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1. A preliminary revision of the genus Muntiacus in the Indo-Australian Archipelago Introduction Sexual differences Sexual cycle Characters of age in the dentitions Age differences in skull measurements Age differences of antlers Age differences in coat Systematic part 2. Revision of the genus Arctogalidia in the Indo-Australian Archipelago. Introduction Key to the greges in the genus Arctogalidia Key to the subspecies of the grex A. t. trivirgata Gregal form A. t. trilineata
The heat stress response is characterized by the presence of heat stress transcription factors (Hsfs) which mediate transcription of heat stress genes. In tomato (Lycopersicon peruvianum) cell cultures the simultaneous expression of four Hsfs, which are either constitutively (HsfA1 and HsfA3) or heat-stress inducible (HsfA2 and HsfB1) expressed, results in a complex network with dynamically changing cellular levels, intracellular localization and functional interactions. In order to examine the relevance of their multiplicity as well as to get more insights into the complexity of the plant heat stress response, the individual tomato Hsfs were investigated with respect to their protein interactions in vitro and in vivo. To this aim, I used pull-down assays as well as yeast assays to study the following aspects: 1. Oligomeric state of Hsfs: the results show that all class A Hsfs (HsfA1, HsfA2 and HsfA3) are trimeric proteins and interact with each other via the oligomerization (HR-A/B) domain. The similarity of their HRA/B regions allows formation of homo- and heterooligomeric complexes between all class A Hsfs. This special property was investigated by mutational studies with HsfA2 indicating that the linker and the HR-B regions are the minimal part required for Hsf/Hsf interactions. The conserved hydrophobic amino acid residues of the HR-B region are most important whereas the amino acid residues of the linker may provide higher flexibility to the HR-B region. Another investigated factor was HsfB1. HsfB1 is a member of class B Hsfs, which are characterized by an oligomerization domain without the 21 amino acid residues linker inserted between the HR-A and HR-B regions. It has a low activator potential and exists exclusively as dimer. HsfB1 can not physically interact with class A Hsfs. However, HsfB1 and HsfA1, binding to adjacent HSE sites, are assumed to cause strong synergistic effects in gene activation. 2. Potential HsfB1 interacting proteins: we searched for HsfB1 interacting proteins by using recombinant His-tagged proteins with HsfB1 as baits in pull-down assays. Histones H2A, H2B and H4 were identified by means of Peptide Mass Finger Printing and N-terminal sequencing analyses. The three histones represent the major proteins in tomato whole cell extracts retrieved by HsfB1. 3. HsfA2/small heat stress proteins (sHsps) interaction: pull-down and yeast two-hybrid assays were used to study the specific interaction of HsfA2 with tomato class II sHsp. This interaction occurs via the oligomerization domain of HsfA2. Other members of the plant Hsp20 family, including class I sHsp, do not interact with HsfA2. Heterooligomers of HsfA2 with class II sHsp may represent precursor forms of the plant higher molecular weight cytoplasmic complexes of heat stress granules, which form during heat stress. The findings presented in this thesis are a contribution to support the concept of a Hsfs network via protein-protein interactions. These data, together with information obtained from other studies, are used to propose a tentative model of the complex Hsfs network controlling the plant heat stress response.
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 heat stress (hs) response is universal to all organisms. As the cell senses increase in temperature, heat stress transcription factors (Hsfs) are activated to upregulate the expression of a number of genes encoding heat stress proteins (Hsp) which act as molecular chaperones to protect cells against heat damages. In higher plants, the phenomenon seems to be unusually complex both at the level of Hsfs and Hsps (e.g., 21 Hsf encoding genes in Arabidopsis and at least 17 in tomato). Upon prolonged hs, another characteristic property of plant cells is the assembly of large cytosolic aggregates called heat stress granules (HSG), which are composed of Hsps, HsfA2, RNA and RNA-binding proteins. The present work was aimed to understand plant hs response using tomato as a model system. To study the function of tomato Hsfs in their native system, we generated transgenic tomato lines altered in expression of HsfA1, HsfA2, and HsfB1. Tomato plants with 10-fold overexpression of HsfA1 (OE plants) were characterised by integration of a single HsfA1 expression cassette, whereas the plants harbouring a tandem inverted repeat (IR) of the cassette showed cosuppression of HsfA1 (CS plants). The lack of HsfA1 expression in CS plants results from posttranscriptional gene silencing connected with the formation of small interfering RNA (siRNA). Under normal growth conditions, major developmental features were similar for wild-type (WT), OE and CS plants. However, in contrast to the former two, CS plants and fruits were extremely sensitive to elevated temperature because hs-induced synthesis of major chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family, the function of tomato HsfA1 is unique as master regulator of induced thermotolerance. On the other hand, maintenance of essential chaperones in CS plants during seed development suggests involvement of other Hsfs and/or transcription factor(s). HsfB1 and HsfA2 transgenic tomato plants, unaffected in thermotolerance, further supported the function of HsfA1 as the major factor regulating hs-inducible genes. Hs87 independent phenotypes of plants with altered expression of HsfB1 indicates developmental role of this Hsf. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmids encoding Hsfs A1, A2 and A3 were well expressed which could function as activators for reporter gene expression. However, in protoplasts derived from CS plants, plasmids encoding HsfA2 and HsfA3 were normally expressed but even higher amounts of HsfA1 expression plasmids were completely silenced. Therefore, silencing of HsfA1 in CS plants was also reproduced in its mesophyll protoplasts. Lacking thermotolerance in CS protoplasts could be restored after transformation with expression plasmids encoding functionally equivalent HsfA2 or HsfA3 resulting in (i) expression of chaperones, (ii) survival of the cells at otherwise lethal temperature, (iii) thermoprotection of firefly luciferase, and (iv) assembly of heat stress granules (HSGs). The strong silencing caused by an IR in CS plants opened the possibility of a broad use of RNAi for gene knock-down also in the transient system of mesophyll protoplasts. Using this technology, we attempted to dissect essential components of thermotolerance and HSG assembly. We demonstrated the previously reported function of chaperones such as Hsp70 and Hsp101, and could discriminate the in vivo chaperone functions of different isoforms of Hsp20 and Hsp70 proteins. Hsp17-CI, Hsp70 (hs-inducible isoforms), and Hsp101 are absolutely essential chaperones for thermotolerance in plants. Furthermore, the results also show that despite Hsp17-CI and -CII being major components of HSG complexes, they are dispensable for assembly of these complexes. Based on these results, it is proposed that in the transient protoplast system an approach with gene-specific IRs can be used to discriminate functions of closely related isoforms among protein-families and to dissect complex protein networks.
