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Seit gezeigt wurde, dass die genetischen Informationen in Form von DNA gespeichert wird, ist das Geheimnis der DNA-Struktur gelöst, der Mechanismus der Gen-Expression und die Rolle der RNA verstanden worden. Das Interesse für die Chemie und die Biologie der Nukleinsäuren ist somit kontinuierlich gewachsen. Besonders interessant ist die RNA, die eine Rolle als ein Vermittler der genetischen Informationen (mRNA) spielt, aber auch als Bote von Aminosäuren (tRNA). Sie ist im Ribosom (rRNA) anwesend, arbeitet als Templat in Telomerasen für DNA-Synthese und hat außerdem wichtige Funktionen in der RNA-Spaltung, z.B. bei Ribozymen wie RNAse P inne. Betreffend bestimmter Spaltstellen in RNA hat auch das Phänomen der siRNA beträchtliche Aufmerksamkeit in diesem Prozess erregt. Der sogenannte RISC-Komplex wird programmiert, einzelsträngige RNA mit hoher Sequenz-Spezifität zu schneiden. Die für die RNA-Interferenz verantwortliche zelluläre Maschinerie ist auch an der Bilbung von MikroRNAs beteiligt. RNA-Interferenz ist heute eines der nützlichsten Werkzeuge in functional genomics geworden. Die große Hoffnung ist, dass es auch vielleicht in der Therapie angewandt werden könnte. Das Thema meiner Doktorarbeit trägt den Titel „Synthesis of Site-Specific Artificial Ribonucleases“. Es beschäftigt sich mit der Entwicklung künstlicher bindungsspezifischer Ribonucleasen. Diese künstlichen Katalysatoren sind im Wesentlichen aus drei Gründen bedeutsam: Zum einen liegt eine mögliche Anwendung in der Affinity-Cleavage (Affinitätsspaltung), eine Technik, die Bindungsstellen von RNA-Liganden durch das kovalente Anbringen eines Reagenzes lokalisiert, das zwischen den Nukleinsäuren schneidet. Zum anderen entsteht die Möglichkeit, neue Werkzeuge für eine gezielte Manipulation großer RNA-Moleküle zu schaffen. Die Vorteile des Ansatzes sind, dass man damit beliebige Zielsequenzen anwählen kann. Das Problem dieser Strategie ist die Notwendigkeit, hohe Genauigkeit im Spaltungssschritt zu erreichen, wie zum Beispiel mit natürlichen Ribozymen. Wichtige Ergebnisse wurden auch während meiner Arbeit erhalten, mit einem Fall von genauer Spaltung zwischen zwei Basen. Der dritte Grund ist die potentielle Anwendung als katalytische antisense-Oligonucleotide in der Chemotherapie. Gegenwärtig existieren zwei Ansätze, unspezifische künstliche RNasen relativ kleiner Größe zu schaffen. Der erste basiert auf Metallkomplexen und führt im Allgemeinen zu höheren Raten. Die Idee ist, ein Metall als elektrophiles Zentrum zur Unterstützung der Transesterfikation zu nutzen. Unter diesen Katalysatoren enthalten die effizientesten Lanthanid-Ionen, Cu2+ und Zn2+. Der zweite Ansatz zielt darauf ab, metallfreie künstliche Ribonucleasen zu entwickeln. Die Vorteile dieser Strategie sind, den Katalysator von der Stabilität der Metallkomplexe, die in vivo problematisch sein könnten, unabhängig zu machen. In diesem Ansatz wird die natürliche Katalyse durch Enzyme simuliert. Zweckmäßige Gruppen mit beschränkter katalytischer Aktivität z.B. als Nucleophile, Säuren oder Basen, werden in einer Weise zusammengesetzt, um Kooperation zu ermöglichen. Potente Katalysatoren können so ohne die Notwendigkeit von Metallen als Cofaktoren erzeugt werden. ...
