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Zwei der wichtigsten Leistungen eines sich entwickelnden Embryos sind der Aufbau des Blutkreislauf- und des Nervensystems. Beide Systeme sind hierarchisch organisierte Strukturen, deren Verzweigungen nahezu alle Teile des Körpers erreichen. Es gibt eine zunehmende Zahl von Hinweisen darauf, dass ihre Entwicklung eng miteinander verknüpft ist, nach ähnlichen Prinzipien verläuft und verwandte molekulare Mechanismen verwendet. Die Entstehung eines funktionellen vaskulären Netzwerks erfordert Signale, die Prozesse wie die Lenkung und die Verzweigung von Gefäßen in den Zielgeweben kontrollieren. Ähnliche Anforderungen werden an wachsende Axone bei der Knüpfung der Verbindungen des Nervensystems während der Embryonalentwicklung gestellt. Einige der Faktoren, die die Lenkung der Axone kontrollieren, spielen auch eine ähnliche Rolle in der vaskulären Entwicklung. Lenkungsmoleküle, die eine Richtungsinformation vermitteln, sind für die Wegfindung der Axone besonders wichtig. Die größte Familie solcher Lenkungsmoleküle wird durch die Semaphorine gebildet. Semaphorine können in acht Klassen unterteilt werden, deren gemeinsames Merkmal eine konservierte Semaphorin-Domäne ist und die unterschieden werden anhand ihrer Klassen-spezifischen carboxyterminalen Domänen. Die Semaphorin-Familie umfasst sowohl sekretierte als auch membrangebundene Proteine. Die am besten charakterisierten hiervon sind die sekretierten Klasse 3 Semaphorine. Eine Kombination von in vitro und in vivo Ansätzen zeigte, dass die Klasse 3 Semaphorine an der Steuerung der Axon- und Dendritenlenkung, der Bildung von Axonbündeln und der neuronalen Migration während der Entwicklung des Nervensystems beteiligt sind. Sie agieren hauptsächlich als repulsiv wirkende Signale, die Axone aus Regionen ausschließen, von den Geweben weg, in denen sie exprimiert sind. Diese Wirkung wird über die Semaphorin-Domäne vermittelt. Verschiedene Hinweise deuten auf eine Beteiligung von Semaphorinen an der Entwicklung des vaskulären Systems. Sowohl homozygote Sema3a- als auch Sema3c-Mausnullmutanten sterben nach der Geburt aufgrund kardiovaskulärer Defekte. Darüber hinaus binden die Rezeptoren für die Klasse 3 Semaphorine, Neuropilin-1 (Nrp-1) und –2 (Nrp-2), einige Isoformen des vaskulären endothelialen Wachstumsfaktors (Vascular Endothelial Growth Factor, VEGF). Neuropilin-1 und Neuropilin-2-defiziente Mäuse und Neuropilin-1/-2-Doppelmutanten weisen Defekte des Gefäßsystems auf, wie z.B. eine Rückbildung der neuralen Vaskularisierung und Abweichungen in der Entwicklung des Herzens und der großen Gefäße. Die membrangebundenen Semaphorine sind bisher nur wenig untersucht, da zuverlässige in vitro Assays fehlen. Somit ist ein genetischer Ansatz der beste Weg, die physiologische Funktion dieser Proteine zu untersuchen. Aus diesen Gründen war die Zielsetzung dieser Arbeit, durch homologe Rekombination in embryonalen Stammzellen eine Mauslinie herzustellen, die ein Nullallel des membrangebundenen Sema5a-Gens trägt. Für diesen Ansatz wurde ein Mitglied der Klasse 5 Semaphorine gewählt, da es nur zwei Mitglieder dieser Klasse im Mausgenom gibt, die weitgehend komplementäre Expressionsmuster aufweisen. Damit unterscheiden sie sich von den anderen Klassen der Semaphorine, deren Mitglieder stark überlappende Expressionsmuster zeigen. Dies verringert die Wahrscheinlichkeit einer gegenseitigen funktionellen Kompensation nach Mutation eines Gens. Die Klasse 5 Semaphorine sind auch deshalb besonders interessant, da sie die einzigen sind, die sowohl in Vertebraten als auch in Invertebraten vertreten sind. Sie sind gekennzeichnet durch sieben carboxyterminale Typ 1-Thrombospondinmodule (TSP) in ihrer extrazellulären Domäne. TSPs wurden ursprünglich in den Proteinen Thrombospondin 1 und 2 gefunden, in denen sie das Auswachsen von Neuriten verschiedener Nervenzelltypen fördern. Dies lässt vermuten, dass Klasse 5 Semaphorine sowohl inhibierende als auch stimulierende Effekte haben könnten, in dem sie unterschiedliche Rezeptoren mit der Semaphorin-Domäne oder der TSPs aktivieren. Das Expressionsmuster von Sema5A und die bekannte Funktion von Semaphorinen in der Ausbildung neuronaler Verbindungen lassen es sinnvoll erscheinen, bei der Untersuchung der mutanten Tiere den Schwerpunkt auf die Entwicklung des Nerven- und des Gefäßsystems zu legen. Aufgrund