Vascular occlusive diseases are one of the leading mortality causes in westernised countries. Occlusions of one of the major arteries can be overcome without devastating consequences provided a timely induction of compensating collateral arteries occurs. Perhaps the most outstanding feature of collateral vessel growth is the proliferation of smooth muscle cells (SMCs). Understanding the molecular mechanisms and identifying key molecular players of SMC proliferation would contribute significantly to the development of efficient therapies to intervene with all processes involving neointima formation, including collateral growth. mRNA and protein coding for co-transcription factor Egr1 were found to be up-regulated in growing collateral vessels 6, 12 or 24 hours following femoral artery ligation in mice. Since Egr1 is required for SMC proliferation in vitro and in vivo and likely to be implicated in the initiation of collateral artery growth, the key signalling mediators regulating Egr1 expression specifically in proliferating vascular SMCs were investigated. Northern blot and Western blot analysis revealed a strong up-regulation of Egr1 within 2 hours of stimulation with PDGF-AB and FGF-2. These two potent SMC mitogens involved in neointima formation were used to stimulate vascular SMCs not only to delineate the regulators of Egr1 expression but also to identify additional key mediators of SMC proliferation. FGF-2 but not PDGF-AB led to a drastic reduction of desmin amount in proliferating SMCs, correlating closely with the phenotypic modulation of SMCs in vivo. Both growth factors triggered a dramatic increase in DNA-synthesis rate with a concomitant loss of p27 exp Kip1. Stimulation with PDGF-AB and FGF-2 triggered a rapid and transient activation of PDGFRβ and FGFR1 respectively, thus providing the basis for activation of down-stream targets. Analysis of an array of signalling pathways demonstrated a strong activation of the Ras-Raf-MEK-ERK cascade in response to both factors as measured by the level of phosphorylation of prominent members MEK, ERK1/2 and c-Myc. SAPK/JNK and p38, which also belong to the superfamily of MAP kinases, did not become activated following stimulation with either PDGF-AB or FGF-2. The analysis of various PKC isoforms identified PKCδ and PKCθ to be the key mediators of PDGF-AB- and FGF-2-induced mitogenesis in proliferating SMCs. Whereas PDGF-AB potently stimulated PKB/Akt with concomitant GSK3β phosphorylation, FGF-2-induced inactivation of GSK3β was independent of PKB/Akt. Specific inhibition in order to evaluate the contribution of individual pathways to Egr1 expression and vascular SMC proliferation revealed that inhibition of the Raf-MEK-ERK module by UO126 completely abolished DNA-synthesis and Egr1 expression without a compensation by alternative pathways. Surprisingly, inhibition of PI3K led to a switch to the mitogenic RafMEK-ERK signalling cascade which resulted in an augmented Egr1 expression. In conclusion, in porcine vascular SMCs, activation of the Ras-Raf-MEK-ERK signalling module appears to be the main prerequisite for Egr1 expression and DNA synthesis induction in response to PDGF-AB and FGF-2 whereas related kinases SAPK/JNK and p38 play no significant role. Inhibition of the PI3K-Akt cascade represents an alternative way to activate ERK1/2 and induce Egr1 expression. Whereas MEK is the central regulator of mitogenic effects in proliferating vascular SMCs, the PI3K-Akt pathway most likely exerts survival function. Inactivation of MEK by its specific inhibitors identified hyperphosphorylation as ayet unknown mechanism of kinase inhibition.
The objective of this study is the avifauna of the North American Green River Formation. Five new Green River bird species as well as several new specimens of already known species are described. * Galliformes: Gallinuloides wyomingensis EASTMAN 1900 A second specimen of the galliform Gallinuloides wyomingensis could be identified. Gallinuloides wyomingensis resembles closely Paraortygoides MAYR 1999, which is known from Messel and the London Clay. The new specimen exhibits characters such as a cup-like cotyla scapularis of the coracoid that clearly indicate that Gallinuloides is a stem-group representative of galliforms. * Eurypygidae: Eoeurypyga olsoni gen. et sp. nov. Eoeurypyga is the only fossil representative of the Eurypygidae. Eoeurypyga and the modern sunbittern Eurypyga helias share the typical long bill, the caudally situated neck and the elongated vertebrae cervicales. Additional synapomorph characters were found. The new species indicates a North American origin for the Eurypygidae. * Messelornithidae: Messelornis nearctica HESSE 1992 The original description of Messelornis nearctica was based on a single specimen. Ten new specimens, described in this study, reveal additional information. Messelornis nearctica shows the same large intraspecific size range as Messelornis cristata HESSE 1988 from Messel, the type species of the genus. * Apodidae: Wyomingcypselus pohli gen. nov. sp. nov. Wyomingcypselus pohli is the first described fossil apodiform bird for North American. Due to characters of the wing, especially the position of the processus musculi extensor metacarpi radialis, Wyomingcypselus is referrred to the Apodidae. * Trogoniformes: unnamed species The Green River birds include a poorly preserved, but apparently heterodactyl specimen, which also resembles trogons in overall appearance. * Primobucconidae: Primobucco mcgrewi BRODKORB 1970 Originally, Primobucco mcgrewi was only known from a partial skeleton consisting of the right wing. Three new specimens could be referred to the species. Primobucco mcgrewi clearly exhibits an anisodactyl foot, which makes the assignment to the zygodactyl Bucconidae highly doubtful. Instead, Primobucco mcgrewi is referrred to the Coraciiformes s.s. Thus, Primobucconidae are the first New World representatives of stem-group Coraciiformes. * ?Leptosomidae: Plesiocathartes wyomingensis sp. nov. and Plesiocathartes major sp. nov. Plesiocathartes wyomingensis and Plesiocathartes major represent the first North American record for the genus. Both species exhibit the diagnostic characters for the Leptosomidae as listed by MAYR (2002a, b). * Primoscenidae: Eozygodactylus americanus gen. et sp. nov. and unnamed species Eozygodactylus americanus is the first North American member of this taxon. Both Eozygodactylus americanus and the unnamed species show the zygodactyl foot and the large processus intermetacarpalis of the carpometacarpus, which are typical for Primoscendiae. Due to differences mainly of the humerus, it was placed in a new genus. Besides the descriptionof new species, the avifauna of the Green River Formatin was studied and compared with the avifauna of Messel. The formations show a high concordance, more than 60 % of the Green River taxa also occur in Messel. Such a high concordance is also found for mammals. This is due to the existence of two landbridges, the Thule landbridge and the de Geer landbridge, between Europe and North America during the early Eocene.
