TY  - THES
A1  - Vellmer, Tim
T1  - New insights into the histone variant H2A.Z incorporation pathway in \(Trypanosoma\) \(brucei\)
T1  - Neue Erkenntnisse zum Einbau der Histonvariante H2A.Z in \(Trypanosoma\) \(brucei\)
N2  - The histone variant H2A.Z is a key player in transcription regulation in eukaryotes. Histone acetylations by the NuA4/TIP60 complex are required to enable proper incorporation of the histone variant and to promote the recruitment of other complexes and proteins required for transcription initiation. The second key player in H2A.Z-mediated transcription is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant. By the time this project started little was known about H2A.Z in the unicellular parasite Trypanosoma brucei. Like in other eukaryotes H2A.Z was exclusively found in the transcription start sites of the polycistronic transcription units where it keeps the chromatin in an open conformation to enable RNA-polymerase II-mediated transcription. Previous studies showed the variant colocalizing with an acetylation of lysine on histone H4 and a methylation of lysine 4 on histone H3. Data indicated that HAT2 is linked to H2A.Z since it is required for acetylation of lyinse 10 on histone H4. A SWR1-like complex and a complex homologous to the NuA4/TIP60 could not be identified yet. This study aimed at identifying a SWR1-like remodelling complex in T. brucei and at identifying a protein complex orthologous to NuA4/TIP60 as well as at answering the question whether HAT2 is part of this complex or not. To this end, I performed multiple mass spectrometry-coupled co-Immunoprecipitation assays with potential subunits of a SWR1 complex, HAT2 and a putative homolog of a NuA4/TIP60 subunit. In the course of these experiments, I was able to identify the TbSWR1 complex. Subsequent cell fractionation and chromatin immunoprecipitation-coupled sequencing analysis experiments confirmed, that this complex is responsible for the incorporation of the histone variant H2A.Z in T. brucei. In addition to this chromatin remodelling complex, I was also able to identify two histone acetyltransferase complexes assembled around HAT1 and HAT2. In the course of my study data were published by the research group of Nicolai Siegel that identified the histone acetyltransferase HAT2 as being responsible for histone H4 acetylation, in preparation to promote H2A.Z incorporation. The data also indicated that HAT1 is responsible for acetylation of H2A.Z. According to the literature, this acetylation is required for proper transcription initiation. Experimental data generated in this study indicated, that H2A.Z and therefore TbSWR1 is involved in the DNA double strand break response of T. brucei. The identification of the specific complex composition of all three complexes provided some hints about how they could interact with each other in the course of transcription regulation and the DNA double strand break response. A proximity labelling approach performed with one of the subunits of the TbSWR1 complex identified multiple transcription factors, PTM writers and proteins potentially involved in chromatin maintenance. Overall, this work will provide some interesting insights about the composition of the complexes involved in H2A.Z incorporation in T. brucei. Furthermore, it is providing valuable information to set up experiments that could shed some light on RNA-polymerase II-mediated transcription and chromatin remodelling in T. brucei in particular and Kinetoplastids in general.