technischer Schwierigkeiten konnte innerhalb der Bearbeitungszeit dieser Doktorarbeit nur der Phänotyp des vaskulären Systems untersucht werden. Die Inaktivierung des Sema5a-Gens wurde durch die Verwendung eines ‚Targeting’-Vektors erreicht, welcher die Exone 4 und 5 des Sema5a-Gens durch eine Neomycin-Selektionskassette ersetzte. Aus 144 untersuchten ES-Zellklonen wurden drei ES-Zellinien mit einem rekombinierten Sema5a-Locus identifiziert. Zwei der positiven Klone wurden zur Herstellung einer chimären Maus durch die Morula-Aggregationsmethode verwendet. Mit einem der Klone konnte eine männliche Chimäre erzeugt werden, die nach Kreuzung mit NMRI-Wildtyptieren die Mutation an die Nachkommen weitergab. Der Verlust der Proteinexpression in homozygoten Sema5a-Mutanten wurde durch Westernblot-Analyse von Zellmembranpräparationen homozygoter Embryonen unter Verwendung eines Antikörpers gegen das zytoplasmatische Ende von Sema5A bestätigt. Dieses Ergebnis bestätigte, dass die Deletion des vierten und fünften Exons des Sema5a-Gens ein Nullallel hervorbringt. Nach Verpaarungen heterozygoter Mutanten konnten keine Neugeborenen identifiziert werden, die homozygot für das mutierte Allel waren. Homozygte Mutanten starben zwischen E11,5 und E12,5 der Embryonalentwicklung, der Verlust von Sema5A ist also embryonal letal. Die Morphologie der homozygoten Tiere zeigte keinen offensichtlichen Unterschied zu den heterozygoten Embryonen oder zu Wildtyp-Geschwistern auf. Frühe embryonale Musterbildungsprozesse in Sema5a-Nullmutanten sind also nicht gestört. Ein Tod bei dieser Entwicklungsstufe deutet auf einen Defekt in der Entwicklung des Blutgefäßsystems hin, da die Embryonalstadien zwischen E9 und E13 besonders wichtig für die Ausbildung dieser Gefäße sind und viele Mutationen, die Herz und Blutgefäßen beeinträchtigen, den Tod der Embryonen in diesem Stadium bewirken. Das embryonale Blutgefäßsystem in E10,5 und E11,5 Embryonen wurde durch immunhistochemische Färbungen ganzer Embryonen unter Verwendung eines spezifischen gegen das Platelet Endothelial Cell Adhesion Molecule (PECAM) gerichteten Antikörpers dargestellt, welches in vaskulären Endothelzellen exprimiert ist. Die allgemeine Architektur des Gefäßsystems war in homo- und heterozygoten Mutanten ähnlich und wies weder an E10,5 noch an E11,5 besondere Abweichungen auf. Es wurden bei der Lage und der Anzahl intersomitischer Gefäße, der Entwicklung der dorsalen Aorta oder der Vaskularisierung der Extremitätenanlagen keine Abweichungen festgestellt. Morphologische Defekte konnten jedoch bei E10,5 in den Verästelungen der Blutgefäße detektiert werden, die von den Hauptvenen der Cranialregion abzweigen. Die Verzweigungen waren geringer ausgeprägt als in heterozygoten oder Wildtyp-Vergleichstieren. Insbesondere zeigte sich eine Verringerung der Anzahl sekundärer und tertiärer Verzweigungen. In dem sich entwickelnden Embryo führt die wiederholte Verzweigung von Ästen der Hauptvenen zu einem hierarchisch gegliederten Netzwerk großer Gefäße in der Region des medialen Kopfes. Während die Ausbildung dieses Netzwerkes in den Sema5a-/--Tieren beeinträchtigt ist, erscheint die Organisation der kleinen Gefäße in den mehr dorsal und peripher gelegenen Regionen des Kopfes normal. In heterozygoten und homozygoten Mutanten bilden die kleineren Gefäße ein dicht verzweigtes Netzwerk. Die Verminderung der Komplexität der größeren Gefäße konnte in allen untersuchten Nullmutanten beobachtet werden. Es variierte jedoch die Penetranz des Phänotyps. In allen Fällen war die Anzahl primärer Verzweigungen unverändert, während die Anzahl der sekundären und der tertiären Verzweigungen zu unterschiedlichen Graden reduziert war. Im Gegensatz dazu zeigte sich im Verzweigungsmuster von heterozygoten Mutanten und beim Wildtyp nur eine geringe Variabilität zwischen individuellen Embryonen. Dies belegt, dass die Verminderung des Verzweigungsgrades größerer Gefäße nicht innerhalb der normalen Variabilität liegt, sondern durch die Inaktivierung des Sema5a-Gens verursacht wird. Dieser Phänotyp ist in späteren Stadien sogar deutlicher ausgeprägt. In E11,5 Embryonen waren die Stämme der großen Blutgefäße in den Nullmutanten weniger komplex und in einigen Fällen trat sogar eine Reduzierung der Anzahl primärer Verzweigungen auf. Diese spätere Verminderung der Anzahl bereits ausgebildeter primärer Verzweigungen