Safety concerns associated with the use of viral vectors in gene therapy applications have attracted considerable attention towards the development of nonviral vectors as alternatives for DNA delivery. While nonviral vectors are commonly not associated with safety problems, they are still very inefficient compared to viral vectors, and require significant improvements to approach the efficiency of their viral counterparts. Meanwhile ligands or single-chain antibody fragments that bind to cell surface receptors for increased and/or specific cellular uptake, endosome escape activities, and nuclear localization sequences (NLSs) to enhance transport of plasmid DNA into the nucleus, have become available that can be incorporated into nonviral vectors to improve their efficacy. However, as gene delivery is a multistep process, the challenge is to incorporate multiple of these functional elements into a single nonviral vector system, while retaining their specific activities. A promising method to attach such entities to plasmid DNA is the use of multifunctional fusion proteins that bind to DNA through a DNA-binding domain. In principle, two types of DNA-binding domains/proteins can be used to anchor additional functional domains or peptides to a plasmid, namely sequence-specific DNA-binding domains, described in the first part of this thesis, or those that bind DNA independent of its sequence, exemplified in the second part of this work by a derivative of the human HMGB2 protein. The first fusion protein constructed and analyzed contained the E. coli LexA repressor as a sequence-specific DNA-binding domain. In addition, this DNA-carrier protein, termed TEL, included a bacterial translocation domain as an integrated endosome escape activity, and human TGF-a for specific targeting to the EGF-receptor (EGFR). TEL was expressed in E. coli and purified under both native and denaturing conditions. Purified, denatured TEL was refolded and subsequently shown to bind specifically to EGFR-expressing cells. However, inclusion of TEL in complexes of plasmid DNA and poly-L-lysine (pL) did not lead to increased gene delivery into EGFR-expressing COS-1 cells. Most likely this was due to the absence of DNA-binding activity of the LexA moiety in TEL. In contrast, native TEL was able to interact specifically with DNA. Nevertheless, since this interaction was rather weak, and refolding of denatured TEL had not resulted in functional activity of all of its protein domains, it seemed unlikely that fusion proteins containing LexA would exhibit gene transfer capabilities superior to those of similar DNA-carrier proteins previously constructed in our group. Further work therefore focused on the use of the E2C-Sp1C protein as an alternative sequencespecific DNA-binding domain. This artificial zinc-finger protein was fused to the single-chain antibody fragment scFv(FRP5), directed against the human ErbB2 growth factor receptor. The resulting 5-E2C fusion protein was expressed in E. coli and purified under native and denaturing conditions. Refolded and native 5-E2C were found to bind specifically to ErbB2-expressing cells, indicating that scFv(FRP5) in 5-E2C was functional in both preparations. In contrast, whereas refolded 5-E2C bound DNA only weakly, significant DNA binding was observed for native 5-E2C. In addition, it could not only be shown that the interaction of native 5-E2C with DNA containing its recognition sequence was specific, but also that this protein was able to bind DNA and recombinant ErbB2 simultaneously, demonstrating the functionality of both domains in native 5-E2C. Despite these encouraging results, the inclusion of native 5-E2C in pL- or polyethyleneimine (PEI)-DNA complexes did not lead to an (5-E2C-specific) enhancement of gene transfer efficiency, irrespective of the presence of the endosome-disruptive reagent chloroquine during transfection. In the second part of this thesis an alternative approach for the development of DNA-carrier proteins for nonviral gene delivery is described, based on human HMGB2, a DNA-binding protein without sequence specificity. HMGB2 contains an acidic C-terminus that has been found to decrease the affinity of the protein for DNA. Therefore, this C-terminal tail was deleted, resulting in an HMGB2-variant consisting of amino acids 1-186. HMGB2186, purified under native conditions from E. coli lysates, was able to interact with DNA and bound to the surface of different cell lines. Importantly, after binding to plasmid DNA HMGB2186 mediated gene delivery into COS-7 cells with higher efficiency than pL. In addition, HMGB2186-mediated gene transfer was strongly enhanced in the presence of chloroquine, indicating that the endocytic pathway was involved in cellular uptake. To improve internalization and intracellular routing of HMGB2186 as a DNA-carrier, a derivative containing the TAT47-57 cell-penetrating peptide (CPP), reported to facilitate cell entry independent of endocytosis, was constructed. Since this peptide also contains an NLS, in addition an HGMB2186-variant containing the SV40-NLS was constructed to investigate the effect of a peptide that has only nuclear localizing properties. Interestingly, the resulting TAT-HMGB2186 and SV40-HMGB2186 fusion proteins displayed DNA-binding activities similar to HMGB2186, but mediated gene delivery into different cell lines clearly more efficiently than the parental molecule. Furthermore, the efficacy of both fusion proteins was enhanced markedly in the presence of chloroquine, an indication that endocytosis was involved in the transfection process mediated by these proteins. This suggests that the increased transfection efficiency observed for TAT-HMGB2186 was more likely due to the NLS function present in the TAT47-57 peptide, rather than to its ‘cell penetrating properties’. Finally, the incorporation of functional peptides derived from human proteins into HMGB2186 was investigated. An uncharged CPP originating from Kaposi-FGF, reported to facilitate efficient cellular uptake of fused protein domains in an endocytosis-independent manner, was fused to HMGB2186 together with the SV40-NLS. Interestingly, the resulting KSV40-HMGB2186 fusion protein bound DNA similarly as previously tested DNA-carrier proteins, but did not mediate enhanced transfection compared to HMGB2186. In addition, the importin-b-binding (IBB) domain derived from human importin-a2 was investigated as a component of a DNA-carrier protein. Since the IBB domain can function as an NLS, it was fused to HMGB2186 resulting in the DNA-carrier protein IBBHMGB2186. Although IBB-HMGB2186 bound DNA in a similar manner as the other HMGB2186-derivatives, gene delivery mediated by IBB-HMGB2186 was only as effective as HMGB2186 mediated transfection, suggesting no significant role of the IBB domain. However, addition of chloroquine resulted in a remarkable enhancement of IBB-HMGB2186-mediated gene transfer, which was now more efficient than with any other HMGB2186-variant tested, and not much lower than gene transfer mediated by PEI, one of the most efficient transfection reagents available to date. To enhance nonviral gene delivery even further, the HMGB2186-based DNA-carrier proteins described in this thesis might now serve as building blocks for novel fusion proteins that include additional complementing activities. In this respect it seems particularly promising that, under conditions of effective end some escape, IBB-HMGB2186, which consists entirely of protein domains of human origin, was the most efficient of all proteins tested in this work.
Studies in particular of the last decade showed that active neurogenesis continuously takes place in the subventricular zone (SVZ) of the lateral ventricles of the adult rodent brain. Neurogenesis in the SVZ leads to migration of neuroblasts within the rostral migratory stream (RMS) and mature neuron formation mainly in the olfactory bulb (OB). According to present understanding, glial cells with astrocytic properties represent the actual adult neural stem cells. The cell types representing the various cellular transition states leading to the formation of mature neurons as well as the mechanisms controlling adult neurogenesis and neuroblast migration are poorly understood. A previous study from this laboratory demonstrated that the ATP-hydrolyzing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is associated with type B cells, the presumptive neural stem cells. NTPDase2 is a protein of the plasma membrane with its catalytic site facing the extracellular space. It hydrolyzes extracellular nucleoside triphosphates to their respective nucleoside diphosphates. This raises the possibility that the signaling pathway via extracellular nucleotides is involved in the control of adult neurogenesis. Neurons as well as glial cells express several subtypes of receptors (P2 receptors) that are responsive to the nucleotides ATP, ADP, UTP, or UDP. P2X receptors are ATP-gated Na+, K+ and Ca2+ permeable ion channels, P2Y receptors are coupled to trimeric G-proteins. In order to probe for a functional role of nucleotides in adult neurogenesis, the present study referred to an in vitro system (neurospheres). Neurospheres produced from isolates of the mouse SVZ and cultured in the presence of EGF and bFGF expressed the neural stem cell marker nestin and also GFAP, S100β, NTPDase2 and tissue non-specific alkaline phosphatase. Neurospheres generated from the cells of the subventricular zone were multipotenital. This was revealed by immunostaining of differentiated cells with markers for astrocytes, neurons and oligodendrocytes. The presence of ecto-nucleotidase was verified by analyzing the free phosphate released from nucleotides. The tissue non-specific form of alkaline phosphatase was the predominant enzyme. Both NTPDase2 and TNAP could be identified by immunocytochemistry and Western blotting. Hydrolysis was not observed for p-nitrophenyl thymidine monophosphate, a substrate of members of the ectonucleotide pyrophosphatase/phosphodiesterase family (NPP1 to NPP3). Since ecto-nucleotidases control the availability of extracellular nucleotide agonists, neurospheres were studied for the potential expression and functional role of nucleotide receptors. Neurospheres responded to extracellular nucleotides with a transient rise in Ca2+ (ATP = ADP > UTP). The rise in Ca2+ was due to P2Y receptors. The Ca2+ response was unaltered in the absence of extracellular Ca2+ and strongly reduced by thapsigargin, a blocker of internal Ca2+ stores. The P2Y1 antagonist MRS2179 strongly reduced the ATP- or ADP-induced increase in Ca2+, suggesting the involvement of a P2Y1 receptor. In addition, suramin and PPADS, non-selective antagonists for P2 receptors, inhibited most of the Ca2+ response. The agonistic activity of UTP and the lack of response to UDP implied the additional presence of a P2Y2 and/or a P2Y4 receptors and the absence of a functional P2Y6 receptor. RT-PCR experiments demonstrated that neurospheres expressed P2Y1 and P2Y2 receptors but not P2Y4 receptor. That the majority of the Ca2+ response to ATP was mediated via P2Y1 receptors was also confirmed by analysis of P2Y1 knockout mice and by application of the P2Y1 receptor-specific antagonist MRS2179. In addition, agonists of P2Y1 and P2Y2 receptors and low concentrations of adenosine augmented cell proliferation inspite of the presence of mitogenic growth factors. Neurosphere cell proliferation was attenuated after application of MRS2179 and in neurospheres from P2Y1 receptor knockout mice. These results infer a nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Taken together these results suggest that P2Y-mediated nucleotidergic signalling is involved in neurosphere function and possibly also in adult neurogenesis in situ.
