TY - INPR A1 - Brenner, Marian A1 - Zink, Christoph A1 - Witzinger, Linda A1 - Keller, Angelika A1 - Hadamek, Kerstin A1 - Bothe, Sebastian A1 - Neuenschwander, Martin A1 - Villmann, Carmen A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - 7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase T2 - eLife N2 - Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain. KW - 7,8-dihydroxyflavone (7,8-DHF) KW - pyridoxal phosphatase (PDXP) KW - vitamin B6 KW - PDXP inhibitors Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350446 ER - TY - JOUR A1 - Dütting, Sebastian A1 - Gaits-Iacovoni, Frederique A1 - Stegner, David A1 - Popp, Michael A1 - Antkowiak, Adrien A1 - van Eeuwijk, Judith M.M. A1 - Nurden, Paquita A1 - Stritt, Simon A1 - Heib, Tobias A1 - Aurbach, Katja A1 - Angay, Oguzhan A1 - Cherpokova, Deya A1 - Heinz, Niels A1 - Baig, Ayesha A. A1 - Gorelashvili, Maximilian G. A1 - Gerner, Frank A1 - Heinze, Katrin G. A1 - Ware, Jerry A1 - Krohne, Georg A1 - Ruggeri, Zaverio M. A1 - Nurden, Alan T. A1 - Schulze, Harald A1 - Modlich, Ute A1 - Pleines, Irina A1 - Brakebusch, Cord A1 - Nieswandt, Bernhard T1 - A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis JF - Nature Communications N2 - Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V. KW - megakaryocytes KW - blood platelets KW - regulatory circuit downstream KW - glycoprotein Ib Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170797 VL - 8 IS - 15838 ER - TY - JOUR A1 - Sander, Bodo A1 - Xu, Wenshan A1 - Eilers, Martin A1 - Popov, Nikita A1 - Lorenz, Sonja T1 - A conformational switch regulates the ubiquitin ligase HUWE1 JF - eLife N2 - The human ubiquitin ligase HUWE1 has key roles in tumorigenesis, yet it is unkown how its activity is regulated. We present the crystal structure of a C-terminal part of HUWE1, including the catalytic domain, and reveal an asymmetric auto-inhibited dimer. We show that HUWE1 dimerizes in solution and self-associates in cells, and that both occurs through the crystallographic dimer interface. We demonstrate that HUWE1 is inhibited in cells and that it can be activated by disruption of the dimer interface. We identify a conserved segment in HUWE1 that counteracts dimer formation by associating with the dimerization region intramolecularly. Our studies reveal, intriguingly, that the tumor suppressor p14ARF binds to this segment and may thus shift the conformational equilibrium of HUWE1 toward the inactive state. We propose a model, in which the activity of HUWE1 underlies conformational control in response to physiological cues—a mechanism that may be exploited for cancer therapy. KW - Medicine KW - Structural Biology KW - Molecular Biophysics KW - HUWE1 KW - HECT Ligase KW - Ubiquitin KW - P14ARF KW - X-Ray Chrystallography KW - Enzyme Regulation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171862 VL - 6 ER - TY - JOUR A1 - Zaho, Huaying A1 - Ghirlando, Rodolfo A1 - Alfonso, Carlos A1 - Arisaka, Fumio A1 - Attali, Ilan A1 - Bain, David L. A1 - Bakhtina, Marina M. A1 - Becker, Donald F. A1 - Bedwell, Gregory J. A1 - Bekdemir, Ahmet A1 - Besong, Tabot M. D. A1 - Birck, Catherine A1 - Brautigam, Chad A. A1 - Brennerman, William A1 - Byron, Olwyn A1 - Bzowska, Agnieszka A1 - Chaires, Jonathan B. A1 - Chaton, Catherine T. A1 - Coelfen, Helmbut A1 - Connaghan, Keith D. A1 - Crowley, Kimberly A. A1 - Curth, Ute A1 - Daviter, Tina A1 - Dean, William L. A1 - Diez, Ana I. A1 - Ebel, Christine A1 - Eckert, Debra M. A1 - Eisele, Leslie E. A1 - Eisenstein, Edward A1 - England, Patrick A1 - Escalante, Carlos A1 - Fagan, Jeffrey A. A1 - Fairman, Robert A1 - Finn, Ron M. A1 - Fischle, Wolfgang A1 - Garcia de la Torre, Jose A1 - Gor, Jayesh A1 - Gustafsson, Henning A1 - Hall, Damien A1 - Harding, Stephen E. A1 - Hernandez Cifre, Jose G. A1 - Herr, Andrew B. A1 - Howell, Elizabeth E. A1 - Isaac, Richard S. A1 - Jao, Shu-Chuan A1 - Jose, Davis A1 - Kim, Soon-Jong A1 - Kokona, Bashkim A1 - Kornblatt, Jack A. A1 - Kosek, Dalibor A1 - Krayukhina, Elena A1 - Krzizike, Daniel A1 - Kusznir, Eric A. A1 - Kwon, Hyewon A1 - Larson, Adam A1 - Laue, Thomas M. A1 - Le Roy, Aline A1 - Leech, Andrew P. A1 - Lilie, Hauke A1 - Luger, Karolin A1 - Luque-Ortega, Juan R. A1 - Ma, Jia A1 - May, Carrie A. A1 - Maynard, Ernest L. A1 - Modrak-Wojcik, Anna A1 - Mok, Yee-Foong A1 - Mücke, Norbert A1 - Nagel-Steger, Luitgard A1 - Narlikar, Geeta J. A1 - Noda, Masanori A1 - Nourse, Amanda A1 - Obsil, Thomas A1 - Park, Chad K A1 - Park, Jin-Ku A1 - Pawelek, Peter D. A1 - Perdue, Erby E. A1 - Perkins, Stephen J. A1 - Perugini, Matthew A. A1 - Peterson, Craig L. A1 - Peverelli, Martin G. A1 - Piszczek, Grzegorz A1 - Prag, Gali A1 - Prevelige, Peter E. A1 - Raynal, Bertrand D. E. A1 - Rezabkova, Lenka A1 - Richter, Klaus A1 - Ringel, Alison E. A1 - Rosenberg, Rose A1 - Rowe, Arthur J. A1 - Rufer, Arne C. A1 - Scott, David J. A1 - Seravalli, Javier G. A1 - Solovyova, Alexandra S. A1 - Song, Renjie A1 - Staunton, David A1 - Stoddard, Caitlin A1 - Stott, Katherine A1 - Strauss, Holder M. A1 - Streicher, Werner W. A1 - Sumida, John P. A1 - Swygert, Sarah G. A1 - Szczepanowski, Roman H. A1 - Tessmer, Ingrid A1 - Toth, Ronald T. A1 - Tripathy, Ashutosh A1 - Uchiyama, Susumu A1 - Uebel, Stephan F. W. A1 - Unzai, Satoru A1 - Gruber, Anna Vitlin A1 - von Hippel, Peter H. A1 - Wandrey, Christine A1 - Wang, Szu-Huan A1 - Weitzel, Steven E A1 - Wielgus-Kutrowska, Beata A1 - Wolberger, Cynthia A1 - Wolff, Martin A1 - Wright, Edward A1 - Wu, Yu-Sung A1 - Wubben, Jacinta M. A1 - Schuck, Peter T1 - A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation JF - PLoS ONE N2 - Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304\(\pm\)0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of \(\pm\)0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies. KW - fluorescence-detected sedimentation KW - size exclusion chromatography KW - field flow fractionation KW - spinco ultracentrifuge KW - aggregation KW - bead models KW - velocity KW - hydrodynamics KW - biopharmaceuticals KW - proteins Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151903 VL - 10 IS - 5 ER - TY - JOUR A1 - van Unen, Jakobus A1 - Stumpf, Anette D. A1 - Schmid, Benedikt A1 - Reinhard, Nathalie R. A1 - Hordijk, Peter L. A1 - Hoffmann, Carsten A1 - Gadella, Theodorus W. J. A1 - Goedhart, Joachim T1 - A New Generation of FRET Sensors for Robust Measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) Activation Kinetics in Single Cells JF - PLoS ONE N2 - G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα\(_{i}\) subunit, and cp173Venus fused to the Gγ\(_{2}\) subunit as acceptor. The Gα\(_{i}\) FRET biosensors constructs are expressed together with Gβ\(_{1}\) from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα\(_{i}\) FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα\(_{i}\) FRET sensor in single cells upon stimulation of several GPCRs, including the LPA\(_{2}\), M\(_{3}\) and BK\(_{2}\) receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation will be valuable for live-cell measurements that probe Gα\(_{i}\) activation. KW - FRET sensors KW - G-protein coupled receptors KW - Förster resonance energy transfer KW - Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation KW - biosensors Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167387 VL - 11 IS - 1 ER - TY - JOUR A1 - Jun, Kyong-Hwa A1 - Gholami, Spedideh A1 - Song, Tae-Jin A1 - Au, Joyce A1 - Haddad, Dana A1 - Carson, Joshua A1 - Chen, Chun-Hao A1 - Mojica, Kelly A1 - Zanzonico, Pat A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Szalay, Aladar A1 - Fong, Yuman T1 - A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter JF - Journal of Experimental & Clinical Cancer Research N2 - Background: Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with Tc-99m pertechnetate scintigraphy and I-124 positron emission tomography (PET). Methods: GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. Tc-99m pertechnetate scintigraphy and I-124 microPET imaging were performed. Results: GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK-45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by Tc-99m pertechnetate scintigraphy and I-124 microPET imaging 2 days after treatment. Conclusions: GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings. KW - oncolytic viral therapy KW - GLV-1 h153 KW - gastric cancer KW - human sodium iodide symporter (hNIS) KW - radioiodine therapy KW - gene therapy KW - expression KW - replication KW - stomach KW - tumors KW - surgery Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117716 SN - 1756-9966 VL - 33 IS - 2 ER - TY - JOUR A1 - Schihada, Hannes A1 - Vandenabeele, Sylvie A1 - Zabel, Ulrike A1 - Frank, Monika A1 - Lohse, Martin J. A1 - Maiellaro, Isabella T1 - A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics JF - Communications Biology N2 - G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands. KW - Fluorescence resonance energy transfer KW - G protein-coupled receptors KW - High-throughput screening Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228592 VL - 1 IS - 105 ER - TY - JOUR A1 - Pfeiffer, Verena A1 - Götz, Rudolf A1 - Xiang, Chaomei A1 - Camarero, Guadelupe A1 - Braun, Attila A1 - Zhang, Yina A1 - Blum, Robert A1 - Heinsen, Helmut A1 - Nieswandt, Bernhard A1 - Rapp, Ulf R. T1 - Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum JF - PLoS ONE N2 - This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures. KW - granule cells KW - hippocampus KW - neurons KW - neuronal dendrites KW - embryos KW - dentate gyrus KW - neuronal differentiation KW - cerebellum Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130304 VL - 8 IS - 3 ER - TY - JOUR A1 - Li, Cong A1 - Deng, Xiaobing A1 - Xie, Xiaowen A1 - Liu, Ying A1 - Friedmann Angeli, José Pedro A1 - Lai, Luhua T1 - Activation of Glutathione Peroxidase 4 as a Novel Anti-inflammatory Strategy JF - Frontiers in Pharmacology N2 - The anti-oxidative enzyme, glutathione peroxidase 4 (GPX4), helps to promote inflammation resolution by eliminating oxidative species produced by the arachidonic acid (AA) metabolic network. Up-regulating its activity has been proposed as a promising strategy for inflammation intervention. In the present study, we aimed to study the effect of GPX4 activator on the AA metabolic network and inflammation related pathways. Using combined computational and experimental screen, we identified a novel compound that can activate the enzyme activity of GPX4 by more than two folds. We further assessed its potential in a series of cellular assays where GPX4 was demonstrated to play a regulatory role. We are able to show that GPX4 activation suppressed inflammatory conditions such as oxidation of AA and NF-κB pathway activation. We further demonstrated that this GPX4 activator can decrease the intracellular ROS level and suppress ferroptosis. Our study suggests that GPX4 activators can be developed as anti-inflammatory or cyto-protective agent in lipid-peroxidation-mediated diseases. KW - arachidonic acid metabolic network KW - GPX4 KW - enzyme activator KW - allosterism KW - drug discovery KW - anti-inflammatory KW - ferroptosis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195985 SN - 1663-9812 VL - 9 IS - 1120 ER - TY - JOUR A1 - Busse, Kathy A1 - Strotmann, Rainer A1 - Strecker, Karl A1 - Wegner, Florian A1 - Devanathan, Vasudharani A1 - Gohla, Antje A1 - Schöneberg, Torsten A1 - Schwarz, Johannes T1 - Adaptive Gene Regulation in the Striatum of RGS9-Deficient Mice JF - PLOS ONE N2 - Background: RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions. Results: Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice. Conclusion: Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions. Significance: Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9-deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice. KW - medium spiny neurons KW - long-term depression KW - dopa-induced dyskinesia KW - adenylyl cyclase KW - Parkinsons disease KW - synaptic plasticity KW - L-3,4-Dihydroxyphenylalanine-induced dyskinesia KW - ampa receptors KW - cholinergic interneurons KW - endocannabinoid release Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117048 VL - 9 IS - 3 ER - TY - THES A1 - El Merahbi, Rabih T1 - Adrenergic-induced ERK3 pathway drives lipolysis and suppresses energy dissipation T1 - Der adrenerge induzierte ERK3-Signalweg verstärkt Lipolyse und unterdrückt Energiedissipation N2 - Obesity-induced diabetes affects over 400 million people worldwide. Obesity is a complex metabolic disease and is associated with several co-morbidities, all of which negatively affect the individual’s quality of life. It is commonly considered that obesity is a result of a positive energy misbalance, as increased food intake and lower expenditure eventually lead to the development of this disease. Moreover, the pathology of obesity is attributed to several genetic and epigenetic factors that put an individual at high risk compared to another. Adipose tissue is the main site of the organism’s energy storage. During the time when the nutrients are available in excess, adipocytes acquire triglycerides, which are released during the time of food deprivation in the process of lipolysis (free fatty acids and glycerol released from adipocytes). Uncontrolled lipolysis is the consequent event that contributes to the development of diabetes and paradoxically obesity. To identify the genetic factors aiming for future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the Extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrate that β-adrenergic stimulation stabilizes ERK3 leading to the formation of a complex with the co-factor MAP kinase-activated protein kinase 5 (MK5) thereby driving lipolysis. Mechanistically, we identify a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Moreover, we shed the light on our pharmacological approach in targeting ERK3/MK5 pathways using MK5 specific inhibitor. Already after 1 week of administering the inhibitor, mice showed signs of improvement of their metabolic fitness as showed here by a reduction in induced lipolysis and the elevation in the expression of thermogenic genes. Taken together, our data suggest that targeting the ERK3/MK5 pathway, a previously unrecognized signaling axis in adipose tissue, could be an attractive target for future therapies aiming to combat obesity-induced diabetes. N2 - Adipositas-induzierter Diabetes betrifft weltweit über 400 Millionen Menschen. Adipositas ist eine komplexe Stoffwechselerkrankung und geht mit mehreren Komorbiditäten einher, die sich alle negativ auf die Lebensqualität der Betroffenen auswirken. Es wird generell angenommen, dass Adipositas aus einem positiven Energieungleichgewicht resultiert, da eine erhöhte Nahrungsaufnahme und ein geringerer Verbrauch zu der Ausbildung dieser Krankheit führen. Darüber hinaus ist die Pathologie von Adipositas auf mehrere genetische und epigenetische Faktoren zurückzuführen, wodurch Individuen einem erhöhtem Risiko ausgesetzt sein können. Das Fettgewebe ist der vorwiegende Energiespeicher des Organismus. In Zeiten eines Nährstoffüberschusses speichern Adipozyten Triglyceride, die im Falle eines Nahrungsmangels durch den Prozess der Lipolyse in Form von freien Fettsäuren und Glycerin freigesetzt werden. Unkontrollierte Lipolyse ist ein Folgeereignis, welches zur Entwicklung von Diabetes und paradoxerweise zu Adipositas beiträgt. Um die genetischen Faktoren zu identifizieren, die in Zukunft therapeutische Angriffspunkte darstellen könnten, haben wir ein Hochdurchsatz-Screening durchgeführt und die extrazellulär regulierte Kinase 3 (ERK3) als Treffer identifiziert. Wir zeigen, dass β-adrenerge Stimulation ERK3 stabilisiert, was zur Bildung eines Komplexes mit dem Cofactor MAP-Kinase-aktivierte Proteinkinase 5 (MK5) führt und dadurch die Lipolyse vorantreibt. Mechanistisch identifizieren wir den Transkriptionsfaktor FOXO1, der dem ERK3/MK5-Signalweg nachgeschaltet ist und die Expression des wichtigsten lipolytischen Enzyms ATGL fördert. Darüber hinaus belegen wir, dass die gezielte Deletion von ERK3 in Maus-Adipozyten die Lipolyse hemmt, aber die Energiedissipation erhöht, den mageren Phänotyp fördert und Diabetes lindert. Außerdem nutzen wir einen pharmakologischen Ansatz durch Verwendung eines MK5 spezifischen Inhibitors, um auf den ERK3/MK5-Signalweg abzuzielen. Bereits eine Woche nach Verabreichung des Inhibitors zeigen Mäuse Anzeichen einer verbesserten metabolischen Fitness, die sich durch einer Verringerung der induzierten Lipolyse und eine verstärkte Expression von thermogenen Genen auszeichnet. Zusammenfassend legen unsere Daten nahe, dass der ERK3/MK5-Signalweg, eine zuvor nicht erkannte Signalachse im Fettgewebe, ein attraktiver Ansatzpunkt für zukünftige Therapien zur Bekämpfung von Adipositas-induziertem Diabetes sein könnte. KW - Metabolism KW - Lipolysis KW - Obesity KW - Adrenalin KW - ATGL KW - Foxo1 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217510 ER - TY - JOUR A1 - Segerer, Gabriela A1 - Hadamek, Kerstin A1 - Zundler, Matthias A1 - Fekete, Agnes A1 - Seifried, Annegrit A1 - Mueller, Martin J. A1 - Koentgen, Frank A1 - Gessler, Manfred A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation JF - Scientific Reports N2 - Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive Pgp\(^{D34N}\) mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation. KW - cell proliferation KW - DNA metabolism KW - lipidomics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181094 VL - 6 ER - TY - JOUR A1 - Sirén, Anna-Leena A1 - Stetter, Christian A1 - Hirschberg, Markus A1 - Nieswandt, Bernhard A1 - Ernestus, Ralf-Ingo A1 - Heckmann, Manfred T1 - An experimental protocol for in vivo imaging of neuronal structural plasticity with 2-photon microscopy in mice JF - Experimental & Translational Stroke Medicine N2 - Introduction Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex. Methods Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45–60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100–200 μm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis. Results Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging. Conclusions Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury. KW - 2-photon microscopy KW - Fluorescence KW - In vivo imaging KW - Neurons KW - Cranial window KW - Mouse model Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96908 UR - http://www.etsmjournal.com/content/5/1/9 ER - TY - JOUR A1 - Mietrach, Nicole A1 - Schlosser, Andreas A1 - Geibel, Sebastian T1 - An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization JF - Acta Crystallographica Section F N2 - The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α‐helices with a melting point of 34.5°C. Size‐exclusion chromatography combined with multi‐angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging‐drop vapor‐diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit‐cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°. KW - ESAT‐6‐like secretion system KW - ESS KW - type VII secretion system KW - EsaA KW - extracellular domain KW - Staphylococcus aureus USA300 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213681 VL - 75 IS - 12 ER - TY - JOUR A1 - Ries, Lena K. A1 - Sander, Bodo A1 - Deol, Kirandeep K. A1 - Letzelter, Marie-Annick A1 - Strieter, Eric Robert A1 - Lorenz, Sonja T1 - Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity JF - Journal of Biological Chemistry N2 - Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin-conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases. KW - ubiquitin KW - ubiquitin ligase KW - ubiquitylation (ubiquitination) KW - post-translational modification KW - enzyme mechanism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226207 VL - 294 IS - 15 ER - TY - JOUR A1 - Brünnert, Daniela A1 - Seupel, Raina A1 - Goyal, Pankaj A1 - Bach, Matthias A1 - Schraud, Heike A1 - Kirner, Stefanie A1 - Köster, Eva A1 - Feineis, Doris A1 - Bargou, Ralf C. A1 - Schlosser, Andreas A1 - Bringmann, Gerhard A1 - Chatterjee, Manik T1 - Ancistrocladinium A induces apoptosis in proteasome inhibitor-resistant multiple myeloma cells: a promising therapeutic agent candidate JF - Pharmaceuticals N2 - The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent. KW - multiple myeloma KW - ancistrocladinium A KW - naphthylisoquinoline alkaloids KW - proteasome inhibitor resistance KW - RNA splicing KW - cellular stress response KW - proteasome subunit beta type-5 (PSMB5) KW - activating transcription factor 4 (ATF4) KW - ataxia teleagiectasia mutated (ATM) KW - H2A histone family member X (H2AX) Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-362887 SN - 1424-8247 VL - 16 IS - 8 ER - TY - JOUR A1 - Busch, Albert A1 - Busch, Martin A1 - Scholz, Claus-Jürgen A1 - Kellersmann, Richard A1 - Otto, Christoph A1 - Chernogubova, Ekaterina A1 - Maegdefessel, Lars A1 - Zernecke, Alma A1 - Lorenz, Udo T1 - Aneurysm miRNA Signature Differs, Depending on Disease Localization and Morphology JF - International Journal of Molecular Science N2 - Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways. KW - AAA KW - miRNA expression KW - pathway analysis KW - histologic diversity KW - popliteal aneurysm Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146422 SN - International Journal of Molecular Science VL - 17 IS - 1 ER - TY - THES A1 - Gschmack, Eva Maria T1 - Anti-Gehirn-Autoantikörper und deren Bedeutung bei Morbus Parkinson T1 - Anti-brain-Autoantibodies and their role in Parkinson's Disease N2 - Hintergrund: Die der Pathogenese von Morbus Parkinson (PD, Parkinson’s disease) zugrunde liegenden Mechanismen sind bis heute nur unvollständig verstanden. Insbesondere ist unklar, durch welche ursächlichen Faktoren Parkinson ausgelöst wird. Bei der HIV-Infektion treten bei vielen Patienten neurologische Störungen auf (HIV-Associated Neurological Disorders, HAND), die in der klinischen Symptomatik und der Lokalisation der betroffenen Gehirnareale dem Morbus Parkinson ähneln. Möglicherweise könnte eine Fehlregulation der Immunantwort eine Rolle als Auslöser beider Erkrankungen spielen. In dieser Arbeit wurde die Autoimmunantwort von PD- und HAND-Patienten und gesunden Kontrollen gegen verschiedene Gehirnhomogenate untersucht, die während der Parkinsonerkrankung in unterschiedlichem Ausmaß geschädigt werden. Das Autoimmun-Signal wurde quantifiziert und prominente Autoantigene wurden identifiziert. Methoden: In dieser Arbeit wurde ein Western-Blot-basiertes Verfahren zum Nachweis von Autoantikörpern gegen Gehirngewebe entwickelt. Dieses Verfahren wurde nach Optimierung mit Plasmaproben von gesunden Kontrollen, PD-Patienten und Patienten mit HIV-Infektion insbesondere an einer Gruppe von 40 Parkinson-Patienten (Durchschnittsalter 65 Jahre, 45 % weiblich) und 40 alters- und geschlechtsgemachten Kontrollen (Durchschnittsalter 62 Jahre, 50 % weiblich) angewendet und die humorale Autoimmunität gegen verschiedene Gehirnareale untersucht. Dazu wurden die verschiedenen Areale (dorsaler Motornucleus des Glossopharynx- und Vagusnervs (dm), Substantia nigra (SN), anteromedialer temporaler Mesocortex (MC), high order sensorische Assoziations- und präfrontale Felder (HC), first oder sensorische Assoziations- und prämotorische Felder, primäre sensorische und motorische Felder (FC)) von post-mortem Gehirnen homogenisiert, auf SDS-Gradienten-Gelen elektrophoretisch aufgetrennt und auf Nitrocellulose geblottet. Die Membranen wurden mit den Plasmen inkubiert und gebundene Autoantikörper immunologisch detektiert. Die Signale wurden qualitativ und quantitativ ausgewertet. Mit Hilfe einer zweidimensionalen Elektrophorese und anschließender Immunfärbung wurden prominente Autoantigene durch Massenspektroskopie identifiziert. Ergebnisse: Mit dem in dieser Arbeit entwickelten Assay lässt sich die humorale Autoimmunantwort gegen Gehirngewebe semiquantitativ bestimmen. In allen untersuchten Proben konnten verschiedene Autoantikörper gegen unterschiedliche Antigene nachgewiesen werden. Der Gesamt-IgG-Gehalt der Plasmen unterscheidet sich weder zwischen PD-Patienten und gesunden Kontrollen, noch zwischen Männern und Frauen signifikant. Weibliche PD-Patienten zeigen signifikant stärkere Signale gegen dm als männliche (p = 0.02, Mann-Whitney-U-Test), der wiederum in jedem Patienten - unabhängig vom Geschlecht - von den untersuchten Hirnarealen signifikant stärker autoimmunologisch erkannt wird, als die übrigen Hirnareale (p < 0.0001, Friedman-ANOVA). In jedem Hirnareal wurden drei Banden besonders häufig erkannt (45, 40 und 37 kDa), jede davon am stärksten im dm (p < 0.0001, Friedman-ANOVA). Die Einzelanalysen der Signalintensitäten zeigt, dass PD-Patienten signifikant weniger Autoreaktivität gegen die 45 kDa-Bande in der SN (p = 0.056), im MC (p = 0.0277) und im FC (p = 0.0188) zeigen, als Kontrollen. Weitere Analysen zeigen, dass männliche PD-Patienten hochsignifikant weniger das 45 kDa-Protein im SN (p < 0.0001), MC (p = 0.0042) und FC (p = 0.0088) erkennen als Kontrollen, wohingegen bei den weiblichen Kontroll- und PD-Plasmen kein Unterschied festzustellen war. Ein weiteres Protein bei 160 kDa wird signifikant unterschiedlich stark in allen Gehirnarealen erkannt (p < 0.0001, Friedman-ANOVA), wobei die stärkste Immunreaktivität gegen FC besteht. Basierend auf dem Nachweis der 45 kDa-Bande aus der SN ergibt sich eine Odds Ratio für das Merkmal Parkinson von 3.38 (CI 1.11 – 10.30). Bei Männern ist diese Odds Ratio sogar 53.12 (CI 2.79 - 1012), bei Frauen 0.44 (CI 0.09 – 2.09). Die Sensitivität dieses Tests liegt bei Männern bei 1 (CI 0.84 – 1), die Spezifität bei 4.41 (0.31 – 0.78). Die negativ prädiktiven Werte liegen in allen Gruppen über 99.15 %. Die Identifizierung der Proteine mittels Massenspektroskopie ergab, dass es sich bei den 37 – 45 kDa Banden um Isoformen oder posttranslational modifizierte Formen des GFAP (glial fibrillary acidic protein), einem Bestandteil von Neurofilamenten v.a. in Astrozyten handelt. Außerdem wurde Fructose-Bisphosphate Aldolase A und Aspartat-Aminotransferase (mitochondriale Isoform 1 Vorläufer), beides Proteine des Kohlenhydrat-Stoffwechsels und der Glykolyse, als weitere Proteine mit ebenfalls 45 kDa identifiziert. Bei dem identifizierten Protein mit dem Molekulargewicht von 160 kDa handelt es sich wahrscheinlich um Dihydropyrimidinase-related protein 2, wie GFAP ebenfalls bei der Bildung des Zytoskeletts beteiligt. Diskussion: Autoantikörper gegen Gehirnantigene sind ein physiologisches Phänomen, das unabhängig von dem Vorliegen einer neurologischen Erkrankung besteht. Gehirnareale, die bei Parkinson besonders stark geschädigt werden, werden von dieser humoralen Autoimmunantwort besonders stark erkannt. Eine vorübergehende Permeabilisierung der Blut-Hirn-Schranke durch Infektion oder Trauma könnte den Zutritt der Autoantikörper zum Gehirn erlauben und so autoreaktive Prozesse in Gang setzen und zum Untergang dopaminerger Neuronen führen. Bei den identifizierten Proteinen handelt es sich um grundlegende Bestandteile eukaryotischer Zellen, was die Hypothese eines Art Beseitigungsmechanismus der Autoantikörper und damit die Aufgabe der Aufrechterhaltung der Homöostase darstellen könnte. Bei männlichen PD Patienten wird die 45 kDa Bande signifikant weniger stark von Auto-IgGs erkannt; dieser Mechanismus könnte somit in den männlichen PD-Patienten vermindert sein. Als Folge wäre die Ablagerung von Zelltrümmern im Gehirn vorstellbar, die dann auch langfristig eine Angriffsfläche für Autoimmunprozesse mit dem Verlust dopaminerger Neuronen bieten könnte. N2 - Background: The pathogenic mechanisms of Parkinson’s disease (PD) are not yet fully understood. Particularly the basic cause of the disease remains unclear. In HIV-Infection many patients show neurologic impairments (HIV-associated neurological disorders, HAND), which are similar in clinic symptoms and localization of the affected brain areas to these observed in Parkinson’s disease. Possibly a dysregulation of immunologic responses might play a role as a cause for PD. In this work autoimmune processes in PD- and HAND-patients as well as in healthy controls against different brain homogenates were measured, which are damaged in variable extent during pathogenesis of PD. The autoimmune-signal was quantified and prominent autoantigens were identified. Methods: In this work we established a Western-Blot-based method to detect autoantibodies against brain homogenates. After the optimization with plasma of healthy controls, PD-patients and HIV-infected patients, the technique was used to measure the humoral autoimmunity against different brain areas in a group of 40 PD-patients (mean age 65, 45 % females), and 40 age- and sex-matched controls (mean age 62 years, 50 % female). Different brain areas (of the dorsal motor nucleus of the glossopharyngeal and vagal nerves (dm), substantia nigra (SN), anteromedial temporal mesocortex (MC), high order sensory association areas and prefrontal fields (HC), first order sensory association areas, premotor areas, as well as primary sensory and motor fields (FC)) of post-mortem brains were homogenized, separated by gel electrophoresis and blotted on a nitrocellulose membrane. The membranes were incubated with plasma and bound antibodies were detected immunologically. The signals were analyzed by quality and quantity. With a two-dimensional electrophoresis and following immune staining prominent autoantigens were identified by mass spectroscopy. Results: With the established assay, the humoral autoimmunity against brain tissue can be analyzed semi-quantitatively. In all investigated samples several autoantibodies against different antigens could be proven. The overall IgG-content did not differ significantly between PD-patients and healthy controls, nor between men and women. Female PD-patients show significant stronger signals against dm than male (p = 0.02, Mann-Whitney-U-test), which was recognized significantly stronger than the