N2  - Die Histonvariante H2A.Z ist ein Schlüsselelement bei der Transkriptionsregulation in Eukaryoten. Histonacetylierungen die vom NuA4/Tip60 Komplex prozessiert werden, sind für den korrekten Einbau der Variante unerlässlich. Darüber hinaus erlauben diese posttranslationellen Modifikationen die Rekrutierung weiterer Proteine und Komplexe die für die Transkription notwendig sind. Ein weiteres Schlüsselelement der mittels H2A.Z regulierten Transkription ist der Komplex zur Umstrukturierung des Chromatins SWR1, welcher das kanonische Histon H2A gegen seine Variante austauscht. Zu Beginn dieses Projektes war der Wissenstand bezüglich der Histonvariante H2A.Z in dem einzelligen Parasiten Trypanosoma brucei limitiert. Wie in anderen eukaryotischen Organismen wurde die Variante ausschließlich an den Startpunkten der polyzistronischen Transkriptionseinheiten gefunden, an denen es für die Öffnung des Chromatins verantwortlich ist und so die Transkription mittels RNAPolymerase II ermöglicht. Vorangegangene Studien konnten zeigen, dass die Variante mit einer Acetylierung des Lysins 10 im Histon H4 und einer Methylierung des Lysins 4 im Histon H3 co-lokalisiert. Einige Daten lieferten den Hinwies, dass die Histon-Acetyltransferase HAT2 mit H2A.Z in Zusammenhang steht, da diese die Acetylierung des Lysins 10 im Hinston H4 prozessiert. Komplexe die in ihrer Funktion dem SWR1 oder dem NuA4/TIP60 Komplex entsprechen, konnten bisher noch nicht gefunden werden. Die vorliegende Arbeit zielt darauf ab Komplexe zu identifizieren, die in ihrer Funktion dem SWR1 sowie dem NuA4/TIP60 Komplex entsprechen. Zudem soll die Frage geklärt werden ob HAT2 Teil eines möglichen NuA4/TIP60 Komplexes ist. In diesem Zusammenhang habe ich mehrere Massenspektrometrie gekoppelte Co-Immunopräzipitationen mit potenziellen Untereinheiten eines SWR1 Komplexes sowie HAT2 und einem Protein welches otholog zu einer NuA4/TIP60 Untereinheit ist, durchgeführt. Im Verlauf dieser Experimente konnte der SWR1 Komplex in T. brucei (TbSWR1) identifiziert werden. Anschließende Zellfraktionierungen sowie Chromatin Immunopräzipitationen gekoppelte Sequenzanalysen konnten bestätigen, dass der identifizierte Komplex für den Einbau der Histonvariante H2A.Z zuständig ist. Darüber hinaus konnten neben diesem Komplex noch zwei weitere Komplexe identifiziert werden, die jeweils die Histonacetyltransferasen HAT1 und HAT2 als Kernkomponenten enthalten. Im Verlauf meiner Arbeit wurden von der Arbeitsgruppe von Nicolai Siegel Daten publiziert die zeigten, dass die Histonacetyltransferase HAT2, in Vorbereitung auf den Einbau von H2A.Z, für die Acetylierung des Histons H4 verantwortlich ist. Im Gegenzug ist HAT1 für die Acetylierung von H2A.Z notwendig, welche wiederum für die korrekte Initiation der Transkription benötigt wird. Damit entspricht die Funktion der Acetylierung von H2A.Z in T. brucei der in der Literatur beschriebenen Funktion. Experimentelle Daten die im Verlauf dieser Arbeit generiert wurden, lieferten einen Hinweis darauf, dass H2A.Z auch an der Reparatur von DNS Doppelstrangbrüchen beteiligt ist. Die Aufschlüsselung der spezifischen Zusammensetzung aller drei Komplexe gab einige Hinweise darauf, wie sie sowohl während der Transkriptionsregulation als auch der Reparatur von DNS Doppelstrangbrüchen miteinander interagieren. Im Zuge einer molekularen Umgebungskartierung, die mit einer der Untereinheiten des TbSWR1 Komplexes durchgeführt wurde, konnten mehrere Transkriptionsfaktoren und Enzyme zur Histonmodifizierung identifiziert werden. Dabei wurden auch einige Proteine identifiziert, welche möglicherweise mit der Umformung des Chromatins in Zusammenhang stehen. Abschließend ist festzuhalten, dass diese Arbeit einige äußerst interessante Einsichten über die Zusammensetzung der Komplexe, die am H2A.Z Einbau in T. brucei beteiligt sind, liefern konnte. Darüber hinaus stellt sie einige wertvolle Informationen zur Verfügung. Diese könnten zur gezielten Planung von Experimenten genutzt werden, um mehr über RNA-Polymerase II vermittelte Transkription und Chromatin Umstrukturierung in T. brucei im speziellen und in Kinetoplastiden im Allgemeinen zu erfahren.