legt nahe, dass der Phänotyp durch eine Rückbildung von Verzweigungen aufgrund möglicher Defizite in deren Reifung und/oder Stabilisierung erfolgt. Die interessanteste Besonderheit der vaskulären Defekte in den Nullmutanten liegt in ihrer regionalen Spezifität. Bis hier ist das Netzwerk großer Gefäße, welches der anterioren Hauptvene entspringt, das einzige Gefäßsystem, in dem Abweichungen entdeckt wurden. Dieses Netzwerk wird durch die strukturelle Umbildung des primären kapillaren Plexuses gebildet. Zwischen E9,5 und E12 sprießen Zweige rostral aus der Hauptvene, um ein hierarchisch organisiertes Netzwerk von Gefäßen zu bilden. Die Umbildung des primären kapillaren Plexus in den mehr rostral und ventral gelegenen Kopfregionen führt zu der Bildung eines hochverzweigten vaskulären Netzwerkes, welches jedoch bei E10,5 noch nicht hierarchisch organisiert erscheint. Die Signale, die für diesen unterschiedlichen Ablauf der Musterbildung während der Entwicklung des Gefäßsystems des Kopfes verantwortlich sind, sind noch unbekannt. Die besonderen Defekte in der stereotypischen Organisation der cranialen Gefäße in Sema5a-Mutanten legt nahe, dass Sema5A eines dieser Signale sein könnte. Es könnte Teil eines Rezeptor/Ligandenkomplexes sein, welcher positionelle Signale für das Verzweigen und das Wachstum großer Gefäße in rostraler Richtung liefert. Sema5A könnte die Bildung von Verzweigungen durch die Regulierung der Wanderung endothelialer Zellen, ihrer Proliferation oder ihrer Interaktion mit unterstützenden Zellen oder der extrazellulären Matrix kontrollieren. Sema5A könnte Teil eines neuen Signalweges sein oder als Teil eines der bekannten Signalwegs wirken, welcher die Entwicklung des Gefäßsystems reguliert. Einer der Signalwege, die essentiell für die Gefäßbildung sind, wird durch VEGF und Angiopoietin (Ang-1) reguliert. Sowohl in VEGF-, als auch in Ang-1-Mutanten ist die Gefäßumbildung im Kopf beeinträchtigt. Insbesondere erscheint das Netzwerk kleiner Gefäße in den Ang-1 Nullmutanten als nur nur teilweise restrukturiert und die großen Gefäße als weniger komplex. Das Verzweigungsmuster der großen Gefäße in den Ang-1- Nullmutanten ähnelt auffallend dem der Sema5a-Nullmutanten. Eine zweite Ähnlichkeit in den Phänotypen von Ang-1- und Sema5a-Mutanten zeigt sich in der Reduzierung der primären Verzweigungen, welche in den Sema5a-Nullmutanten bei E11,5 beobachtet wird. Hier könnte die Verminderung aus einer Rückbildung von Gefäßen resultieren, wie sie auch typischerweise in Mutanten für Ang-1 oder dessen Rezeptor auftritt. Diese Beobachtung legt nahe, dass Sema5A ein neuer Teilnehmer innerhalb des Ang-1-Signalweges ist, welcher die Auswirkung von Ang-1 auf die endothelialen Zellen der großen Gefäße entweder vermittelt oder moduliert und dadurch das spezifische Muster der Blutgefäße des Kopfes beeinflußt. Mit dieser Doktorarbeit wird zum ersten Mal eine funktionelle Untersuchung des Klasse 5 Semaphorins Sema5A vorgestellt. Die phänotypische Untersuchung von Mäusen, die Nullallele für Sema5a-Gens tragen ergab, dass dieses membrangebundene Protein essentiell für die embryonale Entwicklung ist. Es ist an der Musterbildung des Gefäßsystems beteiligt. Seine Aufgabe besteht möglicherweise darin, die Bereitstellung positioneller Signale für die Ausbildung von Gefäßverzweigungen zu gewährleisten. Einige grundlegende Fragen werden durch diesen Phänotyp aufgeworfen. Sowohl die Ursache für die embryonale Sterblichkeit als auch die zellulären Prozesse, welche in den Sema5a-Nullmutanten beeinträchtigt sind, müssen noch beschrieben werden. Unbekannt ist ebenfalls, ob zusätzlich zu der hier beschriebenen Rolle von Sema5A in der Gefäßbildung dieses an der Entwicklung des Nervensystems beteiligt ist. Die ersten Daten über die physiologische Rolle von Sema5A, welche mit dieser Arbeit vorgelegt werden, öffnen den Weg für weitergehende Untersuchungen über die Funktion des Proteins während der Embrionalentwicklung. Das hier erstmals vorgestellte Modellsystem ermöglicht es, Sema5A regulierte zelluläre Mechanismen zu untersuchen. Zusätzlich stellt es ein Werkzeug zur Verfügung, um die funktionelle Beziehung zwischen der Entwicklung des kardiovaskulären Systems und des Nervensystems zu untersuchen. Damit können die Aufgaben der Semaphorin-Proteinfamilie, die an diesen beiden wichtigen Prozessen beteiligt sind, näher charakterisiert werden.