other brain areas in each patient, independently from sex (p < 0.0001, Friedman-ANOVA). In each brain area 3 bands were recognized stronger than others (45, 40 and 37 kDa), each of them the strongest in dm (p < 0.0001, Friedman-ANOVA). The single analysis of the signal intensities show that PD-patients display significantly less autoreactivity against the 45 kDa-band in SN (p = 0.056), MC (p = 0.0277) and FC (p = 0.0188) than controls. Further analyses show that plasma of male PD-patients detect highly significantly less the 45 kDa-protein in SN (p < 0.0001), MC (p = 0.0042) and FC (p = 0.0088) than controls, whereas there was no difference measurable in female control- and PD-plasma. Another protein at 160 kDa is recognized at significantly different intensities in all brain areas (p < 0.0001, Friedman-ANOVA), whereas the strongest immunoreactivity is shown against FC. Based on the detection of the 45 kDa-band from the SN the odds ratio for the feature PD is 3.38 (CI 1.11 – 10.30). In men the odds ratio is even 53.12 (CI 2.79 - 1012), in female 0.44 (CI 0.09 – 2.09). The sensitivity for the test in the group of men displays 1 (CI 0.84 – 1), the specificity is 4.41 (0.31 – 0.78). The negative predictive values exceed 99.15 % in all investigated groups. The identification of the proteins by mass spectroscopy showed that the 37 – 45 kDa-bands might be isoforms of posttranslational modified forms of GFAP (glial fibrillary acidic protein), a part of neurofilaments in astrocytes. Furthermore, fructose-bisphosphate aldolase A and aspartate aminotransferase (mitochondrial isoform 1 precursor), both in the metabolism of carbon hydrates and glycolysis, have been identified for further 45 kDa-bands. The identified protein with a molecular weight of 160 kDa is probably dihydropyrimidinase-related protein 2, like GFAP involved in the formation of the cytoskeleton. Conclusion: The presence of anti-brain-autoantibodies are a physiological occurrence in human plasma, independent of the presence of neurological disease. Brain areas that are damaged prominently during the pathogenesis of PD are recognized stronger by humoral autoimmunity. A temporal permeabilization of the blood brain barrier through infection or trauma might allow autoantibodies access to the brain and initiate autoreactive processes that lead to the demise of dopaminergic neurons. The identified proteins are basic members of eukaryotic cells. This could suggest the hypothesis of sort of a cleaning mechanism of the antibodies, which might have the function of sustaining homeostasis. Male PD patients showed significantly decreased levels of autoreactive antibodies against the 45 kDa band in SN, MC and FC, which could enhance the accumulation of protein debris due to a missing removal mechanism and in long-distance might be responsible for neurodegenerative processes. KW - Parkinson-Krankheit KW - Autoantikörper KW - Parkinson Disease KW - anti-brain autoantibodies KW - GFAP KW - Gliafaserprotein KW - Anti-Gehirn Autoantikörper Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149348 ER - TY - THES A1 - Busch, Martin T1 - Aortic Dendritic Cell Subsets in Healthy and Atherosclerotic Mice and The Role of the miR-17~92 Cluster in Dendritic Cells T1 - Subsets dendritischer Zellen in der Aorta gesunder und atherosklerotischerMäuse und die Rolle des miR-17~92 Clusters in dendritischen Zellen N2 - Atherosclerosis is accepted to be a chronic inflammatory disease of the arterial vessel wall. Several cellular subsets of the immune system are involved in its initiation and progression, such as monocytes, macrophages, T and B cells. Recent research has demonstrated that dendritic cells (DCs) contribute to atherosclerosis, too. DCs are defined by their ability to sense and phagocyte antigens, to migrate and to prime other immune cells, such as T cells. Although all DCs share these functional characteristics, they are heterogeneous with respect to phenotype and origin. Several markers have been used to describe DCs in different lymphoid and non-lymphoid organs; however, none of them has proven to be unambiguous. The expression of surface molecules is highly variable depending on the state of activation and the surrounding tissue. Furthermore, DCs in the aorta or the atherosclerotic plaque can be derived from designated precursor cells or from monocytes. In addition, DCs share both their marker expression and their functional characteristics with other myeloid cells like monocytes and macrophages. The repertoire of aortic DCs in healthy and atherosclerotic mice has just recently started to be explored, but yet there is no systemic study available, which describes the aortic DC compartment. Because it is conceivable that distinct aortic DC subsets exert dedicated functions, a detailed description of vascular DCs is required. The first part of this thesis characterizes DC subsets in healthy and atherosclerotic mice. It describes a previously unrecognized DC subset and also sheds light on the origin of vascular DCs. In recent years, microRNAs (miRNAs) have been demonstrated to regulate several cellular functions, such as apoptosis, differentiation, development or proliferation. Although several cell types have been characterized extensively with regard to the miRNAs involved in their regulation, only few studies are available that focus on the role of miRNAs in DCs. Because an improved understanding of the regulation of DC functions would allow for new therapeutic options, research on miRNAs in DCs is required. The second part of this thesis focuses on the role of the miRNA cluster miR- 17~92 in DCs by exploring its functions in healthy and atherosclerotic mice. This thesis clearly demonstrates for the first time an anti-inflammatory and atheroprotective role for the miR17-92 cluster. A model for its mechanism is suggested. N2 - Atherosklerose ist eine chronisch-entzündliche Erkrankung der arteriellen Gefäßwand und zahlreiche Zellen des Immunsystems, wie zum Beispiel Monozyten, Makrophagen, T und B Zellen sind an der Entstehung und Entwicklung beteiligt. Aktuelle Forschungsergebnisse haben gezeigt, dass auch dendritische Zellen (DCs) zur Atherosklerose beitragen. DCs sind durch ihre Fähigkeit gekennzeichnet, Antigene zu erkennen, aufzunehmen, zu migrieren und andere Immunzellen, wie zum Beispiel T Zellen, zu aktivieren. Auch wenn alle DCs diese funktionellen Merkmale teilen, so sind sie in Bezug auf ihren Phänotyp oder Ursprung eine eher heterogene Gruppe. Zahlreiche Oberflächenmoleküle wurden in der Vergangenheit genutzt, um DCs in lymphatischen und nicht-lymphatischen Geweben zu beschreiben. Allerdings hat sich keines dieser Moleküle als spezifisch und unverwechselbar erwiesen. Die Expression von Oberflächenmolekülen ist sehr variabel und hängt nicht nur vom Aktivierungszustand der DCs, sondern auch vom umliegenden Gewebe ab. Dazu kommt, dass DCs in der Aorta, beziehungsweise im atherosklerotischen Plaque, von designierten Vorläuferzellen, aber auch von Monozyten abstammen können und DCs das Profil ihrer Oberflächenmoleküle, sowie ihre funktionellen Eigenschaften, mit anderen myeloiden Zellen wie Monozyten und Makrophagen teilen. Neuere Arbeiten haben damit begonnen das Repertoire an DCs in der Aorta von gesunden und atherosklerotischen Mäusen zu untersuchen. Da es naheliegt, dass verschiedene DC Untergruppen ganz bestimmte Funktionen ausüben, wird eine detaillierte Beschreibung vaskulärer DCs in der Forschung benötigt. Weil es hierzu allerdings bislang kaum Studien gibt, untersucht der erste Teil dieser Arbeit zum ersten Mal systematisch die in gesunden und atherosklerotischen Mäusen vorkommenden Gruppen an DCs. Sie beschreibt außerdem eine zuvor nicht beachtete DC-Untergruppe und gibt Aufschluss über den Ursprung vaskulärer DCs. In den letzten Jahren wurde gezeigt, dass microRNAs (mirRNAs) zahlreiche zelluläre Vorgänge wie Apoptose, Differenzierung, Entwicklung und Proliferation regulieren. Obwohl viele Zelltypen in Bezug auf die in ihrer Regulation eingebundenen mirRNAs charakterisiert wurden, gibt es nur wenige Studien, die sich mit der Rolle von mirRNAs in DCs beschäftigen. Der zweite Teil dieser Arbeit konzentriert sich auf die Rolle der miRNA Gruppe miR-17~92 in DCs und untersucht deren Rolle in gesunden und atherosklerotischen Mäusen. Diese Arbeit zeigt erstmals eine deutliche anti-inflammatorische und protektive Rolle dieser miRNA und schlägt ein Modell für die entdeckten Mechanismen vor. KW - Aorta KW - Maus KW - Zelle KW - Cluster KW - miRNS KW - Dendritische Zelle KW - Arteriosklerose KW - miR-17~92 KW - dendritic cells KW - atherosclerosis KW - mice KW - murine Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71683 ER - TY - JOUR A1 - Bangalore, Disha M. A1 - Heil, Hannah S. A1 - Mehringer, Christian F. A1 - Hirsch, Lisa A1 - Hemmen, Katharina A1 - Heinze, Katrin G. A1 - Tessmer, Ingrid T1 - Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases JF - Scientific Reports N2 - Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions. KW - atomic-force microscopy KW - base pairs KW - molecular structure KW - crystal structure KW - structural basis KW - repair KW - recognition KW - 8-oxoguanine KW - thymine KW - mismatches Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231338 VL - 10 ER - TY - JOUR A1 - Wippel, Carolin A1 - Maurer, Jana A1 - Fortsch, Christina A1 - Hupp, Sabrina A1 - Bohl, Alexandra A1 - Ma, Jiangtao A1 - Mitchell, Timothy J. A1 - Bunkowski, Stephanie A1 - Brück, Wolfgang A1 - Nau, Roland A1 - Iliev, Asparouh I. T1 - Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage JF - PLoS Pathogens N2 - Abstract Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage. Author Summary Bacterial meningitis is one of the most devastating brain diseases. Among the bacteria that cause meningitis, Streptococcus pneumoniae is the most common. Meningitis predominantly affects children, especially in the Third World, and most of them do not survive. Those that do survive often suffer permanent brain damage and hearing problems. The exact morphological substrates of brain damage in Streptococcus pneumoniae meningitis remain largely unknown. In our experiments, we found that the brain cortex of patients with meningitis demonstrated a loss of synapses (the contact points among neurons, responsible for the processes of learning and memory), and we identified the major pneumococcal neurotoxin pneumolysin as a sufficient cause of this loss. The effect was not direct but was mediated by the brain neurotransmitter glutamate, which was released upon toxin binding by one of the non-neuronal cell types of the brain – the astrocytes. Pneumolysin initiated calcium influx in astrocytes and subsequent glutamate release. Glutamate damaged the synapses via NMDA-receptors – a mechanism similar to the damage occurring in brain ischemia. Thus, we show that synaptic loss is present in pneumococcal meningitis, and we identify the toxic bacterial protein pneumolysin as the major factor in this process. These findings alter our understanding of bacterial meningitis and establish new therapeutic strategies for this fatal disease. KW - synapses KW - brain damage KW - astrocytes KW - neuronal dendrites KW - meningitis KW - glutamate KW - bacterial meningitis KW - neocortex Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130462 VL - 9 IS - 6 ER - TY - THES A1 - Batsching, Sophie Johanna T1 - Behavior under uncontrollable stress in \(Drosophila\) \(melanogaster\) - Learned Helplessness revisited T1 - Verhalten unter nicht kontrollierbarem Stress - Neubetrachtung der Erlernten Hilflosigkeit bei \(Drosophila\) \(melanogaster\) N2 - In order to select the appropriate behavior, it is important to choose the right behavior at the right time out of many options. It still remains unclear nowadays how exactly this is managed. To address this question, I expose flies (Drosophila melanogaster) to uncontrollable stress to study their behavior under restrictive circumstances by using the so-called shock box. Exposing animals to uncontrollable stress may have an impact on subsequent behavior and can last for some time. The animal learns that whatever it does, it cannot change the situation and therefore can develop something called learned helplessness. The term was first conceptualized by two American psychologists Maier and Seligman (1967), who discovered this phenomenon while doing experiments with dogs. They found out that dogs which are exposed to inescapable stress, later fail in a learning task (‘shuttle box’). In this work the walking patterns of three different types of experimental flies, walking in a small dark chamber, were evaluated. Using the triadic design (Seligman and Maier, 1967), flies were either exposed to electric shock randomly (yoked), could turn it off by being active (master) or did not receive punishment at all (control). Master flies were shocked whenever they sat for more than 0.9 seconds. At the same time yoked flies received a shock as well independent of what they were doing, to ensure the same amount of shocks received and to create random punishment pattern for the yoked group. With this so-called no-idleness paradigm flies were conditioned either 10 minutes, which resulted in a short (3 minutes) after-effect, or 20 minutes that turned out to be more stable (10 minutes). In a second part, the behavior during the 20 minute conditioning and a 10 minutes post-test was described in detail. Female flies of the yoked group developed lower activity levels, longer pauses and walked more slowly than master and control flies during conditioning. In the time after the shocks while still in the box, the yoked flies also reduced the frequency and duration of walking bouts as well as their walking speed. Additionally, they took more time to resume walking after the onset of an electric shock than master flies (escape latency) and turned out to make less pauses lasting between 1-1.5 seconds which supports the finding concerning the escape latency. Male flies, tested under the same conditions, showed a slightly weaker after-effect regarding the difference between master and yoked during conditioning and post-test when compared to female flies. When comparing the 20 minutes conditioning with subsequent 10 minutes test in the heat and the shock box in parallel, one finds the same effect: Flies which do not have control over the shocks, lower their activity, make less but longer pauses and walk more slowly than their respective master flies. Despite the similar effect of heat and shock on the flies, some differences between the devices occurred, which can partly be explained by different humidity conditions as well as by different surfaces within the chambers. When the control over the shocks is given back to the yoked flies, it takes them about seven minutes to realize it. One could also show that dopamine levels in the brain were reduced in comparison to flies which did not receive shocks. Yoked flies also were impaired in a place learning task (place learning) and their reaction to light (exit from the box towards the light) directly after conditioning. After characterizing the walking behavior in the chambers, the study deals with the question whether the effects observed in the chambers transfer to different environments. In free walk they only differed from flies which did not receive electric shocks and no effect of uncontrollability was transferred to courtship behavior. Handling as the cause could be excluded. Since handling could be exclude to be the cause of losing the effect, I assumed that the behavior shown in the boxes are context depend. Not only were the after-effects of inescapable shock subject of the current research also the impact of the rearing situation on the response to electric shock was investigated in the present study. Flies which grew up in a single-reared situation turned out to be less affected by inescapable stress in both sexes. In the next part, the first steps to unravel the neuronal underpinning were taken. A mutant – fumin – which is defective in the dopamine re-uptake transporter showed less reaction to inescapable foot shocks, while a mutant for the gene which encodes an adenylate cyclase (rutabaga2080) resulted in a good score during conditioning, but showed no stable after-effect. Downregulating the expression of the adenylate cyclase gene (rutabaga) in different parts of the mushroom bodies showed, that rutabaga is necessary in the α’β’-lobes for expressing the differences between master and yoked flies in the no-idleness paradigm. The study further confirmed previous findings, that rutabaga is needed in operant but not in classical conditioning. As a result, the study could show that not the stimulus itself causes the state of uncontrollability but the fact that the fly learned that it was not in control of the stimulus. This state turned out to be context and time dependent. N2 - Eine wichtige Aufgabe für ein Tier ist es, das passende Verhalten zur richtigen Zeit zu wählen. Heutzutage ist immer noch unklar, wie dieser Prozess exakt abläuft. Zur Untersuchung dieser Frage werden Fliegen (Drosophila melanogaster) in der so genannten Schockbox unkontrollierbarem Stress ausgesetzt um auf diesem Weg Verhaltenskontrolle unter stressigen und stark restriktiven Umständen untersuchen zu können. Wenn Tiere unkontrollierbarem Stress ausgesetzt sind, kann dieser Zustand sowohl langanhaltend sein als auch Einfluss auf das Folgeverhalten haben. Das Tier lernt, dass alle Aktivitäten, die es in dieser Situation unternimmt keinen Einfluss auf die Situation haben. Dadurch kann das Tier einen Zustand der sogenannten Erlernten Hilflosigkeit entwickeln. Dieser Begriff wurde von zwei amerikanischen Psychologen, Maier und Seligman (1976), geprägt, die dieses Phänomen während Experimenten mit Hunden entdeckten und konzipierten. Sie fanden heraus, dass Hunde, die unkontrollierbarem Stress ausgesetzt waren, an einer anschließend gestellten Lernaufgabe scheiterten (‚shuttle-box‘). Gegenstand der vorliegenden Arbeit ist es, das Laufmuster in einer schmalen und kleinen Kammer an drei verschiedenen Versuchsgruppen von Fliegen zu analysieren. Unter Verwendung des sogenannten triadischen Konzepts (Seligman and Maier, 1967) wurden die Fliegen drei unterschiedlichen Situationen ausgesetzt: Zufällige Elektroschocks (Yoked-Gruppe), durch Laufen abschaltbare Elektroschocks (Master-Gruppe) oder keine Bestrafung (Kontroll-Gruppe). Master-Fliegen wurden immer dann geschockt, wenn sie für länger als 0,9 Sekunden saßen. Unabhängig ihres Verhaltens erhielten die Yoked-Fliegen zeitgleich einen Schock um einen zufälligen Bestrafungsreiz zu generieren. Mit diesem so genannten ‚no-idleness‘ (nicht ruhen dürfen) Paradigma wurden die Fliegen entweder zehn Minuten oder 20 Minuten konditioniert. Während eine zehnminütige Konditionierung zu einem kurzen Nacheffekt führte (Nacheffekt von drei Minuten), stellte sich die zwanzigminütige Konditionierung als nachhaltiger heraus (Nacheffekt von zehn Minuten). In einem zweiten Teil der Arbeit wurde das Verhalten der Fliegen sowohl während der zwanzig Minuten andauernden Konditionierung also auch im nachfolgenden zehnminütigen Test im Detail beschrieben. Während der zwanzigminütigen Konditionierung zeigten weibliche Yoked-Fliegen eine geringere Aktivität, saßen länger und liefen langsamer als Master- oder Kontroll-Fliegen. In der Zeit nach den Schocks, zeigten sie immer noch eine verminderte Lauffrequenz sowie kürzere und langsamere Laufphasen. Zusätzlich benötigten sie länger um nach dem Einsetzten eines Elektroschocks loszulaufen (Flucht-Latenzzeit) und machten weniger Kurzpausen die zwischen 1 bis 1,5 Sekunden lang waren. Dies unterstützt das Ergebnis der verlängerten Flucht-Latenzzeit. Männchen, die unter gleichen Bedingungen getestet wurden, wiesen im Vergleich zu weiblichen Fliegen eine leicht abgeschwächte Reaktion bezüglich des Master-Yoked-Unterschieds auf. Wenn die Konditionierung mit dem anschließenden Test in der Schock- und der Hitzekammer gleichzeitig durchgeführt wurde, resultierte dies in vergleichbaren Ergebnissen: Fliegen, die keine Kontrolle über den Reiz haben, vermindern ihr Aktivitätslevel, sitzen seltener aber länger und laufen langsamer als die dazugehörigen Master-Fliegen. Neben der Tatsache, dass ein ähnlicher Effekt auftritt, weisen die Apparaturen dennoch kleine Unterschiede auf. Diese können zu Teilen mit den unterschiedlichen Luftfeuchtigkeitsniveaus als auch durch die Verschiedenheit der Laufoberfläche der jeweiligen Kammern erklärt werden. Wird den Fliegen die Kontrolle über die Schocks zurückgegeben, benötigen sie etwa sieben Minuten um dies zu erkennen. Zudem konnte gezeigt werden, dass die Dopaminkonzentration in den Köpfen, im Vergleich zu Tieren die keine Schocks erhalten haben, vermindert war. Yoked-Fliegen wiesen außerdem unmittelbar nach der Konditionierung Defekte im Ortslernen und in ihrer positiven Reaktion auf Licht auf. Nachdem das Laufverhalten innerhalb der Kammern ausführlich charakterisiert wurde, geht diese Studie darauf ein, ob die Effekte, die in den Kammern gemessen wurden, auch in anderen Umgebungen zu beobachten sind. Im freien Lauf unterschieden sie sich lediglich von Fliegen, die keine Schocks erhalten hatten und es sind keine Auswirkungen durch Kontrollverlust im Paarungsverhalten festzustellen. Da die Handhabung der Tiere als Grund für den Verlust des Nacheffektes ausgeschlossen werden konnte, lässt sich schlussfolgern, dass das Verhalten das in den Kammern gemessen wurde, kontextabhängig ist. Zusätzlich zur Untersuchung der Auswirkungen unausweichlichen Stresses, wurde der Einfluss, der Aufzuchtbedingungen auf die Stress-Antwort in der vorliegenden Studie untersucht. Fliegen, die einzeln aufgezogen wurden, weisen bei beiden Geschlechtern eine verminderte Antwort auf Stress auf. Im darauffolgenden Abschnitt wurden erste Schritte unternommen, um die neuronalen Grundlagen der Erlernten Hilflosigkeit zu untersuchen. Eine Mutante – fumin – die ein defektes Wiederaufnahmetransporter-Gen für Dopamin besitzt, wies eine verminderte Stressantwort auf. Während eine Mutante des Adenylatzyklasegens (rutabaga2080) normale Ergebnisse während der Konditionierung aufzeigten, war im Post-test kein signifikanter Nacheffekt messbar. Das Herunterregulieren des Adenylatcyclasengens (rutabaga), in verschiedenen Teilen der Pilzkörper, zeigte dass die Expression von rutabaga in den α’β’-Loben für die Entwicklung der Erlernten Hilflosigkeit im no-idleness Paradigma benötigt wird. Zudem konnten vorangegangene Studien bestätigt werden, die rutabaga eine Rolle im operanten Lernen jedoch nicht im klassischen Lernen zuordnen. Als Fazit zeigt die Studie, dass nicht der Stressor selbst, sondern die Unkontrollierbarkeit des Stressors der Grund für die Entwicklung der Erlernten Hilflosigkeit darstellt und das Phänomen, innerhalb der hier gewählten Zeitspanne (20 Minuten Stress), kontextabhängig zu sein scheint. KW - Taufliege KW - Stress KW - Verhalten KW - Gelernte Hilflosigkeit KW - Erlernte Hilflosigkeit KW - Learned Helplessness KW - Behavior KW - Drosophila melanogaster Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145416 ER - TY - JOUR A1 - Makbul, Cihan A1 - Kraft, Christian A1 - Grießmann, Matthias A1 - Rasmussen, Tim A1 - Katzenberger, Kilian A1 - Lappe, Melina A1 - Pfarr, Paul A1 - Stoffer, Cato A1 - Stöhr, Mara A1 - Wandinger, Anna-Maria A1 - Böttcher, Bettina T1 - Binding of a pocket factor to Hepatitis B virus capsids changes the rotamer conformation of Phenylalanine 97 JF - Viruses N2 - (1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses. KW - Hepatitis B Virus KW - pocket factor KW - Triton X 100 KW - envelopment KW - maturation signal KW - single strand blocking KW - electron cryo-microscopy KW - isothermal titration calorimetry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248565 SN - 1999-4915 VL - 13 IS - 11 ER - TY - THES A1 - Beitzinger, Christoph T1 - Binding-, Blocking- and Translocation-Processes Concerning Anthrax-Toxin and Related Bacterial Protein-Toxins of the AB7-Family T1 - Charakterisierung von Bindungs-, Blockierungs- und Translokationsprozessen am Anthrax-Toxin und verwanten Toxinen der AB7-Gruppe N2 - Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding. N2 - Bakterielle Protein-Toxine gehören zu den wirksamsten bekannten Toxinen. In vielfältigen Variationen findet man sie in allen Bereichen des Lebens. Einige werden von den Bakterien während einer Infektion freigesetzt und übernehmen einen wichtigen Part in der Pathogenität. Andere werden zu Verteidigungszwecken sekretiert und können in verdorbenen Lebensmitteln gefunden werden. Was die Wirkung binärer Toxine der AB7-Gruppe angeht, so gehören diese zu den potentesten und gefährlichsten Giften weltweit. Selbst kleine Mengen dieser Proteine können schwerste Symptome während einer Infektion mit Bakterien der Gattung Clostridium oder Bacillus verursachen. Abgesehen von der Bedrohung die durch die Toxine ausgeht, zeichnen sie sich durch einen einzigartigen Intoxikationsmechanismus aus. AB7-Toxine sind aus einer porenformenden Domäne B, die als eine Art molekulare Injektionskanüle fungiert, und enzymatisch aktiven Proteinen A zusammengesetzt. Der komplexe Wirkmechanismus ermöglicht es nicht nur Zellen in höchst effektiver Weise abzutöten und sollte deswegen zu Behandlungszwecken untersucht werden, sondern könnte auch als molekulares Werkzeug umfunktioniert werden, um spezielle Zellen mit gewünschten Proteinen zu beladen. Für beide Zwecke (Blockierung und gezielter Transport) ist die Bindung an, und der Transport durch die porenformende Domäne von größter Bedeutung. Die Struktur der B-Domäne ist wichtig um tiefere Einsicht in den Transportprozess der binären Toxine zu ermöglichen. Leider ist es bisher nicht gelungen die Kristallstruktur des Membranproteins protective antigen (PA) von Anthrax oder irgendeiner anderen Bindedomäne eines AB7-Toxins zu lösen. Deshalb wurde in dieser Arbeit ein elektrophysiologischer Ansatz zur Bestimmung der biophysikalischen Konstanten des Prozesses gewählt, die Black-lipid-Bilayer Methode. Zusätzliche Versuche an Zellen und mit Mutanten der Proteine dienen zur Absicherung der in vitro Ergebnisse. Im ersten Teil der Arbeit wird der Translokationsmechanismus, und die mögliche Nutzung dessen als molekulares Werkzeug näher behandelt. Dies erfolgte durch Bindungsstudien an PA und C2II mit Effektoren (sowohl aus der AB7-Familie, als auch nicht näher verwandt). In Kapitel 2 wird beschrieben, dass ein His-Tag es EDIN (von S. aureus) und anderen Effektoren ermöglicht, dass ein Transport durch PA-Poren in vitro und in vivo stattfindet (nicht aber durch C2II). Ebenfalls konnte eine starke Abweichung in der bekannten Spannungsabhängigkeit von PA festgestellt werden, die den Prozess eindeutig mit den Membranpotentialen in Verbindung bringt, die häufig eine wichtige Rolle im Metabolismus spielen. Kapitel 3 fasst zusammen, dass Bindungspartner von PA bestimmte Motive beinhalten, die von Substraten wie Ionen in Elektrolyten, über kleine Moleküle, bis hin zu komplexen Proteinen reichen. Diese Erkenntnisse könnten genutzt werden um Blockersubstanzen zur Behandlung von Anthrax, oder Tags zur Aufnahme durch Anthrax zu designen. Neueste Befunde zu Homologien und Unterschieden zwischen den Komponenten der binären Toxine sind der Kern von Kapitel 4, in dem die Kreuzreaktivität von Anthrax und C2-Toxin analysiert wurde. Die enthaltenen Daten ermöglichen einen tieferen Einblick in die verschiedenen Stufen der Bindung und Translokation des edema factor (EF), des lethal factor (LF) und von C2I an und durch PA und C2II. Im zweiten Teil rückt die Blockierung der Intoxikation in den Fokus. Die Analyse speziell designter Blockersubstanzen für PA wird in Kapitel 5 behandelt. Diese formen einen Porenverschluss, der weiteren Transport von Toxinkomponenten verhindert. Eine besondere Bedeutung könnten diese Substanzen im Zusammenhang mit Multiresitenz bei Anthrax Stämmen (vermutlich in Russland als biologische Waffe hergestellt) zur Verhinderung von Symptomen und der Verlängerung der Zeit spielen, die man hat um neue Antibiotika zu erzeugen. Kapitel 6 beschreibt zum ersten Mal die Blockierung von PA-Poren mittels eines Anti-His Antikörpers von der trans-Seite aus. Interessanterweise trat diese Ionenstärke abhängige Blockierung, auch bei einer Histidin zu Glycin Mutation an der Stelle 310 (vermutete Bindeposition) auf. KW - Bacillus anthracis KW - Translokation KW - Toxin KW - Protective antigen KW - Bindungsprozess KW - Black-lipid-bilayer KW - Bacillus anthracis KW - protective antigen KW - Anthrax Toxin KW - translocation Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70052 ER - TY - JOUR A1 - Bieber, Michael A1 - Schuhmann, Michael K. A1 - Bellut, Maximilian A1 - Stegner, David A1 - Heinze, Katrin G. A1 - Pham, Mirko A1 - Nieswandt, Bernhard A1 - Stoll, Guido T1 - Blockade of platelet glycoprotein Ibα augments neuroprotection in Orai2-deficient mice during middle cerebral artery occlusion JF - International Journal of Molecular Sciences N2 - During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte–platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2\(^{-/-}\)) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα–von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2\(^{-/-}\) mice. During ischemia–reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2\(^{-/-}\) mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke. KW - ischemic penumbra KW - Orai2 KW - glycoprotein receptor Ibα KW - ischemic stroke KW - thrombo-inflammation KW - middle cerebral artery occlusion Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286038 SN - 1422-0067 VL - 23 IS - 16 ER - TY - JOUR A1 - Göbel, Kerstin A1 - Pankratz, Susann A1 - Asaridou, Chloi-Magdalini A1 - Herrmann, Alexander M. A1 - Bittner, Stefan A1 - Merker, Monika A1 - Ruck, Tobias A1 - Glumm, Sarah A1 - Langhauser, Friederike A1 - Kraft, Peter A1 - Krug, Thorsten F. A1 - Breuer, Johanna A1 - Herold, Martin A1 - Gross, Catharina C. A1 - Beckmann, Denise A1 - Korb-Pap, Adelheid A1 - Schuhmann, Michael K. A1 - Kuerten, Stefanie A1 - Mitroulis, Ioannis A1 - Ruppert, Clemens A1 - Nolte, Marc W. A1 - Panousis, Con A1 - Klotz, Luisa A1 - Kehrel, Beate A1 - Korn, Thomas A1 - Langer, Harald F. A1 - Pap, Thomas A1 - Nieswandt, Bernhard A1 - Wiendl, Heinz A1 - Chavakis, Triantafyllos A1 - Kleinschnitz, Christoph A1 - Meuth, Sven G. T1 - Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells JF - Nature Communications N2 - Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein–kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders. KW - blood coagulation KW - factor XII KW - neuroinflammation KW - dendric cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165503 VL - 7 IS - 11626 ER - TY - JOUR A1 - Tilstam, Pathricia V. A1 - Gijbels, Marion J. A1 - Habbeddine, Mohamed A1 - Cudejko, Celine A1 - Asare, Yaw A1 - Theelen, Wendy A1 - Zhou, Baixue A1 - Döring, Yvonne A1 - Drechsler, Maik A1 - Pawig, Lukas A1 - Simsekyilmaz, Sakine A1 - Koenen, Rory R. A1 - de Winther, Menno P. J. A1 - Lawrence, Toby A1 - Bernhagen, Jürgen A1 - Zernecke, Alma A1 - Weber, Christian A1 - Noels, Heidi T1 - Bone Marrow-Specific Knock-In of a Non-Activatable Ikkα Kinase Mutant Influences Haematopoiesis but Not Atherosclerosis in Apoe-Deficient Mice JF - PLOS ONE N2 - Background: The Ikkα kinase, a subunit of the NF-kappa B-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikk alpha mutant knock-in on haematopoiesis and atherosclerosis in mice. Methods and Results: Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (Ikkα(AA/AA) Apoe(-/-)) or with Ikkα(+/+) Apoe(-/-) BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in Ikkα(AA/AA) Apoe(-/-) BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of Ikkα(AA/AA) Apoe(-/-) vs Ikkα(+/+) Apoe(-/-) BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from Ikkα(AA/AA) Apoe(-/-) vs Ikkα(+/+) Apoe(-/-) mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. Conclusion: Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of Ikkα AA mutant BM did not affect atherosclerosis in Apoe(-/-) mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach. KW - NF-KAPPA-B KW - regulatory T cells KW - indoleamine 2,3-dioxygenase KW - dendritic cells KW - gene expression KW - increases atherosclersosis KW - receptor KW - inhibition KW - pathway KW - beta Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117450 VL - 9 IS - 2 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Knobloch, Gunnar A1 - Hoffmann, Axel A1 - Fedorchenko, Oleg A1 - Odersky, Andrea A1 - Lamprecht, Anna‐Karina A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin JF - FEBS Letters N2 - Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca\(^{2+}\)‐CaM complex. KW - calmodulin KW - chronophin KW - CIB1 KW - haloacid dehalogenase KW - pyridoxal phosphatase KW - vitamin B6 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217963 VL - 594 IS - 13 SP - 2099 EP - 2115 ER - TY - JOUR A1 - Calebiro, Davide A1 - Maiellaro, Isabella T1 - cAMP signaling microdomains and their observation by optical methods JF - Frontiers in Cellular Neuroscience N2 - The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains. KW - G protein-coupled receptor KW - cyclic AMP KW - signaling microdomain KW - fluorescence resonance energy transfer KW - neurons Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118252 SN - 1662-5102 VL - 8 ER - TY - JOUR A1 - Maiellaro, Isabella A1 - Lohse, Martin J. A1 - Kitte, Robert J. A1 - Calebiro, Davide T1 - cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons JF - Cell Reports N2 - The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons. KW - cAMP KW - synaptic plasticity KW - PDE KW - octopamine KW - FRET KW - active zone KW - dunce KW - GPCR Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162324 VL - 17 IS - 5 ER - TY - THES A1 - Gotru, Sanjeev Kiran T1 - Cation Homeostasis in Platelets T1 - Kationen-Homöostase in Thrombozyten N2 - Divalent cations are important second messengers triggering various signal transduction events in platelets. Whereas calcium channel blockers have an established antithrombotic effect and the regulation of Ca2+ homeostasis has been elucidated in platelets, the molecular regulation of Mg2+ and Zn2+ homeostasis has not been investigated so far. In the first part of the thesis, the role of -type serine-threonine kinase linked to transient receptor potential cation channel, subfamily M, member 7 (TRPM7) in platelets was investigated. Using Trpm7R/R mice with a point mutation deleting the kinase activity, we showed that the TRPM7 kinase regulates platelet activation via immunoreceptor tyrosine-based activation motif (ITAM), hem(ITAM) and protease-activated receptor (PAR) signaling routes. Furthermore, Trpm7R/R mice were protected from in vivo thrombosis and stroke, thus establishing TRPM7 kinase as a promising anti-thrombotic target. In the second part of the thesis, the role of TRPM7 channel in a megakaryocyte (MK) and platelet-specific knockout mouse, Trpm7fl/fl-Pf4Cre, was investigated. Here, we observed that depending on the type of stimulation, Trpm7fl/fl-Pf4Cre platelets showed either enhanced or inhibited responses. Although Trpm7fl/fl-Pf4Cre mice were thrombocytopenic, no differences to wildtype mice were observed in models of in vivo thrombosis and stroke. The above two studies highlight that inhibition of TRPM7 kinase but not the channel itself (in MKs and platelets) may be a promising anti-thrombotic strategy. Besides TRPM7, we investigated the role of magnesium transporter 1 (MAGT1) in platelet Mg2+ homeostasis and found that MAGT1 primarily regulates receptor-operated calcium entry (ROCE) in platelets specifically upon GPVI activation. This physiological crosstalk is triggered by protein kinase C (PKC) isoforms. Platelets from Magt1-/y mice hyper-reacted to GPVI and thromboxane A2 (TXA2) receptor stimulation in vitro. Consequently, Magt1-/y platelets were found to be pro-thrombotic in disease models of thrombosis and stroke. To compare platelet ITAM-signaling to the immune system, we further investigated the role of MAGT1 in T and B cells. We described the primary role of MAGT1 in mice under pathogen-free conditions. Magt1-/y B cells showed dysregulated Mg2+ and Ca2+ homeostasis upon B-cell receptor activation, thereby altering Syk, LAT, phospholipase C (PLC)2 and PKC phosphorylation. In contrast to human MAGT1-deficient T cells, development and effector functions of mouse Magt1-/y T cells showed no alterations. Finally, in the last part of the thesis, we described methods to measure intracellular free zinc [Zn2+]i in human and mouse platelets with storage pool disease (SPD). We propose to measure the [Zn2+]i status in SPD platelets as a relatively easy diagnostic to screen platelet granule abnormalities. N2 - Zweiwertige Kationen sind wichtige sekundäre Botenstoffe, welche verschiedene Signaltransduktionsereignisse in Thrombozyten initiieren. Zwar wurde die Regulation der Ca2+Homöostase in Blutplättchen bereits aufgeklärt und der Einsatz von Calciumkanalblockern zur antithrombotischen Therapie ausführlich diskutiert, die molekulareRegulation der Mg2+und Zn2+Homöostase in Thrombozyten und Megakaryozyten (MK) wurdebisher jedoch nicht untersucht.Im ersten Teil dieser Thesis wurde die Rolle der -Typ Serin-Threonin Kinase des transienten Rezeptortyp Kation Kanals, Unterfamilie M, 7 (TRPM7) in Thrombozyten untersucht. Unter Verwendung von Trpm7R/RMäusen mit einer Punktmutation in der Kinasedomäne, welche die Aktivität der Kinase blockiert, konnten wir zeigen, dass die TRPM7-Kinase die Thrombozytenaktivierung über Immunorezeptor-Tyrosin-basierte Aktivierungsmotive (ITAM), Hem(ITAM) und Protease-aktivierte Rezeptoren (PAR) reguliert. Trpm7R/RMäuse waren vor in vivoThrombose und Schlaganfall geschützt, was die TRPM7 Kinase als vielversprechendes antithrombotisches Zielprotein etabliert.Im zweiten Teil wurde die Rolle des TRPM7 Kanals in einer Megakaryozyten (MK)-und Plättchen-spezifischen Knockout Maus(Trpm7fl/fl-Pf4Cre) untersucht. Wir konnten zeigen, dass Trpm7fl/fl-Pf4CrePlättchen je nach Art der Stimulation entweder erhöhte oder verminderte Reaktionen zeigten. Obwohl Trpm7fl/fl-Pf4Cre-Mäuse thrombozytopen waren, wurden keine Unterschiede in in vivoThrombosemodellen und Schlaganfall beobachtet. Diese Studien heben hervor, dass die Hemmung der TRPM7 Kinase, aber nicht die des Kanal selbst (inMKs und Plättchen), eine vielversprechende anti-thrombotische Therapie sein könnte. Neben TRPM7 untersuchten wir die Rolle von Magnesium Transporter 1 (MAGT1)in der Mg2+-Homöostase in Thrombozyten und konnten zeigen, dass MAGT1 primär den Rezeptor-gesteuerten Calciuminflux (ROCE) spezifisch nach GPVI Aktivierung reguliert. Dieser physiologische Crosstalk wird durch Proteinkinase C (PKC) Isoformen vermittelt. Thrombozyten von Magt1-/yMäusen reagierten in vitrohyperreaktiv auf GPVI und ThromboxanA2(TXA2) Rezeptor Stimulation. Dementsprechend konnte auch gezeigt werden, dass Magt1-/yPlättchen in Modellen von Thrombose und Schlaganfall pro-thrombotisch wirkten.Um die ITAM-Signalübertragung in Thrombozyten mit der in T und B Zellen zu vergleichen, untersuchten wir die Rolle von MAGT1 in Immunzellen. Wir überprüften die Rolle von MAGT1 in Mäusen unter pathogen-freien Bedingungen. Magt1-/yB Zellen zeigten eine dysregulierte Mg2+und Ca2+Homöostase nach Aktivierung des B Zell Rezeptors, wodurch Syk, LAT, PLCγ2 und PKC Phosphorylierung beeinflusst wurde. Im Gegensatz zu menschlichen MAGT1-defizienten T Zellen, zeigten Magt1-/yT Zellen keine Veränderungen in Entwicklung und Effektorfunktion. Schließlich beschrieben wir im letzten Teil der Arbeit Methoden zur Messung des intrazellulären freien Zinks [Zn2+]iin humanen und murinen Thrombozyten mit Storage-Pool-Defekt (SPD). Wir unterbreiten in dieser Thesis, den [Zn2+]iStatus in SPD Thrombozyten zu messenum nachAnomalien in den Thrombozyten-Granula zu suchen. KW - Thrombozyt KW - Kationen-Homöostase KW - Homöostase KW - Cation Homeostasis KW - Platelets Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176616 ER - TY - THES A1 - Rabie, Tamer T1 - Cellular regulation of platelet glycoprotein VI : in vivo and in vitro studies in mice T1 - Zelluläre Regulation von Plättchen Glykoprotein VI : in vivo und in vitro Studien in der Maus N2 - Platelet interaction with the subendothelium is essential to limit blood loss after tissue injury. However, upon rupture of atherosclerotic plaques, this interaction may result in blood vessel occlusion leading to life threatening diseases such as myocardial infarction or stroke. Among the subendothelial matrix proteins, collagen is considered to be the most thrombogenic component as it directly activates platelets. Platelets interact with collagen, either indirectly through glycoprotein (GP) Ib-V-IX receptor complex, or directly through the major collagen receptor on the platelet surface, GPVI. The work presented here focused on studying the cellular regulation of GPVI. In addition, a possible role for GPVI in thrombus formation induced by atherosclerotic plaque material was investigated and it was found that GPVI plays an important role in this process. Using a recently published mitochondrial injury model, it was found that GPVI contains a cleavage site for a platelet-expressed metalloproteinase. Further studies showed that platelet activation by CRP, or thrombin induced down-regulation of GPIb, but not GPVI. In parallel, cellular regulation of GPV was studied and it was found that GPV is cleaved in vitro by the metalloproteinase ADAM17. In previous studies it was shown that injection of mice with the anti-GPVI mAb, JAQ1, induces GPVI down-regulation, which is associated with a strong, but transient, thrombocytopenia. Using new anti-GPVI mAbs, which bind different epitopes on the receptor, it is shown in this study that GPVI down-regulation occurs in an epitope-independent manner. Further experiments showed that antibody treatment induces a transient, but significant increase in bleeding time. Using different genetically modified mice, it is shown that, upon antibody injection, GPVI is both, shed from the platelet surface and internalized into the platelet. Signaling through the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR chain is essential for both processes, while LAT and PLC2 are essential for the shedding process only. Antibody-induced increase in bleeding time and thrombocytopenia were absent in LAT deficient mice, showing that it is possible to uncouple the associated side effects from the down-regulation process. As antibody-induced GPVI internalization still occurs in LAT and PLC2 deficient mice, this suggests a novel signaling pathway downstream of GPVI that has not been described so far. N2 - Plättchen Interaktion mit dem Subendothel ist für die Blutstillung essentiell. Dies kann jedoch nach dem Aufbrechen atherosklerotischer Plaques zu lebensbedrohlicher Erkrankungen wie Infarkt oder Schlaganfall führen. Kollagen, welches die Plättchen dirket aktiviert, ist der thrombogenste Bestandteil der Extrazellularmatrix (EZM). Die Bindung zwischen Plättchen und Kollagen wird sowohl indirekt durch den Glykoprotein (GP) Ib-V-IX Rezeptorkomplex, als auch direkt durch den Kollagenrezeptor GPVI, auf der Plättchenoberfläche vermittelt. In der vorliegenden Arbeit wurde die zelluläre Regulation von GPVI untersucht. Des Weiteren wurde die Rolle von GPVI in durch atheroklerotisches Plaquematerial induzierter Thrombusbildung studiert. Hierbei wurde festgestellt, dass GPVI eine wichtige Funktion in diesem Prozess spielt. Mittels eines jüngst publizierten mitochondrialen Verletzungsmodels, konnte gezeigt werden, dass GPVI eine Erkennungsstelle für eine in den Plättchen exprimierte Metalloproteinase besitzt. Mehrere Versuche haben gezeigt, dass Plättchenaktivierung durch CRP, und Thrombin zur Runterregulierung von GPIb aber nicht von GPVI führt. Parallellaufende Untersuchungen zeigten, dass GPV durch die Metalloproteinase ADAM17 in vitro abgespalten wird. Vorherige Studien ergaben, dass die in vivo Behandlung von Mäusen mit dem anti-GPVI Antikörper, JAQ1, zur Runterregulierung des Rezeptors führt. Dieses ist mit einer starken, transienten Thrombozytopenie assoziiert. Mittels neu generierte anti-GPVI Antikörper (JAQ2, 3), die unterschiedliche Bindungsstellen auf GPVI erkennen, konnte demonstriert werden, dass die Antikörper vermittele GPVI Runterregulierung Epitop unabhängig ist. Weitere Untersuchungen ergaben, dass Anitkörperinjektion eine transiente Erhöhung der Blutungszeit verursacht. Mittels genetisch modifizierter Mäuse konnte dargestellt werden, dass die Antikörpergabe GPVI sowohl von der Plättchenoberfläche abgespalten, als auch internalisiert wird. Während die Signaltransduktion durch das ITAM Motif der FcR Kette essentiell für beide Prozesse ist, sind LAT und PLC2 nur für das Abspalten wichtig. Antikörper induzierte Erhöhung der Blutungszeit und Thrombozytopenie sind abwesend in LAT-defizienten Mäuse, was zeigt, dass möglicherweise die GPVI Runterregulierung von den assoziierten Nebenwirkungen zu trennen ist. Da die GPVI Runterregulierung in LAT und –PLC2 defizienten Mäusen weiterhin stattfindet, zeigt dies einen neuen GPVI Signalweg, der bisher noch nicht beschrieben wurde. KW - Maus KW - Thrombozyt KW - Glykoproteine KW - Regulation KW - Biologie KW - Plättchen KW - Maus KW - Thrombose KW - Kardiovaskulär KW - maus KW - platelets KW - thrombosis KW - cardiovascular Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-14267 ER - TY - THES A1 - Lorenz, Viola T1 - Cellular regulation of the hemITAM-coupled platelet receptor C-type lectin-like receptor 2 (CLEC-2): In vitro and in vivo studies in mice T1 - Zelluläre Regulation des hemITAM-gekoppelten Thrombozytenrezeptors C-type lectin-like receptor 2 (CLEC-2): In vitro und in vivo Studien in Mäusen N2 - Platelet aggregation at sites of vascular injury is essential to limit posttraumatic blood loss, but may also cause acute ischemic disease states such as myocardial infarction or stroke. Stable thrombus formation requires a series of molecular events involving platelet receptors and intracellular signal transduction, which contribute to adhesion, activation and aggregation of platelets. In this thesis, the cellular regulation of platelet surface receptors and their involvement in thrombus formation was investigated using genetically modified mice. In the first part of the study, the functional relevance of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and of the recently identified hemITAM-bearing C-type lectin-like receptor 2 (CLEC-2) for in vivo thrombus formation was analyzed. Megakaryocyte/ platelet-specific CLEC-2 knock out mice displayed a defective lymphatic development and were protected from occlusive arterial thrombus formation. These phenotypes were more pronounced in mice with a GPVI/CLEC-2 double deficiency. Hemostasis was not compromised in CLEC-2 or GPVI single-deficient animals, as they showed only mildly prolonged tail bleeding times. Combined depletion of both receptors resulted in markedly prolonged bleeding times revealing an unexpected redundant function of the two receptors in hemostasis as well as thrombosis. These findings might have important implications for the development of anti-CLEC-2/ anti-GPVI agents as therapeutics. In the second part, mechanisms underlying the cellular regulation of CLEC-2 were studied. Previous studies have shown that injection of the anti-CLEC-2 antibody INU1 results in complete immunodepletion of platelet CLEC-2 in mice, which is preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. It is demonstrated that INU1-induced CLEC-2 immunodepletion occurs through Src family kinase (SFK)-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with spleen tyrosine kinase (Syk) deficiency, INU1-induced CLEC-2 internalization/ degradation was fully preserved, whereas the associated thrombocytopenia was largely prevented. These results show that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia. Since INU1 IgG induced a pronounced thrombocytopenia, the in vivo effects of monovalent INU1 F(ab) fragments were analyzed. Very unexpectedly, injection of the F(ab) fragments resulted in widespread thrombus formation leading to persistent neurological deficits of the animals. This intravascular thrombus formation is the result of CLEC-2-dependent platelet activation and aggregation. The mechanism underlying the thrombus formation is still unknown and depends potentially on binding of a yet unidentified ligand to F(ab)-opsonized CLEC-2 on platelets. N2 - Die Aggregation von Thrombozyten ist ein essentielle Prozess, um Blutungen nach einer Gefäßverletzung zu stoppen. Sie kann aber auch zu akuten thrombotischen Erkrankungen, wie Herzinfarkt und Schlafanfall, führen. Die Bildung eines stabilen Thrombus ist ein dynamischer Prozess, der ein definiertes Zusammenspiel von thrombozytären Rezeptoren und intrazellulären Signalen benötigt, die zur Adhäsion, Aktivierung und Aggregation der Thrombozyten beitragen. In der hier vorliegenden Dissertation wurde zellulär Regulation von thrombozytären Oberflächenrezeptoren und ihre Beteiligung an der Thromben-Bildung mittels genetisch veränderter Mäuse untersucht. Im ersten Teil der Arbeit wurde die funktionelle Relevanz des immunoreceptor tyrosine-based activation motif (ITAM)-gekoppelten Kollagenrezeptors GPVI und des vor kurzem entdeckten hemITAM-gekoppelten C-type lectin-like receptor 2 (CLEC-2) für die in vivo Thromben-Bildung charakterisiert. Mäuse mit einer Megakaryozyten/ Thrombozyten-spezifischen CLEC-2-Defizienz weisen Defekte in der Entwicklung ihrer lymphatischen Gefäße auf und sind vor arteriellen Gefäßverschlüssen geschützt. Dieser Phänotyp war in Mäusen mit einer Defizient GPVI und CLEC-2 verstärkt. GPVI oder CLEC-2-defiziente Mäuse zeigen eine normale Hämostase, da ihre Schwanzblutungszeiten nur minimal verlängert waren. Die gleichzeitige Defizienz beider Rezeptoren verlängerte die Blutungszeiten der Tiere allerdings erheblich. Das spricht dafür, dass beiden Rezeptoren eine unerwartete redundante Funktion sowohl in der Hämostase als auch während der pathologischen Thromben-Bildung haben. Diese Ergebnisse könnten für die Entwicklung neuer therapeutischen Wirkstoffen, die gegen GPVI und/ oder CLEC-2 gerichtet sind, bedeutsam sein. Im zweiten Teil der Arbeit wurde die zelluläre Regulation von CLEC-2 untersucht. Frühere Studien haben gezeigt, dass die Behandlung von Mäusen mit dem gegen CLEC-2 gerichteten Antikörper INU1 zu einem spezifischen Verlust des Rezeptors in zirkulierenden Thrombozyten führt. Dieser Prozess, der „Immunodepletion“ genannt wird, ist von einer Thrombozytopenie begleitet, die das therapeutische Potential eines solchen Ansatzes reduziert. Im Verlauf der Arbeit konnte gezeigt werden, dass die INU1-abhängige Immunodepletion in vitro und in vivo mittels src family kinase (SFK)-abängiger Internalisierung geschieht. Der Internalisierung ist vermutlich ein intrazellulärer Abbau nachgeschaltet. In Mäusen mit einer spleen tyrosine kinase (Syk) Defizienz war die INU1-abhängige Thrombozytopenie zum größten Teil verhindert, aber die Herabregulation durch Internalisierung von CLEC-2 blieb erhalten. Diese Ergebnisse zeigen, dass CLEC-2 in vitro und in vivo durch Internalisierung von der Thrombozyten-Oberfläche herabreguliert werden kann. Darüber hinaus kann dies von der Antikörper-vermittelten Thrombozytopenie mechanistisch entkoppelt werden. Da die Injektion von INU1 IgG eine starke Thrombozytopenie in der Tieren verursacht, sollen die in vivo Effekte von monovalenten INU1 F(ab) Fragmenten getestet werden. Unerwarteter weise führte die Injektion dieser F(ab) Fragmente zu einer systemischen Thromben-Bildung, die unter anderem neurologische Defizite in den Tieren auslösten. Diese intravaskuläre Thromben-Bildung ist ein Ergebnis von CLEC-2-abhängier Thrombozyten Aktivierung und Aggregation. Der Mechanismus, der dieser Thromben-Bildung zugrunde liegt, ist bisher noch nicht aufgeklärt. Möglicherweise wird er durch die Bindung eines unbekannten Liganden an das F(ab)-gebunde CLEC-2 auf Thrombozyten ausgelöst. KW - Thrombozytenaggregation KW - CLEC-2 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116724 ER - TY - JOUR A1 - Koelmel, Wolfgang A1 - Kuper, Jochen A1 - Kisker, Caroline T1 - Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem JF - Scientific Reports N2 - Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy. KW - structural biology KW - X-ray crystallography Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261644 VL - 11 IS - 1 ER - TY - JOUR A1 - Donat, Ulrike A1 - Rother, Juliane A1 - Schäfer, Simon A1 - Hess, Michael A1 - Härtl, Barbara A1 - Kober, Christina A1 - Langbein-Laugwitz, Johanna A1 - Stritzker, Jochen A1 - Chen, Nanhai G. A1 - Aguilar, Richard J. A1 - Weibel, Stephanie A1 - Szalay, Alandar A. T1 - Characterization of Metastasis Formation and Virotherapy in the Human C33A Cervical Cancer Model JF - PLoS ONE N2 - More than 90% of cancer mortalities are due to cancer that has metastasized. Therefore, it is crucial to intensify research on metastasis formation and therapy. Here, we describe for the first time the metastasizing ability of the human cervical cancer cell line C33A in athymic nude mice after subcutaneous implantation of tumor cells. In this model, we demonstrated a steady progression of lumbar and renal lymph node metastases during tumor development. Besides predominantly occurring lymphatic metastases, we visualized the formation of hematogenous metastases utilizing red fluorescent protein (RFP) expressing C33A-RFP cells. RFP positive cancer cells were found migrating in blood vessels and forming micrometastases in lungs of tumor-bearing mice. Next, we set out to analyze the influence of oncolytic virotherapy in the C33A-RFP model and demonstrated an efficient virus-mediated reduction of tumor size and metastatic burden. These results suggest the C33A-RFP cervical cancer model as a new platform to analyze cancer metastases as well as to test novel treatment options to combat metastases. KW - metastasis KW - renal cancer KW - oncolytic viruses KW - lymph nodes KW - kidneys KW - lung and intrathoracic tumors KW - secondary lung tumors KW - cancer treatment Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119674 SN - 1932-6203 VL - 9 IS - 6 ER - TY - JOUR A1 - Drechsler, Johannes A1 - Groetzinger, Joachim A1 - Hermanns, Heike M. T1 - Characterization of the Rat Oncostatin M Receptor Complex Which Resembles the Human, but Differs from the Murine Cytokine Receptor JF - PLoS One N2 - Evaluation of a pathophysiological role of the interleukin-6-type cytokine oncostatin M (OSM) for human diseases has been complicated by the fact that mouse models of diseases targeting either OSM or the OSM receptor (OSMR) complex cannot fully reflect the human situation. This is due to earlier findings that human OSM utilizes two receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) (type I) and gp130/OSMR (type II), both with wide expression profiles. Murine OSM on the other hand only binds to the gp130/OSMR (type II) receptor complex with high affinity. Here, we characterize the receptor usage for rat OSM. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant and stably transfected Ba/F3 cells) we can clearly show that rat OSM surprisingly utilizes both, the type I and type II receptor complex, therefore mimicking the human situation. Furthermore, it displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the Jak/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, rat disease models would allow evaluation of the relevance of OSM for human biology. KW - in vitro KW - leukemia-inhibitory factor KW - ciliary neurotrophic factor KW - T cell development KW - swiss model KW - fetal liver KW - interleukin-6-type cytokines KW - rheumatoid arthritis KW - signal transduction KW - growth regulator Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133879 VL - 7 IS - 8 ER - TY - JOUR A1 - Drechsler, Johannes A1 - Grötzinger, Joachim A1 - Hermanns, Heike M. T1 - Characterization of the Rat Oncostatin M Receptor Complex Which Resembles the Human, but Differs from the Murine Cytokine Receptor N2 - Evaluation of a pathophysiological role of the interleukin-6-type cytokine oncostatin M (OSM) for human diseases has been complicated by the fact that mouse models of diseases targeting either OSM or the OSM receptor (OSMR) complex cannot fully reflect the human situation. This is due to earlier findings that human OSM utilizes two receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) (type I) and gp130/OSMR (type II), both with wide expression profiles. Murine OSM on the other hand only binds to the gp130/OSMR (type II) receptor complex with high affinity. Here, we characterize the receptor usage for rat OSM. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant and stably transfected Ba/F3 cells) we can clearly show that rat OSM surprisingly utilizes both, the type I and type II receptor complex, therefore mimicking the human situation. Furthermore, it displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the Jak/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, rat disease models would allow evaluation of the relevance of OSM for human biology. KW - Biologie Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78856 ER - TY - THES A1 - Börner, Juliane T1 - Charakterisierung der Phosphorylierungsstellen der Guanylyl Cyklase A, dem Rezeptor für das atriale natriuretische Peptid, mittels Massenspektrometrie T1 - Characterization of the phosphorylation sites of Guanylyl cyclase A, the receptor for atrial natriuretic peptide, by mass spectrometry application N2 - Das ANP/GC-A-System spielt durch die Produktion des sekundären Botenstoffs cGMP eine wichtige Rolle bei der Regulation des Blutdruckes und des Blutvolumens. Bei Patienten mit Herzhypertrophie oder Herzinsuffizienz sind die ANP-Plasmakonzentrationen erhöht, aber die GC-A-vermittelten Effekte stark reduziert, was auf einen Defekt des Signalsystems hinweist. Studien an metabolisch markierten GC-A-überexprimierenden HEK 293-Zellen zeigten, dass der GC-A-Rezeptor im basalen Zustand stark phosphoryliert und die homologe bzw. heterologe Desensitisierung wahrscheinlich mit einer Dephosphorylierung verbunden ist. Die Desensitisierung stellt einen Mechanismus dar, der in vivo zu einem Funktionsverlust des Rezeptors beitragen könnte. Im Rahmen dieser Arbeit konnten mittels Massenspektrometrie sieben Phosphorylierungsstellen in der Kinasehomologen Domäne aus FLAG-GC-A exprimierenden HEK 293-Zellen detektiert werden: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513. Die massenspektrometrische relative Quantifizierung basierend auf der Multiple-Reaction-Monitoring (MRM)-Methode zeigte bei ANP-induzierter, homologer Desensitisierung eine Dephosphorylierung der Phosphorylierungsstellen Ser497, Thr500, Ser502, Ser506, Ser510 und Thr513, was mit bereits publizierten Daten übereinstimmt, und einen starken Anstieg der Phosphorylierung an Ser487. Nach Inkubation mit Angiotensin II, welches eine heterologe Desensitisierung hervorruft, wurde eine Reduzierung aller Phosphorylierungen verzeichnet, die zudem stärker ausgeprägt war als bei der ANP-abhängigen Desensitisierung. Die Funktion der neu identifizierten Phosphorylierung an Ser487 wurde mittels Mutagenese analysiert. Die Substitution des Serins durch Alanin, welche den unphosphorylierten Zustand nachstellt, resultierte in einer Rezeptoraktivität und desensitisierung vergleichbar zum GC-A Wildtyp-Rezeptor. Wurde hingegen Serin gegen Glutamat getauscht, um den phosphorylierten Zustand zu imitieren, konnte der Rezeptor weder aktiviert noch desensitisiert werden. Diese Ergebnisse bestätigen vorherige Studien, dass die GC-A-Rezeptorantwort auf ANP durch die Phosphorylierungen reguliert wird. Allerdings scheint bei der homologen Desensitisierung die Phosphorylierung an der Position Ser487 eine Rolle zu spielen, da sie die Aktivität des Rezeptors inhibiert. Die Identifizierung und Charakterisierung dieser Phosphorylierungsstelle trägt zum Verständnis des Mechanismus der homologen Desensitierung bei. Zusätzlich konnten einige der beschriebenen Phosphorylierungen in Zellsystemen detektiert werden, die die GC-A endogen exprimieren. Dadurch sind unter physiologischen Bedingungen Analysen der Mechanismen möglich, die bei der Aktivierung und Deaktivierung der GC-A involviert sind und somit wichtige pathophysiologische Konsequenzen haben können. N2 - Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3’,5’-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP induced homologous desensitisation of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK 293, revealed that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked homologous desensitisation was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished which is in agreement with published data. However, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. In contrast, the Angiotensin II-provoked heterologous desensitisation resulted in a complete reduction of phosphorylation, which was even more intensive than after ANP-dependend desensitisation. The functional relevance of the newly identified phosphorylation site at serin 487 was analysed by site-directed mutagenesis. The substitution of Ser487 by alanine which mimics dephosphorylation resulted in a receptor which could be activated and desensitised like the GC-A wild-type receptor. However, the substitution by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitisation. These data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitisation seems to involve the phosphorylation of GC-A at Ser487. Identification and characterisation of this phosphorylation site therefore contribute to the understanding of homologous desensitisation. Additionally, some of the detected phosphorylated residues were detected in cells endogenously expressing the GC-A receptor. Therefore future analyses of the mechanisms which are involved in the activation and desensitisation under pathophysiological conditions might be possible. KW - Guanylatcyclase KW - Phosphorylierung KW - Atriales natriuretisches Hormon KW - Massenspektrometrie KW - Guanylyl cyclase A KW - phosphorylation sites KW - atrial natriuretic peptide KW - mass spectrometry Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51914 ER - TY - JOUR A1 - Fischer, Annette A1 - Harrison, Kelly S A1 - Ramirez, Yesid A1 - Auer, Daniela A1 - Chowdhury, Suvagata Roy A1 - Prusty, Bhupesh K A1 - Sauer, Florian A1 - Dimond, Zoe A1 - Kisker, Caroline A1 - Hefty, P Scott A1 - Rudel, Thomas T1 - Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense JF - eLife N2 - Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense. KW - cell-autonomous defense KW - Chlamydia trachomatis KW - deubiquitinase KW - Mcl-1 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171073 VL - 6 IS - e21465 ER - TY - JOUR A1 - Kronhardt, Angelika A1 - Beitzinger, Christoph A1 - Barth, Holger A1 - Benz, Roland T1 - Chloroquine Analog Interaction with C2-and Iota-Toxin in Vitro and in Living Cells JF - Toxins N2 - C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells’ receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa. KW - C2-toxin KW - iota-toxin KW - binding components KW - chloroquine KW - black lipid bilayer KW - aminoquinolinium salts Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168286 VL - 8 IS - 8 ER - TY - THES A1 - Alexander, Stephanie T1 - Collective cancer cell invasion \(in\) \(vivo\): function of β1 and β3 integrins in perivascular invasion and resistance to therapy T1 - Kollektive Tumorzellinvasion \(in\) \(vivo\): Funktion von β1 und β3 Integrinen in perivaskulärer Invasion und Therapieresistenz N2 - Pro-migratory signals mediated by the tumor microenvironment contribute to the cancer progression cascade, including invasion, metastasis and resistance to therapy. Derived from in vitro studies, isolated molecular steps of cancer invasion programs have been identified but their integration into the tumor microenvironment and suitability as molecular targets remain elusive. The purpose of the study was to visualize central aspects of tumor progression, including proliferation, survival and invasion by real-time intravital microscopy. The specific aims were to monitor the kinetics, mode, adhesion and chemoattraction mechanisms of tumor cell invasion, the involved guidance structures, and the response of invasion zones to anti-cancer therapy. To reach deeper tumor regions by optical imaging with subcellular resolution, near-infrared and infrared excited multiphoton microscopy was combined with a modified dorsal skinfold chamber model. Implanted HT-1080 fibrosarcoma and B16/F10 and MV3 melanoma tumors developed zones of invasive growth consisting of collective invasion strands that retained cell-cell contacts and high mitotic activity while invading at velocities of up to 200 μm per day. Collective invasion occurred predominantly along preexisting tissue structures, including blood and lymph vessels, collagen fibers and muscle strands of the deep dermis, and was thereby insensitive to RNAi based knockdown and/or antibody-based treatment against β1 and β3 integrins, chemokine (SDF-1/CXCL12) and growth factor (EGF) signaling. Therapeutic hypofractionated irradiation induced partial to complete regression of the tumor main mass, yet failed to eradicate the collective invasion strands, suggesting a microenvironmentally privileged niche. Whereas no radiosensitization was achieved by interference with EGFR or doxorubicin, the simultaneous inhibition of β1 and β3 integrins impaired cell proliferation and survival in spontaneously growing tumors and strongly enhanced the radiation response up to complete eradication of both main tumor and invasion strands. In conclusion, collective invasion in vivo is a robust process which follows preexisting tissue structures and is mainly independent of established adhesion and chemoattractant signaling. Due to its altered biological response to irradiation, collective invasion strands represent a microenvironmentally controlled and clinically relevant resistance niche to therapy. Therefore supportive regimens, such as anoikisinduction by anti-integrin therapy, may serve to enhance radio- and chemoefficacy and complement classical treatment regimens. N2 - Die Progression von Tumorerkrankungen, einschließlich Tumorinvasion, Metastasierung und Therapieresistenz wird unter anderem durch migrationsfördernde Signale aus der Tumorumgebung vermittelt. Zur bisherigen Aufklärung einzelner Schritte des Tumorinvasions- und Progressionsprogramms trugen dabei wesentlich In-vitro-Studien bei, jedoch erfordert die Darstellung der Relevanz molekularer Zielstrukturen und deren Funktion im Tumormikromilieu die Validierung in geeigneten In-vivo-Tumormodellen. Ziel dieser Studie war, zelluläre und molekulare Mechanismen der Tumorprogression inklusive Proliferation, Überleben und Invasion mittels Echtzeit-Intravitalmikroskopie darzustellen. Untersucht wurden insbesondere die Kinetik und Arten der Tumorzellinvasion, die zugrunde liegenden Adhäsionswege und pro-migratorischen Signale (EGF, SDF-1), beteiligte Leitstrukturen des Tumorstromas, und Strategien, therapeutisch gegen Invasionszonen vorzugehen. Um tiefe Tumorareale mittels subzellulär aufgelöster optischer Bildgebung zu erreichen, wurde nah-infrarote und infrarote Multiphotonenmikroskopie mit einem modifizierten Rückenkammermodell kombiniert. Orthotope Xeno- und Allotransplantate von HT-1080-Fibrosarkom- und B16/F10- oder MV3-Melanomzellen entwickelten dabei ausgeprägte invasive Wachstumszonen bestehend aus kollektiven Invasionssträngen mit intakten Zell-Zell-Kontakten und zeitgleicher Mitoseaktivität, die Geschwindigkeiten von bis zu 200 μm pro Tag erreichten. Diese kollektive Invasion orientierte sich bevorzugt entlang von Funktionsstrukturen der tiefen Dermis wie Blut- und Lymphgefäßen, Kollagenfasern und Muskelsträngen. RNAibasierende Herrunterregulation und/oder Injektion blockierender Antikörper gegen β1 und β3 Integrine, wie auch Inhibition von EGF führten nur zu minimaler Änderung der Invasionseffizienz. Therapeutische hypofraktionierte Bestrahlung induzierte partielle bis komplette Regression der Tumorhauptmasse, nicht jedoch der kollektiven Invasionsstränge, was auf eine kombinierte Invasions- und Resistenznische hinweist. Weder Doxorubicin noch gegen EGFR gerichtete Antikörper steigerten die Radiosensitivität, jedoch führte die simultane Inhibition von β1 und β3 Integrinen zu einer starken Hemmung von Proliferation und Überleben spontan wachsender Tumoren (Anoikis) und verstärkte die Strahlungssensitivität bis hin zum kompletten Verschwinden von sowohl Tumorhauptmasse wie auch Invasionsträngen. Kollektive Invasion ist somit ein wichtiger Invasionsmodus, der sich an vorbestehenden Gewebsstrukturen orientiert und unabhängig von Integrinen und EGF- und SDF-1-Signalen erfolgt. Die kollektiven Stränge entwickeln dabei eine vom Haupttumor verschiedene biologische Reaktion auf Bestrahlung und entsprechen damit einer durch die Mikroumgebung kontrollierten und von Integrinsignalen abhängenden Resistenznische. Somit könnte eine zusätzliche anti- Integrin-Therapie die Effizienz von Bestrahlung und Chemotherapie erhöhen und klassische Behandlungsschemen/-programme ergänzen. KW - Tumorzelle KW - Kollektive Invasion KW - Multiphotonenmikroskopie KW - Integrine KW - collective invasion KW - multiphoton microscopy KW - integrins KW - Invasion KW - Integrine Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85435 ER - TY - JOUR A1 - Hofmann, Elisabeth A1 - Weibel, Stephanie A1 - Szalay, Aladar A. T1 - Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice N2 - Background The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma. Methods PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed. Results GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed. Conclusions CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials. KW - Vaccinia virus KW - Chemotherapy KW - Combination therapy KW - Cyclophosphamide KW - Lung cancer Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-110168 ER - TY - JOUR A1 - Fagan, Jeremy K. A1 - Dollar, Gretchen A1 - Lu, Qiuheng A1 - Barnett, Austen A1 - Jorge, Joaquin Pechuan A1 - Schlosser, Andreas A1 - Pfleger, Cathie A1 - Adler, Paul A1 - Jenny, Andreas T1 - Combover/CG10732, a Novel PCP Effector for Drosophila Wing Hair Formation JF - PLOS ONE N2 - The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh. KW - planar cell polarity KW - RHO-associated kinease KW - convergent extension movements KW - ROK-alpha KW - protein KW - phosphorylation KW - actin KW - gene KW - morphogenesis KW - localization Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115394 SN - 1932-6203 VL - 9 IS - 9 ER - TY - JOUR A1 - Siegel, T. Nicolai A1 - Vasquez, Juan-José A1 - Hon, Chung-Chau A1 - Vanselow, Jens T. A1 - Schlosser, Andreas T1 - Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages N2 - While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei. KW - Ribosom KW - Profilierung KW - Trypanosoma brucei KW - Entwicklung KW - Lebenszyklus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112657 ER - TY - JOUR A1 - Balkenhol, Johannes A1 - Kaltdorf, Kristin V. A1 - Mammadova-Bach, Elmina A1 - Braun, Attila A1 - Nieswandt, Bernhard A1 - Dittrich, Marcus A1 - Dandekar, Thomas T1 - Comparison of the central human and mouse platelet signaling cascade by systems biological analysis JF - BMC Genomics N2 - Background Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. Conclusions Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences. KW - interspecies comparison KW - transcriptome KW - proteome KW - platelet KW - network KW - signaling KW - mouse KW - human KW - interactome KW - cascade Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230377 VL - 21 ER - TY - THES A1 - Toepfer, Franziska Helene T1 - Component selectivity and multistability in a \(Drosophila\) orientation paradigm using incoherent motion stimuli T1 - Komponenten-Selektivität und Multistabilität in einem Orientierungsparadigma für \(Drosophila\) unter Verwendung inkohärenter Bewegungsreize N2 - Visual information is essential for Drosophila to navigate its environment. The visual system of the fly has been studied for many decades and has yielded many insights about vision in general. However, visual information can be ambiguous and the system processing it needs to be able to cope with that. In this study, the visual orientation behavior of Drosophila is challenged by panoramic incoherent motion stimuli to which the fly can respond in three different, equally adaptive ways. The study is conducted in a well-established setup, the so-called flight simulator (Heisenberg and Wolf, 1993), where the fly can control its visual surroundings in stationary flight with its yaw torque, which is simultaneously recorded. The fly can either use one of two incoherently moving panorama patterns or the integrated motion of both as its reference for straight flight. It is observed that flies use all three of these behavioral alternatives for orientation. Previous models of fly motion vision do not predict a bimodal tuning to incoherent wide-field motion stimuli (Joesch et al., 2008, Borst et al., 1995), however, a recent study on blowflies could suggests that they show component selectivity to the individual moving gratings in a compound plaid stimulus (Saleem et al., 2012). Here, it can be shown that the same bimodal tuning manifests in Drosophila, although the stimuli used are different and most of the experiments are conducted in closed loop. It is found that the extent to which the Drosophila expresses this component selectivity in its orientation behavior, i.e. how often it stabilizes a single panorama pattern instead of the integrated motion of both, depends on two properties of the panorama stimuli, pattern contrast and horizontal pattern element distance. Single pattern stabilization decreases with increasing contrast and increasing pattern element distance. In the latter case, it increases again when there are very few horizontal pattern elements, although that appears to be the result of a lack of rivalry between the patterns due to the low number of pattern elements. Both increased pattern contrast and pattern element distance increase the salience of the single pattern elements. A single element in a compound visual stimulus, like a dot within a dot pattern, can be interpreted as a standalone figure or a part of a bigger unit. Previous studies on Drosophila vision have concentrated on how the fly discriminates a figure from the background (Heisenberg and Wolf, 1984, Bahl et al., 2013, Aptekar et al., 2012), but have hardly touched the question of what qualifies a figure or a background (i.e. a panorama) stimulus as such. In the present study, it is observed that, when exposed to incoherent panoramic motion stimuli, the flies prefer to orient themselves towards the average of the two motions when the panorama stimuli possess strong figure features and towards the single patterns when they do not and single pattern elements are therefore less salient. The above-mentioned plaid stimuli are a well-known multistable percept in human psychophysics. Multistability is a property of higher visual systems and considered an indicator of endogenous activity in vision. As Drosophila expresses behavioral multistability in the IPMP, it is evaluated in this respect. The results show several parallels to human multistable perception. For one, the frequency and duration with which a behavior occurs, can be influenced, but the occurrence of the behaviors is non-deterministic and not coupled to the stimulus. It can also be shown that the switches between behaviors do not stem from a rivalry of the two visual hemispheres of the fly, although monocularity does also influence the likelihood with which the behaviors occur. Secondly, like in human perceptual rivalry, individual flies exhibit strong idiosyncrasies regarding the overall durations they spend with the different behaviors and the frequencies with which they switch between them. Finally, the distribution of the durations between the behavioral switches can be fit to the same function as the distribution of percept durations in human multistable perception, the gamma function, although it has a different shape and therefore also differing parameters. The Drosophila mutant radish, which has been shown to have attention-like deficits (van Swinderen and Brembs, 2010, Koenig et al., 2016a), does also express an altered behavior in the IPMP compared to wildtype flies. As these behavioral alterations resemble effects on multistable perception found in humans suffering from ADHD (Amador-Campos et al., 2015) and perceptual multistability is generally considered to be closely related to attention (Leopold and Logothetis, 1999), attentional processes are also very likely to play a role in the flies’ behavior in the IPMP. In conclusion, the visual system of Drosophila is capable disentangle incoherent motion stimuli even if they overlap and cover the entire visual field, i.e. it shows component selectivity of wide-field motion. Whether it uses a single wide-field motion component or the average of two as its reference for straight flight depends on pattern contrast and horizontal pattern element density, which indicates an involvement of a figure-background rivalry. This rivalry and the one between the two wide-field motion components elicit a multistability in the orientation behavior of the fly the temporal dynamics of which partially resemble the temporal dynamics of human multistable perception and which also suggests the involvement of attentional processes. N2 - Visuelle Information ist von wesentlicher Bedeutung für Drosophila um sich in ihrer Umgebung zurecht zu finden. Das visuelle System der Fliege wird seit vielen Jahrzehnten untersucht und hat wichtige Erkenntnisse über Sehen im Allgemeinen hervorgebracht. Visuelle Information kann mehrdeutig sein und das System, das sie verarbeitet muss in der Lage sein damit umzugehen. In dieser Arbeit wird das visuelle Orientierungsverhalten von Drosophila durch inkohärente Großfeldbewegungen herausgefordert, auf die die Fliege auf drei verschiedene, gleich plausible Weisen reagieren kann. Die Studie wird in einem etablierten Versuchsaufbau durchgeführt, dem sogenannten Flugsimulator (Heisenberg and Wolf, 1993), in dem die Fliege im stationären Flug ihren visuellen Input mit ihrem Drehmoment kontrollieren kann. Die Fliege kann entweder eines von zwei sich inkohärent bewegenden Panoramamustern oder deren integrierte Gesamtbewegung als Referenz für ihren Geradeausflug nutzen. Es wird beobachtet, dass die Fliegen alle drei dieser Verhaltens-Alternativen zur Orientierung benutzen. Bisherige Modelle des Bewegungssehens der Fliege sagen keine bimodale Reaktion auf inkohärente Bewegungsreize vorher (Borst et al., 1995, Joesch et al., 2008), allerdings konnte kürzlich eine Studie an Calliphora zeigen, dass diese Komponenten-Selektivität bezüglich der einzelnen Bewegungen der Streifenmuster eines daraus zusammengesetzten Karomusters zeigen (Saleem et al., 2012). Hier kann gezeigt werden, dass diese bimodale Reaktion auch in Drosophila der Fall ist, obwohl die visuellen Stimuli unterschiedlich sind und die meisten Experimente im geschlossenen Regelkreis durchgeführt werden. Des weiteren zeigt sich, dass der Umfang in dem Drosophila diese Komponentenselektivität in ihrem Orientierungsverhalten zeigt, d.h. wie häufig sie ein einzelnes Panoramamuster statt der integrierten Bewegung von beiden stabilisiert, von zwei Eigenschaften des Musters, dem Musterkontrast und dem horizontalen Abstand der einzelnen Musterelemente, abhängt. Die Einzelmusterstabilisierung nimmt mit steigendem Kontrast und steigendem Musterelementabstand ab. Bei letzterem steigt sie wieder, wenn die Anzahl der horizontalen Musterelemente sehr gering ist, allerdings scheint dies das Ergebnis von fehlender Konkurrenz zwischen den Mustern aufgrund der niedrigen Anzahl der Musterelemente zu sein. Sowohl gesteigerter Kontrast und Musterelementabstand steigern das Hervorstechen der einzelnen Musterelemente. Ein einzelnes Element in einem zusammengesetzen visuellen Stimulus, wie ein Punkt in einem Punktemuster, kann sowohl als alleinstehendes Objekt als auch als Teil einer größeren Einheit interpretiert werden. Bisherige Studien am visuellen System von Drosophila haben sich darauf konzentriert, wie die Fliege ein Objekt vom Hintergrund unterscheidet (Aptekar et al., 2012, Bahl et al., 2013, Heisenberg and Wolf, 1984), aber kaum behandelt, was ein Objekt oder einen Hintergrund als solchen definiert. In dieser Studie wird beobachtet, dass die Fliege, wenn sie mit zwei sich inkohärent bewegenden Panoramastimuli konfrontiert wird, es bevorzugt sich am Mittelwert der beiden Bewegungen zu orientieren, wenn diese starke Objekteigenschaften aufweisen und an den Einzelmustern, wenn sie dies nicht der Fall ist und die einzelnen Musterelement daher weniger stark hervorstehen. Die erwähnten Karomusterreize sind ein bekanntes Beispiel multistabiler Wahrnehmung der Humanpsychophysik. Multistabilität ist eine Eigenschaft höherer visueller Systeme und ein Indikator von endogener Aktivität. Da Drosophila Multistabilität in ihrem Verhalten im IPMP aufweist, wird es diesbezüglich ausgewertet. Die Ergebnisse zeigen mehrere Parallelen zur menschlichen multistabilen Wahrnehmung. Zum einen kann die Häufigkeit und die Dauer mit der ein Verhalten auftritt, beeinflusst werden, aber das Auftreten der Verhaltensweisen ist nicht deterministisch und nicht an den Stimulus gekoppelt. Es kann auch gezeigt werden, dass die Wechsel zwischen den Verhaltensweisen nicht von einer Rivalität zwischen den beiden visuellen Hemisphären der Fliege herrühren, obwohl Monokularität die Wahrscheinlichkeit mit der die Verhaltensweisen auftreten, beeinflusst. Zweitens weisen die einzelnen Fliegen, wie auch Menschen in multistabilen Wahrnehmungsparadigmen, starke Idiosynkrasien bezüglich der Gesamtdauer, die sie mit einem Verhalten verbringen und der Häufigkeit mit der sie zwischen diesen hin und her wechseln, auf. Schließlich folgt die Verteilung der Zeitspannen zwischen den Wechseln zwischen den Verhaltensweisen der gleichen Funktion wie die Verteilung der Wahrnehmungsdauern in der menschlichen multistabilen Wahrnehmung, der Gamma-Funktion, obwohl sie eine unterschiedliche Form hat und daher auch andere Parameter. Die Drosophila-Mutante radish, von der gezeigt werden konnte, dass sie aufmerksamkeits-ähnliche Defizite hat (van Swinderen and Brembs, 2010, Koenig et al., 2016a), zeigt im Vergleich mit wildtypischen Fliegen auch im IPMP ein verändertes Verhalten. Da diese Veränderungen Effekten auf multistabile Wahrnehmung ähneln, die bei ADHS-Patienten gefunden wurden (Amador-Campos et al., 2015) und multistabile Wahrnehmung im Allgemeinen als nahe verwandt mit Aufmerksamkeit angesehen wird (Leopold and Logothetis, 1999), spielen Aufmerksamkeitsprozesse sehr wahrscheinlich auch eine Rolle im Verhalten der Fliege im IPMP. Zusammenfassend kann gesagt werden, dass das visuelle System von Drosophila in der Lage ist inkohärente Bewegungsreize sogar dann zu trennen wenn diese vollständig überlappen und das gesamte Sehfeld umfassen, d.h. es weist Komponentenselektivität von Großfeldbewegungen auf. Ob sie eine einzele Großfeldbewegungskomponente oder den Mittelwert von Zweien als Referenz für Geradeausflug nutzt, hängt von Kontrast und Dichte der horizontalen Musterelemente ab, was eine Beteiligung einer Objekt-Hintergrund-Rivalität impliziert. Diese Rivalität und diejenige zwischen den beiden Großfeldbewegungskomponenten lösen eine Multistabilität im Orientierungsverhalten der Fliege aus, deren zeitliche Dynamik teilweise der der menschlichen multistabilen Wahrnehmung entspricht und die die Beteiligung von Aufmerksamkeitsprozessen wahrscheinlich macht. KW - Drosophila KW - Invertebrate vision KW - Transparent motion KW - Component selectivity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-153346 ER - TY - JOUR A1 - Makbul, Cihan A1 - Khayenko, Vladimir A1 - Maric, Hans Michael A1 - Böttcher, Bettina T1 - Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype JF - Microorganisms N2 - Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes. KW - hepatitis B core protein KW - hepatitis B virus KW - peptide inhibitor of envelopment KW - isothermal titration calorimetry KW - electron cryo microscopy KW - low-secretion phenotype mutants KW - peptide microarray Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236720 SN - 2076-2607 VL - 9 IS - 5 ER - TY - JOUR A1 - Tessmer, Ingrid A1 - Melikishvili, Manana A1 - Fried, Michael G. T1 - Cooperative cluster formation, DNA bending and base-flipping by O\(^6\)-alkylguanine-DNA alkyltransferase JF - Nucleic Acids Research N2 - O\(^6\)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O\(^6\)-alkylguanine and O\(^4\)-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of 11 proteins. Longer clusters, predicted by the McGhee-von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (similar to 0, similar to 30 and similar to 60 degrees) that are consistent with models in which each protein induces a bend of similar to 30 degrees. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome. KW - inactivation KW - nucleotide excision-repair KW - atomic-force microscopy KW - noncooperative binding KW - restricition enzymes KW - complex stability KW - stranded DNAs KW - protein KW - chemotherapy KW - AGT Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133949 VL - 40 IS - 17 ER - TY - JOUR A1 - Daryaee, Fereidoon A1 - Chang, Andrew A1 - Schiebel, Johannes A1 - Lu, Yang A1 - Zhang, Zhuo A1 - Kapilashrami, Kanishk A1 - Walker, Stephen G. A1 - Kisker, Caroline A1 - Sotriffer, Christoph A. A1 - Fisher, Stewart L. A1 - Tonge, Peter J. T1 - Correlating drug-target kinetics and in vivo pharmacodynamics: long residence time inhibitors of the FabI enoyl-ACP reductase JF - Chemical Science N2 - Drug-target kinetics enable time-dependent changes in target engagement to be quantified as a function of drug concentration. When coupled to drug pharmacokinetics (PK), drug-target kinetics can thus be used to predict in vivo pharmacodynamics (PD). Previously we described a mechanistic PK/PD model that successfully predicted the antibacterial activity of an LpxC inhibitor in a model of Pseudomonas aeruginosa infection. In the present work we demonstrate that the same approach can be used to predict the in vivo activity of an enoyl-ACP reductase (FabI) inhibitor in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection. This is significant because the LpxC inhibitors are cidal, whereas the FabI inhibitors are static. In addition P. aeruginosa is a Gram-negative organism whereas MRSA is Gram-positive. Thus this study supports the general applicability of our modeling approach across antibacterial space. KW - Staphylococcus aureus KW - antibacterial activity KW - LpxC inhibitors KW - enoyl-ACP reductase inhibitors Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191218 VL - 7 IS - 9 ER - TY - JOUR A1 - Abdali, Narges A1 - Barth, Enrico A1 - Norouzy, Amir A1 - Schulz, Robert A1 - Nau, Werner M. A1 - Kleinekathofer, Ulrich A1 - Tauch, Andreas A1 - Benz, Roland T1 - Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion- Selective Cell Wall Channel JF - PLoS ONE N2 - Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. KW - antibiotics KW - detergents KW - anions KW - corynebacterium diphtheriae KW - membrane potential KW - corynebacteria KW - cell walls KW - permeability Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129989 VL - 8 IS - 10 ER - TY - JOUR A1 - Angelika, Kronhardt A1 - Rolando, Monica A1 - Beitzinger, Christoph A1 - Stefani, Caroline A1 - Leuber, Michael A1 - Flatau, Gilles A1 - Popoff, Michel R. A1 - Benz, Roland A1 - Lemichez, Emmanuel T1 - Cross-Reactivity of Anthrax and C2 Toxin: Protective Antigen Promotes the Uptake of Botulinum C2I Toxin into Human Endothelial Cells JF - PLoS ONE N2 - Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells. KW - Lipid bilayer-membranes KW - Current noise-analysis KW - Matrix protein porin KW - Clostridium-botulinum KW - Lethal factor KW - Edema factor KW - Escherichia-coli KW - Crystal-structure KW - Ion-channel KW - Phenylalanine clamp Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134791 VL - 6 IS - 8 ER - TY - JOUR A1 - Diebold, Mathias A1 - Schönemann, Lars A1 - Eilers, Martin A1 - Sotriffer, Christoph A1 - Schindelin, Hermann T1 - Crystal structure of a covalently linked Aurora-A-MYCN complex JF - Acta Crystallographica N2 - Formation of the Aurora-A–MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A–MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A–MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders. KW - MYCNv KW - neuroblastoma cell KW - proteasome system Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318855 VL - D79 SP - 1 EP - 9 ER - TY - JOUR A1 - Lorenz, Sonja A1 - Bhattacharyya, Moitrayee A1 - Feiler, Christian A1 - Rape, Michael A1 - Kuriyan, John T1 - Crystal Structure of a Ube2S-Ubiquitin Conjugate JF - PLoS ONE N2 - Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a “donor” ubiquitin and a primary amino group of an “acceptor” ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface. KW - crystal structure KW - protein ubiquitination KW - Ubiquitin-conjugating (E2) enzymes KW - Ube2S Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167265 VL - 11 IS - 2 ER - TY - JOUR A1 - Ries, Lena K. A1 - Liess, Anna K. L. A1 - Feiler, Christian G. A1 - Spratt, Donald E. A1 - Lowe, Edward D. A1 - Lorenz, Sonja T1 - Crystal structure of the catalytic C‐lobe of the HECT‐type ubiquitin ligase E6AP JF - Protein Science N2 - The HECT‐type ubiquitin ligase E6AP (UBE3A) is critically involved in several neurodevelopmental disorders and human papilloma virus‐induced cervical tumorigenesis; the structural mechanisms underlying the activity of this crucial ligase, however, are incompletely understood. Here, we report a crystal structure of the C‐terminal lobe (“C‐lobe”) of the catalytic domain of E6AP that reveals two molecules in a domain‐swapped, dimeric arrangement. Interestingly, the molecular hinge that enables this structural reorganization with respect to the monomeric fold coincides with the active‐site region. While such dimerization is unlikely to occur in the context of full‐length E6AP, we noticed a similar domain swap in a crystal structure of the isolated C‐lobe of another HECT‐type ubiquitin ligase, HERC6. This may point to conformational strain in the active‐site region of HECT‐type ligases with possible implications for catalysis. Significance Statement The HECT‐type ubiquitin ligase E6AP has key roles in human papilloma virus‐induced cervical tumorigenesis and certain neurodevelopmental disorders. Here, we present a crystal structure of the C‐terminal, catalytic lobe of E6AP, providing basic insight into the conformational properties of this functionally critical region of HECT‐type ligases. KW - dimerization KW - domain swapping KW - E3 enzyme KW - UBE3A KW - X‐ray crystallography Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214812 VL - 29 IS - 6 SP - 1550 EP - 1554 ER - TY - THES A1 - Gerold, Kay T1 - CTLA4 and CLEC16A in Type 1 Diabetes - Looking behind the association T1 - CTLA4 und CLEC16A in Typ 1 Diabetes - Ein Blick hinter die Assoziation N2 - Type 1 diabetes is an autoimmune disease that leads to the destruction of insulin-producing pancreatic beta cells and consequently to hyperglycemia. In the last 60 years, the prevalence of type 1 diabetes has been increasing constantly and is predicted to continue rising. About 80% of the disease risk is attributable to the genetic variation. Thanks to genome wide association studies the number of known disease-associated polymorphisms climbed from five to 53 in the last 10 years. As these studies reveal possible candidate genes but not underlying mechanisms we strove to take the next step and explore the association of two genes suggested by these studies with type 1 diabetes. As a method of choice we decided to use lentiviral RNAi in non obese diabetic (NOD) mice, a widely-used model for type 1 diabetes, introducing a shRNA directed against the target message into the genome of this mouse strain via a lentivirus. This allowed us to study the partial loss-of-function of the target gene within the context of diabetes, directly seeing its effect on autoimmune mechanisms. In this thesis we examined two different genes in this manner, Ctla4 and Clec16a. A type 1 diabetes associated polymorphism in the CTLA4 gene had been found to alter the splicing ratio of its variants soluble CTLA-4 (sCTLA-4) and full length CTLA-4, the associated allele producing less sCTLA-4 than the protective allele. We mimicked this effect by specifically targeting the sCtla4 mRNA via lentiviral RNAi in the NOD model. As a result we could confirm the reduction of sCTLA-4 to accelerate type 1 diabetes development. Furthermore we could show a function of sCTLA-4 in regulatory T cells, more specifically at least partly in their ability to modulate costimulation by antigen presenting cells. The second candidate gene, Clec16a was targeted with the shRNA in a way that was designed to knock down most splice variants. As the gene function and the effect of the associated SUMMARY 10 polymorphism was unknown, we reasoned this method to be feasible to investigate its role in type 1 diabetes. The knockdown of Clec16a in NOD mice resulted in an almost complete protection from diabetes development that could be attributed to T cells dysfunction. However, as expression patterns and a study of the Drospophila orthologue suggested a possible role of CLEC16A in antigen presentation we also examined antigen presenting cells in the thymus and periphery. Although we did not detect any effect of the knockdown on peripheral antigen presenting cells, thymic epithelial cells were clearly affected by the loss of CLEC16A, rendering them more activated and shifting the ratio of cortical to medullary epithelial cells in favor of cortical cells. We therefore suggest a role of CLEC16A in the selection of T cells, that needs, however, to be further investigated. In this thesis we provided a feasible and fast method to study function of genes and even of single splice variants within the NOD mouse model. We demonstrate its usefulness on two candidate genes associated with type 1 diabetes by confirming and unraveling the cause of their connection to the disease. N2 - Typ 1 Diabetes ist eine Autoimmunerkrankung, bei der es zur Zerstörung von pankreatischen beta-Zellen und daraus folgend zu einer Hyperglykämie kommt. In den letzten 60 Jahren stieg die Diabetes Prävalenz stetig an und Studien sagen voraus, dass sich dieser Trend in Zukunft noch stärker fortsetzen wird. Man geht davon aus, dass ca. 80% des Erkrankungsrisikos für autoimmunen Diabetes genetischer Natur sind. Dank Genom-weiter Assoziationsstudien wurde dieser Beitrag gerade in den letzten zehn Jahren immer weiter aufgeklärt und bis heute wurden 53 mit Typ 1 Diabetes assozierte Polymorphismen identifiziert. Da diese Studien es nur leisten können, mögliche Kandidatengene aufzuzeigen, allerdings keine Aussagen über die zugrunde liegenden Krankheitsmechanismen machen können, haben wir es uns zum Ziel gesetzt diesen nächsten Schritt zu gehen und zwei der durch diese Studien vorgeschlagenen Gene auf ihre Rolle in der Typ 1 Diabetes Ätiologie zu untersuchen. Unsere Methode der Wahl war die lentivirale RNA Interferenz im Mausmodell der nonobese diabetic mouse (NOD). Via lentiviralen Vektoren wird die Information für eine shRNA, die an die mRNA des Zielgenes bindet, in das Empfängergenom integriert. Die daraus folgende Herabregulierung der Ziel-mRNA erlaubt es uns den Effekt dieser fehlenden Geninformation auf die Immunregulation zu analysieren. Auf diese Weise wurden in dieser Thesis zwei Kandidatengene untersucht, Ctla4 und Clec16a. Der mit Typ 1 Diabetes assoziierte Polymorphismus im CTLA4 Gen verursacht eine Verschiebung im Splice Verhältnis der beiden Isoformen im Menschen, soluble CTLA-4 (sCTLA-4) und full length CTLA-4, zu Gunsten der full length Variante. Im NOD Mausmodell konnte diese Verschiebung durch eine Einführung einer gegen sCtla4 gerichteten shRNA nachgeahmt werden. In Folge dessen konnten wir bestätigen, dass eine eduzierung der sCTLA-4 Variante die Typ 1 Diabetes Entwicklung beschleunigt. Zudem ZUSAMMENFASSUNG 12 konnten wir eine Rolle von sCTLA-4 in der Funktion von regulatorischen T Zellen, genauer in deren Fähigkeit die Kostimulation durch Antigen präsentierenden Zellen zu modulieren, zeigen. Bei dem zweiten Gen, das in dieser Thesis untersucht wurde handelte sich um Clec16a. Es wurde von einer shRNA herunterreguliert, die den Großteil der Varianten abdeckt, da die Funktion des Genes, sowie die Auswirkungen des assoziierten Polymorphismus unbekannt waren. Der Knockdown von Clec16a in der NOD Maus verursachte einen fast vollständigen Schutz vor Diabetes, der im weiteren Verlauf den T Zellen zugerechnet werden konnte. Allerdings hatten das Expressionsmuster, sowie eine Studie am Drosophila Ortholog ema eine Rolle von CLEC16A in Antigen präsentierenden Zellen impliziert. Folglich untersuchten wir die Möglichkeit, dass diese Zellgruppe in der Peripherie oder im Thymus durch den CLEC16A Mangel beeinträchtigt sein könnten. Tatsächlich wies die Zellgruppe, die im Thymus für die Selektion von T Zellen zuständig ist einen erhöhten Aktivierungsstatus auf, was auf eine modifizierte T Zell Selektion hindeuten könnte. Mit dieser Arbeit konnten wir eine praktikable und schnelle Methode, für die funktionelle Analyse von Genen und sogar einzelnen Splice Varianten, aufzeigen. Wir konnten ihren Nutzen weiterhin an zwei mit Typ 1 Diabetes assoziierten Kandidatengenen unter Beweis stellen, indem wir so die Assoziation bestätigen und Licht auf die zugrunde liegenden Mechanismen werfen konnten. KW - Diabetes mellitus KW - Typ 1 KW - Molekulargenetik KW - Type 1 Diabetes Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-66617 ER - TY - JOUR A1 - Wagner, Nicole A1 - Mott, Kristina A1 - Upcin, Berin A1 - Stegner, David A1 - Schulze, Harald A1 - Ergün, Süleyman T1 - CXCL12-abundant reticular (CAR) cells direct megakaryocyte protrusions across the bone marrow sinusoid wall JF - Cells N2 - Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation. KW - megakaryocytes KW - microvasculature KW - CXCL12-abundant reticular (CAR)-cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234180 SN - 2073-4409 VL - 10 IS - 4 ER - TY - JOUR A1 - Stritt, Simon A1 - Nurden, Paquita A1 - Favier, Remi A1 - Favier, Marie A1 - Ferioli, Silvia A1 - Gotru, Sanjeev K. A1 - van Eeuwijk, Judith M.M. A1 - Schulze, Harald A1 - Nurden, Alan T. A1 - Lambert, Michele P. A1 - Turro, Ernest A1 - Burger-Stritt, Stephanie A1 - Matsushita, Masayuki A1 - Mittermeier, Lorenz A1 - Ballerini, Paola A1 - Zierler, Susanna A1 - Laffan, Michael A. A1 - Chubanov, Vladimir A1 - Gudermann, Thomas A1 - Nieswandt, Bernhard A1 - Braun, Attila T1 - Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg\(^{2+}\) homeostasis and cytoskeletal architecture JF - Nature Communications N2 - Mg\(^{2+}\) plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg\(^{2+}\)]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7\(^{fl/fl-Pf4Cre}\)) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7\(^{fl/fl-Pf4Cre}\) MKs, which is rescued by Mg\(^{2+}\) supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice. KW - Cytoskeleton KW - homeostasisIon channels KW - thrombopoiesis Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173843 VL - 7 ER - TY - JOUR A1 - Busch, Martin A1 - Westhofen, Thilo C. A1 - Koch, Miriam A1 - Lutz, Manfred B. A1 - Zernecke, Alma T1 - Dendritic Cell Subset Distributions in the Aorta in Healthy and Atherosclerotic Mice JF - PLoS ONE N2 - Dendritic cells (DCs) can be sub-divided into various subsets that play specialized roles in priming of adaptive immune responses. Atherosclerosis is regarded as a chronic inflammatory disease of the vessel wall and DCs can be found in non-inflamed and diseased arteries. We here performed a systematic analyses of DCs subsets during atherogenesis. Our data indicate that distinct DC subsets can be localized in the vessel wall. In C57BL/6 and low density lipoprotein receptor-deficient (Ldlr−/−) mice, CD11c+ MHCII+ DCs could be discriminated into CD103− CD11b+F4/80+, CD11b+F4/80− and CD11b−F4/80− DCs and CD103+ CD11b−F4/80− DCs. Except for CD103− CD11b− F4/80− DCs, these subsets expanded in high fat diet-fed Ldlr−/− mice. Signal-regulatory protein (Sirp)-α was detected on aortic macrophages, CD11b+ DCs, and partially on CD103− CD11b− F4/80− but not on CD103+ DCs. Notably, in FMS-like tyrosine kinase 3-ligand-deficient (Flt3l−/−) mice, a specific loss of CD103+ DCs but also CD103− CD11b+ F4/80− DCs was evidenced. Aortic CD103+ and CD11b+ F4/80− CD103− DCs may thus belong to conventional rather than monocyte-derived DCs, given their dependence on Flt3L-signalling. CD64, postulated to distinguish macrophages from DCs, could not be detected on DC subsets under physiological conditions, but appeared in a fraction of CD103− CD11b+ F4/80− and CD11b+ F4/80+ cells in atherosclerotic Ldlr−/− mice. The emergence of CD64 expression in atherosclerosis may indicate that CD11b+ F4/80− DCs similar to CD11b+ F4/80+ DCs are at least in part derived from immigrated monocytes during atherosclerotic lesion formation. Our data advance our knowledge about the presence of distinct DC subsets and their accumulation characteristics in atherosclerosis, and may help to assist in future studies aiming at specific DC-based therapeutic strategies for the treatment of chronic vascular inflammation. KW - flow cytometry KW - monocytes KW - diet KW - cell staining KW - DAPI staining KW - aorta KW - macrophages Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119907 SN - 1932-6203 VL - 9 IS - 2 ER - TY - JOUR A1 - Beitzinger, Christoph A1 - Bronnhuber, Annika A1 - Duscha, Kerstin A1 - Riedl, Zsuzsanna A1 - Huber-Lang, Markus A1 - Benz, Roland A1 - Hajos, György A1 - Barth, Holger T1 - Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin JF - PLoS ONE N2 - Background Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric \(PA_{63}\) binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the \(PA_{63}\)-channel in a dose dependent way. Methodology/Principal Findings Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the \(PA_{63}\)-channel in the µM range, when both, inhibitor and \(PA_{63}\) are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of \(PA_{63}\)-channel function also efficiently block intoxication of the cells by the combination lethal factor and \(PA_{63}\) in the same concentration range as they block the channels in vitro. Conclusions/Significance These results strongly argue in favor of a transport of lethal factor through the \(PA_{63}\)-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax. KW - intoxication KW - chloroquine KW - toxins KW - anthrax KW - cell membranes KW - lipid bilayer KW - macrophages KW - membrane potential Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130097 VL - 8 IS - 6 ER - TY - THES A1 - Drechsler, Johannes T1 - Determination of the hypertrophic potential of Oncostatin M on rat cardiac cells and the characterisation of the receptor complexes utilised by rat Oncostatin M T1 - Erforschung des hypertrophen Potentials von Oncostatin M auf Ratten-Herzzellen und die Charakterisierung der Rezeptorkomplexe, welche von Ratten-Oncostatin M genutzt werden N2 - Interleukin-6 (IL-6), oncostatin M (OSM), leukaemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) are members of the IL-6-type cytokine family that is characterised by sharing the common receptor subunit gp130. While the involvement of these polypeptides in cell differentiation, cell survival, proliferation, apoptosis, inflammation, haematopoiesis, immune response and acute phase reaction has already been demonstrated, the description of their role in development and progression of cardiac hypertrophy is still rather limited. A model has been postulated that declares the transient expression of IL-6-type cytokines as protective, while a continuous cardiac secretion of these proteins seems to be rather harmful for the heart. Within the first part of the study (results 4.1, 4.2 and 4.3) it was shown that OSM induces hypertrophy of primary neonatal rat cardiomyocytes (NRCM), just as its related cytokines LIF, CT-1 and hIL-6/hsIL-6R (hsIL-6R, human soluble IL-6 receptor). Regarding the hypertrophic potentials the LIFR/gp130 utilising cytokines (hLIF, hOSM and hCT-1) are stronger inducers than the OSMR/gp130 utilising mOSM. Human IL-6/hsIL-6R which signals via a gp130 homodimer has the weakest hypertrophic effect. The thorough analysis of typical signalling pathways initiated by IL-6-type cytokines revealed that STAT3 phosphorylation at Y705 seems to be the most important hypertrophy promoting pathway. In addition and in contrast to published work, we clearly demonstrate that classical IL-6 signalling (upon pure IL-6 treatment) has no hypertrophic effect on cardiomyocytes, because they lack sufficient amounts of the membrane-bound IL-6R. This is also true for neonatal rat cardiac fibroblasts (NRCFB). Since these cells can also influence cardiac hypertrophy, signalling pathways and target genes were additionally examined in NRCFB in response to OSM, LIF and IL-6/sIL-6R. One of the key findings of this thesis is the selective change in expression of cytokines and receptors of the IL-6 family in both cell types upon IL-6-type cytokine stimulation. A striking difference between NRCM and NRCFB is the fact that the target gene induction in NRCM is of similar duration upon mOSM and hIL-6/hsIL-6R treatment, while hIL-6/hsIL-6R is capable of promoting the induction of OSMR and IL-6 significantly longer in NRCFB. By searching for transcription factors or intermediate cytokines which could be responsible for this difference, a strong correlation between increased Il6 transcription and amount of mRNA levels for C/EBPβ and C/EBPδ was observed in response to IL-6/sIL-6R stimulation. Interestingly, mOSM also mediates the induction of C/EBPβ and δ, but the initiation is significantly less efficient than in response to IL-6/sIL-6R. Therefore, we assume that mOSM stimulation fails to reach threshold values required for a prolonged IL-6 secretion. Since we additionally observe a slight IL-6R mRNA upregulation in NRCFB, we assume that the combination of IL-6, LIF, C/EBPβ, C/EBPδ and IL-6R expression might be responsible for the observed different kinetics with which IL-6 and OSM stimulate NRCFB. In addition to the aforementioned proteins, members of the renin-angiotensin system seem to support the IL-6-type cytokine mediated hypertrophy. Since it has already been shown that angiotensin II vice versa induces IL-6 expression in NRCM and NRCFB, this enhanced expression of AT1α and ACE could be of crucial interest for the hypertrophy supporting phenotype. The second part of the presented work dealt with the characterisation of the receptor complexes of rat OSM. The central question of this analysis was, whether rOSM, just like mOSM, only binds the type II (OSMR/gp130) receptor complex or is able to utilise the type II and type I (LIFR/gp130) receptor complex. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant, and generation of stably transfected Ba/F3 cells expressing the newly cloned rat OSMR/gp130 or LIFR/gp130 receptor complex) we can clearly show that rat OSM surprisingly utilises both, the type I and type II receptor complex. Therefore it closely mimics the human situation. Furthermore, rOSM displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the JAK/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, the results obtained in the last section of this thesis clearly suggest that rat disease models would allow evaluation of the relevance of OSM for human biology much better than murine models. N2 - Interleukin-6 (IL-6), Oncostatin M (OSM), Leukämie inhibierender Faktor (LIF) und Cardiotrophin-1 (CT-1) sind Mitglieder der IL-6-Typ Zytokin-Familie, welche durch die gemeinsame Nutzung der Rezeptoruntereinheit gp130 charakterisiert ist. Während eine Beteiligung dieser Proteine bei Zelldifferenzierung, Zellüberleben, Proliferation, Apoptose, Entzündung, Hämatopoese, Immunantwort und Akut-Phase-Reaktion bereits gezeigt wurde, ist die Beschreibung ihrer Rolle bei der Entstehung und dem Fortschreiten der kardialen Hypertrophie deutlich limitierter. Es wurde bereits ein Modell postuliert, nach dem die kurzzeitige Expression dieser Zytokine schützend wirkt, während eine andauernde kardiale Sekretion eher schädlich für das Herz zu sein scheint. Im ersten Teil der Arbeit (Ergebnisse 4.1, 4.2 und 4.3) konnte gezeigt werden, dass OSM wie auch seine verwandten Zytokine LIF, CT-1 und hIL-6/hsIL-6R (hsIL-6R, humaner löslicher IL-6 Rezeptor) Hypertrophie-induzierend auf primäre neonatale Ratten-Kardiomyozyten (NRCM) wirkt. Hinsichtlich ihres hypertrophen Potentials sind die Zytokine, welche über LIFR/gp130 signalisieren (hLIF, hOSM und hCT-1), die stärkeren Induktoren im Vergleich zu mOSM, welches den OSMR/gp130 Rezeptorkomplex bindet. Die Stimulation mit humanem IL-6/hsIL-6R hatte hingegen die schwächste hypertrophe Wirkung. Unsere genaue Analyse der typischen IL-6-Typ Zytokin vermittelten Signalwege enthüllte die Phosphorylierung von STAT3 an Y705 als offenkundig wichtigsten hypertrophen Weg. Zusätzlich dazu konnten wir auch zeigen, dass klassisches IL-6 Signalling (ohne sIL-6R) keinen hypertrophen Einfluss auf NRCM hat, da diesen Zellen ausreichende Mengen des membranständigen IL-6R fehlen. Diese Beobachtung steht in klarem Kontrast zu bereits publizierten Arbeiten. In den ebenfalls untersuchten neonatalen Ratten-Kardiofibroblasten (NRCFB) verhält es sich, was den IL-6R angeht, genauso wie in NRCM. Da auch diese Zellen eine kardiale Hypertrophie mit beeinflussen können, wurden in ihnen die gleichen Signalwege und Zielgene nach Stimulation mit OSM, LIF und IL-6/sIL-6R untersucht. Die selektive Expressionsregulation von Zytokinen und Rezeptoren der IL-6-Familie in beiden Zelltypen nach IL-6-Typ Zytokin Stimulation ist hierbei einer unserer wichtigsten Befunde. Ein gravierender Unterschied zwischen NRCM und NRCFB besteht darin, dass die mOSM und hIL-6/hsIL-6R vermittelte Geninduktion in NRCM von vergleichbarer Dauer ist, wohingegen sie sich in NRCFB unterscheidet. Bei der Suche nach Transkriptionsfaktoren oder intermediären Zytokinen, welche für diesen Unterschied verantwortlich sein könnten, beobachteten wir nach IL-6/sIL-6R Stimulation eine deutliche Korrelation zwischen der Il6-Transkription und den mRNA Mengen von C/EBPβ und C/EBPδ. Auch OSM ist in der Lage beide Transkriptionsfaktoren zu induzieren, jedoch viel ineffizienter als IL-6/sIL-6R. Wir vermuten, dass mOSM einen bestimmten Schwellenwert, der für die verlängerte IL-6 Sekretion benötigt wird, nicht erreicht. Da wir zusätzlich noch eine schwache Zunahme der IL-6R mRNA in NRCFB beobachten konnten, gehen wir davon aus, dass die Expression von IL-6, LIF, C/EBPβ, C/EBPδ und IL-6R für die unterschiedlichen Kinetiken, mit denen IL-6 und OSM NRCFB stimulieren, verantwortlich sein dürfte. Es scheinen auch Mitglieder des Renin-Angiotensin-Systems die IL-6-Typ Zytokin vermittelte Hypertrophie zu unterstützen. Da schon gezeigt wurde, dass Angiotensin II reziprok die IL-6 Expression induziert, könnte diese verstärkte Synthese von AT1α und ACE von größter Bedeutung für den Hypertrophie-unterstützenden Phänotyp sein. Der zweite Teil der Arbeit (4.4) beschäftigte sich mit der Charakterisierung der Rezeptorkomplexe des Ratten-OSM. Die zentrale Frage hierbei bestand darin, ob rOSM wie mOSM nur den Typ II (OSMR/gp130) Rezeptorkomplex bindet, oder wie das hOSM sowohl den Typ II als auch den Typ I (LIFR/gp130) Rezeptorkomplex benutzen kann. Mit Hilfe unterschiedlicher experimenteller Strategien (knock-down der OSMR Expression durch RNA-Interferenz, LIFR-Blockade durch antagonistisches LIF-05, und die Generierung von stabil transfizierten Ba/F3-Zellen, welche die hierzu klonierten OSMR/gp130 oder LIFR/gp130 Rezeptorkomplexe der Ratte exprimieren) konnten wir eindeutig zeigen, dass Ratten-OSM überraschenderweise beide Rezeptorkomplexe benutzt. In dieser Hinsicht verhält sich es sich wie das humane Homolog. Des Weiteren besitzt Ratten-OSM Kreuz-Spezies-Aktivität und stimuliert humane und murine Zellen. Das Signal-Potential von rOSM ist dem von humanem OSM auf Zellen unterschiedlichen Ursprungs sehr ähnlich. Das Zytokin ist befähigt JAK/STAT, MAP Kinase und PI3K/Akt Signalwege potent zu aktivieren. Deshalb deuten die Daten des zweiten Teils dieser Arbeit darauf hin, dass Krankheitsmodelle in Ratten die Evaluierung der Relevanz des OSM für die humane Biologie deutlich besser widerspiegeln würden als murine Modelle. KW - Interleukin 6 KW - Leukaemia-inhibitory factor KW - Herzhypertrophie KW - Interleukin 6 KW - Leukaemia-inhibitory factor KW - Oncostatin M KW - gp130 KW - LIFR KW - OSMR KW - Kardial Hypertrophy KW - Receptor Preference KW - Ratte Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85215 ER - TY - THES A1 - ElBashir, Rasha T1 - Development of New Mass Spectrometry-based Methods for the Analysis of Posttranslational Modifications T1 - Entwicklung neuer massenspektrometrischer Methoden für die Analyse posttranslationaler Proteinmodifikationen N2 - Posttranslational modifications (PTMs) play a crucial role in many cellular processes. They are reversible, dynamic, and highly regulated events that alter the properties of proteins and increase their functional diversity. The identification and quantification of PTMs are critical for deciphering the molecular mechanisms of PTMs-related biological processes and disease treatment and prevention. Two of the most common and important PTMs that regulate many protein functions are acetylation and phosphorylation. An important role of acetylation is the regulation of DNA/RNA-protein interactions. A prominent example for this are histones, whose tail regions are lysine-rich and can be highly acetylated at their N-terminal domain. In spite of the utmost importance of this PTM, methods that allow the accurate measuring the site-specific acetylation degree are missing. One of the challenges in quantifying the acetylation degree at an individual lysine residue of the histones N-termini is the occurrence of multiple lysines in close proximity. Herein, we describe the development of the ”Fragment Ion Patchwork Quantification,” a new mass spectrometry-based approach for the highly accurate quantification of sites-pecific acetylation degrees. This method combines 13C1-acetyl derivatization on the protein level, proteolysis by low-specificity proteases and quantification on the fragment ion level. Acetylation degrees are determined from the isotope patterns of acetylated b and y ions. We have shown that this approach allows determining the site-specific acetylation degrees of all lysine residues for all core histones of Trypanosoma brucei. In addition, we demonstrate the use of this approach to identify the substrate sites of histone acetyltransferases and to monitor the changes in acetylation of the histones of canonical nucleosome and transcription start site nucleosomes. Phosphorylation is one of the most common and most important PTMs. The analysis of the human genome showed that there are about 518 kinases and more than 500,000 phosphorylation sites are believed to exist in the cellular proteome. Protein phosphorylation plays a crucial role in signaling many different cell processes, such as intercellular communication, cell growth, differentiation of proliferation and apoptosis. Whereas MS-based identification and relative quantification of singly phosphorylated peptides have been greatly improved during the last decade, and large-scale analysis of thousands of phosphopeptides can now be performed on a routine-base, the analysis of multi-phosphorylated peptides is still lagging vastly behind. The low pKa value of phosphate group and the associated negative charge are considered the major source of the problems with the analysis of multi-phosphorylated peptides. These problems include the formation of phosphopeptide-metal complexes during liquid chromatography (e.g. Fe 3+), which leads to a drastic deterioration of the chromatographic properties of these peptides (peak tailing), the decreased ionization efficiencies of phosphorylated peptides compared to their unphosphorylated counterparts, the labile nature of phosphate during CID/HCD fragmentation, and the unsuitability of low-charged phosphopeptides for ETD fragmentation are the most important factors that hinder phosphorylation analysis by LC-MS/MS. Here we aimed to develop a method for improving the identification of multi-phosphorylated peptides as well as the localization of phosphorylation sites by charge-reversal derivatization of the phosphate groups. This method employs a carbodiimide-mediated phosphoramidation to converted the phosphates to stable aromatic phosphoramidates. This chemical modification of phosphosite(s) reversed the negative charge of the phosphate group(s) and increased the number of the positive charges within the phosphopeptide. This modification prevented the formation of phosphopeptide-metal ion complexes that dramatically decreases or completely diminishes the signal intensity of protonated phosphopeptides, specifically multi-phosphorylated peptides. Furthermore, the increased net charge the (phospho-)peptides made them suitable for ETD fragmentation, which generated a high number of fragment ions with high intensities that led to a better phosphopeptide identification and localization of phosphosite(s) with high confidence. N2 - Posttranslationale Modifikationen (PTMs) spielen eine entscheidende Rolle in vielen zellulären Prozessen. Sie sind reversible, dynamische und hochregulierte Ereignisse, die die Proteineneigenschaften verändern und ihre funktionale Diversität erhöhen. Die Identifizierung und Quantifizierung von PTMs sind wesentlich für die Entschlüsselung der molekularen Mechanismen von PTM-regulierten biologischen Prozessen und für ein besseres Verständnis der Rolle posttranslationaler Modifikationen bei einer Vielzahl von Krankheiten. Zwei der bedeutendsten PTMs, welche die Funktion unzähliger Proteine regulieren sind die Acetylierung an Lysin-Resten und die Phosphorylierung an Serin-, Threonin- und Tyrosinresten. Im Rahmen dieser Arbeit wurden eine neue Methode zur Bestimmung des positionsspezifischen Acetylierungsgrades, sowie verbesserte Methoden für die Analyse der Phosphorylierung mittels Flüssigchromatographie-gekoppelter Tandem Massenspektrometrie entwickelt. Wir haben eine neue MS-basierte Methode (”Fragment Ion Patchwork Quantification”) entwickelt, welche es erlaubt die Acetylierungsgrade an individuellen Positionen mit hoher Genauigkeit zu messen. Diese Methode kombiniert die 13C1- Acetylderivatisierung von intakte Proteine, die Proteolyse durch Proteasen mit niedriger Spezifität, und die Quantifizierung auf dem MS2-Level. Die Acetylierungsgrade werden aus den Isotopenmustern von acetylierten b- und y-Ionen bestimmt. Obwohl unsere Methode zur Quantifizierung der positionsspezifischen Acetylierungsgrade auf jedes beliebige Protein angewandt werden kann, stand bei der Methodenentwicklung die Analyse der Histonacetylierung aufgrund