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 Na+/proline transporter of E. Coli (PutP) is responsible for the uptake of proline which is subsequently used not only as a carbon and nitrogen source and a constituent of proteins but also as a particularly effective osmoprotectant. However, for a long time there was little known about the single steps in the reaction cycle of this transporter and only few details about its structure-function relationship are available. Aim of the present work was to achieve a deeper understanding about the kinetic properties of the Na+/proline transporter and to get insights into the structure-function relationship of the substrate binding. To answer these questions different techniques were used. By using the novel SSM technique combining the preparation of PutP proteoliposomes it was possible to demonstrate for the first time the electrogenic substrate binding to PutP transporter. Due to rapid solution exchange measurements on the SSM it was additionally possible to obtain time resolved information about the kinetic details of the cytoplasmic substrate binding sites which were not available by previous steady state and equilibrium binding measurements. Pre-steady-state charge translocation was observed after rapid addition of one or both of the cosubstrates Na+ and/or proline to the PutP-WT proteoliposomes adsorbed on the SSM. Thereby it was possible to link the observed electrical signals with the binding activity of PutP. The observed Na+ and/or proline induced charge displacement were assigned to an electrogenic Na+ and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k > 50 s-1 proceeding the rate limiting step of the reaction cycle. Furthermore, based on the kinetic analysis of the electrical signals obtained from the measurements of PutP on SSM, the following characteristics of the substrates binding in PutP were deduced: (1) both Na+ and proline can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails; while at sufficiently high concentrations, each substrate can bind in the absence of the other; (2) substrate binding is electrogenic not only for Na+, but also for the uncharged cosubstrate proline. The charge displacement associated with Na+ binding and proline binding is of comparable size and independent of the presence of the respective cosubstrate. In addition, it was concluded that Na+ accesses its binding site through a high-field access channel resulting in a charge translocation, whereas the binding of the electroneutral proline induces a conformation alteration involving the displacement of charged amino acid residue(s) of the protein; (3) Na+ and proline binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate; (4) proline binding proceeds in a two step process: low affinity (~ 0.9 mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition; (5) membrane impermeable PCMBS inhibits both Na+ and proline binding to the inside-out orientated PutP transporter, indicating that rather than selectively blocking a specific binding site, PCMBS probably locks the enzyme in an inactive state. The possible targets for this SH-reagent are cysteines 281 and 344 located close to the cytoplasmic surface of the protein. Beyond it, transient electrical currents of PutP were also observed on the BLM after rapid addition of proline in the presence of Na+. This was possible by combining the conventional BLM technique with high-speed flash-photolysis of caged-proline. Indeed the signals on the BLM indicate the detection of a different underlying reaction process in comparison to the data achieved by the SSM technique. This has paved the way for supplemental information about the reaction cycle since it was possible to assign the flash-photolysis BLM signals to the proline binding step followed by the internalization of Na+ and proline into the liposome. Thereby it was found, that the presence of Na+ is indispensable and the time constant for the process is ~ 63 ms. Moreover, structure-function information about the Na+ and proline binding sites of PutP was obtained by investigating the functionally important amino acid residues Asp55, Gly63 and Asp187 with site-directed mutagenesis and the combined SSM technique. One finding is that the mutated proteins PutP-D55C and PutP-G63C showed no activity on the SSM. Therefore, it can be assumed that either both Asp55 and Gly63 are crucial for the structure of PutP protein, or they are located at or close to the Na+ and proline binding sites. Furthermore, the results obtained from PutP-D187N and PutP-D187C mutants on SSM suggest that Asp187 of PutP is likely to be involved in the Na+ binding at the cytoplasmic side of the backward running carrier. Taken together the results of the present work have substantially broadened the known picture of the Na+/proline transporter PutP thereby several steps of the reaction cycle were elucidated, and moreover, valuable insights into the structure-function relationship of the transporter have become available.
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 ...
Ubiquitylation is a three-step process, which results in the attachment of the small protein ubiquitin (Ub) to lysine residues on a substrate protein. SUMO proteins are ubiquitin (Ub)-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. During my PhD I isolated and characterized SUMO1 and SUMO2 binding motifs. Using Yeast Two Hybrid system, bioinformatics and NMR spectroscopy we defined a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a β-strand that could bind in parallel or anti-parallel orientation to the β2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions. Mutation of the SUMO interacting motif of TTRAP (TRAFS and TNF receptor associated protein) influences both its localization and dynamic behaviour in living cells. Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitylated targets and control numerous biological processes including receptor trafficking, DNA repair, virus budding and gene transcription. They themselves undergo UBD-dependent monoubiquitylation, which promotes intramolecular binding of the UBD to the attached Ub and consequently leads to their functional inhibition. During the second part of my PhD I could show that, in contrast to the established ubiquitylation pathway, the presence of UBDs allows the monoubiquitylation of host protein independently of classical E3 ligases. UBDs of different types including UBA, UIM, UBM, NFZ and UBZ, can directly cooperate with E2 Ub-conjugating enzymes to promote monoubiquitylation of their host proteins. Using FRET technology I verified that the E2 enzyme and the substrate directly interact in cells. Moreover, UBD-containing proteins Stam2 and Sts2 promote self-ubiquitylation and not ubiquitylation of other targets or form polyUb chains from free Ub. Our study revealed a yet unappreciated role of E2 enzymes in ubiquitylation reactions of UBD containing proteins.
Active neurogenesis continuously takes place in the dentate gyrus of the adult mammalian brain. The dentate gyrus of the adult rodent hippocampus contains an astrocytelike cell population that is regarded as residual radial glia. These cells reside with their cell bodies in the subgranular layer (SGL). Radial processes traverse the granule cell layer (GCL) and form bushy ramifications in the inner molecular layer (IML). The residual radial glial cells apparently represent neuronal progenitor cells that can give rise to functionally integrated granule cells. To date the cellular and molecular events driving a subpopulation of these cells into neurogenesis as well as the cellular transition states are poorly understood. The present study shows, that in the mouse dentate gyrus, this cell type selectively expresses surfacelocated ATPhydrolyzing activity and is immunopositive for nucleoside triphosphate diphosphohydrolase 2 (NTPDase2). NTPDase2 is an ectoenzyme and hydrolyzes extracellular nucleoside triphosphates such as ATP or UTP to their respective nucleoside diphosphates. The enzyme becomes expressed in the hippocampus during late embryogenesis from E17 onwards, and is thus not involved in early brain development. Its embryonicpattern of expression mirrors dentate migration of neuroblasts and the formation of the primary and finally the tertiary dentate matrix. NTPDase2 is also expressed by a transient population of cortical radial glia from late embryonic development until postnatal day 5. NTPDase2 can be employed as a novel markerfor defining cellular transition states along the neurogenic pathway. It is associated with subpopulations of GFAP and nestinpositive cells. These intermediate filaments are typically expressed by the progenitor cells of the dentate gyrus. In addition there is a considerable overlap with doublecortinand PSANCAM positive cells. The expression of the microtubuleassociated protein doublecortin and of PSANCAM which are expressed by migrating neuroblasts is indicative of a transition of progenitors to a neural phenotype or an immature form of granule cell. NTPDase2 is no longer associated with young neurons and with maturegranule cells, as indicated by the lack of doubleimmunostaining for III tubulin and NeuN, respectively. Furthermore, β S100positive astrocytes do not express NTPDase2 validating that NTPDase2 is also not associated with later stages of gliogenesis. Experiments with the Sphase marker bromodeoxyuridine (BrdU) demonstrate that NTPDase2positive cell proliferate. Postmitotic BrdU-labeled cells preferentially acquire an NTPDase2positive phenotype. Many of these cells were also positive for GFAP. The contribution of BrdUlabeled cells positive for NTPDase2 increased with time from 2 h to 72 h, validating a strong association of NTPDase2 with proliferating cells of the dentate gyrus. The colocalization studies with various markers and the results of the experiments suggestthat NTPDase2 is associated with cell types of varying maturation states but not with mature neurons or astrocytes. Studies on the formation of neurospheres from the dentate gyrus validate previous data suggesting that the hippocampal progenitors have little capacity for self renewal in vitro. In situ hybridization results indicate the presence of one of the metabotropic purinergic receptor subtypes (the P2Y1 receptor) within the adult neurogenic regions, the dentate gyrus and the lateral walls of the lateral ventricles. A patchclamp analysis demonstrates the presence of functional ionotropic nucleotide receptor (P2X receptors) in progenitor cells expressing nestin promotordriven GFP. They suggest that the signaling pathway via extracellular nucleotides and nucleotide receptors may play a role in the control of adult hippocampal neurogenesis.