The technique of site-specific fluorescence labelling with Tetramethylrhodaminemaleimide (TMRM) in combination with two electrode voltage-clamp technique (TEVC), an approach that has been named voltage clamp fluorometry (VCF), has been used in this work to study the Na,K-ATPase. The TMRM dye has the ability to attach covalently to cysteine residues and it responds to changes in the hydrophobicity of its local environment. We exploited this property using a construct of the Na-pump in which the native, extracellularly accessible cysteines were removed and cysteine residues were introduced by site-directed mutagenesis in specific positions of the Na-pump. In this way it was possible to detect site-specific conformational rearrangements of the Na-pump in a time-resolved fashion within a native membrane environment. In particular this technique allows to resolve reactions with low electrogenicity that cannot be satisfactorily analyzed with purely electrophysiological techniques and to identify the conformations of the enzyme under specific ionic composition of the measuring buffers. We used VCF to study the influence that several cations like Na+, K+, NMG+, TEA+ and BTEA+ exert on the distribution of the Na,K-ATPase between several enzymatic intermediates and on some of the reactions related to cation transport. To this end we utilized the mutants N790C in the loop M5-M6 and the mutant E307C, T309C, L311C and E312C in the loop M3-M4. From the correspondence of the fluorescence changes with the activation and inhibition of pumping current, by K+ and ouabain respectively, and from the fact that in Na+/Na+ exchange conditions the voltage distribution of charge movement and fluorescence changes evoked by voltage jumps are in reasonable agreement we conclude that through the fluorescence signals measured from these mutants, we can indeed monitor conformational changes linked to transport activity of the enzyme. For the mutants N790 and L311, it was found that the Na+ dependence of the amplitude and kinetics of the fluorescence signal associated with the E1P-E2P transition is in agreement with the prediction of an access channel model describing the regulation of the access of extracellular Na+ to its binding site. In particular for the mutants E307 and T309 it was found that in Na+/Na+ exchange conditions, the conformational change tracked by the fluorescence was much slower than the charge relaxation at hyperpolarized potentials while the kinetics was very similar at depolarized potentials. This implies that at hyperpolarized potentials the conformational change connected to the E1P-E2P transition does not give a large contribution to the electrogenicity of the process which is also consistent with the access channel model. On the mutant N790C it was found that the external pH does not seem to have any effect on the E1P-E2P equilibrium even if it seems to modulate the fluorescence quantum yield of the dye. Fluorescence quenching experiments with iodide and D2O indicate that at hyperpolarized potentials the local environment of the mutant N790C, experiences a small change in the accessibility to water without major changes in the local electrostatic field ...
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics, and play primary roles in the homeostasis of intracellular pH, cellular Na+ content and cell volume. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of such Na+/H+ antiporters. NhaA is the essential Na+/H+ antiporter for pH and Na+ homeostasis (at alkaline pH) in Escherichia coli and many other enterobacteria. NhaA is an electrogenic Na+/H+ antiporter that exchanges 2H+ for 1Na+ (or Li+). NhaA shares with many other prokaryotic and eukaryotic antiporters a very strong dependence on pH. In order to achieve three-dimensional structure of NhaA, the previously described NhaA protein preparation was modified: (i) the wild type bacterial strain (TA16) used for homologous over-expression of NhaA was replaced with a delta nhaA strain (RK20). As a result, the purity and homogeneity of the sample was significantly improved; (ii) the previously two-step purification procedure was shortened to a single step affinity chromatography purification; (iii) a wide-range screening of crystallisation conditions, more than 20,000, was performed; (iv) a Seleno-L-methionine (SeMet) NhaA derivative was produced in order to solve the phases during structure determination. In parallel, attempts of production and crystallisation of co-complexes composed of NhaA and antibody fragments have been made. Four different monoclonal antibodies were available against NhaA. Selected antibody fragments were produced and the stability of the complex analysed. Here, the crystal structure of the pH down-regulated secondary transporter NhaA of Escherichia coli is presented at 3.45 Å resolution. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. A possible mechanism is proposed: the binding of charged substrates causes electric imbalance inducing movements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry. The structure represents a novel fold that provides two major insights: it reveals the structural basis for the mechanism of Na+/H+ exchange and its unique regulation by pH in NhaA and in many other similar antiporters. Furthermore, it is also important for the understanding of the architecture of membrane proteins in general. However, although many aspects of the ion-translocation mechanism and pH regulation are clarified by the NhaA structure, higher resolution structures with Li+ or Na+ bound are required for understanding the ligand binding and the translocation mechanism at the atomic level. The alkaline pH-induced conformation is essential to further understand the pH-control and proton access to the binding site.
The generation of O2- by NADPH oxidaes was mainly attributed to immune cells that kill invading bacteria or cancer cells. But importantly, in the past several years, several homologs of the catalytic subunit gp91phox (Nox2) of the phagocytic NADPH oxidase have been identified in non-immune cells and tissues. Superoxide production derived from NADPH oxidaes has been shown to play a role not only in host defense but also in defined signaling cascades mediating growth and apoptosis. The aim of this work was to study the expression and the regulation of the”new” Nox isoforms in rat renal mesangial cells (MC). In particular the following results were achieved. 1) mRNA’s for both Nox1 and Nox4 were detected by RT-PCR. 2) Nox1 mRNA levels were increased upon exposure to basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and fetal calf serum (FCS) in a time- and dose-dependent manner. Exposure of MC to bFGF and FCS increased also basal production of reactive oxygen species (ROS) by MC. By contrast, Nox4 mRNA levels were not significantly affected by bFGF treatment, but were markedly down-regulated by PDGF and FCS. 3) To study the regulation of Nox1 on the protein level, an anti-Nox1 antibody was generated and characterized using affinity chromatography. Up-regulation of Nox1 expression by growth factors was confirmed also on the protein level. 4) Based on the already known cDNA sequence for Nox1, the transcriptional start site was determined by the “gene RACE” technique. 2547 bp of the genomic sequence of the 5´-flanking region of the Nox1 gene were cloned and sequenced using the „Genome-Walking“ method. To study the regulation of Nox1 transcription functional Nox1 promoter/luciferase fusions were be established. MC were transiently transfected with different promoter/luciferase constructs and stimulated with growth factors. By measuring luciferase activity it was determined that growth factors induced the Nox1 transcription and that the Nox1 core promoter is sufficient for the activation. 5) By measurement of superoxide radicals and analysis of Nox1 mRNA expression by quantitative RT-PCR (TaqMan) as well as protein level by Western blotting it could be shown that treatment of MC with NO donors inhibited the expression of Nox1 in a time- and dose-dependent manner. Moreover, using activators and inhibitors of the soluble guanylyl cyclase (sGC) it could be shown, that the activation of sGC mediates the effect of NO on Nox1 expression. However, NO had no inhibitory effect on Nox1 promoter activity. Experiments with the inhibitor of transcription, actinomycin D, suggest that NO-mediated regulation of Nox1 is triggered probably via post-transcriptional mechanisms. Nox4 is regulated on the mRNA levels in a similar manner as Nox1. 6) To analyze the sub-cellular localization of the Nox isoforms, coding sequences for Nox1 and Nox4 were fused together with green fluorescent protein into the pEGFP-N1 demonstrated that both isoforms are localized predominantly in the plasma membrane, but also in the perinuclear region and cytoplasm. However, the localization of Nox1 in the plasma membrane was more pronounced. 7) In addition to Nox1 and Nox4, mRNA of the newly identified NOXA1 that is a homolog of the p67phox subunit of NADPH oxidase was detected in MC by RT-PCR.
