Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p

Långsprötad silverfisk i museer, bibliotek och arkiv i Sverige

Det finns flera syften med denna rapport. Ett är att klargöra utbredningen av långsprötad silverfisk i Sverige, i såväl privatbostäder som i museer, arkiv och bibliotek. Den långsprötade silverfiskens biologi redogörs det också för i detalj utifrån befintlig litteratur. Dels för att synliggöra potentiella angreppspunkter för framtida bekämpningsåtgärder, dels för att utforma en biologiskt understödd riskbedömning för verksamheter som arbetar med samlingar och arkiv. Väldigt knapphändig information har tidigare funnits för beskrivning av typiska silverfiskskador i samlingar och arkiv. Dokumentationen i denna rapport bidrar därför till en ökad förståelse kring hur svårupptäckta skadorna kan vara och synliggör potentiella risker med att angrepp lämnas utan åtgärd. Ytterligare frågeställningar som berörs gäller artens fenologi i Sverige, dess fortsatta spridning samt skadepotential

Långsprötad silverfisk i museer, bibliotek och arkiv i Sverige

Det finns flera syften med denna rapport. Ett är att klargöra utbredningen av långsprötad silverfisk i Sverige, i såväl privatbostäder som i museer, arkiv och bibliotek. Den långsprötade silverfiskens biologi redogörs det också för i detalj utifrån befintlig litteratur. Dels för att synliggöra potentiella angreppspunkter för framtida bekämpningsåtgärder, dels för att utforma en biologiskt understödd riskbedömning för verksamheter som arbetar med samlingar och arkiv. Väldigt knapphändig information har tidigare funnits för beskrivning av typiska silverfiskskador i samlingar och arkiv. Dokumentationen i denna rapport bidrar därför till en ökad förståelse kring hur svårupptäckta skadorna kan vara och synliggör potentiella risker med att angrepp lämnas utan åtgärd. Ytterligare frågeställningar som berörs gäller artens fenologi i Sverige, dess fortsatta spridning samt skadepotential

Diffuse binding of Zn2+ to the denatured ensemble of Cu/Zn superoxide dismutase 1

The stability and structural properties of the metalloprotein superoxide dismutase 1 (SOD1) are found to depend critically on metal ions. Native SOD1 monomers coordinate one structural Zn2+ and one redox‐active Cu2+/1+ to the active site. To do this, the Zn2+ ions need to interact with the SOD1 protein on the denatured side of the folding barrier, prior to the formation of the folding nucleus. In this study, we have examined at residue level the nature of this early Zn2+ binding by NMR studies on the urea denatured‐state of SOD1. Nearly complete backbone chemical shift assignments were obtained in 9 M urea at physiological pH, conditions at which NMR studies are scarce. Our results demonstrate that SOD1 is predominantly unstructured under these conditions. Chemical‐shift changes upon Zn2+ titration show that denatured SOD1 retains a significant affinity to Zn2+ ions, even in 9 M urea. However, the Zn2+ interactions are not limited to the native metal‐binding ligands in the two binding sites, but are seen for all His residues. Moreover, the native Cu2+/1+ ligand H46 seems not to bind as well as the other His residues, while the nearby non‐native H43 does bind, indicating that the binding geometry is relaxed. The result suggests that the Zn2+‐binding observed to catalyze folding of SOD1 in physiological buffer is initiated by diffuse, non‐specific coordination to the coil, which subsequently funnels by ligand exchange into the native coordination geometry of the folded monomer. Altogether, this diffuse binding is a result with fundamental implications for folding of metalloproteins in general

Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227–245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130–148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227–245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed

Subtle structures with not-so-subtle functions: A dataset of arthropod constructs and their host plants