Shrew-1 wurde bei der Suche invasivitätsassoziierter Gene mittels eines DDRT-PCR-Ansatzes aus invasiven Zellen isoliert. Wie computergestützte Analysen der Sequenz ergaben, wies das bis dahin unbekannte Protein keinerlei Ähnlichkeiten mit bereits bekannten Proteinen auf und homologe Proteine wurden bisher nur in Vertebraten gefunden. Expressionsanalysen mit einem GFP-markierten shrew-1 zeigten, dass es an der basolateralen Plasmamembran lokalisiert, wo es mit dem E-Cadherin vermittelten Adhäsions-Komplex kolokalisiert. Eine Integration in diesen Komplex geschieht höchstwahrscheinlich durch direkte Interaktion mit β-Catenin. Ein weiteres Molekül das als potenzieller Interaktionspartner von shrew-1 identifiziert wurde und das in der Literatur oft als Tumorsuppressor diskutiert wird, ist Caveolin-1. Ferner konnten Überexpressionexperimente bereits zeigen, dass shrew-1 die Invasivität von HT1080-Zellen erhöhen kann. Das Ziel dieser Arbeit war es, zum einen mit Hilfe des Hefe-Split-Ubiquitin-Systems eine Interaktion von shrew-1 und Caveolin-1 zu bestätigen und zum anderen neue Interaktionspartner zu identifizieren, die helfen könnten, die Rolle von shrew-1 in invasiven Vorgängen zu erklären. Um eine mögliche Verbindung von shrew-1 und einem neuen Interaktionspartner in Bezug auf die Zellinvasivität zu untersuchen, sollten sowohl shrew-1 als auch der potenzielle Interaktionspartner mittels RNAi ausgeschaltet werden. Mit Hilfe des Split-Ubiquitin-Systems war es möglich, die Interaktion zwischen shrew-1 und caveolin-1 zu bestätigen und zu zeigen, dass diese durch die zytoplasmatische Domäne von shrew-1 vermittelt wird. Weiterhin konnte CD147 als neuer Interaktionpartner identifiziert werden. Eine Interaktion beider Proteine konnte ferner mit Hilfe des Bimolekularen-Fluoreszens-Komplementations-Systems (BIFC), des Fluoreszens-Resonanz-Energie-Transfers (FRET) und Coimmunoprezipitationen bestätigt werden. Die Interaktion von shrew-1 und CD147 scheint allerdings abhängig vom zellulären Kontext zu sein, wie die FRET-Analysen vermuten lassen. So konnte nämlich mit diesen Analysen eine starke Interaktion in MCF7-Zellen gezeigt werden, wohingegen die Interaktion in MDCK-Zellen schwächer war. Einer der auffälligsten Unterschiede dieser beiden Zelllinien im Bezug auf diese Interaktion könnte sein, dass MCF7-Zellen im Gegensatz zu MDCK-Zellen kein Caveolin-1 exprimieren. Caveolin-1 konnte seinerseits als Interaktionspartner von shrew-1 mit Hilfe des Hefe-Split-Ubiquitin-Systems bestätigt werden und andererseits wurde von einer anderen Arbeitsgruppe eine Interaktion von CD147 mit Caveolin-1 publiziert. Um dies näher zu untersuchen, wurde Caveolin-1 in MCF7-Zellen exprimiert und die FRET-Analysen in diesen wiederholt. Wie vermutet kam es zu einer Reduktion der Interaktion in Caveolin-1 exprimierenden MCF7-Zellen. CD147 ist neben vielen anderen Funktionen auch maßgeblich an der Regulation von Matrix-Metalloproteinasen beteiligt und kann somit die Invasivität von Zellen beeinflussen. Um einen Einfluß von shrew-1 und CD147 auf die Invasivität zu untersuchen, wurden beide Proteine mittels RNAi in HeLa-Zellen ausgeschaltet. Nachdem ein negativer Einfluss dieses Ansatzes auf das Proliferationsverhalten der Zellen ausgeschlossen werden konnte, wurde ein möglicher Effekt auf die Invasivität der Zellen untersucht. Durch die Analyse in Matrigel-Invasionsassays konnte gezeigt werden, dass das unabhängige Ausschalten beider Proteine die Invasivität der Zellen auf 35-55% im Vergleich zu Kontrollzellen reduziert. Die Ergebnisse dieser Arbeit untermauern die Annahme, dass shrew-1 eine Rolle bei invasiven Vorgängen spielt und weisen darauf hin, dass dies möglicherweise durch eine Interaktion mit CD147 geschieht. Die Interaktion mit CD147 und damit eine mögliche Funktion von shrew-1 bei invasiven Vorgängen scheinen dabei abhängig vom zellulären Kontext zu sein.
Membranes are essential for life, because a cell must separate itself from the environment to keep its molecules from dissipating away and also must keep out foreign molecules that disturb them or their cell components. However, the cell must communicate with the environment and adapt to the external conditions, needs to pump in nutrients and release toxic products of its metabolism. Membrane proteins present in the membranes of the cell and cell organelles, help the cell to gather information about the environment and perform various biological processes. Membrane proteins perform a wide range of biological functions including respiration, signal transduction and transport. Despite their high importance in biological function, only few structures have been determined because of the difficulties in producing high amounts of membrane proteins and obtaining good quality crystals. This Ph. D. thesis involves the study of different kinds of cytochrome oxidases and a membrane anchored cytochrome oxidase electron donor. Though structures of many cytochrome oxidases are known to date, there exist many different types of oxidases in different organisms, which help the organism to survive under unfavorable environmental conditions. The structural differences between these terminal oxidases which make the organism to survive in extreme environments are unclear. To investigate these, structures of different types of oxidases are necessary. Therefore, we are interested in revealing the structural details of different types of oxidases. The different types of oxidase I worked with were the caa3 HiPIP:oxygen oxidoreductase from Rhodothermus marinus, the aa3-type quinol oxidase from Acidianus ambivalens and bd-type quinol oxidase from three different organisms (Escherichia coli, Bacillus thermodenitrificans and Aquifex aeolicus). Besides the protein from E. coli all other proteins are from thermophilic organisms from which the proteins obtained are generally believed to be highly stable. The presence of a high content of charged amino acids that enhances the occurrence of salt bridges contributes to the stability of thermophilic proteins. ....