KW  - Chromatinremodelling
KW  - Histone
KW  - Transkription
KW  - Chromatinremodeling
KW  - Histones
KW  - Variants
KW  - Complexes
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257960
ER  - 
TY  - JOUR
A1  - Eiring, Patrick
A1  - McLaughlin, Ryan
A1  - Matikonda, Siddharth S.
A1  - Han, Zhongying
A1  - Grabenhorst, Lennart
A1  - Helmerich, Dominic A.
A1  - Meub, Mara
A1  - Beliu, Gerti
A1  - Luciano, Michael
A1  - Bandi, Venu
A1  - Zijlstra, Niels
A1  - Shi, Zhen-Dan
A1  - Tarasov, Sergey G.
A1  - Swenson, Rolf
A1  - Tinnefeld, Philip
A1  - Glembockyte, Viktorija
A1  - Cordes, Thorben
A1  - Sauer, Markus
A1  - Schnermann, Martin J.
T1  - Targetable conformationally restricted cyanines enable photon-count-limited applications
JF  - Angewandte Chemie Internationale Edition
N2  - Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.
KW  - biology
KW  - super-resolution microscopy
KW  - conformational restriction
KW  - cyanine dyes
KW  - DNA nanotechnology
KW  - fluorescent dyes
KW  - single-molecule fluorescence spectroscopy
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256559
VL  - 60
IS  - 51
ER  - 
TY  - JOUR
A1  - Leverkus, Alexandro B.
A1  - Thorn, Simon
A1  - Gustafsson, Lena
A1  - Noss, Reed
A1  - Müller, Jörg
A1  - Pausas, Juli G.
A1  - Lindenmayer, David B.
T1  - Environmental policies to cope with novel disturbance regimes–steps to address a world scientists’ warning to humanity
JF  - Environmental Research Letters
N2  - No abstract available.
KW  - global change
KW  - novel disturbance
KW  - regime shift
KW  - forest management
KW  - risk management
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254180
SN  - 1748-9326
VL  - 16
IS  - 2
ER  - 
TY  - JOUR
A1  - Heidrich, Lea
A1  - Pinkert, Stefan
A1  - Brandl, Roland
A1  - Bässler, Claus
A1  - Hacker, Hermann
A1  - Roth, Nicolas
A1  - Busse, Annika
A1  - Müller, Jörg
A1  - Friess, Nicolas
T1  - Noctuid and geometrid moth assemblages show divergent elevational gradients in body size and color lightness
JF  - Ecography
N2  - Previous macroecological studies have suggested that larger and darker insects are favored in cold environments and that the importance of body size and color for the absorption of solar radiation is not limited to diurnal insects. However, whether these effects hold true for local communities and are consistent across taxonomic groups and sampling years remains unexplored. This study examined the variations in body size and color lightness of the two major families of nocturnal moths, Geometridae and Noctuidae, along an elevational gradient of 700 m in Southern Germany. An assemblage-based analysis was performed using community-weighted means and a fourth-corner analysis to test for variations in color and body size among communities as a function of elevation. This was followed by a species-level analysis to test whether species occurrence and abundance along an elevation gradient were related to these traits, after controlling for host plant availability. In both 2007 and 2016, noctuid moth assemblages became larger and darker with increasing elevation, whereas geometrids showed an opposite trend in terms of color lightness and no clear trend in body size. In single species models, the abundance of geometrids, but not of noctuids, was driven by habitat availability. In turn, the abundance of dark-colored noctuids, but not geometrids increased with elevation. While body size and color lightness affect insect physiology and the ability to cope with harsh conditions, divergent trait–environment relationships between both families underline that findings of coarse-scale studies are not necessarily transferable to finer scales. Local abundance and occurrence of noctuids are shaped by morphological traits, whereas that of geometrids are rather shaped by local habitat availability, which can modify their trait–environment-relationship. We discuss potential explanations such as taxon-specific flight characteristics and the effect of microclimatic conditions.