Colorectal cancer is one of the most cause of cancer and death in Western societies. Recently, histone deacetylase inhibitors (HDIs), which regulate transcription through modification of chromatin structure, received considerable interest on the ground of they ability to stop the growth and induce cell death in colon cancer tumours, representing a promising transcriptional cancer therapy. This kind of cancer initiates with an activating mutation in the Wnt cascade, allowing the nuclear import of ß-catenin binding to LEF/TCF. This induces the overexpression of growthpromoting oncogenes affecting the cell cycle arrest, lineage-specific cell differentiation and apoptosis processes. In addition, ß-catenin also participates in cell-cell adhesion via interactions with E-cadherin, which can be repressed by families of transcription factors Snail and ZEB. This, and gain of vimentin has been closely correlated with local invasion and metastasis since they avoid the induction of apoptosis through the loss of cell anchorage, a phenomenon called anoikis. In this process the inactivation of the kinases Src an FAK provoking disruption of focal adhesion complexes through is involved. LAQ824 is a HDAC inhibitor derivative of hydroxamic acid, which present antitumor effect in colon and other cancer cells. The aim of this study is to analyse the effect of LAQ824 in cell proliferation, apoptosis, motility and tumour invasion in a colon carcinoma model based on the adenoma-carcinoma sequence descrying trough which pathways LAQ824 is able to cause these effects. Here I demonstrate for the first time that a HDAC inhibitor, LAQ824, induces detachmentinduced cell death of colon cancer cell lines HCT116 and HT-29, a phenomenon called anoikis, in a caspase-dependent and p53-independent manner. In this process the component of the Wnt signalling pathway ß-catenin is involved. Furthermore LAQ824 upregulates the adhesion molecule E-cadherin expression in these cell lines independently of its repressor Snail, but probably mediated by the repressor ZEB. In addition LAQ824-induced anoikis is caused by disruption of focal adhesion complexes through inhibition of the activity of the kinases FAK and Src inhibiting cell motility indicating a strong antimetastatic potential for LAQ824.
The chemiosmotic theory suggested by Peter Mitchell (Mitchell, 1961, Nature 191:144-148; see Mitchell, 1979, Science 206:1148-1159 for review) postulated that the energy released upon the oxidation of electron donor substrates is transiently stored as electrochemical proton potential, delta-p across energy-transducing membranes, which acts then as the driving force for the ATP synthesis. Membrane protein complexes can both generate and utilise a transmembrane electrochemical proton potential, either by transmembrane proton transfer or by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane. The dihaem-containing membrane protein complex quinol:fumarate reductase (QFR) from the anaerobic epsilon-proteobacterium Wolinella succinogenes apparently combines both of these mechanisms (Haas et al, 2005, Biochemistry 44:13949-13961; Lancaster et al, 2005, PNAS 102:18860–18865; Mileni et al, 2005, Biochemistry 44:16718-16728; Madej et al, 2006, EMBO J 25:4963-4970). QFR is the terminal enzyme of anaerobic fumarate respiration that allows bacteria to use fumarate as the terminal electron acceptor (Kröger, 1978, Biochim Biophys Acta 505:129-45; Lancaster, 2004, In: Respiration in Archaea and Bacteria Volume 1:57-85). QFR couples the two-electron reduction of fumarate to succinate to the two-electron oxidation of quinol to quinone. QFR contains two haem b groups bound by the transmembrane subunit C, which are termed the ‘proximal haem’, bP, and the ‘distal haem’, bD, according to the relative proximity to the hydrophilic subunits A and B (Lancaster et al, 1999, Nature 402:377-85). The two-electron transfer via the two haem groups has been proposed (Lancaster, 2002, Biochimica et Biophysica Acta 1565:215-231) and demonstrated (Madej et al, 2006, EMBO J 25:4963-4970) to be coupled to a compensatory, parallel transfer of two protons via a transmembrane proton transfer pathway. The two most prominent constituents of the proposed pathway were suggested to be the haem bD ring C propionate and the side chain of amino-acid residue Glu C180, after which the proton transfer pathway was named the ‘E-pathway’ (Lancaster, 2002, Biochimica et Biophysica Acta 565:215-231). The essential role of Glu C180 was supported by site-directed mutagenesis and structural and functional characterization of the enzyme E180Q, where the Glu C180 was replaced with a Gln residue (Lancaster et al, 2005, PNAS 102:18860–18865). Moreover, multiconformer continuum electrostatics (MCCE) calculations (Haas and Lancaster 2004, Biophys J 87:4298-4315) and Fouriertransformed infrared (FTIR) spectroscopy experiments (Haas et al, 2005, Biochemistry 44:13949-13961) indicated the Glu C180 side chain to undergo a combination of a conformational change and protonation upon haem reduction. The contribution of haem bD propionate is less clear, however, a combination of 13C labelling of the haem propionates with redox-induced FTIR