ihrer herausragenden Bedeutung bei der Regulation der Genexpression im Vordergrund. Wir haben gezeigt, dass mit dieser Methode die Bestimmung der positionsspezifischen Acetylierungsgrade an allen Lysin Resten aller Core-Histone von Nukleosomhistone von Trypanosoma brucei möglich ist. Darüber hinaus haben wir diese Methode angewandt, um die Substrat-Positionen von Histon Acetyltransferasen zu identifizieren und um quantitative Veränderungen der Acetylierung an Histonen aus kanonischen Nukleosomen sowie Nukleosomen an Transkriptionsstartstellen zu analysieren. Phosphorylierung ist eine der häufigsten und wichtigsten posttranslational Proteinmodifikationen. Im Verlauf des Sequenzierung des humanen Genoms wurden 518 Gene für Proteinkinasen entdeckt und es wird angenommen, dass im zellulären Proteom mehr als 500 000 Phosphorylierungsstellen existieren. Die Proteinphosphorylierung spielet eine entscheidende Rolle in der Signalisierung vieler verschiedener Zellprozesse wie zum Beispiel der interzellulären Kommunikation, dem Zellwachstum, der Differenzierung der Proliferation und der Apoptose. Während bei der massenspektrometrie-basierte Identifizierung und relativen Quantifizierung von einfach phosphorylierten Peptiden in den letzten große Fortschritte erzielt wurden, und die Analyse tausender Phosphopeptide mittlerweile häufig routinemäßig durchgeführt werden kann, bereitet die massenspektrometrische Analyse merhfach phosphorylierter Peptide nach wie vor große Probleme. Der niedrige pKa-Wert der Phosphatgruppe, und die damit einhergehende negative Ladung ist die Hauptursache für die Probleme bei der Analyse merhfach phosphorylierter Peptide. Die mehrfache negative Ladung dieser Peptide führt zu einer ausgeprägten Neigung zur Komplexbildung mit mehrwertigen Metallionen (wie z.B. Fe3+), welche zu einer drmatischen Verschlechterung der chromatographischen Eigenschaften dieser Peptide führt (Peak Tailing), zu einer Verschlechterung der Ionisierungseffizienz, und zu einem ungewöhnlich niedrigen Protonierungsgrad im Positivionen-Modus, welcher diese Peptide für eine Fragmentierung mittels ETD ungeeignet macht. Im Rahmen dieser Arbeit wurde mittels chemischer Modifikation der Phosphatgruppe versucht sowohl die Detektion von mehrfach-phosphorylierten Peptiden, als auch die Lokalisierung von Phosphorylierungsstellen zu verbessern. Hierfür wurden die Phosphatgruppen unter Verwendung des Aktivierungsreagenzes EDC in hydrolysestabile, aromatische Phosphoramidate überführt. Die durch diese Modifikation erzielte Ladungsumkehr führt wie erwartet zu einer verbesserten Signalintensität bei den entsprechend modifizierten Phosphopeptiden, sowie zu einem verbesserten Fragmentierungsverhalten bei ETD, und somit letztlich zu einer verbesserten Lokalisierbarkeit der Phosphatgruppe inerhalb des Peptids. KW - LC-MS KW - Posttranslationale Änderung KW - Acetylierung KW - Phosphorylierung KW - Quantifizierung KW - Mass Spectrometry KW - PTMs KW - Acetylation KW - Quantitation KW - Phosphorylation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-153731 ER - TY - THES A1 - Neagoe, Raluca Alexandra Iulia T1 - Development of techniques for studying the platelet glycoprotein receptors GPVI and GPIb localisation and signalling T1 - Entwicklung von Methoden zur Untersuchung zur der Lokalisation und Signaltransduktion der Thrombozytenrezeptoren GPVI und GPIb N2 - Platelets play an important role in haemostasis by mediating blood clotting at sites of blood vessel damage. Platelets, also participate in pathological conditions including thrombosis and inflammation. Upon vessel damage, two glycoprotein receptors, the GPIb-IX-V complex and GPVI, play important roles in platelet capture and activation. GPIb-IX-V binds to von Willebrand factor and GPVI to collagen. This initiates a signalling cascade resulting in platelet shape change and spreading, which is dependent on the actin cytoskeleton. This thesis aimed to develop and implement different super-resolution microscopy techniques to gain a deeper understanding of the conformation and location of these receptors in the platelet plasma membrane, and to provide insights into their signalling pathways. We suggest direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) as the best candidates for imaging single platelets, whereas expansion microscopy (ExM) is ideal for imaging platelets aggregates. Furthermore, we highlighted the role of the actin cytoskeleton, through Rac in GPVI signalling pathway. Inhibition of Rac, with EHT1864 in human platelets induced GPVI and GPV, but not GPIbα shedding. Furthermore, EHT1864 treatment did not change GPVI dimerisation or clustering, however, it decreased phospholipase Cγ2 phosphorylation levels, in human, but not murine platelets, highlighting interspecies differences. In summary, this PhD thesis demonstrates that; 1) Rac alters GPVI signalling pathway in human but not mouse platelets; 2) our newly developed ExM protocol can be used to image platelet aggregates labelled with F(ab’) fragments N2 - Thrombozyten, spielen in der Hämostase eine entscheidende Rolle, indem sie die Blutstillung bei Gefäßverletzung vermitteln. Sie sind jedoch auch an pathologischen Prozessen wie zum Beispiel der Thrombose und Entzündungen beteiligt. Bei einer Gefäßverletzung spielen zwei Glykoproteinrezeptoren eine wichtige Rolle bei der Adhäsion und Aktivierung von Thrombozyten: der GPIb-IX-V-Komplex und GPVI. GPIb-IX-V bindet an den von-Willebrand-Faktor und GPVI an Kollagen. Dies initiiert eine Signalkaskade, die zu einer Änderung der Morphologie der Thrombozyten führt, welche vom Aktin-Zytoskelett abhängig ist. Ziel dieser Doktorarbeit war die Entwicklung und Anwendung verschiedener hochauflösender Mikroskopietechniken, um ein tieferes Verständnis der Konformation und Lokalisation dieser Rezeptoren in der Plasmamembran der Thrombozyten zu erlangen und Einblicke in ihre Signalwege zu gewinnen. Hierbei etablierten wir dSTORM und die structured illumination microscopy (SIM) als die geeignetsten Methoden für die mikroskopische Untersuchung einzelner Thrombozyten, während die Expansionsmikroskopie (ExM) ideal für die Darstellung von Thrombozytenaggregaten ist. Darüber heben unsere Ergebnisse zur Funktion von Rac im GPVI Signalweg die wichtige Rolle des Aktin-Zytoskeletts hervor. Die Hemmung von Rac mit EHT1864 in menschlichen Thrombozyten induzierte das Abscheiden (shedding) von GPVI und GPV, nicht jedoch von GPIbα. Darüber hinaus blieb die GPVI Dimerisierung und GPVI-Clusterbildung durch EHT1864-Behandlung unverändert, jedoch verringerte sich die Phosphorylierung der Phospholipase Cγ2 in humanen, aber nicht in murinen Thrombozyten, was Unterschiede zwischen den Spezies aufzeigt. Zusammenfassend zeigen die Ergebnisse dieser Doktorarbeit, dass; 1) Rac den GPVI Signalweg in humanen aber nicht in murinen Thrombozyten beeinflusst; 2) unser neu entwickeltes ExM-Protokoll zur Darstellung von F(ab’)-Fragment markierten Thrombozytenaggregaten verwendet werden kann. KW - Platelet-Membranglykoprotein p62 KW - Platelets KW - Microscopy KW - GPVI KW - Rac1 Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313064 ER - TY - JOUR A1 - Lorenzin, Francesca A1 - Benary, Uwe A1 - Baluapuri, Apoorva A1 - Walz, Susanne A1 - Jung, Lisa Anna A1 - von Eyss, Björn A1 - Kisker, Caroline A1 - Wolf, Jana A1 - Eilers, Martin A1 - Wolf, Elmar T1 - Different promoter affinities account for specificity in MYC-dependent gene regulation JF - eLife N2 - Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells. KW - MYC KW - promoter affinity KW - human KW - mathematical modeling KW - mouse KW - ChIP-sequencing KW - MIZ1 KW - cancer biology KW - cell biology KW - WDR5 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162913 VL - 5 ER - TY - JOUR A1 - Imam, Nasir A1 - Choudhury, Susobhan A1 - Heinze, Katrin G. A1 - Schindelin, Hermann T1 - Differential modulation of collybistin conformational dynamics by the closely related GTPases Cdc42 and TC10 JF - Frontiers in Synaptic Neuroscience N2 - Interneuronal synaptic transmission relies on the proper spatial organization of presynaptic neurotransmitter release and its reception on the postsynaptic side by cognate neurotransmitter receptors. Neurotransmitter receptors are incorporated into and arranged within the plasma membrane with the assistance of scaffolding and adaptor proteins. At inhibitory GABAergic postsynapses, collybistin, a neuronal adaptor protein, recruits the scaffolding protein gephyrin and interacts with various neuronal factors including cell adhesion proteins of the neuroligin family, the GABAA receptor α2-subunit and the closely related small GTPases Cdc42 and TC10 (RhoQ). Most collybistin splice variants harbor an N-terminal SH3 domain and exist in an autoinhibited/closed state. Cdc42 and TC10, despite sharing 67.4% amino acid sequence identity, interact differently with collybistin. Here, we delineate the molecular basis of the collybistin conformational activation induced by TC10 with the aid of recently developed collybistin FRET sensors. Time-resolved fluorescence-based FRET measurements reveal that TC10 binds to closed/inactive collybistin leading to relief of its autoinhibition, contrary to Cdc42, which only interacts with collybistin when forced into an open state by the introduction of mutations destabilizing the closed state of collybistin. Taken together, our data describe a TC10-driven signaling mechanism in which collybistin switches from its autoinhibited closed state to an open/active state. KW - autoinhibition KW - fluorescence resonance energy transfer (FRET) KW - gephyrin KW - guanine nucleotide exchange factor (GEF) KW - inhibitory postsynapse KW - Rho GTPase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-282816 SN - 1663-3563 VL - 14 ER - TY - JOUR A1 - Liu, Ruiqi A1 - Friedrich, Mike A1 - Hemmen, Katherina A1 - Jansen, Kerstin A1 - Adolfi, Mateus C. A1 - Schartl, Manfred A1 - Heinze, Katrin G. T1 - Dimerization of melanocortin 4 receptor controls puberty onset and body size polymorphism JF - Frontiers in Endocrinology N2 - Xiphophorus fish exhibit a clear phenotypic polymorphism in puberty onset and reproductive strategies of males. In X. nigrensis and X. multilineatus, puberty onset is genetically determined and linked to a melanocortin 4 receptor (Mc4r) polymorphism of wild-type and mutant alleles on the sex chromosomes. We hypothesized that Mc4r mutant alleles act on wild-type alleles by a dominant negative effect through receptor dimerization, leading to differential intracellular signaling and effector gene activation. Depending on signaling strength, the onset of puberty either occurs early or is delayed. Here, we show by Förster Resonance Energy Transfer (FRET) that wild-type Xiphophorus Mc4r monomers can form homodimers, but also heterodimers with mutant receptors resulting in compromised signaling which explains the reduced Mc4r signaling in large males. Thus, hetero- vs. homo- dimerization seems to be the key molecular mechanism for the polymorphism in puberty onset and body size in male fish. KW - fluorescence lifetime imaging microscopy KW - Förster Resonance Energy Transfer KW - Mc4r KW - puberty KW - Xiphophorus Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-354261 SN - 1664-2392 VL - 14 ER - TY - JOUR A1 - Thein, Marcus A1 - Bonde, Mari A1 - Bunikis, Ignas A1 - Denker, Katrin A1 - Sickmann, Albert A1 - Bergström, Sven A1 - Benz, Roland T1 - DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates N2 - Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a singlechannel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species. KW - Medizin Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75809 ER - TY - JOUR A1 - Hupp, Sabrina A1 - Förtsch, Christina A1 - Wippel, Carolin A1 - Ma, Jiangtao A1 - Mitchell, Timothy J. A1 - Iliev, Asparouh I. T1 - Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin JF - Journal of Molecular Biology N2 - The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought. KW - pore-forming toxin KW - cholesterol-dependent cytolysin KW - actin KW - membrane KW - pneumolysin Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132297 VL - 425 IS - 3 ER - TY - JOUR A1 - Soehnlein, Oliver A1 - Drechsler, Maik A1 - Döring, Yvonne A1 - Lievens, Dirk A1 - Hartwig, Helene A1 - Kemmerich, Klaus A1 - Ortega-Gómez, Almudena A1 - Mandl, Manuela A1 - Vijayan, Santosh A1 - Projahn, Delia A1 - Garlichs, Christoph D. A1 - Koenen, Rory R. A1 - Hristov, Mihail A1 - Lutgens, Esther A1 - Zernecke, Alma A1 - Weber, Christian T1 - Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes JF - EMBO Molecular Medicine N2 - We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical \((inflammatory/Gr1^{hi})\) or non-classical \((resident/Gr1^{lo})\) monocytes to dissect their differential role in atheroprogression under high-fat diet (HFD). Apolipoprotein E-deficient \((Apoe^{-/-})\) mice lacking classical but not non-classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1-neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene-deficient \(Apoe^{-/-}\) mice, adoptive transfer of gene-deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or \(CX_3CR1\) in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment. KW - hypercholeterolemia KW - CCR2 KW - atherosclerosis KW - chemokine KW - accumulation KW - subsets KW - inflammatory sites KW - fractalkine KW - marcophages KW - mobilization KW - monocyte KW - recruitment KW - bone-marrow KW - atheriosclerotic lesions KW - hyperlipedemic mice Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122204 SN - 1757-4676 VL - 5 ER - TY - JOUR A1 - Maurer, Jana A1 - Hupp, Sabrina A1 - Bischoff, Carolin A1 - Foertsch, Christina A1 - Mitchell, Timothy J. A1 - Chakraborty, Trinad A1 - Iliev, Asparouh I. T1 - Distinct neurotoxicity profile of listeriolysin O from \(Listeria\) \(monocytogenes\) JF - Toxins N2 - Cholesterol-dependent cytolysins (CDCs) are protein toxins that originate from Gram-positive bacteria and contribute substantially to their pathogenicity. CDCs bind membrane cholesterol and build prepores and lytic pores. Some effects of the toxins are observed in non-lytic concentrations. Two pathogens, \(Streptococcus\) \(pneumoniae\) and \(Listeria\) \(monocytogenes\), cause fatal bacterial meningitis, and both produce toxins of the CDC family—pneumolysin and listeriolysin O, respectively. It has been demonstrated that pneumolysin produces dendritic varicosities (dendrite swellings) and dendritic spine collapse in the mouse neocortex, followed by synaptic loss and astrocyte cell shape remodeling without elevated cell death. We utilized primary glial cultures and acute mouse brain slices to examine the neuropathological effects of listeriolysin O and to compare it to pneumolysin with identical hemolytic activity. In cultures, listeriolysin O permeabilized cells slower than pneumolysin did but still initiated non-lytic astrocytic cell shape changes, just as pneumolysin did. In an acute brain slice culture system, listeriolysin O produced dendritic varicosities in an NMDA-dependent manner but failed to cause dendritic spine collapse and cortical astrocyte reorganization. Thus, listeriolysin O demonstrated slower cell permeabilization and milder glial cell remodeling ability than did pneumolysin and lacked dendritic spine collapse capacity but exhibited equivalent dendritic pathology. KW - medicine KW - listeriolysin O KW - meningitis KW - acute slices KW - variocosities KW - dendritic spines Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172130 VL - 9 IS - 1 ER - TY - JOUR A1 - Atak, Sinem A1 - Langlhofer, Georg A1 - Schaefer, Natascha A1 - Kessler, Denise A1 - Meiselbach, Heike A1 - Delto, Carolyn A1 - Schindelin, Hermann A1 - Villmann, Carmen T1 - Disturbances of ligand potency and enhanced degradation of the human glycine receptor at affected positions G160 and T162 originally identified in patients suffering from hyperekplexia JF - Frontiers in Molecular Neuroscience N2 - Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. KW - mutations KW - trafficking KW - domain KW - hyperekplexia KW - loop B KW - side chain properties KW - ligand potencies KW - Cys-loop receptor KW - glycine receptor KW - site KW - activation KW - binding KW - channel KW - mechanisms KW - dominant KW - startle Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144818 VL - 8 IS - 79 ER - TY - THES A1 - Sauer, Markus T1 - DNA-Bindungseigenschaften von Mitgliedern der p53 Familie T1 - DNA binding properties of members of the p53 family N2 - Ein sehr wichtiger Tumorsuppressor ist der Transkriptionsfaktor p53, der Zellschicksals-Entscheidungen wie Zellzyklus-Arrest und programmierten Zelltod (Apoptose) kontrolliert. Die Wirkung von p53 und von seinen Familienmitgliedern p63 und p73 beruht überwiegend auf der Fähigkeit, als Transkriptionsfaktoren die Genexpression zu regulieren. Die DNA-Bindung an Promotoren von Zielgenen ist dabei von grundlegender Bedeutung und wird durch die hoch konservierte zentrale DNA-Bindungs-Domäne und den Carboxy-Terminus bestimmt. In dieser Arbeit wurden die DNA-Bindungseigenschaften von p53 und verschiedener Carboxy-terminalen p73 Isoformen untersucht. In „electrophoretic mobility shift assay” (EMSA) Experimenten bildeten p53 und p73gamma nur schwache Sequenz-spezifische DNA-Komplexe, wohingegen p73alpha, beta und delta die DNA deutlich stärker banden. Die schwache DNA-Bindung von p53 und p73gamma kann durch mehrfach positiv geladene Carboxy-Termini erklärt werden, die über eine Sequenz-unabhängige DNA-Bindung ein Gleiten entlang der DNA ermöglichen. Die Deletion der Carboxy-terminalen Domäne (CTD) von p53 („p53delta30“) verstärkte dementsprechend die Sequenz-spezifische DNA-Bindung in vitro und seine Übertragung auf p73alpha („p73alpha+30“) schwächte sie ab. Mittels „fluorescence recovery after photobleaching“ (FRAP) Experimenten konnte in lebenden Zellen eine Verminderung der intra-nukleären Mobilität von p53 und p73alpha+30 durch die CTD gezeigt werden, die aus der Sequenz-unabhängigen DNA-Bindung resultiert. Zusätzlich reduzierte die CTD die Sequenz-spezifische DNA-Bindung von p53 an den p21 (CDKN1A) Promotor. Das Spektrum der regulierten Zielgene wurde in einer Genom-weiten Genexpressions-Analyse nicht durch die CTD verändert, sondern maßgeblich durch das Protein-Rückgrat von p53 beziehungsweise p73 bestimmt. Allerdings verminderte die CTD das Ausmaß der Transkriptions-Regulation und hemmte die Induktion von Zellzyklus-Arrest und Apoptose. Die mehrfach positiv geladene CTD in p53 besitzt demzufolge eine negativ regulatorische Wirkung, die in den wichtigsten p73 Isoformen alpha, beta und delta fehlt. Die zentrale DNA-Bindungs-Domäne trägt durch elektrostatische Wechselwirkungen zwischen H1-Helices (Aminosäurereste 177 bis 182) unterschiedlicher p53 Monomere zu kooperativer DNA-Bindung und zu Zellschicksals-Entscheidungen bei. Anhand von Mutanten, die unterschiedlich starke H1-Helix-Interaktionen ermöglichen, konnte gezeigt werden, dass starke Interaktionen die Bindung an Promotoren von pro-apoptotischen Genen verstärkte, wohingegen die Bindung an anti-apoptotische und Zellzyklus-blockierende Gene unabhängig von der Interaktions-Stärke war. Diese Unterschiede in der Promotor-Bindung ließen sich nicht auf eine veränderte zelluläre Lokalisation der Mutanten zurückführen, da alle Mutanten überwiegend nukleär lokalisiert waren. Eine an Serin 183 Phosphorylierungs-defekte Mutante von p53 bildete stabile DNA-Komplexe, entsprechend einer Mutante mit starker H1-Helix-Interaktion, und trans-aktivierte pro-apoptotische Promotoren stärker als Mutanten, die Phosphorylierung von p53 an Serin 183 simulieren. Da zusätzlich bekannt ist, dass Serin 183 mit der H1-Helix wechselwirkt, könnte diese Phosphorylierung einen physiologischen Mechanismus zur Regulation der H1-Helix-Interaktion und damit des Zellschicksals darstellen. Zusammenfassend ließ sich zeigen, dass sowohl die Interaktions-Stärke zweier DNA-Bindungs-Domänen als auch die elektrische Ladung des Carboxy-Terminus die DNA-Bindungseigenschaften von p53 Familienmitgliedern bestimmen und so Zellschicksals-Entscheidungen der p53 Familie beeinflussen. N2 - A very important tumour suppressor is the transcription factor p53 that controls cell fate decisions like cell cycle arrest and programmed cell death (apoptosis). The effects of p53 and its family members p63 and p73 are mainly based on their transcription factor activities to regulate gene expression. The DNA binding to promoters of target genes is of fundamental importance for their functionality and is determined by the highly conserved core DNA binding domain and the carboxy-terminus. In this thesis the DNA binding properties of p53 and different carboxy-terminal p73 isoforms were examined. In electrophoretic mobility shift assays (EMSA) p53 and p73gamma formed only weak sequence-specific protein-DNA-complexes while p73alpha, beta and delta bound considerably stronger to DNA. A highly positively charged carboxy-terminus can explain the weak DNA binding of p53 and p73gamma by enabling sequence-nonspecific DNA binding leading to sliding on DNA. According to this the deletion of the carboxy-terminal domain (CTD) of p53 („p53delta30“) reinforced DNA binding in vitro, and its fusion to p73alpha („p73alpha+30“) attenuated it. In living cells the CTD reduced intranuclear mobility of p53 and p73alpha+30 in fluorescence recovery after photobleaching (FRAP) experiments by mediating sequence-nonspecific binding to DNA. In addition, the CTD reduced sequence-specific occupancy of the p21 (CDKN1A) promoter by p53 in vivo. In an unbiased genome-wide gene expression analysis the spectrum of target genes was not changed by the presence of the CTD, but mainly determined by the p53 and p73 protein backbone, respectively. However, the CTD diminished the level of target gene activation and inhibited the induction of cell cycle arrest and apoptosis. As a result, the highly positively charged carboxy-terminus of p53 exhibits a negative regulatory effect that is missing in the most important p73 isoforms alpha, beta and delta. The core DNA binding domain adds to cooperative DNA binding and cell fate decisions by electrostatic interactions between H1 helices (residues 177 to 182) of different p53 monomers. Strong H1 helix interactions increased binding to promoters of pro-apoptotic genes, whereas binding to anti-apoptotic and proliferation inhibiting genes was independent of the interaction strength as shown by mutants with different strengths of the H1 helix interactions. These differences in promoter binding were not caused by different cellular localizations of the mutants as they were all predominantly localized to the nucleus. A serine 183 phosphorylation-defective mutant of p53 formed stable protein-DNA-complexes, comparable to a mutant with strong H1 helix interactions, and trans-activated pro-apoptotic promoters stronger than mutants that mimicked p53 phosphorylated on serine 183. Due to the fact that serine 183 interacts with the H1 helix, these data suggest that phosphorylation of serine 183 is a physiological mechanism to regulate H1 helix interactions and thereby cell fate decisions. In summary, it was shown that both the interaction strength of two DNA binding domains and the electrostatic charge of the CTD define the DNA binding properties of p53 family members and thereby influence cell fate decisions of the p53 family. KW - Protein p53 KW - DNS-Bindung KW - Protein p73 KW - Transkriptionsfaktor KW - Apoptosis KW - FRAP KW - Gleiten KW - Carboxy-Terminus KW - H1-Helix KW - FRAP KW - sliding KW - carboxy terminus KW - H1 helix Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-35083 ER - TY - JOUR A1 - Pauls, Dennis A1 - Hamarat, Yasmin A1 - Trufasu, Luisa A1 - Schendzielorz, Tim M. A1 - Gramlich, Gertrud A1 - Kahnt, Jörg A1 - Vanselow, Jens A1 - Schlosser, Andreas A1 - Wegener, Christian T1 - Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate JF - European Journal of Neuroscience N2 - Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well‐characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE ), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD ) in global neuropeptide processing and selected peptide‐regulated behaviours in Drosophila . We found that a deficiency in dCPD results in C‐terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD ‐encoding gene silver in the larva causes lethality, and leads to deficits in starvation‐induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide‐regulated behaviour in Drosophila . dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE. KW - direct muss spectrometric profiling KW - friut fly behaviour KW - M14 carboxypeptidasses KW - peptidomoics KW - protein processing Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-204863 VL - 50 IS - 9 ER - TY - JOUR A1 - Kalleda, Natarajaswamy A1 - Amich, Jorge A1 - Arslan, Berkan A1 - Poreddy, Spoorthi A1 - Mattenheimer, Katharina A1 - Mokhtari, Zeinab A1 - Einsele, Hermann A1 - Brock, Matthias A1 - Heinze, Katrin Gertrud A1 - Beilhack, Andreas T1 - Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens JF - Frontiers in Microbiology N2 - Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4\(^+\) or CD8\(^+\) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b\(^+\) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b\(^+\) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions. KW - corticosteroids and cyclophosphamide KW - aspergillus fumigatus KW - CD11b+ myeloid cells KW - immune cell recruitment Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165368 VL - 7 IS - 1107 ER - TY - THES A1 - Storim, Julian T1 - Dynamic mapping of the immunological synapse in T cell homeostasis and activation T1 - Dynamische Untersuchung der immunologischen Synapse während T-Zellhomöostase und -aktivierung N2 - Polarity and migration are essential for T cell activation, homeostasis, recirculation and effector function. To address how T cells coordinate polarization and migration when interacting with dendritic cells (DC) during homeostatic and activating conditions, a low density collagen model was used for confocal live-cell imaging and high-resolution 3D reconstruction of fixed samples. During short-lived (5 to 15 min) and migratory homeostatic interactions, recently activated T cells simultaneously maintained their amoeboid polarization and polarized towards the DC. The resulting fully dynamic and asymmetrical interaction plane comprised all compartments of the migrating T cell: the actin-rich leading edge drove migration but displayed only moderate signaling activity; the mid-zone mediated TCR/MHC induced signals associated with homeostatic proliferation; and the rear uropod mediated predominantly MHC independent signals possibly connected to contact-dependent T cell survival. This “dynamic immunological synapse” with distinct signaling sectors enables moving T cells to serially sample antigen-presenting cells and resident tissue cells and thus to collect information along the way. In contrast to homeostatic contacts, recognition of the cognate antigen led to long-lasting T cell/DC interaction with T cell rounding, disintegration of the uropod, T cell polarization towards the DC, and the formation of a symmetrical contact plane. However, the polarity of the continuously migrating DC remained intact and T cells aggregated within the DC uropod, an interesting cellular compartment potentially involved in T cell activation and regulation of the immune response. Taken together, 3D collagen facilitates high resolution morphological studies of T cell function under realistic, in vivo-like conditions. N2 - Zellpolarität und Migration sind essentielle Voraussetzungen für T Zellaktivierung und homöostase sowie für Rezirkulation, und Effektorfunktionen. Um unter homöostatischen bzw. aktivierenden Bedingungen die Koordi¬nation von Polarisation und Migration von T Lymphozyten, die mit dendritischen Zellen (DC) interagieren, zu untersuchen, wurde ein Kollagenmatrix-Model mit niedriger Kollagendichte für konfokale Zeitraffermikro¬skopie und die hochaufgelöste Rekonstruktion fixierter Proben genutzt. Bei kurzen (5-15 min), migratorischen homöostatischen Kontakten behielten voraktivierte T-Zel¬len ihr amöboide Polarisation bei, während sie sich gleichzeitig Richtung DC polarisierten. Die hieraus resultie¬rende, dynamische und asymmetrische Kontaktflä¬che bestand aus allen Kompartimenten der migrierenden T-Zelle: Der F-Aktin-reiche vordere Zellpol („leading edge“) sorgte für Vor¬schub, hatte aber nur einen geringen Anteil an der Singaltransduktion; im mittleren Bereich („mid-zone“) waren MHC/TCR-abhängige Signale mit homöostatischer Proliferation assozi¬iert; und im als Uropod bezeichneten hintere Zellpol fanden sich vor allem MHC-unabhän¬gige Signale, die möglicherweise im Zusammenhang mit kontaktabhängigem Überleben stehen. Diese „dynamische immuno¬logische Synapse“ mit ihren Signaltransduktionsbereichen versetzt wan¬dernde T-Zellen in die Lage, nacheinander Kontakt zu mehreren antigenpräsen¬tierenden oder gewebsspezifischen Zellen aufzunehmen und so Informationen „im Vorbeigehen“ zu sammeln. Im Gegensatz zu homöostatischen Kontakten führte die Bindung des spezifischen Antigens zu langlebigen T Zellen/DC Kontakten, die mit der Abrundung der T Zelle und der Pola¬risation Richtung DC, der Auflösung ihres Uropods sowie der Ausbildung einer symmetri¬schen Kontaktfläche einher gin¬gen. Die Polarität der währenddessen fortge¬setzt migrierenden DC blieb dem gegenüber erhal¬ten und T-Zellen akkumulierten im DC-Uropod, einem interes¬santen Zellkompartiment, dass an T Zell-aktivierung und der Regu¬lation der Immunantwort beteiligt sein könnte. Zusammenge¬fasst ermöglicht das 3D Kollagenmatrix-Modell die hoch aufgelöste morphologische Untersu¬chung von T-Zell¬funk¬tionen unter realistischen, in vivo-artigen Bedingungen. KW - T-Lymphozyt KW - Dendritische Zelle KW - Zellmigration