TY - JOUR A1 - Abbas, Ioana M. A1 - Vranic, Marija A1 - Hoffmann, Holger A1 - El-Khatib, Ahmed H. A1 - Montes-Bayón, María A1 - Möller, Heiko Michael A1 - Weller, Michael G. T1 - Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR⁺ JF - International Journal of Molecular Sciences N2 - Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others. KW - hepcidin-25 KW - copper KW - nickel KW - copper complex KW - ATCUN motif KW - metal complex KW - MS KW - NMR structure KW - metal peptide KW - metalloprotein KW - metallopeptide KW - isomerization KW - racemization KW - purity KW - reference material Y1 - 2018 U6 - https://doi.org/10.3390/ijms19082271 SN - 1422-0067 SN - 1661-6596 VL - 19 IS - 8 PB - Molecular Diversity Preservation International CY - Basel ER - TY - GEN A1 - Abbas, Ioana M. A1 - Vranic, Marija A1 - Hoffmann, Holger A1 - El-Khatib, Ahmed H. A1 - Montes-Bayón, María A1 - Möller, Heiko Michael A1 - Weller, Michael G. T1 - Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR⁺ T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 701 KW - hepcidin-25 KW - copper KW - nickel KW - copper complex KW - ATCUN motif KW - metal complex KW - MS KW - NMR structure KW - metal peptide KW - metalloprotein KW - metallopeptide KW - isomerization KW - racemization KW - purity KW - reference material Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-427926 SN - 1866-8372 IS - 701 ER - TY - THES A1 - Abd Allah Salem, Mohamed T1 - Comparative and systemic metabolomic analysis of the model plant Arabidopsis thaliana after perturbing the essential Target of Rapamycin (TOR) pathway Y1 - 2018 ER - TY - JOUR A1 - Abdirashid, Hashim A1 - Lenhard, Michael T1 - Say it with double flowers JF - Journal of experimental botany N2 - Every year, lovers world-wide rely on mutants to show their feelings on Valentine's Day. This is because many of the most popular ornamental flowering plants have been selected to form extra petals at the expense of reproductive organs to enhance their attractiveness and aesthetic value to humans. This so-called 'double flower' (DF) phenotype, first described more than 2000 years ago (Meyerowitz et al., 1989) is present, for example, in many modern roses, carnations, peonies, and camellias. Gattolin et al. (2020) now identify a unifying explanation for the molecular basis of many of these DF cultivars. KW - ABCE model KW - APETALA2 KW - double flowers KW - flower development KW - homoeotic KW - mutants KW - microRNA172 Y1 - 2020 U6 - https://doi.org/10.1093/jxb/eraa109 SN - 0022-0957 SN - 1460-2431 VL - 71 IS - 9 SP - 2469 EP - 2471 PB - Oxford Univ. Press CY - Oxford ER - TY - THES A1 - Adamla, Frauke T1 - Polyglutamine- and aging-dependent aberrancies in transcription and translation Y1 - 2015 ER - TY - GEN A1 - Adhikari, Rishi Ram A1 - Glombitza, Clemens A1 - Nickel, Julia C. A1 - Anderson, Chloe H. A1 - Dunlea, Ann G. A1 - Spivack, Arthur J. A1 - Murray, Richard W. A1 - D’Hondt, Steven A1 - Kallmeyer, Jens T1 - Hydrogen utilization potential in subsurface sediments T2 - Frontiers in microbiology N2 - Subsurface microbial communities undertake many terminal electron-accepting processes, often simultaneously. Using a tritium-based assay, we measured the potential hydrogen oxidation catalyzed by hydrogenase enzymes in several subsurface sedimentary environments (Lake Van, Barents Sea, Equatorial Pacific, and Gulf of Mexico) with different predominant electron-acceptors. Hydrogenases constitute a diverse family of enzymes expressed by microorganisms that utilize molecular hydrogen as a metabolic substrate, product, or intermediate. The assay reveals the potential for utilizing molecular hydrogen and allows qualitative detection of microbial activity irrespective of the predominant electron-accepting process. Because the method only requires samples frozen immediately after recovery, the assay can be used for identifying microbial activity in subsurface ecosystems without the need to preserve live material. We measured potential hydrogen oxidation rates in all samples from multiple depths at several sites that collectively span a wide range of environmental conditions and biogeochemical zones. Potential activity normalized to total cell abundance ranges over five orders of magnitude and varies, dependent upon the predominant terminal electron acceptor. Lowest per-cell potential rates characterize the zone of nitrate reduction and highest per-cell potential rates occur in the methanogenic zone. Possible reasons for this relationship to predominant electron acceptor include (i) increasing importance of fermentation in successively deeper biogeochemical zones and (ii) adaptation of H(2)ases to successively higher concentrations of H-2 in successively deeper zones. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 447 KW - hydrogenase KW - tritium assay KW - deep biosphere KW - microbial activity KW - Lake Van KW - Barents Sea KW - Equatorial Pacific KW - Gulf of Mexico Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-407678 ER - TY - JOUR A1 - Aga-Barfknecht, Heja A1 - Soultoukis, George A. A1 - Stadion, Mandy A1 - Garcia-Carrizo, Francisco A1 - Jähnert, Markus A1 - Gottmann, Pascal A1 - Vogel, Heike A1 - Schulz, Tim Julius A1 - Schürmann, Annette T1 - Distinct adipogenic and fibrogenic differentiation capacities of mesenchymal stromal cells from pancreas and white adipose tissue JF - International journal of molecular sciences N2 - Pancreatic steatosis associates with beta-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals. KW - MSCs KW - fatty pancreas KW - WAT KW - lineage commitment KW - transcriptomics KW - miRNAs Y1 - 2022 U6 - https://doi.org/10.3390/ijms23042108 SN - 1422-0067 VL - 23 IS - 4 PB - Molecular Diversity Preservation International CY - Basel ER - TY - THES A1 - Agarwal, Pallavi T1 - Functional characterization of ROS-responsive genes, ANAC085 and ATR7, in Arabidopsis thaliana Y1 - 2023 ER - TY - JOUR A1 - Agne, Stefanie A1 - Preick, Michaela A1 - Straube, Nicolas A1 - Hofreiter, Michael T1 - Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set JF - Frontiers in Ecology and Evolution N2 - A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material. KW - target capture KW - type specimens KW - molecular species identification KW - museum specimens KW - cross-species capture Y1 - 2022 U6 - https://doi.org/10.3389/fevo.2022.909846 SN - 2296-701X VL - 10 PB - Frontiers Media S.A. CY - Lausanne, Schweiz ER - TY - GEN A1 - Agne, Stefanie A1 - Preick, Michaela A1 - Straube, Nicolas A1 - Hofreiter, Michael T1 - Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1293 KW - target capture KW - type specimens KW - molecular species identification KW - museum specimens KW - cross-species capture Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-574600 SN - 1866-8372 IS - 1293 ER - TY - THES A1 - Agrawal, Shreya T1 - Engineering the isoprenoid pathway for molecular farming and effect of tRNA(Glu) manipulation on tetrapyrrole biosynthesis Y1 - 2018 ER - TY - THES A1 - Ahmad Abadi, Mohammad T1 - Development and application of novel genetic transformation technologies in maize (Zea mays L.) T1 - Entwicklung und Anwendung neuer genetischer Transformationstechnologien im Mais (Zea Mays L.) N2 - Plant genetic engineering approaches are of pivotal importance to both basic and applied research. However, rapid commercialization of genetically engineered crops, especially maize, raises several ecological and environmental concerns largely related to transgene flow via pollination. In most crops, the plastid genome is inherited uniparentally in a maternal manner. Consequently, a trait introduced into the plastid genome would not be transferred to the sexually compatible relatives of the crops via pollination. Thus, beside its several other advantages, plastid transformation provides transgene containment, and therefore, is an environmentally friendly approach for genetic engineering of crop plants. Reliable in vitro regeneration systems allowing repeated rounds of regeneration are of utmost importance to development of plastid transformation technologies in higher plants. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. In one part of this work, a novel tissue culture and plant regeneration system was developed that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. Also, protocols were established for (i) the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants. Furthermore, several selection methods were tested for developing a plastid transformation system in maize. However, stable plastid transformed maize plants could not be yet recovered. Possible explanations as well as suggestions for future attempts towards developing plastid transformation in maize are discussed. Nevertheless, these results represent a first essential step towards developing chloroplast transformation technology for maize, a method that requires multiple rounds of plant regeneration and selection to obtain genetically stable transgenic plants. In order to apply the newly developed transformation system towards metabolic engineering of carotenoid biosynthesis, the daffodil phytoene synthase (PSY) gene was integrated into the maize genome. The results illustrate that expression of a recombinant PSY significantly increases carotenoid levels in leaves. The beta-carotene (pro-vitamin A) amounts in leaves of transgenic plants were increased by ~21% in comparison to the wild-type. These results represent evidence for maize to have significant potential to accumulate higher amounts of carotenoids, especially beta-carotene, through transgenic expression of phytoene synthases. Finally, progresses were made towards developing transformation technologies in Peperomia (Piperaceae) by establishing an efficient leaf-based regeneration system. Also, factors determining plastid size and number in Peperomia, whose species display great interspecific variation in chloroplast size and number per cell, were investigated. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. Investigating plastid morphology in Peperomia species with giant chloroplasts, plasmatic connections between chloroplasts (stromules) were observed under the light microscope and in the absence of tissue fixation or GFP overexpression demonstrating the relevance of these structures in vivo. Furthermore, bacteria-like microorganisms were discovered within Peperomia cells, suggesting that this genus provides an interesting model not only for studying plastid biology but also for investigating plant-microbe interactions. N2 - Pflanzliche Gentechnik spielt sowohl in der Grundlagenforschung als auch der Biotechnologie eine große Rolle. Allerdings bringt die landwirtschaftliche Nutzung gentechnisch veränderter Pflanzen (GM) ökologische Umweltrisiken mit sich, wie z.B. die Kreuzung GM Pflanzen mit sexuell kompatiblen Verwandten durch Fremdbestäubung. Gegenüber den Kerntransformanden haben Plastidtransformanden für die biotechnologische Nutzung große Vorteile, unter anderem da die Vererbung des Plastidgenoms bei höheren Angiospermen ausschließlich maternal geschieht. Somit kann ein Gentransfer transplastomischer Pflanzen über Pollen ausgeschlossen werden. Zuverlässige in-vitro-Regenerationssysteme, die wiederholte Regenerationsrunden erlauben, sind von großem Wert für die Etablierung der Plastidentransformationstechnologie. Trotz Sein die Hauptgetreidenahrungsmittel der Welt, Zerealie Pflanzen gehören zu schwierigsten in der Gewebekultur zu handeln, die Annäherungen der genetischen Technik streng begrenzt. Im Mais werden hauptsächlich junge zygotische Embryonen für die Herstellung der Regenerations-kompetenten Kalluskulturen benutzt. Der Arbeitsaufwand dafür ist hoch und die Prozedur schwierig und von den Gewächshausbedingungen abhängig. Im Rahmen dieser Arbeit wurden neue Gewebekultursysteme für Mais etabliert, welches junge Blattgewebe nutzt und somit unabhängig von Embryonen und Gewächshaus ist. Weiterhin wurden die aus Blättern gebildeten Kalluskulturen für die Generierung der genetisch veränderten Maispflanzen benutzt. Ebenso wurden verschiedene Selektionsmethoden für die Entwicklung eines Plastidentransformationssystems in Mais getestet. Jedoch konnten keine transplastomischen Maispflanzen erhalten werden. Sowohl die möglichen Ursachen als auch Vorschläge für weiterführende Versuche diesbezüglich werden im Rahmen dieser Arbeit diskutiert. Dennoch stellt diese