experiments (Mileni et al, 2005, Biochemistry 44:16718-16728) and MCCE calculations (Haas and Lancaster, 2004, Biophys J 87:4298-4315) support a change in protonation, possibly accompanied by a change in environment upon haem reduction. These experiments and their results strongly support the existence of the ‘E-pathway’ which is transiently open during the reduction of the haem groups and blocked in the oxidized state of the enzyme (Lancaster, 2002b, Biochim Biophys Acta 1565:215-231). All available crystal structures of the QFR, however, are those of the oxidized enzyme. Therefore, it is advantageous to perform simulations of various redox states of the enzyme to determine for instance, how the side-chain of Glu C180 and haem bD ring C propionate behave upon changes of the redox states of the haem groups and why is the ‘E-pathway’ blocked in the oxidized state of the enzyme. Although the distal haem ring C propionate and Glu C180 were identified as the most prominent components of the proton transfer pathway, it was not clear, on the basis of the structure, how proton transfer could occur between them. In addition, two constituents are not enough to span the membrane region and the additional participants in the proton transfer pathway must be identified. Since an atomistic investigation of proton transfer in this system is not yet possible experimentally, I used available theoretical methods such as classical molecular dynamics (MD) simulation (Alder and Wainwright, 1959, J Phys Chem 31:459-466; McCammon et al, 1977, Nature 267:585-590) and Q-HOP molecular dynamics (Q-HOP MD) simulation (Lill and Helms, 2001, J Chem Phys 115:7993-8005) to investigate the postulated mechanism of electron coupled proton transfer in QFR. MD simulations allowed us to move away from static difference pictures obtained from FTIR experiments and MCCE calculations. The advantage of the MD simulations over the experiments and the simulations performed so far is that the time-dependent properties could now be analyzed. The behaviour of various residues and their side-chains and any environmental changes may be directly observed during MD simulations. Although classical MD simulations cannot be used to study proton transfer reactions, they can provide information on formation of configurations that would allow either direct proton transfer between donor and acceptor residues or indirect proton transfer mediated by water molecules. To avoid the static protonation of residues which is inherent in classical MD simulations, Q-HOP MD simulations were performed which explicitly describe proton transfer reactions by allowing the change of the protonation state of residues ‘on the fly’. The structures obtained after classical molecular dynamics simulations ....
The ABC protein ABCE1, also called HP68 or RNase L inhibitor (RLI), is one of the most conserved proteins in evolution. It is universally expressed in eukaryotes and archaea, where ABCE1 is essential for life. ABCE1 plays a crucial role in translation initiation and ribosome biogenesis, however, the molecular mechanism of ABCE1 remains unclear. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines predicted to coordinate iron-sulfur (Fe-S) clusters. To analyze the function of ABCE1, the hyperthermophilic crenarchaeote Sulfolobus solfataricus was chosen as a model system. S. solfataricus ABCE1 was overexpressed homologously in S. solfataricus and heterologously in E. coli. Noteworthy, for tagged-protein production in S. solfataricus a novel expression system based on a virus shuttle vector was established. This is the first example for a successful overexpression and purification of isolated full-length ABCE1. For the first time it was shown that ABCE1 indeed bears biochemical properties of an ABC protein even though it has unique features. Remarkably, the nucleotide binding domains (NBDs) of ABCE1 bound ATP and AMP, but were functionally non-equivalent in ATP hydrolysis. Mutations of conserved residues in the second NBD led to a hyperactive ATPase, which implies an intramolecular mechanism of dimer formation. Truncation of the Fe-S cluster domains did not influence ATPase activity. The Fe-S clusters of ABCE1 were analyzed by biophysical and biochemical methods. As presented in this study, ABCE1 harbors two essential diamagnetic [4Fe-4S]2+ clusters, one ferredoxin-like cluster formed by cysteines at position 4/5/6/7 and one unique ABCE1 cluster formed by cysteines at position 1/2/3/8. ABCE1 was found to be associated with RNA after purification from S. solfataricus and bound ribosomal RNA in vitro. In addition, ABCE1 showed homo-oligomerization and appeared to form a hexameric complex of ~440 kDa, which was RNase sensitive. Archaeal ABCE1 associated with ribosomes, however, the unique Fe-S clusters of ABCE1 were not required for this interaction. Although archaeal ABCE1 assembled with ribosomes and ribosomal RNA, ABCE1 proved not to be essential for translation in S. solfataricus and did not interact with archaeal initiation factors. Nevertheless, the ABCE1 gene is one of the few genes conserved between archaea and eukaryotes and fulfills a universal task, which needs further characterization.