G-protein coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and possess a signature motif of seven transmembrane helices. The endothelin B (ETB) receptor is a member of rhodopsin like GPCR family. It plays an important role in vasodilation and is found in the membranes of the endothelial cells enveloping blood vessels. Knowledge of the three-dimensional structure of G-protein coupled receptors in general would significantly add to our understanding of their molecular mechanisms and would be useful in the search for new specific drugs. However, three-dimensional structural analysis will require milligram quantities of pure and homogeneous protein. This dissertation is a study of the production, biochemical characterization and preliminary structural studies of the human ETB G-protein coupled receptor. The present work aimed at elucidating the structure and mechanistic details of function of the receptor by using a combination of X-ray crystallographic and NMR methods for collecting structural data. To obtain homogenous and monodisperse receptor protein preparation for structural and functional studies, we implemented the baculovirus expression system for the production of ETB receptor for the present work. The two step affinity purification ensured capture of full-length receptor. Silver stained SDS-PAGE of the purified receptor-ligand complex indicated greater than 90% protein purity. Based on previous reports, we used the high affinity ligand (endothelin -1) binding to the receptor for co-crystallization of receptor-ligand complex by locking the receptor in the activated conformation. As a prerequisite for 3D crystallization trials, the stability of the detergent solubilized receptor-ligand complex was assessed with respect to pH, temperature and time. Receptor-ligand complex did not show any degradation and aggregation over 6 days at 4°C and 18°C. Interestingly, change of pH suggested that receptor-ligand complex is unstable at lower pH due to possible charge induced conformational changes. In our work, we introduced the idea of using fluorophore labeled ligand for simple visual recognition of the receptor-ligand complex during purification and crystallization. On the other hand, we alternatively used biotinylated endothelin-1 to produce an adequate amount of ligand bound receptor complex, thus ensuring homogeneity of the purified complex for use in structural studies. Thus far, preliminary crystals have been obtained for both the unlabelled ET-1 and fluorophore labeled ET-1 complexed with ETB receptor. Moreover, we performed the systematic investigation of the protein/peptide binding partner for the receptor-ligand complex with the chief aims of stabilizing structure and increasing the possibilities of 3D-crystal contacts. Thus subsequent to formation of receptor-ligand complex, the additional in vitro formation of a ternary arrestin-receptor-ligand complex was also attempted for use in structural studies. We successfully demonstrated that arrestin mutant (R169E) forms a tight complex with ETB receptor regardless of its phosphorylation state. A second approach to get insight into the ETB receptor ligand binding site relied on the use of spin isotope labeled ET-1 ligand peptide by employing solid state MAS NMR method. Preliminary data provided compelling evidence that the C-terminal region of the peptide is immobilized in an ordered environment and presumably bound to the receptor. This indicates that the approach is feasible, although there are difficulties in sample preparation for further spectral measurements and data collection which are currently being discussed in ongoing investigations. At this point of our research work, we initiated a collaborative effort to obtain high yields of pure, active receptor without post translational modifications, from an E. coli cell lysate based in vitro expression system. We successfully optimized the production of homogenous and monodisperse endothelin B receptor in mg amounts. Thus this could potentially provide an alternative source of high quality receptor production in large quantities for immediate crystallization trials. Thus we hope that the results from these investigations can be applied in a more general sense to the production and crystallization of other G protein-coupled receptors.