G protein-coupled receptors (GPCRs) comprise the largest membrane protein family and play an essential role in signal transduction through the cell membrane. They are currently the targets of approximately 50 % of the pharmaceuticals on the market (Klabunde and Hessler, 2002). However, only one high-resolution GPCR structure has been determined up to now, that of bovine rhodopsin (Palczewski et al., 2000). The GPCR activation and regulation mechanisms are still unknown and other GPCR structures are thus required. MePNet (Membrane Protein Network) was a European consortium dedicated to structural studies of GPCRs. The approach was to produce 100 GPCRs in three expression systems (Escherichia coli, Pichia pastoris and Semliki Forest Virus infected mammalian cells) in order to select at each step of the process (production, solubilization, purification) the constructs that fulfilled quantity and quality (functionality) requirements for crystallization trials. In our team, we screened 38 of the 100 targets in P. pastoris. For each receptor, the clone with the highest production level was identified by dot-blot. The size and homogeneity of each receptor were then analyzed by Western-blot. The human adenosine A2A receptor showed a well-defined and pronounced single band and was thus selected for further characterization. The adenosine A2A receptor is a GPCR mainly localized in the central nervous system and, as it antagonizes dopaminergic activity, it has great potential as a drug target for the treatment of Parkinson’s disease. Functional characterization by binding assays with the specific antagonist [3H]-ZM241385 demonstrated a Bmax of 56 +/- 3 pmol/mg i.e. pmol of binder per milligram of total membrane protein, and a KD of 0.40 +/- 0.02 nM. Receptor production was then improved by lowering the induction temperature, decreasing the induction time and adding DMSO to the medium. For large-scale production, fermention reached around 300 g cells (wet weight)/L culture, which provided 43 mg of functional receptor in membranes per liter of culture. Functional solubilization was achieved with dodecyl-β-D-maltoside and the soluble yield was increased to 70-80 % of the membrane content by addition of cholesteryl hemisuccinate and increasing the ionic strength. The receptor was successfully purified via Ni-NTA and monomeric avidin chromatography in the presence of the antagonist ZM241385. This strategy produced a pure, homogeneous and stable receptor preparation with functionality demonstrated by radioligand binding assays. The total receptor yield after purification was routinely around 20 % of the membrane functional receptor content and 2 g of membranes provided 4 mg of pure receptor for crystallization trials. GPCRs are very difficult targets for crystallization, and co-crystallization with antibody fragments has been shown to be a successful method for crystallization of membrane proteins. In order to develop such a tool for the adenosine A2A receptor, a single-chain Fv (scFv) fragment specific to the purified receptor was selected by phage display. The receptor was functionally immobilized on the surface of streptavidin beads and after two rounds of selection, 6 different phages were identified several times. After production in E. coli and purification via Ni-NTA affinity chromatography, 4 out of the 6 scFv fragments were sufficiently enriched to be tested by ELISA. For the ELISA, the receptor was functionally immobilized via the biotinylation domain of the construct in a 96-well streptavidin-coated plate. The antibody fragments binding to the receptor were identified based on interaction with HRP-conjugated protein L. One scFv fragment gave a positive ELISA signal 10 fold above background and titration of the scFv fragment binding to the receptor was specific and saturable. However no complex of scFv fragment and receptor was observed on gel filtration. In order to have a more sensitive detection method, the scFv fragment was labeled with fluorescein: a complex was then observed up on gel filtration but the binding appeared to be non-specific. A pull-down assay with immobilized non-labeled scFv fragment finally confirmed the specificity of the binding, but also the low affinity of the interaction. Affinity maturation of this specific scFv fragment by a random mutagenesis and selection process should improve this parameter in order to obtain an adapted tool for co-crystallization.
Die Verarbeitung von Informationen im zentralen Nervensystem beruht auf dem Zusammenspiel von erregender und hemmender Neurotransmission. Die Übertragung von Signalen zwischen Neuronen erfolgt chemisch über die Ausschüttung von Neurotransmittern an spezialisierten Kontaktstellen, den Synapsen. Glyzin und gamma-Aminobuttersäure (GABA) sind die bedeutendsten inhibitorischen Neurotransmitter im zentralen Nervensystem von Säugern, welche Rezeptoren vom Glyzin- (GlyR) und GABAA-Typ (GABAAR) aktivieren. Diese ligandengesteuerten Ionenkanäle sind in postsynaptischen Membranen angereichert und mit intrazellulären Proteinen assoziiert. Die Rekrutierung der Rezeptoren in postsynaptischen Domänen ist ein an das zytoplasmatisch lokalisierte Protein Gephyrin gekoppelter Prozess. So bindet Gephyrin spezifisch an die intrazelluläre Domäne der beta-Untereinheit des GlyR (GlyR beta) und bildet für die Verankerung des Rezeptors ein gerüstartiges Netzwerk unterhalb der synaptischen Membran. Die gezielte Inaktivierung des Gephyrin-Gens führt in Mäusen zu einem postnatal letalen Phänotyp und zu dem Verlust der synaptischen Anreicherung des GlyR und bestimmter GABAA-Rezeptoren auf zellulärer Ebene. Gephyrin ist ein 93 kDa großes Protein, das nicht nur im zentralen Nervensystem (ZNS), sondern auch in anderen Organen wie Leber und Niere exprimiert wird, in denen es an der Synthese des Molybdän-Kofaktors von Oxido-Reduktasen beteiligt ist. Das Gephyrin-Protein wird durch 30 Exons codiert, von denen zehn als sogenannte Kassetten alternativ gespleißt werden können. Die bestuntersuchte Spleißvariante besitzt 736 Aminosäuren und ist in eine N- und eine C-terminale Domäne (Aminosäuren 1-181 bzw. 318-736) sowie eine zentrale Linker-Domäne unterteilt. Die N- und die C-terminalen Bereiche von Gephyrin sind den Proteinen MogA und MoeA aus E. coli homolog und werden daher auch als G-Domäne (N-terminal) bzw. E-Domäne (C-terminal) bezeichnet. In kristallographischen Untersuchungen wurde gezeigt, dass die G- und E-Domänen zur Tri- bzw. Dimerisierung befähigt sind. Diese speziellen Oligomerisierungseigenschaften der beiden Gephyrindomänen bilden