Two distinct mechanisms contribute to the development of blood vessels: vasculogenesis, which is the de novo formation of vascular structures from progenitor cells, and angiogenesis, the formation of new blood vessels from pre-existing ones.
Angiogenesis is a highly ordered and carefully regulated multi-step process, during which the precise spatio-temporal interaction between endothelial and mural cells, i.e. smooth muscle cells and pericytes, is prerequisite for the formation of a functional blood vessel. The crosstalk between these two latter cell ty pes is mediated indirectly by various
secreted growth factors, and directly through cell-cell and cell-matrix interactions. The secretory epidermal growth factor-like protein 7 (EGFL7) has been implicated to
play an important role in the regulation of smooth muscle and endothelial cell recruitment and vascular tube formation. However, in-depth investigation of the underlying molecular mechanism has so far been hampered by the lack of functional recombinant EGFL7. In this study for the first time full length EGFL7 was successfully expressed as a His 6- tagged fusion protein from insect cells using the Baculovirus expression vector system. Recombinant EGFL7 was purified in a two-step protocol involving ion metal affinity chromatography and gel filtration. Furthermore, recombinant EGFL7 was
purified from human embryonic kidney EBN A 293 cells using a similar approach, allowing the production of high amounts of recombinant EGFL7 protein in its native state, with proper post-translational processing and full biological activity. Detailed analysis of the post-translational processing of recombinant EGFL7 and EGFL7-mutants revealed extensive proteolytic processing by protein convertases both at the N- and the C-terminus, the latter being prerequisite for EGFL7 secretion. Furthermore, secreted EGFL7 protein was shown to bind to the extracellular matrix and the responsible heparin-binding domain of EGFL7 was mapped to its N-terminal
portion. Purified recombinant EGFL7 protein was tested for its functionality using cell migration assays, cell proliferation studies and in vivo matrigel studies in mice. In the
modified Boyden chamber migration assay, recombinant EGFL7 proteins inhibited PDGF-BB-induced smooth muscle cell migration. Moreover, recombinant EGLF7 proteins strongly inhibited PDGF-BB-induced proliferation of smooth muscle cells, while it did not affect VEGF induced proliferation of endothelial cells. When applied in the in vivo matrigel plug assay, EGFL7 proteins induced a strong pro-angiogenic response, comparable with that of VEGF on an equimolar basis. Moreover, EGFL7 expression was strongly induced in endothelial cells in response to VEGF stimulation. These novel findings demonstrate the important function of EGFL7 in angiogenesis and are well in line with previous results. They demonstrate a cell specific action of EGFL7 on the different cell types involved in vessel formation, which is a prerequisite for a regulatory function in cell-to-cell crosstalk. Based on the results described here, the following model can be proposed: VEGF, a known strong initiator of angiogenesis, induces endothelial cell proliferation and migration, allowing the
escape from the comparatively rigid structure of a functional vessel to form an angiogenic sprout. At the same time VEGF induces the expression of EGFL7 in endothelial cells. EGFL7 is expressed, proc essed and secreted from these cells. While EGFL7 has no known effect on endothelial cells, it inhibits smooth muscle cell proliferation and migration, providing a mechanism to prevent pre-mature stabilization of the forming vessel. The availability of purified recombinant EGFL7 will be helpful in the detailed characterization of the underlying molecular mechanism of EGFL7 action, including the identification of the putative EGFL7 receptor, and will allow - together with knock-out experiments in mice - the exploration of the additional biological functions of EGFL7. Moreover, considering the strong pro-angiogenic effect of EGFL7 in vivo, it would be also of a great therapeutic interest to investigate its role in the development of tumor vasculature. The insights into these molecular mechanisms might provide a novel approach for the development of anti tumor therapies.
Analysis of coding principles in the olfactory system and their application in cheminformatics
(2007)
Unser Geruchssinn vermittelt uns die Wahrnehmung der chemischen Welt. Im Laufe der Evolution haben sich in unserem olfaktorischen System Mechanismen entwickelt, die wahrscheinlich optimal auf die Erfüllung dieser Aufgabe angepasst sind. Die Analyse dieser Verarbeitungsstrategien verspricht Einblicke in effiziente Algorithmen für die Kodierung und Verarbeitung chemischer Information, deren Entwicklung und Anwendung dem Kern der Chemieinformatik entspricht. In dieser Arbeit nähern wir uns der Entschlüsselung dieser Mechanismen durch die rechnerische Modellierung von funktionellen Einheiten des olfaktorischen Systems. Hierbei verfolgten wir einen interdisziplinären Ansatz, der die Gebiete der Chemie, der Neurobiologie und des maschinellen Lernens mit einbezieht.