The research presented in this thesis characterizes U2AF homology motifs (UHM) and their interactions with UHM ligand motifs (ULM) in the context of splicing regulation. UHM domains are a subgroup of RNA recognition motifs (RRM) originally discovered in the proteins U2AF65 and U2AF35. Whereas canonical RRMs are usually involved in binding of RNA, UHM domains bind tryptophan containing linear protein motifs (ULM) instead. In the first article, we analyze the complex network of interactions between splicing factors and RNA that initiate the assembly of the spliceosome at the 3´ splice site of an intron. The protein U2AF65 binds a pyrimidine-rich element in introns and recruits U2snRNP by binding its protein component SF3b155. My contribution was to define the binding site of the protein U2AF65 to the intrinsically unstructured N-terminus of the scaffolding protein SF3b155. I could show that the UHM domain of U2AF65 recognizes a ULM in SF3b155, and that this binding site is not overlapping with the binding sites of other splicing factors, like p14, to SF3b155. As the U2AF65-UHM:SF3b155-ULM interaction is mutually exclusive with an interaction between U2AF65-UHM and a ULM in the splicing factor SF1, which was reported to initially recognize the branch point sequence, my results provide the molecular details on how SF3b155 replaces SF1 during spliceosomal reorganizations. In the second article, we show that overexpression of the UHM domain of the splicing factor SPF45 induces exon 6 skipping in the pre-mRNA of Fas (CD95/APO-1). I provide evidence for in vitro binding of SPF45-UHM to ULM sequences in the splicing factors U2AF65, SF1, and SF3b155. I crystallized free and SF3b155-bound SPF45 UHM and solved both structures by X-ray crystallography. The analysis of the complex interface and sequence differences in the ULMs allowed me to design mutations of SPF45-UHM, which selectively inhibit binding to distinct ULMs. After assessing the ULM binding properties in vitro, we could show that the activity of SPF45-UHM in influencing the splicing pattern of Fas relies on interactions with SF3b155 and/or SF1, but that an interaction with U2AF65 is dispensable. A mechanism for the activity of SPF45-UHM could thus be engaging in ULM interactions and thus interfering with the network of interactions that initiate the assembly of the spliceosome at the 3´splice site, as described above. In the third article, we describe an unusual flexible homodimerization mode of the UHM in the splicing factor Puf60, which enables simultaneous interactions with ULM sequences on other splicing factors. I could show that the NMR relaxation properties of Puf60-UHM are inconsistent with a model of a rigid dimer, but rather indicate a dimerization via a flexible linker. I identified a flexible loop in the peptide backbone of Puf60-UHM, and showed that mutiation of acidic residues in this loop impairs the dimerization. To analyze the dimerization interface in further detail, I solved the structure of Puf60-UHM by X-ray crystallography. The acidic residues in the flexible loop of one UHM dimer subunit mediate the dimerization by contacting basic residues on the β-sheet surface of the other dimer subunit. Differences in the four dimer interfaces observed for the eight molecules in the asymmetric unit of the crystal support the model of an undescribed, flexible mode of dimerization, and thus complement the NMR relaxation data. Furthermore, I could show that the Puf60-UHM dimer and U2AF65-UHM contact different ULM sequences on the SF3b155 N-terminus in vitro, thus providing a possible explanation for the mutual cooperative activation of Puf60 and U2AF65 in splicing assays described in the literature. The fourth article is a review about recent research on the recognition of DNA double strand breaks (DSB) by covalent histone modifications. The p53 binding protein 1 (53BP1) is a DSB sensor and a checkpoint protein for mitosis. Recent crystallographic evidence indicates that 53BP1 recognizes DSB sites by binding histone H4 dimetylated at lysine 20 (H4-K20). We provide a comprehensive overview of the atomic resolution structures that revealed how proteins can specifically recognize histone tail modifications, especially methylated lysines, to read the information stored in what is called the histone code.
By adopting a variety of shapes, proteins can perform a wide number of functions in the cell, from being structural elements or enabling communication with the environment to performing complex enzymatic reactions needed to sustain metabolism. The number of proteins in the cell is limited by the number of genes encoding them. However, several mechanisms exist to increase the overall number of protein functions. One of them are post-translational modifications, i.e. covalent attachment of various molecules onto proteins. Ubiquitin was the first protein to be found to modify other proteins, and, faithful to its evocative name, it is involved in nearly all the activities of a cell. Ubiquitylation of proteins was believed for a long time only to be responsible for proteasomal degradation of modified proteins. However, with the discovery of various types of ubiquitylation, such as mono-, multiple- or poly-ubiquitylation, new functions of this post-translational modification emerged. Mono-ubiquitylation has been implicated in endocytosis, chromatin remodelling and DNA repair, while poly-ubiquitylation influences the half-life of proteins or modulates signal transduction pathways. DNA damage repair and tolerance are example of pathways extensively regulated by ubiquitylation. PCNA, a protein involved in nearly all types of DNA transaction, can undergo both mono- and poly-ubiquitylation. These modifications are believed to change the spectrum of proteins that interact with PCNA. Monoubiquitylation of PCNA is induced by stalling of replication forks when replicative polymerases (pols) encounter an obstacle, such as DNA damage or tight DNA-protein complexes. It is believed that monoubiquitylation of PCNA stimulates the exchange between replicative pols to one of polymerases that can synthesize DNA across various lesions, a mechanism of damage tolerance known as translesion synthesis (TLS). Our work has helped to understand why monoubiqutylation of PCNA favours this polymerase switch. We have identified two novel domains with the ability to bind Ub non-covalently. These domains are present in all the members of Y polymerases performing TLS, and were named Ub-binding zinc finger (UBZ) (in polη and polκ) and Ub-binding motif (UBM) (in polι and Rev1). We have shown that these domains enable Y polymerases to preferentially gain access to PCNA upon stalling of replication, when the action of translesion polymerases is required. While the region of direct interaction between Y pols and PCNA had been known (BRCT domain in Rev1 and PIP box motif (PIP) in three others members), we propose that Ub-binding domains (UBDs) in translesion Y pols enhance the PIP- or BRCT-domain-mediated interaction between these polymerases and PCNA by binding to the Ub moiety attached onto PCNA. Following these initial studies, we have also discovered that Y polymerases themselves undergo monoubiquitylation and that their UBDs mediate this modification. This auto-ubiquitylation is believed to lead to an intramolecular interaction between UBD and Ub attached in cis onto the UBD-containing protein. We have mapped monoubiquitylation sites in polη in the C-terminal portion of the protein containing the nuclear localization signal (NLS) and the PIP box. Beside PIP, the NLS motif is also involved in direct interaction of polη with PCNA. Based on these findings, we propose that monoubiquitylation of either NLS or PIP masks them from potential interaction with PCNA. Lastly, using several functional assays, we have demonstrated the importance of all these three motifs in the C-terminus of polη (UBZ, NLS and PIP) for efficient TLS. We have also constructed a mimic of monoubiquitylated polη by genetically fusing polη with Ub. Interestingly, this chimera is deficient in TLS as compared to the wild-type protein. Altogether, these studies demonstrate that the C-terminus of polη constitutes a regulatory module involved in multiple-site interaction with monoubiquitylated PCNA, and that monoubiquitylation of this region inhibits the interaction between polη and PCNA. Our work has also revealed that the UBDs of Y pols as well as of other proteins implicated in DNA damage repair and tolerance, such as the Werner helicase-interacting protein 1 (Wrnip1), are required for their proper sub-nuclear localization. All these proteins localize to discrete focal structures inside the nucleus and mutation of their UBDs results in inability to accumulate in these foci. Interestingly, by exchanging UBDs between different proteins we have learned that each UBD seems to have a distinct functional role, surprisingly not limited to Ubbinding ability. In fact, swapping the UBZ of Wrnip1 with the UBM of polι abolished the localization of Wrnip1 to foci despite preserving the Ub-binding ability of the chimeric protein. In summary, this work provides an overview of how post-translation modification of proteins by Ub can regulate several DNA transactions. Firstly, key regulators (e.g. PCNA) can be differentially modified by Ub. Secondly, specialized UBDs (e.g. UBM, UBZ) embedded only in a subset of proteins act as modules able to recognize these modifications. Thirdly, by means of mediating auto-ubiquitylation, UBDs can modulate the behaviour of host proteins by allowing for either in cis or in trans Ub-UBD interactions.