KW - Immunologische Synapse KW - T-Zellaktivierung KW - T-Zellhomöostase KW - Kollagen KW - T lymphocyte KW - dendritic cell KW - immunological synapse KW - migration Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70114 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Müller, Meike A1 - Adelfinger, Marion A1 - Weibel, Stephanie A1 - Grummt, Friedrich A1 - Zimmermann, Martina A1 - Bitzer, Michael A1 - Heisig, Martin A1 - Zhang, Qian A1 - Yu, Yong A. A1 - Chen, Nanhai G. A1 - Stritzker, Jochen A1 - Lauer, Ulrich M. A1 - Szalay, Aladar A. T1 - Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68 JF - PLOS ONE N2 - Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man. KW - Breast-tumors KW - Nude-mice KW - In-vivo KW - Cancer KW - Inhibitor KW - Tissue KW - Agent KW - COX-2 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135319 VL - 6 IS - 7 ER - TY - JOUR A1 - Weider, Matthias A1 - Wegener, Amélie A1 - Schmitt, Christian A1 - Küspert, Melanie A1 - Hillgärtner, Simone A1 - Bösl, Michael R. A1 - Hermans-Borgmeyer, Irm A1 - Nait-Oumesmar, Brahim A1 - Wegner, Michael T1 - Elevated in vivo levels of a single transcription factor directly convert satellite glia into oligodendrocyte-like cells JF - PLoS Genetics N2 - Oligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dorsal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies. KW - peripheral nervous system KW - Hirschsprung disease KW - spinal-cord KW - boundary cap KW - differentiation KW - stem cells KW - factor Sox10 KW - mouse model KW - expression KW - Olig2 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144123 VL - 11 IS - 2 ER - TY - THES A1 - Mony Nair, Rahul T1 - Elucidating ubiquitin recognition by the HECT-type ubiquitin ligase HUWE1 T1 - Studien zur Ubiquitinerkennung durch die HECT-Typus Ubiquitinligase HUWE1 N2 - The small protein modifier ubiquitin is at the heart of an immensely versatile posttranslational modification system that orchestrates countless physiological and disease-associated cellular processes. Key to this versatility are the manifold modifications that can be assembled from ubiquitin “building blocks” and are associated with specific functional outcomes for the modified substrates. In particular, ubiquitin molecules can form polymeric chains of distinct lengths and linkage types that give rise to distinct chain conformations, thereby providing recognition sites for specific signaling receptors/effectors. The class of E3 enzymes (ubiquitin ligases) provides critical specificity determinants in ubiquitin linkage formation; it is therefore crucial to unravel precisely how E3 enzymes operate in order to understand the structural basis of ubiquitin signaling and exploit these insights for therapeutic benefit. Overexpression and deregulation of the HECT-type ubiquitin ligase HUWE1 is implicated in several different cancer types and neurodegenerative disorders. It is largely unknown which factors control the ubiquitin modifications formed by HUWE1, how the catalytic HECT domain interacts with functionally distinct ubiquitin molecules (donor, acceptor and regulatory ubiquitin molecules) and which conformational transitions enable these interactions during ubiquitin chain formation. One aim of this study was to structurally elucidate the recognition of donor ubiquitin by the HECT domain of HUWE1. To this end I utilized a ubiquitin activity-based probe to reconstitute a proxy for a donor ubiquitin-linked conjugate of the HECT domain of HUWE1 and determined its structure by X-ray crystallography. This structure reveals that the donor ubiquitin binds to the C-lobe of HUWE1 in the same way as NEDD4-type ligases, corroborating the idea that HECT ligases utilize a conserved mode of donor ubiquitin recognition. independent of their linkage and substrate specificities. With the help of biochemical analyses, I also validated specific features of the structure, in particular the positioning of the C-terminal tail of the ligase, which was known to be critical for activity. In the newly determined structure, which reflects an “L-shaped”, active state of the HECT domain, this tail is fully resolved and coordinated at the N-lobe-C-lobe interface. I defined residues that are critical for this coordination and showed that they are also essential for the activity of HUWE1, including auto-ubiquitination, free ubiquitin chain formation, and substrate ubiquitination. Furthermore, I discovered that the N-lobe of HUWE1 harbors a ubiquitin-binding exosite similar to NEDD4-type ligases and E6AP. My in-vitro activity and binding assays show that HUWE1 uses the exosite for isopeptide bond formation, but that it is dispensable for thioester bond formation. The binding assays further show that the donor ubiquitin loaded HECT domain binds an additional ubiquitin molecule at the exosite more tightly than the apo HECT domain, which possibly suggests allosteric communication between the two sites. Finally, I showed that the ubiquitin activity-based probe (ubiquitin-propargylamine) can label the catalytic cysteine of HUWE1 and NEDD4-type with close to quantitative turn- over, while it does not react with the HECT domain of the evolutionarily more divergent E6AP. The determinants underlying these differential reactivities remain to be explored. Taken, together my results significantly enhance our mechanistic understanding of the catalytic domain of HUWE1 and pinpoint linchpins for therapeutic interventions with the activity of this disease-relevant enzyme. N2 - Der kleine Proteinmodifikator Ubiquitin ist das Herzstück eines immens vielseitigen posttranslationalen Modifikationssystems, das unzählige physiologische und krankheitsassoziierte zelluläre Prozesse orchestriert. Der Schlüssel zu dieser Vielseitigkeit sind die vielfältigen Modifikationen, die sich aus Ubiquitin-"Bausteinen" zusammensetzen lassen und mit spezifischen funktionellen Ergebnissen für die modifizierten Substrate verbunden sind. Insbesondere können Ubiquitin-Moleküle Ketten unterschiedlicher Länge und Verknüpfungstypen bilden, die zu unterschiedlichen Kettenkonformationen führen und dadurch Erkennungsstellen für spezifische Signalrezeptoren/-effektoren bieten. Die Klasse der E3-Enzyme (Ubiquitin-Ligasen) liefert kritische Spezifitätsdeterminanten für die Bildung von Ubiquitin-Bindungen; daher ist es entscheidend, die genaue Funktionsweise der E3-Enzyme zu entschlüsseln, um die strukturelle Grundlage der Ubiquitin-Signalisierung zu verstehen und diese Erkenntnisse für therapeutische Anwendungen zu nutzen. Die Überexpression und Deregulierung der Ubiquitin-Ligase HUWE1 aus der Klasse der HECT-E3-Ligasen ist an mehreren verschiedenen Krebsarten und neurodegenerativen Erkrankungen beteiligt. Es ist weitgehend unbekannt, welche Faktoren durch die von HUWE1 gebildeten Ubiquitin-Modifikationen kontrolliert werden, wie die katalytische HECT-Domäne mit funktionell unterschiedlichen Ubiquitin-Molekülen (Donor-, Akzeptor- und regulatorische Ubiquitin-Moleküle) interagiert und welche Konformationsübergänge diese Interaktionen während der Ubiquitin-Kettenbildung ermöglichen. Ein Ziel dieser Studie war es, die Erkennung des Donor-Ubiquitin-Moleküls durch die HECT-Domäne von HUWE1 strukturell aufzuklären. Zu diesem Zweck verwendete ich eine ´ubiquitin activity-based probe´, um ein Konjugat der HUWE1-HECT-Domäne mit einem Donor-Ubiquitin-Molekül zu rekonstitutieren und die Struktur mittels Röntgenkristallographie zu bestimmen. Diese Struktur zeigte, dass das Donor-Ubiquitin-Molekül auf die gleiche Weise an den C-lobe von HUWE1 bindet wie die Klasse der NEDD4-Ligasen, was die Idee bestätigt, dass HECT-Ligasen einen vergleichbaren Mechanismus bei der Donor-Ubiquitin-Erkennung verwenden, unabhängig von ihrer Bindung und Substratspezifität. Mit Hilfe biochemischer Analysen validierte ich auch spezifische Merkmale der Struktur, insbesondere die Positionierung des C-terminal tail der Ligase, der entscheidend für die Aktivität ist. In der neu bestimmten Struktur, die einen "L-förmigen", aktiven Zustand der HECT-Domäne widerspiegelt, ist der C-terminal tail an der Grenzfläche von N-lobe und C-lobe vollständig aufgelöst und koordiniert. Ich konnte Seitenketten festmachen, die für diese Koordination kritisch sind, und habe gezeigt, dass sie auch für die Aktivität von HUWE1 wesentlich sind, einschließlich der Auto-Ubiquitinierung, der freien Ubiquitin-Kettenbildung und der Substrat-Ubiquitinierung. Darüber hinaus entdeckte ich, dass der N-lobe von HUWE1 eine Ubiquitin-bindende exosite aufweist, ähnlich wie für die Klasse der NEDD4-Ligasen und E6AP. Meine in vitro Aktivitäts- und Bindungstests ergaben, dass HUWE1 die exosite für die Bildung von Isopeptidbindungen verwendet, diese aber für die Bildung von Thioesterbindungen entbehrlich ist. Die Bindungstests zeigten ferner, dass die Donor-Ubiquitin-beladene HECT-Domäne ein zusätzliches Ubiquitin-Molekül an der exosite stärker bindet als die Apo-HECT-Domäne, was möglicherweise auf eine allosterische Kommunikation zwischen den beiden Ubiquitin-Bindestellen hindeutet. Schließlich zeigte ich, dass die ´ubiquitin activity-based probe´ (Ubiquitin-Propargylamin) das katalytische Cystein von HUWE1 und NEDD4 mit nahezu quantitativem Umsatz markieren kann, während es nicht mit der HECT-Domäne des evolutionär stärker divergierenden E6AP reagiert. Die Faktoren, die diesen unterschiedlichen Reaktivitäten zugrunde liegen, müssen noch erforscht werden. Zusammengenommen verbessern meine Ergebnisse unser mechanistisches Verständnis der katalytischen Domäne von HUWE1 und geben uns die Dreh- und Angelpunkte für therapeutische Interventionen mit der Aktivität dieses krankheitsrelevanten Enzyms an die Hand. KW - HUWE1 KW - Ubiquitin-PA Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221030 ER - TY - JOUR A1 - Ponnuswamy, Padmapriya A1 - Schröttle, Angelika A1 - Ostermeier, Eva A1 - Grüner, Sabine A1 - Huang, Paul L. A1 - Ertl, Georg A1 - Hoffmann, Ulrich A1 - Nieswandt, Bernhard A1 - Kuhlencordt, Peter J. T1 - eNOS Protects from Atherosclerosis Despite Relevant Superoxide Production by the Enzyme in apoE\(^{-/-}\) Mice JF - PLoS One N2 - Background: All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial-(L/E) and platelet/endothelial-(P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme. Principal Findings: Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE\(^{-/-}\)/eNOS\(^{-/-}\)), while P/E-interactions did not differ, compared to apoE\(^{-/-}\). eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE\(^{-/-}\) vessels. Conclusion: Overt plaque formation, increased vascular inflammation and L/E-interactions are associated with significant reduction of superoxide production in apoE\(^{-/-}\)/eNOS\(^{-/-}\) vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE\(^{-/-}\) atherosclerosis but does not negate the enzyme's strong protective effects. KW - platelet adhesion KW - lesion formation KW - nitric oxide synthase KW - endothelial cell interactions KW - double knockout mice KW - apolipoprotein E KW - deficient mice KW - in vivo KW - accelerated atherosclerosis KW - leukocyte adhesion Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134866 VL - 7 IS - 1 ER - TY - JOUR A1 - Chubanov, Vladimir A1 - Ferioli, Silvia A1 - Wisnowsky, Annika A1 - Simmons, David G. A1 - Leitzinger, Christin A1 - Einer, Claudia A1 - Jonas, Wenke A1 - Shymkiv, Yuriy A1 - Gudermann, Thomas A1 - Bartsch, Harald A1 - Braun, Attila A1 - Akdogan, Banu A1 - Mittermeier, Lorenz A1 - Sytik, Ludmila A1 - Torben, Friedrich A1 - Jurinovic, Vindi A1 - van der Vorst, Emiel P. C. A1 - Weber, Christian A1 - Yildirim, Önder A. A1 - Sotlar, Karl A1 - Schürmann, Annette A1 - Zierler, Susanna A1 - Zischka, Hans A1 - Ryazanov, Alexey G. T1 - Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival JF - eLife N2 - Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood. KW - signalling pathways Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164987 VL - 5 ER - TY - THES A1 - Geisenhof [geb. Trinkwalder], Michaela T1 - Erforschung des Schicksals des Mittelkörpers anhand der ZF1-Methode T1 - Investigating the Fate of the Midbody after Cytokinesis N2 - Bei der Teilung einer Zelle werden das Genom und die Zellbestandteile zwischen zwei Tochterzellen aufgeteilt. Dies erfordert verschiedene fein aufeinander abgestimmte Vorgänge. Unter anderem ist eine proteinreiche Struktur beteiligt, die 1891 entdeckt wurde: der Mittelkörper. In vorliegender Arbeit wurden gezielt gekennzeichnete Mittelkörperproteine analysiert und verschiedene Phasen des Transports unterschieden. Es erfolgten erstmals Messungen unter Nutzung der ZF1-Methode. Zudem wird anhand der ZF1-Technik nachgewiesen, dass im Rahmen der Zellteilung die Trennung der interzellulären Brücke zu beiden Seiten des Mittelkörpers stattfindet, woraufhin dieser nach extrazellulär abgegeben wird und über einen der Phagozytose ähnlichen und von Aktin abhängigen Mechanismus von einer Tochterzelle oder unverwandten Nachbarzelle aufgenommen wird. N2 - In animals, the midbody coordinates the end of cytokinesis. Using the ZF1-mediated degradation technique it is shown that midbodies are released outside the cell in C. elegans embryos. Furthermore it is shown that midbodies are released after abscission cuts on both sides of the midbody and that released midbodies are internalized via actin-driven phagocytosis. KW - Mitose KW - mitosis KW - Phagozytose KW - phagocytosis KW - Mittelkörper KW - midbody KW - Abszission KW - C. elegans KW - abscission Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-182199 ER - TY - JOUR A1 - Schanbacher, Constanze A1 - Bieber, Michael A1 - Reinders, Yvonne A1 - Cherpokova, Deya A1 - Teichert, Christina A1 - Nieswandt, Bernhard A1 - Sickmann, Albert A1 - Kleinschnitz, Christoph A1 - Langhauser, Friederike A1 - Lorenz, Kristina T1 - ERK1/2 activity is critical for the outcome of ischemic stroke JF - International Journal of Molecular Sciences N2 - Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke. KW - ERK1/2 KW - tMCAO KW - ischemic stroke KW - RKIP Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-283991 SN - 1422-0067 VL - 23 IS - 2 ER - TY - THES A1 - Aigner, Max T1 - Establishing successful protocols and imaging pipelines for Expansion Microscopy in murine blood platelets T1 - Etablierung erfolgreicher Protokolle zur Probenpräparation und Bildgebung für die ‚Expansion Microscopy‘ in murinen Thrombozyten N2 - Platelets play an important role in the body, since they are part of the hemostasis system, preventing and stopping blood loss. Nevertheless, when platelet or coagulation system function are impaired, uncontrolled bleedings but also irreversible vessel occlusion followed by ischemic tissue damage can occur. Therefore, understanding platelet function and activation, mechanisms which are controlled by a variety of platelet membrane receptors and other factors is important to advance out knowledge of hemostasis and platelet malfunction. For a complete picture of platelet function and their modulating behavior it is desired to be able to quantify receptor distributions and interactions of these densely packed molecular ensembles in the membrane. This challenges scientists for several reasons. Most importantly, platelets are microscopically small objects, challenging the spatial resolution of conventional light microscopy. Moreover, platelet receptors are highly abundant on the membrane so even super-resolution microscopy struggles with quantitative receptor imaging on platelets. With Expansion microscopy (ExM), a new super-resolution technique was introduced, allowing resolutions to achieve super-resolution without using a super-resolution microscope, but by combining a conventional confocal microscopy with a highly processed sample that has been expanded physically. In this doctoral thesis, I evaluated the potential of this technique for super-resolution platelet imaging by optimizing the sample preparation process and establishing an imaging and image processing pipeline for dual-color 3D images of different membrane receptors. The analysis of receptor colocalization using ExM demonstrated a clear superiority compared to conventional microscopy. Furthermore, I identified a library of fluorescently labeled antibodies against different platelet receptors compatible with ExM and showed the possibility of staining membrane receptors and parts of the cytoskeleton at the same time. N2 - Thrombozyten spielen eine wichtige Rolle im Körper, denn als Teil des Gerinnungssystems, sind sie daran beteiligt Blutverlust vorzubeugen und zu stoppen. Gleichwohl können sie bei Störungen des Gerinnungssystems zu unkontrollierbaren Blutungen und auch durch Aggregation zu kardiovaskulären Ereignissen, wie Herzinfarkt und Schlaganfällen führen. Für ein besseres Verständnis von Hämostase und Gerinnungsstörungen ist es deshalb nötig die Funktion und Aktivierung von Thrombozyten zu verstehen, welche durch eine Vielzahl von Membranrezeptoren und anderen Faktoren gesteuert wird. Eine Methode, um weitere Einblicke in diese Prozesse zu bekommen ist die mikroskopische Darstellung von Rezeptorverteilungen auf der Zellmembran und deren Interaktionen. Dies zu realisieren ist aus verschiedenen Gründen anspruchsvoll. Der mikroskopisch kleine Durchmesser der Thrombozyten macht es konventioneller Lichtmikroskopie schwer, einzelne Rezeptoren auf der Membran darzustellen. Außerdem befinden sich sehr viele Rezeptoren dicht gepackt auf der Membran, sodass sogar superhochauflösende Mikroskope Schwierigkeiten haben, die Rezeptoren quantitativ zu beurteilen. Mit ‚Expansion microscopy‘ (ExM) wurde eine relativ junge superhochaufösende Technik auf Thrombozyten angewendet. Diese Technik erreicht Auflösungen vergleichbar mit sogenannten ‚super-resolution‘ Mikroskopen, ohne die Benutzung selbiger, sondern durch die Kombination von konfokaler Mikroskopie mit einer physikalisch expandierten Probe. In dieser Arbeit evaluierte ich das Potential dieser Technik für superhochauflösende Bilder von Thrombozytenrezeptoren und optimierte die Probenvorbereitung, sodass zweifarbige 3D Bilder von verschiedenen Membranrezeptoren möglich waren. Die Ergebnisse der Kolokationsanalyse zeigten einen deutlich vergrößerten Dynamikumfang durch ExM. Außerdem katalogisierte ich fluoreszenzmarkierte Antikörper gegen verschiedene Thrombozyten Rezeptoren bezüglich ihrer Tauglichkeit mit ExM und zeigte, dass es möglich ist Membranrezeptoren und Bestandteile des Zytoskeletts gleichzeitig zu färben. KW - Expansion Microscopy KW - platelets KW - Mikroskopie KW - Microscopy Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-309003 ER - TY - JOUR A1 - Wippel, Carolin A1 - Förtsch, Christina A1 - Hupp, Sabrina A1 - Maier, Elke A1 - Benz, Roland A1 - Ma, Jiangtao A1 - Mitchell, Timothy J A1 - Iliev, Asparouh I T1 - Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue JF - The Journal of Infectious Diseases N2 - Background Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis. Many cellular effects of pneumolysin appear to be calcium dependent. Methods  Live imaging of primary mouse astroglia exposed to sublytic amounts of pneumolysin at various concentrations of extracellular calcium was used to measure changes in cellular permeability (as judged by lactate dehydrogenase release and propidium iodide chromatin staining). Individual pore properties were analyzed by conductance across artificial lipid bilayer. Tissue toxicity was studied in continuously oxygenated acute brain slices. Results  The reduction of extracellular calcium increased the lytic capacity of the toxin due to increased membrane binding. Reduction of calcium did not influence the conductance properties of individual toxin pores. In acute cortical brain slices, the reduction of extracellular calcium from 2 to 1 mM conferred lytic activity to pathophysiologically relevant nonlytic concentrations of pneumolysin. Conclusions  Reduction of extracellular calcium strongly enhanced the lytic capacity of pneumolysin due to increased membrane binding. Thus, extracellular calcium concentration should be considered as a factor of primary importance for the course of pneumococcal meningitis. " KW - bacteria Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139356 VL - 204 IS - 6 ER - TY - JOUR A1 - Rohleder, Florian A1 - Huang, Jing A1 - Xue, Yutong A1 - Kuper, Jochen A1 - Round, Adam A1 - Seidman, Michael A1 - Wang, Weidong A1 - Kisker, Caroline T1 - FANCM interacts with PCNA to promote replication traverse of DNA interstrand crosslinks JF - Nucleic Acids Research N2 - FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair. KW - genome integrity KW - repair and replication KW - FANCM KW - proliferating cell nuclear antigen (PCNA) Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175401 VL - 44 IS - 7 ER - TY - THES A1 - Balakrishnan, Ashwin T1 - Fast molecular mobility of β\(_2\)-adrenergic receptor revealed by time-resolved fluorescence spectroscopy T1 - Schnelle molekulare Beweglichkeit des β\(_2\)-adrenergen Rezeptors durch zeitaufgelöste Fluoreszenzspektroskopie N2 - G-protein- coupled receptors (GPCRs) are the largest family of membrane confined receptors and they transduce ligand binding to downstream effects. Almost 40% of the drugs in the world target GPCRs due to their function, albeit knowing less about their activation. Understanding their dynamic behaviour in basal and activated state could prove key to drug development in the future. GPCRs are known to exhibit complex molecular mobility patterns. A plethora of studies have been and are being conducted to understand the mobility of GPCRs. Due to limitations of imaging and spectroscopic techniques commonly used, the relevant timescales are hard to access. The most commonly used techniques are electron paramagnetic resonance or double electronelectron resonance, nuclear magnetic resonance, time-resolved fluorescence, single particle tracking and fluorescence recovery after photobleaching. Among these techniques only fluorescence has the potential to probe live cells. In this thesis, I use different time-resolved fluorescence spectroscopic techniques to quantify diffusion dynamics / molecular mobility of β2-adrenergic receptor (β2-AR) in live cells. The thesis shows that β2-AR exhibits mobility over an exceptionally broad temporal range (nanosecond to second) that can be linked to its respective physiological scenario. I explain how β2-AR possesses surprisingly fast lateral mobility (~10 μm²/s) associated with vesicular transport in contrast to the prior reports of it originating from fluorophore photophysics and free fluorophores in the cytosol. In addition, β2-AR has rotational mobility (~100 μs) that makes it conform to the Saffman-Delbrück model of membrane diffusion unlike earlier studies. These contrasts are due to the limitations of the methodologies used. The limitations are overcome in this thesis by using different time-resolved fluorescence techniques of fluorescence correlation spectroscopy (FCS), time-resolved anisotropy (TRA) and polarisation resolved fullFCS (fullFCS). FCS is limited to microsecond to the second range and TRA is limited to the nanosecond range. fullFCS complements the two techniques by covering the blind spot of FCS and TRA in the microsecond range. Finally, I show how ligand stimulation causes a decrease in lateral mobility which could be a hint at cluster formation due to internalisation and how β2-AR possesses a basal oligomerisation that does not change on activation. Thus, through this thesis, I show how different complementary fluorescence techniques are necessary to overcome limitations of each technique and to thereby elucidate functional dynamics of GPCR activation and how it orchestrates downstream signalling. N2 - G¬Protein¬gekoppelte Rezeptoren (GPCRs) sind die größte Familie der Membran¬Rezeptoren und durch Bindung von Liganden leiten sie extrazlluläre Signal in das Innere der Zelle weiter. Fast 40% der Medikamente auf der Welt zielen aufgrund ihrer Funktion auf GPCRs ab, obwohl man relative wenig über ihre Aktivierung weiß. Das Verständnis ihres dynamischen Verhaltens im basalen und aktivierten Zustand könnte sich in Zukunft als Schlüssel zur Medikamentenentwicklung erweisen. GPCRs sind dafür bekannt, dass sie komplexe molekulare Bewegungsmuster aufweisen. Eine Fülle von Studien wurden und werden durchgeführt, um die Beweglichkeit von GPCRs zu verstehen. Aufgrund der Einschränkungen der gängigen bildgebenden und spektroskopischen Techniken sind die relevanten Zeitskalen nur schwer messbar. Die am häufigsten verwendeten Techniken sind die paramagnetische Elektronenresonanz oder die Doppel¬Elektron¬Elektron¬Resonanz, die magnetische Kernresonanz, die zeitaufgelöste Fluoreszenz, die Einzelpartikelverfolgung und die Fluoreszenzwiederherstellung nach Photobleichung. Unter diesen Techniken haben nur die Fluoreszenz¬basierten Techniken das Potential, lebende Zellen zu untersuchen. In dieser Arbeit werden verschiedene zeitaufgelöste fluoreszenzspektroskopische Techniken zur Quantifizierung der Diffusionsdynamik oder molekularen Mobilität des β2¬adrenergen Rezeptors (β2¬AR) in lebenden Zellen verwendet. Diese Arbeit zeigt, dass β2-AR eine Beweglichkeit über einen außergewöhnlich breiten, zeitlichen Bereich (Nanosekunde bis Sekunde) aufweist, der mit dem jeweiligen physiologischen Szenario verknüpft werden kann. Es wird gezeigt, wie β2¬AR eine überraschend schnelle, laterale Bewegung (~10 μm²/s) besitzt, welche mit vesikulärem Transport in Verbindung gebracht werden kann. Im Gegensatz zu früheren Berichten, wonach die beobachtete Komponente von der Photophysik der Fluorophore und freien Fluorophoren im Zytosol abstammt. Zusätzlich weist β2¬AR eine Rotationsbeweglichkeit (~100 μs) auf, welche es ¬ im Gegensatz zu früheren Studien ¬ dem Saffman¬Delbrück¬Modell der Membrandiffusion zuordnen lässt. Dieser Unterschied ist auf die Beschränkungen der verwendeten Techniken zurückzuführen. Die Einschränkungen werden in dieser Arbeit durch die Verwendung verschiedener zeitaufgelöster Fluoreszenztechniken überwunden, z. B. der Fluoreszenzkorrelationsspektroskopie (FCS) im Bereich von mehreren hundert Nanosekunden bis Sekunden, der zeitaufgelösten Anisotropie (TRA) im Nanosekundenbereich und der polarisationsaufgelösten FullFCS (FullFCS), die die zeitlich Lücke zwischen FCS und TRA schließt. Zuletzt wird eine Abnahme der lateralen Beweglichkeit durch Ligandenstimulation gezeigt, was ein Hinweis auf Clusterbildung aufgrund von Internalisierung sein könnte, und dass β2¬AR eine basale Oligomerisierung aufweist, die sich bei Aktivierung nicht ändert. Zusammenfassend kann man sagen, dass verschiedene komplementäre Fluoreszenztechniken notwendig sind, um die Einschränkungen der einzelnen Techniken zu überwinden und dadurch die funktionelle Dynamik der GPCR¬Aktivierung und deren Bedeutung für die nachgeschaltete Signalübertragung aufzuklären. KW - Fluorescence correlation spectroscopy KW - GPCR KW - time-resolved anisotropy KW - adrenergic receptor KW - homoFRET Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250856 ER - TY - JOUR A1 - Reuter, Isabel A1 - Jäckels, Jana A1 - Kneitz, Susanne A1 - Kuper, Jochen A1 - Lesch, Klaus-Peter A1 - Lillesaar, Christina T1 - Fgf3 is crucial for the generation of monoaminergic cerebrospinal fluid contacting cells in zebrafish JF - Biology Open N2 - In most vertebrates, including zebrafish, the hypothalamic serotonergic cerebrospinal fluid-contacting (CSF-c) cells constitute a prominent population. In contrast to the hindbrain serotonergic neurons, little is known about the development and function of these cells. Here, we identify fibroblast growth factor (Fgf)3 as the main Fgf ligand controlling the ontogeny of serotonergic CSF-c cells. We show that fgf3 positively regulates the number of serotonergic CSF-c cells, as well as a subset of dopaminergic and neuroendocrine cells in the posterior hypothalamus via control of proliferation and cell survival. Further, expression of the ETS-domain transcription factor etv5b is downregulated after fgf3 impairment. Previous findings identified etv5b as critical for the proliferation of serotonergic progenitors in the hypothalamus, and therefore we now suggest that Fgf3 acts via etv5b during early development to ultimately control the number of mature serotonergic CSF-c cells. Moreover, our analysis of the developing hypothalamic transcriptome shows that the expression of fgf3 is upregulated upon fgf3 loss-of-function, suggesting activation of a self-compensatory mechanism. Together, these results highlight Fgf3 in a novel context as part of a signalling pathway of critical importance for hypothalamic development. KW - Fgf-signalling KW - Serotonin KW - Dopamine KW - Hypothalamus KW - Central nervous system Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200749 VL - 8 ER - TY - JOUR A1 - Mielich-Süss, Benjamin A1 - Wagner, Rabea M. A1 - Mietrach, Nicole A1 - Hertlein, Tobias A1 - Marincola, Gabriella A1 - Ohlsen, Knut A1 - Geibel, Sebastian A1 - Lopez, Daniel T1 - Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus JF - PLoS Pathogens N2 - Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen. KW - flotillin KW - scaffold protein KW - Staphylococcus aureus KW - type VII secretion system Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170035 VL - 13 IS - 11 ER - TY - JOUR A1 - Piselli, Claudio A1 - Benz, Roland T1 - Fosmidomycin transport through the phosphate‐specific porins OprO and OprP of Pseudomonas aeruginosa JF - Molecular Microbiology N2 - The Gram‐negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen, responsible for many hospital‐acquired infections. The bacterium is quite resistant toward many antibiotics, in particular because of the fine‐tuned permeability of its outer membrane (OM). General diffusion outer membrane pores are quite rare in this organism. Instead, its OM contains many substrate‐specific porins. Their expression is varying according