wahrscheinlich die Grundlage für die Entstehung von Gephyrin-Clustern sowie eines hexagonalen Gephyrin-Gerüstes. Dieses Gerüst stellt den Verknüpfungspunkt zwischen Rezeptoren und dem Zytoskelett dar und ermöglicht somit die effiziente Clusterbildung und die zielgerichtete Anordnung einer großen Anzahl inhibitorischer Rezeptoren. In der vorliegenden Arbeit sollten die Rolle dieser beiden Domänen bei der Bildung membranassoziierter Gephyrinaggregate und die molekularen Mechanismen der Clusterbildung des Gephyrinmoleküls untersucht werden. Zu diesem Zweck wurden durch zielgerichtete Mutagenese unterschiedliche Gephyrin-Mutanten hergestellt, um die Fähigkeit der Oligomerisierung der G- und E-Domäne gezielt zu modifizieren. Dadurch sollte die Bedeutung der Oligomerisierung hinsichtlich der Aggregat- bzw. Clusterbildung untersucht werden. Außerdem sollten die Wechselwirkungen zwischen Gephyrin und anderen Proteinen und deren Einfluss auf die synaptische Lokalisation analysiert werden. Für diese Untersuchungen wurden auf der Basis von Röntgenstruktur-Daten spezifische Aminosäurereste an den bei der Oligomerisierung beteiligten Kontaktstellen ausgetauscht. In der G-Domäne wurden zu diesem Zweck vier separate Aminosäuren des Trimer-Interface durch Arginin ersetzt (GephRRRR). Analog hierzu wurden in der EDomäne einzelne Aminosäuren durch Arginin bzw. Glutamat substituiert (GephRER), um dadurch eine Dimersierung zu verhindern. Für die Kassette C5’ wird angenommen, dass deren Vorhandensein die Interaktion zwischen Gephyrin und GlyR beeinträchtigt, wodurch GlyR aus GABAergenen Synapsen ausgeschlossen wird. Daher wurde der Einfluss dieser Gephyrin-Spleißvariante (GephC5’), die zu einer Peptidinsertion innerhalb der G-Domäne führt, und einer Gephyrin-Mutante (Gephmut), die den Verlust der Wechselwirkung mit dem GlyR bedingt, auf die Aggregatbildung von Gephyrinoligomeren untersucht. Bei dem Konstrukt Gephmut wurden, basierend auf Daten von Röntgenstrukturuntersuchungen, neun Aminosäuren (713-721) am Cterminalen Ende der E-Domäne durch den homologen Bereich des bakteriellen MoeA Proteins aus E. coli ersetzt. Zunächst wurden die einzelnen isolierten Domänen mittels Gelfiltration hinsichtlich ihres Oligomerisierungsverhaltens untersucht. Die Mutationen wurden hierzu in verkürzte Proteine eingeführt, bei denen nur die G- bzw. die E-Domäne exprimiert wurden. Diese Konstrukte wurden daher als GRRRR, GC5’ bzw. ERER und Emut bezeichnet. Bei diesen zeigte sich, dass die G-Domäne des Gephyrin-Wildtyps zu trimeren Proteinkomplexen oligomerisiert. Im Gegensatz hierzu war die Mutante GRRRR nicht in der Lage, Trimere zu bilden. Das Einfügen der C5’-Kassette führte ebenfalls zu einer Störung der Trimerisierung. Gelfiltrationsexperimente mit der E-Domäne ergaben, dass die mutierte Domäne ERER, im Gegensatz zum Wildtyp-Konstrukt, keine Dimere ausbildet. Bisherige Studien haben jedoch gezeigt, dass das Emut Polypeptid zur Dimerisierung befähigt ist. Das Oligomerisierungsverhalten des kompletten Gephyrin-Proteins wurde mittels blauer nativer Gelelektrophorese (BN-PAGE) analysiert. Für die hier beschriebenen Untersuchungen mit BN-PAGE wurde rekombinantes Gephyrin in Xenopus laevis Oozyten heterolog exprimiert. Die Analyse ergab, dass Wildtyp Gephyrin nativ als Hexamer vorliegt, welches durch ansteigende Konzentrationen des Detergenzes Natriumdodecylsulfat (SDS) in Trimere, Dimere und Monomere zerfällt. Sowohl GephRRRR und GephC5’ liegen nativ fast ausschließlich als Dimere vor, während GephRER nur trimere Aggregate formt. Die entsprechende Doppelmutante mit Mutationen in Gund E-Domäne war wie erwartet nur noch als Monomer existent. Die als Kontrolle eingesetzte Glyzinrezeptor-Bindungsmutante Gephmut bildete, ebenso wie der Wildtyp, Hexamere aus. Daraus folgt, dass die Oligomere der G- bzw E-Domäne Zwischenprodukte der Hexamerbildung darstellen. Die Analyse der Oligomerisierungseigenschaften der Mutanten wurde nachfolgend in humanen embryonalen Nierenzellen (HEK 293T) untersucht. Nach heterologer Expression von Wildtyp Gephyrin in HEK 293T-Zellen formen sich große, charakteristische Gephyrinaggregate. Die Oligomerisierungs-Mutanten GephRRRR, GephRER und GephC5’ aggregierten jedoch nicht, sondern waren diffus im Zytoplasma verteilt. Die wiederum als Kontrolle eingesetzte Bindungsmutante Gephmut hingegen wies eine normale Aggregation auf. Diese Ergebnisse bestätigen die grundlegende Rolle der Oligomerisierung von G- und E- Domänen für die Aggregatbildung von Gephyrin. Mittels GST-Pulldown und Kolokalisationsanalysen in HEK Zellen wurde die Wechselwirkung der Gephyrinmutanten mit der GlyR beta, dem Motorkomplexprotein Dynein light chain-1 (Dlc-1) und dem Guanin-Nukleotid-Austauschfaktor Collybistin (Cb) untersucht. Beide Ansätze weisen darauf hin, dass die Trimerisierung der G-Domäne an der Interaktion von Gephyrin mit Dlc-1 und die Dimerisierung der E-Domäne bei der Bindung an GlyR beta und Cb beteiligt ist. Die Mutante Gephmut zeigte in beiden Fällen einen totalen Verlust der Bindungsfähigkeit sowohl an das GlyR beta Bindungsmotiv als auch an Cb. Der Einbau der C5’ Kassette in Gephyrin scheint jedoch nicht dessen Bindung an den GlyR zu beeinflussen. Für die Analyse der Clusterbildung und des zielgerichteten Transports in Neuronen wurden Wildtyp und mutiertes Gephyrin in hippocampalen und spinalen Primärkulturen der Ratte exprimiert. Zur Überprüfung einer synaptischen Lokalisation wurde Gephyrin gemeinsam mit dem vesikulären inhibitorischen Aminosäure-Transporter (VIAAT), einem präsynaptischen Marker-Protein, detektiert. In beiden Kulturen wies Gephyrin eine punktartige Verteilung in den Neuriten auf und wurde gezielt an Synapsen angereichert. Im Kontrast dazu zeigten alle Oligomerisierungsmutanten, GephRRRR, GephC5’ und GephRER keine Ausbildung von Clustern sondern eine diffuse Verteilung im Zellkörper und in Dendriten. Das Konstrukt Gephmut wies jedoch Clusterbildung und eine punktförmige Verteilung auf. Diese Daten belegen, dass die Oligomerisierung der G- wie auch der E-Domänen für die Clusterbildung und synaptische Lokalisation von Gephyrin unerlässlich ist. Die Wechselwirkung mit dem GlyR und/oder Collybistin ist ebenfalls für die Anreicherung in der Synapse erforderlich, nicht jedoch für die Bildung der Gephyrin-Cluster. Die dargestellten Ergebnisse belegen die Rolle der spezifischen Oligomerisierungseigenschaften der G- und E-Domäne für die Ausbildung des hexagonalen Gephyringerüstes und dessen grundlegende Bedeutung für die spezifische Anreicherung von Gephyrin an inhibitorischen Synapsen in Neuronen.