Arbeit den ersten wesentlichen Schritt für die Entwicklung eines Plastidentransformationssystems in Mais vor. In einem zweiten Teil dieses Projekts wird die erfolgreiche Integration der Narzissen Phytoene Synthase in das Maisgenom durch das neu entwickelte nukleäre Transformationssystem gezeigt. Dadurch konnte eine signifikante Steigerung um 17% des Gesamtcarotinoid- und 21% des Beta-Carotengehalts in Maisblättern beobachtet werden. Schließlich wurden Fortschritte für die Entwicklung eines Transformationssystems für Peperomia (Piperaceae) durch die Etablierung eines Regenerationssystems aus Blättern gemacht. Außerdem wurden Faktoren, die die Plastidengröße und –zahl bestimmen, untersucht. Diese Ergebnisse geben Hinweise darauf, dass die Organellengröße und –zahl eher gewebespezifisch als in Abhängigkeit vom Plastidentyp reguliert wird. KW - Mais KW - genetische Manipulation KW - Regeneratin KW - Plastid KW - Maize KW - Genetic transformation KW - Regeneration KW - Plastid Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-14572 ER - TY - THES A1 - Al Fadel, Frdoos T1 - Influence of sphingosine 1-phosphate and its receptor modulators on the development of liver fibrosis Y1 - 2018 ER - TY - THES A1 - Albers, Philip T1 - Funktionelle Charakterisierung des bakteriellen Typ-III Effektorproteins HopZ1a in Nicotiana benthamiana T1 - Functional characterization of the bacterial type-III effector protein HopZ1a in Nicotiana benthamiana N2 - Um das Immunsystem der Pflanze zu manipulieren translozieren gram-negative pathogene Bakterien Typ-III Effektorproteine (T3E) über ein Typ-III Sekretionssystem (T3SS) in die pflanzliche Wirtszelle. Dort lokalisieren T3Es in verschiedenen subzellulären Kompartimenten, wo sie Zielproteine modifizieren und so die Infektion begünstigen. HopZ1a, ein T3E des Pflanzenpathogens Pseudomonas syringae pv. syringae, ist eine Acetyltransferase und lokalisiert über ein Myristolierungsmotiv an der Plasmamembran der Wirtszelle. Obwohl gezeigt wurde, dass HopZ1a die frühe Signalweiterleitung an der Plasmamembran stört, wurde bisher kein mit der Plasmamembran assoziiertes Zielprotein für diesen T3E identifiziert. Um bisher unbekannte HopZ1a-Zieleproteine zu identifizieren wurde im Vorfeld dieser Arbeit eine Hefe-Zwei-Hybrid-Durchmusterung mit einer cDNA-Bibliothek aus Tabak durchgeführt, wobei ein nicht näher charakterisiertes Remorin als Interaktor gefunden wurde. Bei dem Remorin handelt es sich um einen Vertreter der Gruppe 4 der Remorin-Familie, weshalb es in NbREM4 umbenannt wurde. Durch den Einsatz verschiedener Interaktionsstudien konnte demonstriert werden, dass HopZ1a mit NbREM4 in Hefe, in vitro und in planta wechselwirkt. Es wurde ferner deutlich, dass HopZ1a auf spezifische Weise mit dem konservierten C-Terminus von NbREM4 interagiert, das Remorin jedoch in vitro nicht acetyliert. Analysen mittels BiFC haben zudem ergeben, dass NbREM4 in Homodimeren an der Plasmamembran lokalisiert, wo auch die Interaktion mit HopZ1a stattfindet. Eine funktionelle Charakterisierung von NbREM4 ergab, dass das Remorin eine spezifische Rolle im Immunsystem der Pflanze einnimmt. Die transiente Expression in N. benthamiana induziert die Expression von Abwehrgenen sowie einen veränderten Blattphänotyp. In A. thaliana wird HopZ1a über das Decoy ZED1 und das R-Protein ZAR1 erkannt, was zur Auslösung einer starken Hypersensitiven Antwort (HR von hypersensitive response) führt. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass ZAR1 in N. benthamiana konserviert ist, NbREM4 jedoch nicht in der ETI als Decoy fungiert. Mit Hilfe einer Hefe-Zwei-Hybrid-Durchmusterung mit NbZAR1 als Köder konnten zwei Proteine, die Catalase CAT1 und der Protonenpumpeninteraktor PPI1, als Interaktoren von NbZAR1 identifiziert werden, welche möglicherweise in der Regulation der HR eine Rolle spielen. Aus Voruntersuchungen war bekannt, dass NbREM4 mit weiteren, nicht näher charakterisierten Proteinen aus Tabak interagieren könnte. Eine phylogenetische Einordnung hat gezeigt, dass es sich um die bekannte Immun-Kinase PBS1 sowie zwei E3-Ubiquitin-Ligasen, NbSINA1 und NbSINAL3, handelt. PBS1 interagiert