KW  - insects
KW  - color lightness
KW  - body size
KW  - elevation
KW  - habitat availability
KW  - flight characteristics
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256694
VL  - 44
IS  - 8
ER  - 
TY  - JOUR
A1  - Panzer, Sabine
A1  - Zhang, Chong
A1  - Konte, Tilen
A1  - Bräuer, Celine
A1  - Diemar, Anne
A1  - Yogendran, Parathy
A1  - Yu-Strzelczyk, Jing
A1  - Nagel, Georg
A1  - Gao, Shiqiang
A1  - Terpitz, Ulrich
T1  - Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates
JF  - Frontiers in Molecular Biosciences
N2  - Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.
KW  - black yeast
KW  - photoreceptor
KW  - microbial rhodopsins
KW  - optogenetics
KW  - proton channel
KW  - membrane trafficking
KW  - fungal rhodopsins
KW  - Aureobasidium
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249248
SN  - 2296-889X
VL  - 8
ER  - 
TY  - JOUR
A1  - Shityakov, Sergey
A1  - Skorb, Ekaterina V.
A1  - Förster, Carola Y.
A1  - Dandekar, Thomas
T1  - Scaffold Searching of FDA and EMA-Approved Drugs Identifies Lead Candidates for Drug Repurposing in Alzheimer’s Disease
JF  - Frontiers in Chemistry
N2  - Clinical trials of novel therapeutics for Alzheimer’s Disease (AD) have consumed a significant amount of time and resources with largely negative results. Repurposing drugs already approved by the Food and Drug Administration (FDA), European Medicines Agency (EMA), or Worldwide for another indication is a more rapid and less expensive option. Therefore, we apply the scaffold searching approach based on known amyloid-beta (Aβ) inhibitor tramiprosate to screen the DrugCentral database (n = 4,642) of clinically tested drugs. As a result, menadione bisulfite and camphotamide substances with protrombogenic and neurostimulation/cardioprotection effects were identified as promising Aβ inhibitors with an improved binding affinity (ΔGbind) and blood-brain barrier permeation (logBB). Finally, the data was also confirmed by molecular dynamics simulations using implicit solvation, in particular as Molecular Mechanics Generalized Born Surface Area (MM-GBSA) model. Overall, the proposed in silico pipeline can be implemented through the early stage rational drug design to nominate some lead candidates for AD, which will be further validated in vitro and in vivo, and, finally, in a clinical trial.
KW  - scaffold search
KW  - approved drugs
KW  - drug repurposing
KW  - alzheimer's disease
KW  - chemical similarity
KW  - molecular modeling
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248703
SN  - 2296-2646
VL  - 9
ER  - 
TY  - JOUR
A1  - Britz, Sebastian
A1  - Markert, Sebastian Matthias
A1  - Witvliet, Daniel
A1  - Steyer, Anna Maria
A1  - Tröger, Sarah
A1  - Mulcahy, Ben
A1  - Kollmannsberger, Philip
A1  - Schwab, Yannick
A1  - Zhen, Mei
A1  - Stigloher, Christian
T1  - Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy
JF  - Frontiers in Neuroanatomy
N2  - At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.
KW  - FIB-SEM
KW  - 3D reconstruction
KW  - neuroanatomy
KW  - IL2 branching
KW  - amphids
KW  - Caenorhabditis elegans (C. elegans)
KW  - dauer
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249622
SN  - 1662-5129
VL  - 15
ER  - 
TY  - JOUR
A1  - Kuhlemann, Alexander
A1  - Beliu, Gerti
A1  - Janzen, Dieter
A1  - Petrini, Enrica Maria
A1  - Taban, Danush
A1  - Helmerich, Dominic A.
A1  - Doose, Sören
A1  - Bruno, Martina
A1  - Barberis, Andrea
A1  - Villmann, Carmen
A1  - Sauer, Markus
A1  - Werner, Christian
T1  - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
JF  - Frontiers in Synaptic Neuroscience
N2  - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.
KW  - super-resolution microscopy (SRM)
KW  - click-chemistry
KW  - dSTORM
KW  - GABA-A receptor
KW  - genetic code expansion
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035
SN  - 1663-3563
VL  - 13
ER  - 
TY  - JOUR
A1  - Borges, Alyssa R.
