucleotides, Paper V showed that MTH1 also degrades methylated nucleotides.

    MTHFD2 is responsible for providing one-carbon units for nucleotide synthesis in cancer cells. As MTHFD2 is present in cancer cells but not in healthy cells, targeting the enzyme would make it possible to selectively kill cancer cells. Paper VI presents the first structure of MTHFD2, along with the first inhibitor of the protein. This information provides a starting point for the development of potent and selective MTHFD2 inhibitors.

    The botulinum neurotoxin from the bacterium Clostridium Botulinum is the causative agent of the deadly disease botulism. The action of the botulinum neurotoxin on nerve cells results in paralysis, and is life-threatening if the patient is not helped with breathing support. However, low doses of the neurotoxin are used as a successful treatment for several medical conditions, such as involuntary spasms. In Paper VII the structure of two proteins, P47 and OrfX2, encoded in the gene cluster of a botulinum neurotoxin, were determined. The structures resembled tubular lipid-binding proteins, previously only found in eukaryotes. The proteins were also found to be able to bind lipids. This work gives new insight into the structure and function of this group of proteins, which help the deadly botulinum neurotoxins.

  • 299.
    Gustafsson, Robert
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berntsson, Ronnie P-A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Umeå University, Sweden.
    Martínez-Carranza, Markel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    El Tekle, Geniver
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Odegrip, Richard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johnson, Eric A.
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster2017Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, nr 22, s. 3781-3792Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

  • 300. Gustafsson, Tomas N.
    et al.
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Lu, Jun
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Holmgren, Arne
    Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 47Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.

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