Orthopoxviruses are large DNA viruses that replicate within the cytoplasm of infected cells encoding over a hundred different proteins. The orthopoxviral 68k ankyrin‐like protein (68k‐ank) is highly conserved among orthopoxviruses, and this study aimed at elucidating the function of 68k‐ank. The 68k‐ank protein is composed of four ankyrin repeats (ANK) and an F‐box‐like domain; both motifs are known proteinprotein interaction domains. The F‐box is found in cellular F‐box proteins (FBP), crucial components of cellular E3 ubiquitin (Ub) ligases. With yeast‐two‐hybrid screens and subsequent co‐immunoprecipitation analyses, it was possible to identify S‐phase kinase‐associated protein 1a (Skp1a) as a cellular counterpart of 68k‐ank via binding to the F‐box‐like domain. Additionally, Cullin‐1 was co‐precipitated, suggesting the formation of a viral‐cellular SCF E3 Ub ligase complex. Modified Vaccinia virus Ankara (MVA) ‐ being attenuated and unable to replicate in most mammalian cell lines due to a block in morphogenesis – nevertheless, expresses its complete genetic information attributing to its properties as promising vector vaccine. Conservation of 68k‐ank as the only ANK protein encoded by MVA implied a substantial role of this viral factor. Hence, its function in the viral life cycle was assessed by studying a 68k‐ank knock‐out MVA. A mutant phenotype manifested in nonpermissive mammalian cells characterized by a block succeeding viral early gene expression and by a reduced ability of the virus to shutoff host protein synthesis. Studies with MVA encoding a 68k‐ank F‐box‐like domain truncated protein revealed that viral‐cellular SCF complex formation and maintenance of viral gene expression are two distinct, unrelated functions fulfilled by 68k‐ank. Moreover, K1, a well‐described VACV host range factor of the ANK protein family, is able to complement 68k‐ank function. This suggests that gene expression of MVA putatively depends on the ANKs encoded in 68k‐ank. In addition to the important findings in vitro, first virulence studies with the mouse pox agent, ectromelia virus (ECTV) deleted of the 68k‐ank ortholog (C11) suggested that this factor contributes to ECTV virulence in vivo.
In mitochondrial respiration, the soluble protein cytochrome c accepts an electron from the membrane bound cytochrome bc1. The interaction between cytochrome bc1 and cytochrome c is highly transient in nature, enabling turnover numbers greater than 160 s-1. Yeast cytochrome bc1 has been successfully crystallised with bound cytochrome c with the help of an antibody fragment (Lange and Hunte 2002; Solmaz and Hunte 2008). In all crystal structures of the complex, the homodimeric cytochrome bc1 binds only one cytochrome c, with the binding site located on subunit cytochrome c1. Univalent cytochrome c binding is correlated with conformational changes of the Rieske protein head domain and subunit QCR6p. The interface of the complex is small. The haem moieties are centrally located in a mainly non-polar contact site that includes a cation–! interaction and is surrounded by complementary charged residues. The crystal structure is in agreement with the general architecture of the interfaces of transient redox complexes and also reveals several interesting features unique to the cytochrome bc1. On the basis of the crystal structures, an extensive thermodynamic and kinetic characterisation of the interaction was carried out in this work to challenge the static snapshot of the bound proteins in the crystal structure as the relevant physiological electron transfer. The thermodynamic parameters of the interaction between the redox partners were determined using isothermal titration calorimetry (ITC). The association constant for cytochrome bc1 and cytochrome c in oxidised state under physiological ionic strength of 120 mM at 25 °C, was determined to be 5 " 103 M-1 by direct ITC titration. So, the partners interact with an affinity of 200 #M. In spite of the low affinity the complex has a life time ($ = 1/koff) of 5 #second, sufficiently long to enable the theoretically calculated electron transfer rates of 1.0 " 106 to 2.6 " 107 s%1 with a lifetime ($ = 1/rate) of 1-0.04 μseconds and experimentally determined rate of 7.7 " 104 s%1 with a lifetime of 13 μseconds. The low affinity makes it difficult to ascertain the stoichiometry of binding. The enthalpy of the interaction is endothermic, which is consistent with the nature of an interface where hydrophobic interactions are dominant. The enthalpy and entropy is 3.6 kJmol-1 and 83 kJmol-1K-1, respectively. The importance of key interface residues was also investigated. The role of the interface residue G89 of cytochrome c which might have a role in the dissociation of the complex has been probed by site-directed mutagenesis. The interface contains a cation-! interaction between F230 of cytochrome bc1 and R19 of cytochrome c, which is thought to provide the specificity to the interaction between the otherwise promiscuous partners. To analyse the role of this interaction pair in electron transfer, F230L and F230W mutants were used to measure direct electron transfer rates by flash photolysis and steady state kinetics. The findings indicate that another ! system can work as functional substitution of F230, while deleting the ! system has a deleterious effect on the complex formation. The inability of F230L to achieve the transient and steady state turnover rates as wild type protein indicates a scenario where the variant achieves an altered bound state with inefficient electron