Transport of proteins into or across cellular membranes is mediated by the conserved and ubiquitous Sec-machinery. The Sec-homologue in the inner membrane of Escherichia coli is SecYEG. Sec-mediated insertion of numerous membrane proteins is aided by YidC, another protein integral to the inner membrane of Escherichia coli. YidC fulfils in addition the integration of a variety of membrane proteins Sec-independently. It belongs to a conserved but structurally uncharacterised family of proteins important for membrane protein biogenesis and comprises homologues in mitochondria and chloroplasts. By modification of a former crystallisation protocol two-dimensional crystals of SecYEG were grown in presence of the signal sequence peptide of LamB. Recording of structural data by electron cryo-microscopy and calculation of a difference structure comparing a former SecYEG projection structure with the one of SecYEG crystallised in presence of the substrate revealed several new and vacant densities. These hint to signal peptide binding close to the translocation pore and to significant rearrangements in proximity to the lateral exit site for transmembrane domains in SecYEG. The difference structure suggests that dimeric SecYEG is an asymmetric molecule consisting of one active and one inactive SecYEG monomer. Detergent removal from a mixture of purified YidC and lipids produced two-dimensional crystals that were highly dependent on the ionic strength and lipid composition for their growth. Electron cryo-microscopy on the frozen-hydrated crystals and image processing visualised structural details at about 10 Å resolution. Averaging two alternative projection structures in p2 and p121_a symmetry, respectively, yielded essentially the same features. Four YidC monomers form one unit cell (dimensions 82 x 71 Å, included angle 85 ° and 90 °, respectively) and seem to be arranged as two sets of dimers integrated in an anti-parallel fashion into the membrane. An area of low density in the centre of each YidC monomer resembles possibly a constriction of the membrane, which could have particular relevance for the integration of substrate proteins into the lipid bilayer.
A detailed understanding of how potassium channels function is crucial e. g. for the development of drugs, which could lead to novel therapeutic concepts for diseases ranging from diabetes to cardiac abnormalities. An improved understanding of channel structure may allow researchers to design medication that can restore proper function of these channels. This is particularly important for KCNQ channels, since four out of five family members are involved in human inherited disease. In addition to structure and function relationships the determinants which govern assembly of KCNQ subunits are decisive to understand the physiological role of the KCNQ channel family members. Many details of KCNQ channel assembly remain incompletely understood. Previous work has shown that the subunit-specific heteromerisation between KCNQ subunits is determined by a ~115 amino acid-long subunit interaction domain (si) within the C-terminus (Schwake et al., 2003). Recently, Jenke et al. (2003) proposed that the C-terminal domains in eag and erg K+ channels act as sites which drive tetramerization. From their ability to form coiled coils, these domains were referred to as tetramerizing coiled-coil (TCC) sequences. Jenke et al. also pointed out that KCNQ channels contain bipartite TCC motifs within their C-termini, exactly within the si domain, which is responsible for the subunit-specific interaction pattern. The first part of this thesis was dedicated to determine the individual role of these TCC domains on homomeric and heteromeric channel formation in order to further characterize the molecular determinants of KCNQ channel assembly. In the second part of this thesis cystein-scanning mutagenesis was employed, followed by thiol-specific modification using MTS reagents to screen more than 20 residues in the S3-S4 linker region and in the S4 transmembrane domain of the KCNQ1 channel to gain information about residue accessibility, the functional effects of thiol-modifying reagents (MTSES), and effects of crosslinking selected pairs of Cys residues by Cd+ ions, which could be used for testing model predictions based upon known Kv channel structures from the literature. According to homology modelling based on the Kv1.2 structure it was attempted to determine the proximity of individual residues from different transmembrane segments using the metal bridge approach (crosslinking by Cd+ ions). This led us to derive structural constraints for interactions between the S4 voltage sensor and adjacent transmembrane segments of KCNQ1. Similar studies have previously been performed on the Shaker K+ channel, which has served as a paradigm for structure-function research of voltage-gated K+ channels for a long time, but little is known for KCNQ channels concerning their similarity to published K+ channel structures.