mit NbREM4 an der Plasmamembran und phosphoryliert das Remorin innerhalb des intrinsisch ungeordneten N-Terminus. Mittels Massenspektrometrie konnten die Serine an Position 64 und 65 innerhalb der Aminosäuresequenz von NbREM4 als PBS1-abhängige Phosphorylierungsstellen identifiziert wurden. NbSINA1 und NbSINAL3 besitzen in vitro Ubiquitinierungsaktivität, bilden Homo- und Heterodimere und interagieren ebenfalls mit dem N-terminalen Teil von NbREM4, wobei sie das Remorin in vitro nicht ubiquitinieren. Aus den in dieser Arbeit gewonnenen Ergebnissen lässt sich ableiten, dass der bakterielle T3E HopZ1a gezielt mit dem Tabak-Remorin NbREM4 an der Plasmamembran interagiert und über einen noch unbekannten Mechanismus mit dem Immunsystem der Pflanze interferiert, wobei NbREM4 möglicherweise eine Rolle als Adapter- oder Ankerprotein zukommt, über welches HopZ1a mit weiteren Immunkomponenten interagiert. NbREM4 ist Teil eines größeren Immunnetzwerkes, zu welchem die bekannte Immun-Kinase PBS1 und zwei E3-Ubiquitin-Ligasen gehören. Mit NbREM4 konnte damit erstmalig ein membranständiges Protein mit einer Funktion im Immunsystem der Pflanze als Zielprotein von HopZ1a identifiziert werden. N2 - In order to manipulate the plant's immune system, gram-negative pathogenic bacteria inject type-III effector proteins (T3E) via a type III secretion system (T3SS) into the plant host cell. Inside the cell, T3Es localize to different subcellular compartments, where they modify target proteins and thereby promote the infection. HopZ1a, a T3E of the plant pathogen Pseudomonas syringae pv. syringae is an acetyltransferase and localizes to the plasma membrane. Although it has been shown that HopZ1a interferes with early signal transduction at the plasma membrane, no dedicated plasma membrane-associated target protein has been identified so far. To identify unknown HopZ1a target proteins, a yeast two-hybrid screening using a cDNA library from tobacco was performed in advance of this work. The screen identified a previously uncharacterized remorin-family protein as a putative interactor of HopZ1a. Using phylogenetic analyses, the remorin could be classified as a group 4 remorin family member and therefore was renamed NbREM4. By using different interaction studies, it has could be demonstrated that HopZ1a interacts with NbREM4 in yeast, in vitro, and in planta. It also became evident that HopZ1a specifically interacts with the conserved C-terminus of NbREM4 but does not acetylate it. BiFC analyses showed that NbREM4 localizes in homodimers at the plasma membrane, and NbREM4 interacts with HopZ1a in this subcellular compartment. From preliminary studies it was known that NbREM4 may interact with other uncharacterized proteins from tobacco. A phylogenetic analysis revealed the immune kinase NbPBS1 and two E3 ubiquitin ligases, NbSINA1 and NbSINAL3, as putative NbREM4 interacting proteins. Analysis showed that NbPBS1 interacts with NbREM4 at the plasma membrane and phosphorylates the Remorin within the intrinsically disordered N-terminus. By means of mass spectrometry, serines at position 64 and 65 within the amino acid sequence of NbREM4 were identified as PBS1-dependent phosphorylation sites. NbSINA1 and NbSINAL3 have in vitro ubiquitination activity and also interact with the N-terminal part of NbREM4, but do not ubiquitinate it. It has already been shown that, in Arabidopsis thaliana, HopZ1a is recognized by the R protein ZAR1. In the presence of the effector, ZAR1 induces a strong hypersensitive response (HR) of the cell. In this study it could be confirmed that ZAR1 is conserved in Nicotiana benthamiana and is also responsible for the recognition of HopZ1a. In addition, a yeast two-hybrid screen revealed the catalase CAT1 and the proton pump interactor PPI1 as putative NbZAR1-interacting proteins, possibly contributing to the downstream activation of HR. From the results obtained in this work, it can be deduced that the bacterial T3E HopZ1a specifically interacts with the Remorin NbREM4 at the plasma membrane and interferes with the immune system via a yet unknown mechanism. NbREM4 is part of a larger immune network that includes NbPBS1 and two E3 ligases. With NbREM4, the first membrane-associated target protein of HopZ1a could have been identified. KW - Pseudomonas syringae KW - Remorin KW - HopZ1a KW - PBS1 KW - pflanzliches Immunsystem KW - Pseudomonas syringae KW - Remorin KW - HopZ1a KW - PBS1 KW - plant immune system Y1 - 2018 ER - TY - GEN A1 - Albers, Philip A1 - Uestuen, Suayib A1 - Witzel, Katja A1 - Bornke, Frederik T1 - Identification of a novel target of the bacterial effector HopZ1a T2 - Phytopathology N2 - The plant pathogen Pseudomonas syringae is a gram-negative bacterium which infects a wide range of plant species including important crops plants. To suppress plant immunity and cause disease P.syringae injects type-III effector proteins (T3Es) into the plant cell cytosol. In this study, we identified a novel target of the well characterized bacterial T3E HopZ1a. HopZ1a is an acetyltransferase that was shown to disrupt vesicle transport during innate immunity by acetylating tubulin. Using a yeast-two-hybrid screen approach, we identified a REMORIN (REM) protein from tobacco as a novel HopZ1a target. HopZ1a interacts with REM at the plasma membrane (PM) as shown by split-YFP experiments. Interestingly, we found that PBS1, a well-known kinase involved in plant immunity also interacts with REM in pull-down assays, and at the PM as shown by BiFC. Furthermore, we confirmed that REM is phosphorylated by PBS1 in vitro. Overexpression of REM provokes the upregulation of defense genes and leads to disease-like phenotypes pointing to a role of REM in plant immune signaling. Further protein-protein interaction studies reveal novel REM binding partners with a possible role in plant immune signaling. Thus, REM might act as an assembly hub for an immune signaling complex targeted by HopZ1a. Taken together, this is the first report describing that a REM protein is targeted by a bacterial effector. How HopZ1a might mechanistically manipulate the plant immune system through interfering with REM function will be discussed. Y1 - 2018 SN - 0031-949X SN - 1943-7684 VL - 108 IS - 10 PB - American Phytopathological Society CY - Saint Paul ER - TY - JOUR A1 - Albers, Philip A1 - Üstün, Suayib A1 - Witzel, Katja A1 - Kraner, Max Erdmund A1 - Börnke, Frederik T1 - A Remorin from Nicotiana benthamiana Interacts with the Pseudomonas Type-III Effector Protein HopZ1a and is Phosphorylated by the Immune-Related Kinase PBS1 JF - Molecular Plant-Microbe Interactions N2 - The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed. KW - bacterial pathogenesis KW - defense signaling pathways KW - effectors KW - elicitors KW - HopZ1a KW - MAMPs KW - PAMPs KW - PBS1 KW - Pseudomonas syringae KW - remorin KW - type-3 secretion Y1 - 2019 U6 - https://doi.org/10.1094/MPMI-04-19-0105-R SN - 0894-0282 SN - 1943-7706 VL - 32 IS - 9 SP - 1229 EP - 1242 PB - Amer phytopathological SOC CY - ST Paul ER - TY - JOUR A1 - Alberti, Federica A1 - Gonzalez, Javier A1 - Paijmans, Johanna L. A. A1 - Basler, Nikolas A1 - Preick, Michaela A1 - Henneberger, Kirstin A1 - Trinks, Alexandra A1 - Rabeder, Gernot A1 - Conard, Nicholas J. A1 - Muenzel, Susanne C. A1 - Joger, Ulrich A1 - Fritsch, Guido A1 - Hildebrandt, Thomas A1 - Hofreiter, Michael A1 - Barlow, Axel T1 - Optimized DNA sampling of ancient bones using Computed Tomography scans JF - Molecular ecology resources N2 - The prevalence of contaminant microbial DNA in ancient bone samples represents the principal limiting factor for palaeogenomic studies, as it may comprise more than 99% of DNA molecules obtained. Efforts to exclude or reduce this contaminant fraction have been numerous but also variable in their success. Here, we present a simple but highly effective method to increase the relative proportion of endogenous molecules obtained from ancient bones. Using computed tomography (CT) scanning, we identify the densest region of a bone as optimal for sampling. This approach accurately identifies the densest internal regions of petrous bones, which are known to be a source of high-purity ancient DNA. For ancient long bones, CT scans reveal a high-density