A1  - Link, Fabian
A1  - Engstler, Markus
A1  - Jones, Nicola G.
T1  - The Glycosylphosphatidylinositol Anchor: A Linchpin for Cell Surface Versatility of Trypanosomatids
JF  - Frontiers in Cell and Developmental Biology
N2  - The use of glycosylphosphatidylinositol (GPI) to anchor proteins to the cell surface is widespread among eukaryotes. The GPI-anchor is covalently attached to the C-terminus of a protein and mediates the protein’s attachment to the outer leaflet of the lipid bilayer. GPI-anchored proteins have a wide range of functions, including acting as receptors, transporters, and adhesion molecules. In unicellular eukaryotic parasites, abundantly expressed GPI-anchored proteins are major virulence factors, which support infection and survival within distinct host environments. While, for example, the variant surface glycoprotein (VSG) is the major component of the cell surface of the bloodstream form of African trypanosomes, procyclin is the most abundant protein of the procyclic form which is found in the invertebrate host, the tsetse fly vector. Trypanosoma cruzi, on the other hand, expresses a variety of GPI-anchored molecules on their cell surface, such as mucins, that interact with their hosts. The latter is also true for Leishmania, which use GPI anchors to display, amongst others, lipophosphoglycans on their surface. Clearly, GPI-anchoring is a common feature in trypanosomatids and the fact that it has been maintained throughout eukaryote evolution indicates its adaptive value. Here, we explore and discuss GPI anchors as universal evolutionary building blocks that support the great variety of surface molecules of trypanosomatids.
KW  - cell surface proteome
KW  - evolution
KW  - GPI-anchor
KW  - Kinetoplastea
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249253
SN  - 2296-634X
VL  - 9
ER  - 
TY  - JOUR
A1  - Osmanoglu, Özge
A1  - Khaled AlSeiari, Mariam
A1  - AlKhoori, Hasa Abduljaleel
A1  - Shams, Shabana
A1  - Bencurova, Elena
A1  - Dandekar, Thomas
A1  - Naseem, Muhammad
T1  - Topological Analysis of the Carbon-Concentrating CETCH Cycle and a Photorespiratory Bypass Reveals Boosted CO\(_2\)-Sequestration by Plants
JF  - Frontiers in Bioengineering and Biotechnology
N2  - Synthetically designed alternative photorespiratory pathways increase the biomass of tobacco and rice plants. Likewise, some in planta–tested synthetic carbon-concentrating cycles (CCCs) hold promise to increase plant biomass while diminishing atmospheric carbon dioxide burden. Taking these individual contributions into account, we hypothesize that the integration of bypasses and CCCs will further increase plant productivity. To test this in silico, we reconstructed a metabolic model by integrating photorespiration and photosynthesis with the synthetically designed alternative pathway 3 (AP3) enzymes and transporters. We calculated fluxes of the native plant system and those of AP3 combined with the inhibition of the glycolate/glycerate transporter by using the YANAsquare package. The activity values corresponding to each enzyme in photosynthesis, photorespiration, and for synthetically designed alternative pathways were estimated. Next, we modeled the effect of the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle (CETCH), which is a set of natural and synthetically designed enzymes that fix CO₂ manifold more than the native Calvin–Benson–Bassham (CBB) cycle. We compared estimated fluxes across various pathways in the native model and under an introduced CETCH cycle. Moreover, we combined CETCH and AP3-w/plgg1RNAi, and calculated the fluxes. We anticipate higher carbon dioxide–harvesting potential in plants with an AP3 bypass and CETCH–AP3 combination. We discuss the in vivo implementation of these strategies for the improvement of C3 plants and in natural high carbon harvesters.
KW  - CO2-sequestration
KW  - photorespiration
KW  - elementary modes
KW  - synthetic pathways
KW  - carboxylation
KW  - metabolic modeling
KW  - CETCH cycle
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249260
SN  - 2296-4185
VL  - 9
ER  -