transfer pathways and higher edge-to-edge distance. The role of supernumerary subunit QCR6p in complex formation was investigated by steady state kinetics measurements. Subunit QCR6p does not interact directly with cytochrome c but is positioned in such a way that it could electrostatically steer cytochrome c in a reactive ensemble. The highly acidic and disordered N-terminus of QCR6p could interact with a patch of conserved lysine residues on cytochrome c. The role of subunit QCR6p has been assessed using QCR6p deleted cytochrome bc1 and a lysine variant of cytochrome c. The results show that QCR6p not only affects the kinetics of the interaction but is also important for the stability of cytochrome bc1. The kinetic and thermodynamic data obtained during this study provide evidence for the functional importance of non-catalytic cytochrome bc1 subunit QCR6p, show that the entropy driven interaction is indeed of low affinity and highly transient in nature and indicate that the interface is well suited to ensure the high turnover of the electron transfer chain where cytochrome c interacts with multiple partners using overlapping interfaces. The suggested role of the cation-! interaction as a highly specific interaction has been validated.
In this thesis, the structure of the C-terminal domain of presenilin-1, the catalytic component of the y-secretase complex, is investigated by NMR spectroscopy. The ysecretase complex has a definitive role in the pathogenic development of Alzheimer's disease, in that it mediates the cleavage of aprecursor to create the amyloid ß peptide. Aggregates of amyloid ß which form amyloid plaques are the most overt clinieal feature observed in the post-mortem brains of Alzheimer's patient. In addition, many of the mutations found in the aggressive early onset familial Alzheimer's disease have been linked to presenilin-1, highlighting its importance in disease progression and deeming it an important target for investigation. One of the greatest challenges for the structural investigation of the y-secretase components is their low expression yields in cell-based systems. We therefore applied continuous-exchange cell-free expression to obtain sufficient amounts of protein for our structural studies. An added benefit of the cell-free expression system is the freedom to incorporate any desired combination of stable-isotope labels directly into sampies. We were therefore able to develop a labeling scheme which targets the amino acid composition of transmembrane a-helices, allowing us to simplify an assignment procedure whieh tends to be cumbersome and diffieult for most a-helical transmembrane proteins. The y-secretase complex is a member of the intramembrane cleaving proteases which, as their name implies, cleave their transmembrane substrates within the bilayer. Single particle analysis of the y-secretase (1) as weil as crystal structures of rhomboid (2) and S2P (3) have revealed the presence of hydrophilie po res within the membrane where catalysis occurs. In light of evidence that certain elements of CTF reside in close proximity or even contribute to the formation of the hydrophilic pore, we chose to study the structure of CTF in mieelles, whieh may be better suited to accommodate CTF in isolation as compared with solid membranes in the absence of the other y-secretase components. The structure of CTF was solved to 1.7 A (backbone r.m.s.d) and revealed the presence of unusual features, including a partially membrane-spanning helix which situates the catalytic asparte at its N-terminus in what would be the center of the membrane where catalysis is proposed to occur, as weil as a severely kinked helix which is partially embedded beneath the surface of the membrane (P6). Interestingly, similar features have been observed in the crystal structure of the GlpG rhomboid. In addition, a soluble helix was found in the long N-terminal loop of CTF which until now has been described as unstructured. The first part of the thesis is designed to provide an introduction to Alzheimer's disease, the role of y-secretase and its presenilin-l catalytic component in disease progression, as weil as cell-free expression and liquid-state NMR techniques involved in the structural investigation of membrane proteins. In chapter 2, the reader is familiarized with the history, the clinical manifestation, and biochemical features of Alzheimer's disease. The chapter goes further to describe the role of the y-secretase complex and its individual components in disease progression and substrate processing. Chapter 3 focuses more specifically on presenilin-l in the context of the newly emerging class of intramembrane proteases. In chapter 4, attention is shifted to the cell-free expression system with special focus on the expression of membrane proteins, and chapter 5 explores the various liquid-state NMR techniques that were required for the characterization of CTF. The second part of the thesis is cumulative and contains original research, method, and review articles that were produced during the course of study. Chapter 6 explores the various techniques and innovations used to study membrane proteins using continuous exchange cell-free expression coupled with NMR spectroscopy. In chapter 7, a new technique, transmembrane segment targeted labeling, is described as a tool that facilitates the backbone assignment of transmembrane proteins which display severe overlap in NMR spectra. Chapter 8 presents the novel NMR structure of the C-terminal fragment of presenilin-l solved in SOS micelles.