The construction of shelters on plants by arthropods might influence other organisms via changes in colonization, community richness, species composition and functionality. Arthropods, including beetles, caterpillars, sawflies, spiders, and wasps often interact with host plants via the construction of shelters, building a variety of structures such as leaf ties, tents, rolls, and bags; leaf and stem galls, and hollowed out stems. Such constructs might have both an adaptive value in terms of protection (i.e., serve as shelters) but may also exert a strong influence on terrestrial community diversity in the engineered and neighboring hosts via colonization by secondary occupants. While different traits of the host plant (e.g., physical, chemical and architectural features) may affect the potential for ecosystem engineering by insects, such effects have been, to a certain degree, overlooked. Further analyses of how plant traits affect the occurrence of shelters may thus enrich our understanding of the organizing principles of plant-based communities. This dataset includes more than a thousand unique records of ecosystem engineering by arthropods, in the form of structures built on plants. All records have been published in the literature, and span both natural structures (90.6% of the records) and structures artificially created byresearchers (9% of the records). The data were gathered between 1932 and 2021, across more than 50 countries and several ecosystems, ranging from polar to tropical zones. Besides data on host plants and engineers, we aggregated data on the type of constructs and the identity of inquilines using these structures. This dataset highlights the importance of these subtle structures for the organization of terrestrial arthropod communities, enabling hypotheses testing in ecological studiesaddressing ecosystem engineering and facilitation mediated by constructs.Fil: Pereira, Cássio Cardoso. Universidade Federal de Minas Gerais; BrasilFil: Novais, Samuel. Instituto de Ecología; MéxicoFil: Barbosa, Milton. Universidade Federal de Minas Gerais; BrasilFil: Negreiros, Daniel. Universidade Federal de Minas Gerais; BrasilFil: Gonçalves Souza, Thiago. Universidade Federal de Pernambuco; BrasilFil: Roslin, Tomas. Swedish University Of Agricultural Sciences; SueciaFil: Marquis, Robert. University of Missouri; Estados UnidosFil: Marino, Nicholas. Universidade Federal do Rio de Janeiro; BrasilFil: Novotny, Vojtech. Biology Centre of the Academy of Sciences of the Czech Republic; República ChecaFil: Orivel, Jerome. Universite de Guyane; GuyanaFil: Sui, Shen. New Guinea Binatang Research Center; GuineaFil: Aires, Gustavo. Universidade Federal de Pernambuco; BrasilFil: Antoniazzi, Reuber. University of Texas at Austin; Estados UnidosFil: Dáttilo, Wesley. Instituto de Ecología; MéxicoFil: Breviglieri, Crasso. Universidade Estadual de Campinas; BrasilFil: Busse, Annika. Bavarian Forest National Park; AlemaniaFil: Gibb, Heloise. La Trobe University. Department Of Ecology, Environment And Evolution; AustraliaFil: Izzo, Thiago. Universidade Federal do Mato Grosso do Sul; BrasilFil: Kadlec, Tomas. Czech University Of Life Sciences Prague; República ChecaFil: Kemp, Victoria. Queen Mary University of London; Reino UnidoFil: Kersch Becker, Monica. University of Alabama at Birmingahm; Estados UnidosFil: Knapp, Michal. Czech University Of Life Sciences Prague; República ChecaFil: Kratina, Pavel. Queen Mary University of London; Reino UnidoFil: Luke, Rebecca. Royal Holloway University of London; Reino UnidoFil: Majnari, Stefan. University Of Zagreb, Faculty Of Science; CroaciaFil: Maritz, Robin. University of the Western Cape; SudáfricaFil: Martins, Paulo Mateus. Universidade Federal de Pernambuco; BrasilFil: Mendesil, Esayas. Jimma University; EtiopíaFil: Michalko, Jaroslav. Slovak Academy of Sciences; EslovaquiaFil: Mrazova, Anna. Biology Centre of the Academy of Sciences of the Czech Republic; República ChecaFil: Peri, Mirela Serti. University Of Zagreb. Faculty Of Science; CroaciaFil: Petermann, Jana. University Of Salzburg. Department Of Biosciences; AustriaFil: Ribeiro, Sérvio. Universidade Federal de Ouro Preto; BrasilFil: Sam, Katerina. University of Missouri; Estados UnidosFil: Trzcinski, M. Kurtis. University of British Columbia; CanadáFil: Vieira, Camila. Universidade Federal de Uberlândia; BrasilFil: Westwood, Natalie. University of British Columbia; CanadáFil: Bernaschini, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Carvajal, Valentina. Universidad de Caldas; ColombiaFil: González, Ezequiel. Czech University of Life Sciences Prague; República ChecaFil: Jausoro, Mariana. Universidad Nacional de Chilecito. Departamento de Ciencias Basicas y Tecnologicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Kaensin, Stanis. New Guinea Binatang Research Center; GuineaFil: Ospina, Fabiola. Universidad de Caldas; ColombiaFil: Pérez, Jacob Cristóbal. Universidad Autónoma del Estado de México; MéxicoFil: Quesada, Mauricio. Universidad Autónoma del Estado de México; MéxicoFil: Rogy, Pierre. University of British Columbia; CanadáFil: Srivastava, Diane S.. University of British Columbia; CanadáFil: Szpryngiel, Scarlett. The Swedish Museum of Natural History; SueciaFil: Tack, Ayco J. M.. Stockholms Universitet; SueciaFil: Teder, Tiit. University of Tartu; EstoniaFil: Videla, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Viljur, Mari Liis. University of Tartu; EstoniaFil: Koricheva, Julia. Royal Holloway University of London; Reino UnidoFil: Fernandes, Geraldo Wilson Afonso. Universidade Federal de Minas Gerais; BrasilFil: Romero, Gustavo Q.. Universidade Estadual de Campinas; BrasilFil: Cornelissen, Tatiana. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi