Rational design of selective anion receptors

The new family of the anion receptors based on oligoureas with varied flexibility was developed and studied. The preparation of the urea chains containing two different units in various sequences was elaborated. The complete sets of four cyclic trimers and six tetramers based on the two units were prepared. Their conformational and complexation properties were studied with NMR spectroscopy and X-ray structure determinations, their behaviour towards various anions was evaluated and compared. The synthesis and the same studies were performed also with four different cyclic hexamers. During these studies the remarkable templation by two halide anions was observed.

Die Rolle von Ephrin-B2 in der Invasion maligner Gliome

Das Glioblastoma multiforme zählt zu den häufigsten glialen Neoplasien des Menschen und weist zudem unter den Gliomen die höchste Malignität auf. Glioblastompatienten haben trotz aggressiver therapeutischer Ansätze eine mittlere Überlebenszeit von weniger als einem Jahr. Die diffuse Invasion in das umliegende Hirngewebe ist einer der Hauptgründe für die Rezidivbildung und die infauste Prognose von Glioblastompatienten. Neuere Untersuchungen lassen vermuten, dass die starke Invasion auch einer der Gründe für die beobachtete anti-angiogene Resistenz bei der Behandlung von Glioblastomen ist. Das bidirektionale EphB/Ephrin-B-System wurde bei der axonalen Wegfindung als Vermittler repulsiver Signale identifiziert und auch im Zusammenhang der Migration und Invasion von Zellen überprüft. In der vorliegenden Arbeit sollte daher die Funktion der bidirektionalen Eph- und Ephrin-Signaltransduktion in Bezug auf die Glioblastominvasion und Progression untersucht werden. rn Genetische und epigenetische Untersuchungen der EphB/Ephrin-B-Familie in einer Kohorte von Gliompatienten unterschiedlicher Malignitätsgrade identifizierten Ephrin-B2 als mögliches Tumorsuppressorgen. In Übereinstimmung damit führte die Inaktivierung von Ephrin-B2 in einem murinen Gliommodell zu einer verstärkten Invasion und einem erhöhtem Tumorwachstum in vivo. Dies konnte in verschiedenen Invasion-Assays in vitro bestätigt werden. Weiterhin zeigten unsere Untersuchungen, dass Ephrin-B2 transkriptionell durch das hypoxische Mikromilieu HIF-1α-vermittelt reprimiert wird. Da HIF-1α als transkriptioneller Aktivator Ephrin-B2 nicht direkt reprimieren kann, wurden potentielle HIF-1α-regulierte Repressoren untersucht, die für die Ephrin-B2 Herunterregulation verantwortlich sein könnten. Dabei wurde anhand von Ephrin-B2-Promotoranalysen und ChIP-Assays ZEB2 als HIF-1α-induzierbarer Repressor von Ephrin-B2 identifiziert. Zur Bestätigung der Hypothese, dass ZEB2 ein wichtiger Regulator der Tumorinvasion ist, wurden humane ZEB2-Knockdown-Glioblastomzellen generiert und in vitro sowie in vivo untersucht. Im Hinblick auf mögliche therapeutische Anwendungen wurden die ZEB2-Knockdown-Glioblastomzellen zusätzlich im Zusammenhang anti-Angiogenese-induzierter Invasion analysiert. Der Verlust von ZEB2 führte dabei zu einer verringerten Glioblastominvasion und Progression in einem Maus-Xenograft Modell. Die Behandlung der Tumoren mit dem anti-VEGF-Antikörper Avastin resultierte in einer stark erhöhten Invasion, die durch die Inaktivierung von ZEB2 und der dadurch reaktivierten repulsiven Signale von Ephrin-B2 wieder aufgehoben werden konnte. Zusammenfassend konnte in der vorliegenden Arbeit erstmals gezeigt werden, dass Ephrin-B2 als Tumorsuppressor in Gliomen agiert und durch verschiedene Mechanismen wie der genetischen und epigenetischen Kontrolle, aber auch der HIF-1α-vermittelten, ZEB2-abhängigen Repression inaktiviert wird. Dies resultiert in einer Blockade repulsiver Signale, so dass Tumorzellen diffus in das Parenchym und zu den Blutgefäßen migrieren können. Der in dieser Arbeit neu identifizierte Signalweg stellt ein attraktives therapeutisches Ziel zur Inhibition der Tumorzellinvasion dar und ermöglicht darüber hinaus der Ausbildung von Resistenzen gegenüber anti-angiogener Behandlung entgegenzuwirken. rn

Functional characterisation of in vitro synthesised G-protein coupled receptors in polymersomes

Membrane proteins play an indispensable role in physiological processes. It is, therefore, not surprising that many diseases are based on the malfunction of membrane proteins. Hence membrane proteins and especially G-protein coupled receptors(GPCRs)- the largest subfamily- have become an important drug target. Due to their high selectivity and sensitivity membrane proteins are also feasible for the detection of small quantities of substances with biosensors. Despite this widespread interest in GPCRs due to their importance as drug targets and biosensors there is still a lack of knowledge of structure, function and endogenous ligands for quiet a few of the previously identified receptors.rnBottlenecks in over-expression, purification, reconstitution and handling of membrane proteins arise due to their hydrophobic nature. Therefore the production of reasonable amounts of functional membrane proteins for structural and functional studies is still challenging. Also the limited stability of lipid based membrane systems hampers their application as platforms forrnscreening applications and biosensors.rnIn recent years the in vitro protein synthesis became a promising alternative to gain better yields for expression of membrane proteins in bio-mimetic membrane systems. These expression systems are based on cell extracts. Therefore cellular effects on protein expression are reduced. The open nature of the cell-free expression systems easily allows for the adjustment of reactionrnconditions for the protein of interest. The cell-free expression in the presence of bio-mimetic membrane systems allows the direct incorporation of the membrane proteins and therefore skips the time-consuming purification and reconstitution processes. Amphiphilic block-copolymers emerged as promising alternative for the less stable lipid-based membrane systems. They, likernlipids, form membraneous structures in aqueous solutions but exhibit increased mechanical and chemical stability.rnThe aim of this work was the generation of a GPCR-functionalised membrane system by combining both promising alternatives: in vitro synthesis and polymeric membrane systems. This novel platform should be feasible for the characterisation of the incorporated GPCR. Immunodetection of Dopamine receptor 1 and 2 expressed in diblock- and triblock-polymersomes demonstrated the successful in vitro expression of GPCRs in polymeric membranes. Antibodyrnbinding studies suggested a favoured orientation of dopamine receptors in triblockpolymersomes.rnA dopamine-replacement assay on DRD2-functionalised immobilised triblockpolymersomes confirmed functionality of the receptor in the polymersomes. The altered binding curve suggests an effect of the altered hydrophobic environment presented by the polymer membrane on protein activity.

Induction of RAGE shedding by activation of G-protein-coupled receptors

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNAmediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimers disease and diabetes-induced inflammation.