outermost layer, which has been routinely removed and discarded prior to DNA extraction. For almost all long bones investigated, we find that targeted sampling of this outermost layer provides an increase in endogenous DNA content over that obtained from softer, trabecular bone. This targeted sampling can produce as much as 50-fold increase in the proportion of endogenous DNA, providing a directly proportional reduction in sequencing costs for shotgun sequencing experiments. The observed increases in endogenous DNA proportion are not associated with any reduction in absolute endogenous molecule recovery. Although sampling the outermost layer can result in higher levels of human contamination, some bones were found to have more contamination associated with the internal bone structures. Our method is highly consistent, reproducible and applicable across a wide range of bone types, ages and species. We predict that this discovery will greatly extend the potential to study ancient populations and species in the genomics era. KW - ancient DNA KW - computer tomography KW - palaeogenomics KW - paleogenetics KW - petrous bone Y1 - 2018 U6 - https://doi.org/10.1111/1755-0998.12911 SN - 1755-098X SN - 1755-0998 VL - 18 IS - 6 SP - 1196 EP - 1208 PB - Wiley CY - Hoboken ER - TY - THES A1 - Aleksandrova, Krasimira T1 - Understanding the link between obesity and colorectal cancer BT - the role of biomarkers of iflammation, immunity and metabolic dysfunction Y1 - 2020 ER - TY - THES A1 - Alhajturki, Dema T1 - Characterization of altered inflorescence architecture in Arabidopsis thaliana BG-5 x Kro-0 hybrid T1 - Charakterisierung veränderter Blütenstandarchitektur in Arabidopsis thaliana BG-5 x Kro-0-Hybrid N2 - A reciprocal cross between two A. thaliana accessions, Kro-0 (Krotzenburg, Germany) and BG-5 (Seattle, USA), displays purple rosette leaves and dwarf bushy phenotype in F1 hybrids when grown at 17 °C and a parental-like phenotype when grown at 21 °C. This F1 temperature-dependent-dwarf-bushy phenotype is characterized by reduced growth of the primary stem together with an increased number of branches. The reduced stem growth was the strongest at the first internode. In addition, we found that a temperature switch from 21 °C to 17 °C induced the phenotype only before the formation of the first internode of the stem. Similarly, the F1 dwarf-bushy phenotype could not be reversed when plants were shifted from 17 °C to 21 °C after the first internode was formed. Metabolic analysis showed that the F1 phenotype was associated with a significant upregulation of anthocyanin(s), kaempferol(s), salicylic acid, jasmonic acid and abscisic acid. As it has been previously shown that the dwarf-bushy phenotype is linked to two loci, one on chromosome 2 from Kro-0 and one on chromosome 3 from BG-5, an artificial micro-RNA approach was used to investigate the necessary genes on these intervals. From the results obtained, it was found that two genes, AT2G14120 that encodes for a DYNAMIN RELATED PROTEIN3B and AT2G14100 that encodes a member of the Cytochrome P450 family protein CYP705A13, were necessary for the appearance of the F1 phenotype on chromosome 2. It was also discovered that AT3G61035 that encodes for another cytochrome P450 family protein CYP705A13 and AT3G60840 that encodes for a MICROTUBULE-ASSOCIATED PROTEIN65-4 on chromosome 3 were both necessary for the induction of the F1 phenotype. To prove the causality of these genes, genomic constructs of the Kro-0 candidate genes on chromosome 2 were transferred to BG-5 and genomic constructs of the chromosome 3 candidate genes from BG-5 were transferred to Kro-0. The T1 lines showed that these genes are not sufficient alone to induce the phenotype. In addition to the F1 phenotype, more severe phenotypes were observed in the F2 generations that were grouped into five different phenotypic classes. Whilst seed yield was comparable between F1 hybrids and parental lines, three phenotypic classes in the F2 generation exhibited hybrid breakdown in the form of reproductive failure. This F2 hybrid breakdown was less sensitive to temperature and showed a