to growth conditions and virulence. Phosphate limitations, as well as pathogenicity factors, result in the induction of the two mono‐ and polyphosphate‐specific porins, OprP and OprO, respectively, together with an inner membrane uptake mechanism and a periplasmic binding protein. These outer membrane channels could serve as outer membrane pathways for the uptake of phosphonates. Among them are not only herbicides, but also potent antibiotics, such as fosfomycin and fosmidomycin. In this study, we investigated the interaction between OprP and OprO and fosmidomycin in detail. We could demonstrate that fosmidomycin is able to bind to the phosphate‐specific binding site inside the two porins. The inhibition of chloride conductance of OprP and OprO by fosmidomycin is considerably less than that of phosphate or diphosphate, but it can be measured in titration experiments of chloride conductance and also in single‐channel experiments. The results suggest that fosmidomycin transport across the OM of P. aeruginosa occurs through OprP and OprO. Our data with the ones already known in the literature show that phosphonic acid‐containing antibiotics are in general good candidates to treat the infections of P. aeruginosa at the very beginning through a favorable OM transport system. KW - fosmidomycin KW - lipid bilayer membrane KW - OprO KW - OprP KW - porin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238905 VL - 116 IS - 1 SP - 97 EP - 108 ER - TY - JOUR A1 - Bothe, Sebastian A1 - Hänzelmann, Petra A1 - Böhler, Stephan A1 - Kehrein, Josef A1 - Zehe, Markus A1 - Wiedemann, Christoph A1 - Hellmich, Ute A. A1 - Brenk, Ruth A1 - Schindelin, Hermann A1 - Sotriffer, Christoph T1 - Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97 JF - Communications Chemistry N2 - Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. Here, we describe a biolayer interferometry-based fragment screen targeting the N-domain of p97 and demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97. KW - fragment screening KW - AAA+ ATPase p97 KW - biosensor Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300821 VL - 5 IS - 1 ER - TY - THES A1 - Valchanova, Stamatova Ralitsa T1 - Functional analysis of the murine cytomegalovirus genes m142 and m143 N2 - Human cytomegalovirus (HCMV) infection causes clinical symptoms in immunocompromised individuals such as transplantant recipients and AIDS patients. The virus is also responsible for severe complications in unborn children and young infants. The species specificity of HCMV prevents the direct study of mechanisms controlling the infection in animal models. Instead, the murine cytomegalovirus (MCMV) is used as a model system. Human and murine CMVs have large double-stranded DNA genomes, encoding nearly 170 genes. About 30% of the genes are committed to essential tasks of the virus. The remaining genes are involved in virus pathogenesis or host interaction and are dispensable for virus replication. The CMV genes are classified in gene families, based on sequence homology. In the present work, the function of two genes of the US22 gene family was analyzed. The MCMV genes m142 and m143 are the only members of this family that are essential for virus replication. These genes also differ from the remaining ten US22 gene family members in that they lack 1 of 4 conserved sequence motifs that are characteristic of this family. The same conserved motif is missing in the HCMV US22 family members TRS1 and IRS1, suggesting a possible functional homology. To demonstrate an essential role of m142 and m143, the genes were deleted from the MCMV genome, and the mutants were reconstituted on complementing cells. Infection of non-complementing cells with the deletion mutants did not result in virus replication. Virus growth was rescued by reinsertion of the corresponding genes. Cells infected with the viral deletion mutants synthesized reduced amounts of viral DNA, and viral late genes were not expressed. However, RNA analyses showed that late transcripts were present, excluding a role of m142 and m143 in regulation of gene transcription. Metabolic labelling experiments showed that total protein synthesis at late times postinfection was impaired in cells infected with deletion mutants. Moreover, the dsRNA-dependent protein kinase R (PKR) and its target protein, the translation initiation factor 2α (eIF2α) were phosphorylated in these cells. This suggested that the m142 and m143 are required for blocking the PKR-mediated shut-down of protein synthesis. Expression of the HCMV gene TRS1, a known inhibitor of PKR activation, rescued the replication of the deletion mutants, supporting the observation that m142 and m143 are required to inhibit this innate immune response of the host cell. N2 - Die Infektion mit dem humanen Cytomegalovirus (HCMV) kann bei immunsupprimierten Personen wie Transplantatempfängern oder AIDS Patienten, aber auch bei Neugeborenen klinische Symptome hervorrufen. Die Spezies-Spezifität des humanen CMV lässt keine Untersuchung viraler Mechanismen im Tiermodell zu, jedoch steht mit dem murinen CMV (MCMV) ein geeignetes und verbreitetes Modell zur Verfügung. Beide CMVs besitzen große doppelsträngige DNA Genome, die ca. 170 Gene beinhalten. Hiervon sind ca. 30% essentiell für die virale Replikation. Die anderen Gene sind für die Pathogenesse und Interaktion mit den Wirtszellen von Bedeutung. Die Gene des CMV werden auf Grund von Sequenzhomologien in Familien gruppiert. In der vorliegenden Arbeit wird die Funktion der Gene m142 und m143 des MCMV analysiert. Beide Gene sind die einzigen für die Virusreplikation essentiellen Mitglieder der US22 Genfamilie. Darüber hinaus unterscheiden sie sich von den anderen 10 US22 Mitgliedern darin, daß ihnen eine von vier konservierten Sequenzmotiven fehlt. Dieses fehlende Motiv kommt auch bei den HCMV US22 Mitgliedern TRS1 und IRS1 nicht vor, was einen möglichen Hinweis auf eine funktionelle Homologie gibt. Um die essentielle Rolle der m142 und m143 Gene zu belegen, wurden letztere aus dem MCMV Genom entfernt und die Virusmutanten auf komplimentierenden Zellen rekonstituiert. Die Infektion nicht komplimentierender Zellen mit den Virusmutanten erzeugte keine Infektion, konnte jedoch mit der Reinsertion der Gene wieder hergestellt werden. Infizierte Zellen, die mit den Virusmutanten infiziert wurden, produzierten geringere Mengen viraler DNA. Obwohl die Expression später viraler Gene nicht stattfand, konnten späte virale Transkripte nachgewiesen und somit eine Rolle von m142 und m143 bei der Regulation der viralen Transkription ausgeschlossen werden. In Experimenten, in denen Zellen metabolisch markiert wurden, wurde gezeigt, daß die Gesamtproteinsynthese zu späten Zeitpunkten nach Infektion mit den Virusmutanten gehemmt war. Des weiteren wurde eine Phosphorylierung der dsRNA-abhängigen Proteinkinase R (PKR) sowie des Zielproteins, des Translations Initiationsfaktors 2α (eIF2α), nachgewiesen. Dies läßt vermuten, daß m142 und m143 die PKR-vermittelte Stillegung der Proteinsynthese verhindern. Durch Expression des HCMV TRS1 Gens, einem bekannten Inhibitor der PKR-Aktivierung, konnte die Replikation der Virusmutanten wieder hergestellt werden. Dies unterstützt die Ansicht, daß m142 und m143 für die Inhibition der Angeborenen Immunanwort der infizierten Wirtszelle erforderlich sind. KW - Maus KW - Cytomegalie-Virus KW - Genanalyse KW - murine cytomegalovirus KW - essential genes KW - US22 gene family KW - PKR KW - protein synthesis shut down KW - innate immune response Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-20215 ER - TY - JOUR A1 - Mambretti, Egle M. A1 - Kistner, Katrin A1 - Mayer, Stefanie A1 - Massotte, Dominique A1 - Kieffer, Brigitte L. A1 - Hoffmann, Carsten A1 - Reeh, Peter W. A1 - Brack, Alexander A1 - Asan, Esther A1 - Rittner, Heike L. T1 - Functional and structural characterization of axonal opioid receptors as targets for analgesia JF - Molecular Pain N2 - Background Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in naïve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. Results Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala\(^2\), N-MePhe\(^4\), Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in naïve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using β-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of β-arrestin-2 to the membrane followed by a β-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization. Conclusion MOPs are present and functional in the axonal membrane from naïve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia. KW - µ-Opioid receptor KW - hypertonic solution KW - fentanyl KW - calcitonin gene-related peptide KW - DAMGO KW - internalization KW - peripheral nerve KW - ultrastructure Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145917 IS - 12 ER - TY - THES A1 - Schneider, Johannes T1 - Functional diversification of membrane microdomains in Bacillus subtilis T1 - Funktionale Diversifizierung von Membran-Mikrodomänen in Bacillus subtilis N2 - Eukaryotic cells are considered as evolutionary complex organisms because they possess organelles that enable them to regulate the spatio-temporal organization of cellular processes. Spatio-temporal organization of signal transduction cascades occurs in eukaryotic cells via organization of membrane-associated microdomains or lipid rafts. Lipid rafts are nanoscale-sized domains in the plasma membrane that are constituted by a specific set of lipids and proteins and harbor a number of proteins related to signal transduction and trafficking. The integrity of lipid rafts is important for the assembly and functional coordination of a plethora of signaling networks and associated processes. This integrity is partially mediated by a chaperone protein called flotillin. Disruption of lipid raft integrity, for example via depletion or overproduction of flotillin, alters raft-associated signal transduction cascades and causes severe diseases like Alzheimer’s, Parkinson’s disease or cardiovascular disease. It was traditionally assumed that a sophisticated compartmentalization of cellular processes like the one exhibited in lipid rafts was exclusive to eukaryotic cells and therefore, lipid rafts have been considered as a hallmark in the evolution of cellular complexity, suggesting that prokaryotic cells were too simple organisms to organize such sophisticated membrane platforms. However, it was recently discovered that bacteria are also able to organize Functional Membrane Microdomains (FMMs) in their cellular membrane that are able to organize and catalyze the functionality of many diverse cellular processes. These FMMs of bacterial membranes contain flotillin-like proteins which play important roles in the organization of FMM-associated cellular processes. In this dissertation I describe the structural and biological significance of the existence of two distinct flotillin proteins, FloA and FloT, in the FMMs of the bacterial model Bacillus subtilis. Localization studies, proteomic data and transcriptomic analyses show that FloA and FloT are individual scaffold proteins that activate different regulatory programs during bacterial growth. Using the tractable bacterial model system, I show that the functionality of important regulatory proteins, like the protease FtsH or the signaling kinases KinC, PhoR and ResE, is linked to the activity of FMMs and that this is a direct consequence of the scaffold activity of the bacterial flotillins. FloA and FloT distribute heterogeneously along the FMMs of B. subtilis thereby generating a heterogeneous population of FMMs that compartmentalize different signal transduction cascades. Interestingly, diversification of FMMs does not occur randomly, but rather in a controlled spatio-temporal program to ensure the activation of given signaling networks at the right place and time during cell growth. N2 - Eukaryotische Zellen werden als evolutionär komplexe Organismen betrachtet, weil sie Organellen besitzen, mit denen sie die raum-zeitliche Organisation von zellulären Prozessen steuern können. Die räumliche und zeitliche Organisation von Signalwegen in eukaryotischen Zellen erfolgt durch die Abgrenzung von membran-assoziierten Mikrodomänen oder Lipid Rafts. Lipid Rafts sind wenige Nanometer große Felder in der Plasmamembran, die aus einem spezifischen Set von Lipiden und Proteinen zusammengesetzt sind und eine Reihe von für die Signaltransduktion und den Proteintransfer erforderlichen Proteine enthalten. Die Integrität der Lipid Rafts ist wichtig um zahlreiche Signalwege und damit assoziierte Prozesse zu verbinden und funktional zu koordinieren. Diese Integrität wird zum Teil von einem Chaperon-Protein namens Flotillin vermittelt. Eine Beeinträchtigung der Integrität der Lipid Rafts, z.B. aufgrund eines Mangels an Flotillin oder einer Überproduktion von Flotillin, verändert Raft-assoziierte Signalwege und verursacht schwere Erkrankungen wie Alzheimer, Parkinson oder kardiovaskuläre Erkrankungen. Bislang wurde angenommen, dass eine so anspruchsvolle Kompartimentierung zellulärer Prozesse wie im Falle der Lipid Rafts ausschließlich in eukaryotischen Zellen vorkommt. Lipid Rafts galten daher als Meilenstein in der Evolution der zellulären Komplexität und prokaryotische Zellen als zu einfache Organismen, um solch komplexe Plattformen in der Membran einzurichten. Vor kurzem wurde jedoch herausgefunden, dass Bakterien ebenfalls in der Lage sind, Funktionale Mikrodomänen in der Membran (FMMs) zu formen, die viele verschiedene zelluläre Prozesse organisieren und katalysieren können. Diese FMMs in bakteriellen Membranen enthalten Flotillin-ähnliche Proteine, die wichtige Aufgaben bei der Organisation von FMM-assoziierten Prozessen übernehmen. In dieser Dissertation beschreibe ich die strukturelle und biologische Signifikanz des Vorkommens der beiden verschiedenen Flotillin-Proteine FloA und FloT in den FMMs des bakteriellen Modellorganismus Bacillus subtilis. Lokalisationsstudien, proteomische Daten und transkriptomische Analysen demonstrieren, dass FloA und FloT individuelle Gerüstproteine sind, die während des Bakterienwachstums verschiedene regulatorische Programme aktivieren. Mit Hilfe des zugänglichen bakteriellen Modellorganismus zeige ich, dass die Funktionsweise von wichtigen regulatorischen Proteinen, wie z.B. der Protease FtsH oder der Signalwegskinasen KinC, PhoR und ResE, an die Aktivität der FMMs gebunden ist, und dass dies eine direkte Folge der stützenden Tätigkeit der bakteriellen Flotilline ist. FloA und FloT sind unterschiedlich in den FMMs von B. subtilis verteilt, wodurch sie eine heterogene Population von FMMs erzeugen, die verschiedene Signalwege abgrenzen kann. Interessanterweise erfolgt die Diversifizierung der FMMs nicht zufällig, sondern durch ein räumlich und zeitlich kontrolliertes Programm, um die Aktivierung von bestimmten Signalwegen am richtigen Ort und zur richtigen Zeit während des Zellwachstums sicherzustellen. KW - Heubacillus KW - Plasmamembran KW - Diversifikation KW - FMMs KW - Bacillus subtilis KW - Flotillin KW - Lipid Rafts Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127569 ER - TY - THES A1 - Khayenko, Vladimir T1 - Functional peptide-based probes for the visualization of inhibitory synapses T1 - Funktionelle peptidbasierte Sonden zur Visualisierung von hemmenden Synapsen N2 - Short functional peptidic probes can maximize the potential of high-end microscopy techniques and multiplex imaging assays and provide new insights into normal and aberrant molecular, cellular and tissue function. Particularly, the visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques and relies either on genetic manipulation or on antibodies that underperform in tissue immunofluorescence. Starting from an endogenous activity-related ligand of gephyrin, a universal marker of the inhibitory post-synapse, I developed a short peptidic multivalent binder with exceptional affinity and selectivity to gephyrin. By tailoring fluorophores to the binder, I have obtained Sylite, a probe for the visualization of inhibitory synapses, with an outstanding signal-to-background ratio, that bests the “gold standard” gephyrin antibodies both in selectivity and in tissue immunofluorescence. In tissue Sylite benefits from simplified handling, provides robust synaptic labeling in record-short time and, unlike antibodies, is not affected by staining artefacts. In super-resolution microscopy Sylite precisely localizes the post-synapse and enables accurate pre- to post-synapse measurements. Combined with complimentary tracing techniques Sylite reveals inhibitory connectivity and profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the periaqueductal gray brain region. Lastly, upon probe optimization for live cell application and with the help of novel thiol-reactive cell penetrating peptide I have visualized inhibitory synapses in living neurons. Taken together, my work provided a versatile probe for conventional and super-resolution microscopy and a workflow for the development and application of similar compact functional synthetic probes. N2 - Kurze funktionelle peptidische Sonden können das Potenzial von High-End-Mikroskopietechniken und Multiplex-Imaging-Assays maximieren und neue Erkenntnisse über normale und abweichende Molekulare-, Zelluläre- und Gewebefunktionen liefern. Insbesondere die Visualisierung inhibitorischer Synapsen erfordert eine Anpassung des Protokolls an verschiedene Probentypen und Bildgebungsverfahren und ist entweder auf genetische Manipulationen oder auf Antikörper angewiesen, die in der Gewebeimmunfluoreszenz unterdurchschnittlich abschneiden. Ausgehend von einem endogenen aktivitätsbezogenen Liganden von Gephyrin, einem universellen Marker der hemmenden Postsynapse, habe ich einen kurzen peptidischen multivalenten Binder mit außergewöhnlicher Affinität und Selektivität zu Gephyrin entwickelt. Durch die Anpassung von Fluorophoren an das Bindemittel habe ich Sylite erhalten, eine Sonde für die Visualisierung inhibitorischer Synapsen mit einem hervorragenden Signal-Hintergrund-Verhältnis, das die "Goldstandard"-Gephyrin-Antikörper sowohl in der Selektivität als auch in der Gewebe-Immunfluoreszenz übertrifft. Im Gewebe profitiert Sylite von einer vereinfachten Handhabung, bietet eine robuste synaptische Markierung in rekordverdächtig kurzer Zeit und wird im Gegensatz zu Antikörpern nicht durch Färbungsartefakte beeinträchtigt. In der Super-Resolution-Mikroskopie lokalisiert Sylite präzise die Post-Synapse und ermöglicht genaue Messungen von Prä- zu Postsynapse. In Kombination mit ergänzenden Tracing-Techniken deckt Sylite die hemmende Konnektivität auf und erstellt Profile der hemmenden Eingänge und Synapsengrößen von erregenden und hemmenden Neuronen in der periaquäduktalen Grau Hirnregion. Schließlich habe ich nach Optimierung der Sonde für die Anwendung in lebenden Zellen und mit Hilfe eines neuartigen thiolreaktiven zelldurchdringenden Peptids hemmende Synapsen in lebenden Neuronen visualisiert. Insgesamt lieferte meine Arbeit eine vielseitige Sonde für konventionelle und superauflösende Mikroskopie und einen Arbeitsablauf für die Entwicklung und Anwendung ähnlicher kompakter funktioneller synthetischer Sonden. KW - Fluoreszenzsonde KW - Peptidsynthese KW - Neurowissenschaften KW - Inhibitorische Synapse KW - Gephyrin KW - Peptide KW - Fluorescent probes KW - Neuroscience KW - Inhibitory synapse KW - Super-Resolution Microscopy KW - Tissue staining Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320438 ER - TY - THES A1 - Erro, Alejandro Berna T1 - Generation and Characterization of Stromal Interaction Molecule 2 (STIM2)-deficient Mice T1 - Generierung und Charakterisierung von Stromal Interaction Molecule 2 (STIM2)-defizienten Mäusen N2 - An increase in cytosolic Ca2+ levels ([Ca2+]i) is a key event that occurs downstream of many signaling cascades in response to an external stimulus and regulates a wide range of cellular processes, including platelet activation. Eukaryotic cells increase their basal [Ca2+]i allowing extracellular Ca2+ influx into the cell, which involves different mechanisms. Store-operated Ca2+ entry (SOCE) is considered the main mechanism of extracellular Ca2+ influx in electrically non-excitable cells and platelets, and comprises an initial Ca2+ depletion from intracellular Ca2+ stores prior to activation of extracellular Ca2+ influx. Although the close relation between Ca2+ release from intracellular stores and extracellular Ca2+ influx was clear, the nature of the signal that linked both events remained elusive until 2005, when Stromal Interaction Molecule 1 (STIM1) was identified as an endoplasmic reticulum (ER) Ca2+ sensor essential for inositol (1,4,5)-trisphosphate (IP3)-mediated SOCE in vitro. However, the function of its homologue STIM2 in Ca2+ homeostasis was in general unknown. Therefore, mice lacking STIM2 (Stim2-/-) were generated in this work to study initially STIM2 function in platelets and in cells of the immune system. Stim2-/- mice developed normally in size and weight to adulthood and were fertile. However, for unknown reasons, they started to die spontaneously at the age of 8 weeks. Unexpectedly, Stim2-/- mice did not show relevant differences in platelets, revealing that STIM2 function is not essential in these cells. However, STIM2 seems to be involved in mammary gland development during pregnancy and is essential for mammary gland function during lactation. CD4+ T cells lacking STIM2 showed decreased SOCE. Our data suggest that STIM2 has a very specific function in the immune system and is involved in Experimental Autoimmune Encephalomyelitis (EAE) at early stages of the disease progression. Stim2-/- neurons were also defective in SOCE. Surprisingly, our results evidenced that STIM2 participates in mechanisms of neuronal damage after ischemic events in brain. This is the first time that the involvement of SOCE in ischemic neuronal damage has been reported. This finding may serve as a basis for the development of novel neuroprotective agents for the treatment of ischemic stroke, and possibly other neurodegenerative disorders in which disturbances in cellular Ca2+ homeostasis are considered a major pathophysiological component. N2 - Der Anstieg des cytosolischen Ca2+-Spiegels ([Ca2+]i) ist ein Schlüsselereignis, das vielen Signalkaskaden, durch extrazellulären Stimulus ausgelöst werden, nachgeschalten ist, und eine große Reihe zellulärer Prozesse reguliert, z.B. die Aktivierung von Blutplättchen. Eukaryotische Zellen erhöhen ihren basalen ([Ca2+]i) durch Einstrom von extrazellulärem Ca2+ in die Zelle hinein, was durch verschiedene Mechanismen geschehen kann. Store-operated Ca2+-entry (SOCE), wird als der Hauptmechanismus für den Einstrom von extrazellulärem Ca2+ in nicht elektrisch-erregbaren Zellen sowie Plättchen angesehen und beinhaltet einen initialen Ca2+-Ausstrom aus intrazellulären Speichern der dem Ca2+-Einstrom aus der Extrazellulärraum vorrausgeht. Obwohl die Beziehung zwischen Ca2+-Ausstrom aus intrazellulären Speichern und extrazellulärem Ca2+-Einstrom über die Plasmamembran viele Jahre bekannt war, so blieb doch das beide Ereignisse verknüpfende Element unbekannt. Im Jahre 2005 jedoch wurde Stromal Interaction Molecule 1 (STIM1) als Ca2+-Sensor des endoplasmatischen Retikulums (ER) und als essentieller Bestandteil für inositol(1,4,5)-triphosphat (IP3)-vermittelten SOCE in vitro identifiziert. Die Funktion seines Homologs, STIM2, in der Ca2+ Homeostase blieb jedoch unklar. Aus diesem Grund generierten wir STIM2-defiziente (Stim2-/-) Mäuse um die Funktion dieses Proteins in Blutplättchen und Immunzellen untersuchen zu können. Bis zum Erwachsenenalter entwickelten sich Stim2-/- Mäuse normal in Bezug auf Größe und Gewicht und waren fertil. Jedoch sterben die Tiere spontan aus unbekannten Gründen, beginnend ab einem Alter von 8 Wochen. Unerwarteter Weise, zeigten Stim2-/- Mäuse keine maßgeblichen Funktionsunterschiede in Plättchen, was eine essentielle Funktion von STIM2 in diesen Zellen ausschließt. Jedoch scheint STIM2 in die Entwicklung der Brustdrüsen während der Schwangerschaft involviert und essentiell für die Brustdrüsenfunktion während der Säugephase zu sein. Darüberhinaus zeigten STIM2 defiziente CD4+ T-Zellen einen verminderten SOCE. Weiter deuten unsere Daten auf eine spezifische Funktion von STIM2 im Immunsystem hin, mit einem Einfluss auf die frühen Phasen und das Fortschreiten der Experimentellen Autoimmun-Enzephalomyelitis (EAE). Stim2-/- Neuronen wiesen ebenso wie CD4+ T-Zellen einen gestörten SOCE auf. Desweiteren belegen unsere Ergebnisse, dass STIM2 überrascherweise an den Neuronen zerstörenden Mechanismen nach ischämischen Ereignissen des Gehirns mitwirkt. Dies ist die erste Studie, die von einer Beteiligung von SOCEan ischämischen neuronalen Schäden berichtet. Diese Entdeckungen können vielleicht als Basis für die Entwicklung neuer neuroprotektiver Medikamente bei ischämischen Schlaganfall dienen - und möglicherweise auch bei anderen neurodegenerativen Erkrankungen, bei denen Störungen der zellulären Ca2+ Homöostase als hauptsächliche pathophysologische Komponente angesehen werden. KW - Calcium-bindende Proteine KW - Intrazellulärraum KW - Thrombozyt KW - Knockout KW - STIM2 KW - SOC KW - SOCE KW - STIM2 KW - SOC KW - SOCE KW - Store-Operated KW - knockout Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47301 ER - TY - JOUR A1 - Beck, Sarah A1 - Stegner, David A1 - Loroch, Stefan A1 - Baig, Ayesha A. A1 - Göb, Vanessa A1 - Schumbutzki, Cornelia A1 - Eilers, Eva A1 - Sickmann, Albert A1 - May, Frauke A1 - Nolte, Marc W. A1 - Panousis, Con A1 - Nieswandt, Bernhard T1 - Generation of a humanized FXII knock-in mouse-A powerful model system to test novel anti-thrombotic agents JF - Journal of Thrombosis and Haemostasis N2 - Background Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. Objective The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. Methods A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. Results These hF12\(^{KI}\) mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12\(^{KI}\) mice in an arterial thrombosis model without affecting bleeding times. Conclusion These data establish the newly generated hF12\(^{KI}\) mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors. KW - hemostasis, KW - blood coagulation KW - factor XII KW - animal models KW - thrombosis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259567 VL - 19 IS - 11 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 - Tretter, Verena A1 - Mukherjee, Jayanta A1 - Maric, Hans-Michael A1 - Schindelin, Hermann A1 - Sieghart, Werner A1 - Moss, Stephen J. T1 - Gephyrin, the enigmatic organizer at GABAergic synapses JF - Frontiers in Cellular Neuroscience N2 - GABA(A) receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor beta subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABA(A) receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABA(A) receptor alpha 1, alpha 2, and alpha 3 subunits. Gephyrin-binding to GABA(A) receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity. KW - scaffolding protein gephyryrin KW - containing GABA(A) receptors KW - GABA(A) receptors KW - inhibitory synapse KW - gamma-aminobutyric-acid KW - receptor-beta subunits KW - molybdenum cofactor biosynthesis KW - temporal-lobe epilepsy KW - cultured hippocampal-neurons KW - exchange factor collybistin KW - rat spinal-cord KW - glycine KW - gephyrin KW - receptor clustering KW - synapse formation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133356 VL - 6 IS - 23 ER - TY - JOUR A1 - Rauschenberger, Vera A1 - Piro, Inken A1 - Kasaragod, Vikram Babu A1 - Hörlin, Verena A1 - Eckes, Anna-Lena A1 - Kluck, Christoph J. A1 - Schindelin, Hermann A1 - Meinck, Hans-Michael A1 - Wickel, Jonathan A1 - Geis, Christian A1 - Tüzün, Erdem A1 - Doppler, Kathrin A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation JF - Frontiers in Molecular Neuroscience N2 - Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera. KW - glycine receptor KW - autoantibodies KW - glycosylation KW - extracellular domain KW - adsorption Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304206 VL - 16 ER - TY - JOUR A1 - Wiessler, Anna-Lena A1 - Talucci, Ivan A1 - Piro, Inken A1 - Seefried, Sabine A1 - Hörlin, Verena A1 - Baykan, Betül B. A1 - Tüzün, Erdem A1 - Schaefer, Natascha A1 - Maric, Hans M. A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor β–targeting autoantibodies contribute to the pathology of autoimmune diseases JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Background and Objectives Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit–binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. Methods In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Results Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Discussion Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization. KW - autoantibody (aAb) KW - glycine receptor (GlyR) KW - stiff-person syndrome (SPS) KW - clinical neurology KW - movement disorders KW - progressive encephalitis with rigidity and myoclonus (PERM) Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349958 VL - 11 IS - 2 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Schlötzer, Jan A1 - Hadamek, Kerstin A1 - Yuan-Chen, Natalia A1 - Alwahsh, Mohammad A1 - Hollmann, Robert A1 - Fratz, Stefanie A1 - Yesilyurt-Gerhards, Dilan A1 - Frankenbach, Tina A1 - Engelmann, Daria A1 - Keller, Angelika A1 - Kaestner, Alexandra A1 - Schmitz, Werner A1 - Neuenschwander, Martin A1 - Hergenröder, Roland A1 - Sotriffer, Christoph A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Glycolytic flux control by drugging phosphoglycolate phosphatase JF - Nature Communications N2 - Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates. KW - phosphoglycolate phosphatase KW - glycolytic flux control KW - intrinsic metabolism Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300928 VL - 13 IS - 1 ER -