dose-dependent effect of the loci involved in F1 phenotype. The severest class of hybrid breakdown phenotypes was observed only in the population of backcross with the parent Kro-0, which indicates a stronger contribution of the BG-5 allele when compared to the Kro-0 allele on the hybrid breakdown phenotypes. Overall, the findings of my thesis provide a further understanding of the genetic and metabolic factors underlying altered shoot architecture in hybrid dysfunction. N2 - Die reziproke Kreuzung der zwei A. thaliana-Akzessionen Kro-0 aus Krotzenburg (Deutschland) sowie BG-5 aus Seattle (USA) manifestiert sich in einem Zwergbusch-Phänotyp in den F1 Hybriden bei 17 °C. Dagegen zeigen die Nachkommen bei 21 °C einen Phänotyp, der den Eltern ähnelt. Somit handelt es sich bei dieser Kreuzung um einen temperaturabhängigen Phänotyp. Dieser ist gekennzeichnet durch einen gestörten Wuchs des Primärstammes sowie einer vermehrten Anzahl an gebildeten Seitenzweigen. Das gestörte Wachstum des Hauptsprosses ist am gravierendsten rund um das 1. Internodium der Pflanzen. Es konnte gezeigt werden, dass durch einen Temperaturwechsel von 21 °C auf 17 °C der Phänotyp nur induziert werden kann vor der Bildung des 1. Internodiums. Im Gegenzug dazu ist ebenfalls die Rettung des parentalen Phänotyps nur möglich vor der Bildung des 1. Internodiums am Hauptspross. Des Weiteren zeigten metabolische und hormonelle Analysen der F1 Hybriden eine signifikante Erhöhung von Anthozyanen, Kaempferol, Salizylsäure, Jasmonsäure sowie Abscisinsäure. In Vorarbeiten wurde der Phänotyp bereits mit zwei verschiedenen Loci verknüpft, einer befindet sich auf Chromosom 2 der Elternlinie Kro-0, der andere auf Chromosom 3 von BG-5. Mittels eines micro-RNA Versuches konnte ich zeigen, dass die zwei Gene AT2G14120 DYNAMIN RELATED PROTEIN3B und CYP705A13, welches zur Familie des Zytochrom P450 gehört, auf dem Chromosom 2 von Kro-0 involviert sind. Auf Chromosom 3 von BG-5 gehören die Gene CYP76C8P (AT3G61035) sowie MICROTUBULE-ASSOCIATED65-4 (AT3G60840) zu den verantwortlichen Genen. Es wurden genomische Konstrukte der Kro-0-Kandidatengene auf BG-5 übertragen sowie ebenfalls genomische Konstrukte der Chr3-Kandidatengene auf Kro-0 übertragen. Die T1-Linien bewiesen keines dieser Gene als allein ausreichend. Zusätzlich zu dem F1-Phänotyp wurden in den F2-Generationen, die in fünf verschiedene phänotypische Klassen eingeteilt waren, schwerwiegendere Phänotypen beobachtet. Während die Samenausbeute zwischen F1-Hybriden und Elternlinien vergleichbar war, zeigten drei phänotypische Klassen in der F2-Generationen einen Hybridabbau in Form von Fortpflanzungsversagen. Dieser F2-Hybridabbau war weniger temperaturempfindlich und zeigte einen dosisabhängigen Effekt, basierend auf der genetischen Architektur der am F1-Phänotyp beteiligten Loci. Die schwerste Klasse von hybriden Abbauphänotypen wurde nur in der Population von Rückkreuzungen mit dem Elternteil Kro-0 beobachtet, was einen stärkeren Beitrag des BG-5-Allels im Vergleich zu dem Kro-0-Allel auf den hybriden Abbauphänotypen anzeigt. Insgesamt liefern die Ergebnisse meiner Dissertation ein weiteres Verständnis der genetischen und metabolischen Faktoren, die der veränderten Sprossarchitektur bei hybrider Dysfunktion zugrunde liegen. KW - hybrid incompatibility KW - hybride Inkompatibilität KW - hybrid breakdown KW - Hybridzerfall KW - altered shoot branching KW - veränderte Triebverzweigung Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-420934 ER - TY - JOUR A1 - Alker, Wiebke A1 - Schwerdtle, Tanja A1 - Schomburg, Lutz A1 - Haase, Hajo T1 - A Zinpyr-1-based Fluorimetric Microassay for Free Zinc in Human Serum JF - International journal of molecular sciences N2 - Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual’s zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body’s zinc status, and its suitability as a future biomarker for an individual’s zinc status. KW - zinc KW - free zinc KW - serum KW - biomarker KW - fluorescent probe KW - Zinypr-1 Y1 - 2019 U6 - https://doi.org/10.3390/ijms20164006 SN - 1661-6596 SN - 1422-0067 VL - 20 IS - 16 PB - MDPI CY - Basel ER -