On the molecular basis of novel anti-inflammatory compounds and functional leukocyte responses
(2006)
Inflammation is a complex pathophysiological event that can be triggered by activation of a number of distinct activation pathways eventually leading to the release of pro-inflammatory molecules and enzymes. Among all cells involved in inflammatory processes, neutrophils, monocytes and platelets are of major relevance. Activation of leukocytes occurs via binding of agonists to distinct GPCRs leading to activation of G proteins and proximate signaling cascades. In short, GPCR activation by pro-inflammatory agonists such as fMLP, PAF or LTB4 leads to activation of G proteins that are associated with the receptor at the cytosolic side of the plasma membrane. G proteins consist of a Gα- and a Gβγ-subunit which are associated in the inactive state. In this state, G proteins bind GDP. Upon activation, GDP is replaced by GTP that results in the dissociation of the Gα- from the Gβγ-subunit. Both subunits are capable of activating distinct PLC-β isoenzymes that catalyze the turnover of PtdIns(4,5)P2 into the second messengers Ins(1,4,5)P3 and DAG. Every GPCR holds a distinct pattern of associated G proteins which preferentially activate distinct PLC-β isoenzymes. Ca2+ channels within the SR/ER-membrane function as specific receptors for Ins(1,4,5)P3. Ligation of Ins(1,4,5)P3 to this receptor causes a release of Ca2+ from intracellular stores into the cytosol that is subsequently followed by the influx of Ca2+ e through channels in the plasma membrane. Ca2+ represents an important signaling molecule, involved in the regulation of cellular processes and enzymes that mediate inflammatory events such as ROS formation and the release of degradative enzymes. 5-LO and COXs are involved in the biosynthesis of pro-inflammatory eicosanoids and catalyze the turnover of AA into LTs and PGs, respectively. Both enzymes play pivotal roles in the initiation and maintenance of allergic diseases and inflammatory processes. LTB4 is regarded as a potent chemotactic and chemokinetic substance, whereas the cysteinyl-LTs cause smooth muscle contraction and increased vascular permeability. Therefore, 5-LO inhibitors are assumed to possess therapeutic potential for the treatment of diseases related to inflammation. Besides the intervention with 5-LO activity, inhibition of COX-activity is an effective way to suppress inflammatory reactions. The two COX isoenzymes, namely COX-1 and COX-2 show different patterns in terms of tissue expression and sensitivity towards inhibitors. COX-1 is supposed to be constantly expressed whereas COX-2 expression is upregulated at sites of inflammation. The extract of H. perforatum is commonly used for the treatment of mild to moderate depressive disorders, accompanied by a moderate profile of side effects. The extract´s efficacy as an antidepressant can be traced back to the content of the phloroglucinol hyperforin which represents the most abundant lipophilic constituent. However, in folk medicine hypericum extracts are additionally used for the treatment of inflammatory disorders such as rheumatoid arthritis or inflammatory skin diseases. In fact, it was shown that hypericum extracts and hyperforin possess anti-inflammatory potential. Hyperforin was described as a dual inhibitor of 5-LO and COX-1. The phloroglucinols MC and S-MC from M. communis significantly differ from the molecular structure of hyperforin. Hyperforin represents a monomeric prenylated derivative whereas MS and S-MC are non-prenylated oligomeric compounds. To date, the anti-inflammatory potential of SM and S-MC has not been investigated in detail. So far, solely antioxidant activity was attributed to MC and S-MC that indeed might qualify them as anti-inflammatory drugs. The phloroglucinols MC, S-MC and hyperforin are potent inhibitors of ROS formation and HLE release. However, any inhibitory potential of these compounds was only observed when cells were activated by GPCR agonists such as fMLP or PAF. In contrast, when cells were stimulated under circumvention of G protein-associated signaling cascades, the abovementioned inhibitors were not effective at all. In leukocytes, [Ca2+]i plays a pivotal role in signal transduction and regulation of the indicated pro-inflammatory cellular functions. We were able to show that MC, S-MC and hyperforin inhibited GPCR-mediated Ca2+ mobilization with approximately the same potency as the above-mentioned leukocyte responses. However, all of the indicated phloroglucinols were ineffective when cells were stimulated with ionomycin. Since ionomycin as well as GPCR agonists exert their effects by mobilizing Ca2+ i, it seems conceivable that MC, S-MC and hyperforin somehow interfere with G protein-associated signaling pathways. In order to investigate PLC as a potential target of hyperforin, the effects of hyperforin were compared to those of the broad spectrum PLC inhibitor U-73122. We found that both inhibitors acted in a comparable manner in terms of agonist-induced Ca2+ mobilization and in regard of the manipulation of basal Ca2+ levels in unstimulated cells. In this respect, significant differences between hyperforin and U-73122 were obvious for inhibition of total PLC activity in vitro. Thus, U-73122 blocked PLC activity whereas hyperforin was ineffective in this respect. This might indicate that only certain PLC isoenzymes are affected by hyperforin. Alternatively, other components within G protein-associated signaling pathways such as G proteins itself or the Ins(1,4,5)P3 receptor must be taken into account as putative targets of hyperforin. We were able to introduce MC and S-MC as novel dual inhibitors of 5-LO and COX-1. Interestingly, such a pattern was also described for hyperforin. MC and S-MC turned out to be direct inhibitors of 5-LO, based on the fact that they inhibit 5-LO not only in intact cells but also as purified enzyme in vitro. For MC and S-MC, great discrepancies were observed between the IC50 values concerning 5-LO inhibition and the concentrations that exert the antioxidative effects. It seems probable that 5-LO inhibition is not related to reduction of the active site iron as a result of the antioxidant activity of MC and S-MC but rather to direct interference with the 5-LO enzyme. The capability of MC and S-MC to suppress COX-1 activity seems not to be a unique effect of these phloroglucinols because for COX-1, the IBPC, present in both MC and S-MC, turned out to be the most active compound. ....
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.
21 Hsfs belonging to classes A, B and C were identified in Arabidopsis following the sequencing of its genome. 1.) Cloning of full length and CTD chimeric constructs followed by transient reporter assays in tobacco protoplast using GUS fusion constructs of the promoters of Hsp17.4-CI, synthetic (HSE9) and APX2 showed Hsfs A1a, A1b, A1d, A1e, A2, A3 and A9 to be active. CTDs of Hsfs A7a, A7b and HsfC1 had activity but they showed poor DNA binding in reporter assays. Hsfs A1a, A1b, A1d, A1e, A2 and A3 were able to induce the expression of endogenous Hsps in tomato protoplasts. Interesting differences in promoter selectivity were observed for several Hsfs. 2.) RT-PCR and microarray analysis showed the Hsfs to be differentially expressed depending on tissue, abiotic and biotic stress, hormone and developmental s ge. Interesting patterns of coexpressed Hsfs were observed under different stresses and developmental stages. 3.) HsfA1b was found to be active on the plasmid borne PHsf:GUS reporters of Hsfs A1d, A2, A4a, A7b and B4 when tested in tobacco mesophyll protoplasts. Hsfs A1d, A2, A4a, A7b and B4 when tested in tobacco mesophyll protplasts. HsfA2 was inactive on PHsfA:GUS. HsfB1 showed repression of endogenous activity on several PHsf:GUS reporter constructs. 4.) The transcriptional regulation under heat stress and promoter organization of HsfA2 and FtSH4 (a metalloprotease gene oriented in a head to head fashion with HsfA2 in the Arabidopsis genome, sharing a common promoter region) was studied. The transcripts of FtSH4 and HsfA2 coaccumulated under heat stress. HsfA1b was active on PHsfA2:GUS and PFtSH4:GUS. Hsf binding sites on the intergenic region were determined using promoter deletion constructs in tobacco and Arabidopsis protoplasts. A bidirectional regulation of HsfA2 and FtSH4 by HsfA1b was observed in tobacco protoplast. 5.) Microarray analysis of a HsfA2 T-DNA insertion line vs. wild type Col-0 under heat stress conditions led to identification of a subset of target genes to be severely affected in the absence of HsfA2. Apart from several Hsps (heat stressproteins) and APX2 (Ascorbate peroxidase 2, oxidative stress scavenger), several other unknown genes are affected. APX2 was the most severely affected among them. HsfA2 was able to induce the transcription from its target gene promoters in fusion to GUS in transient reporter assays in tobacco protoplast. The HSE cluster to which HsfA2 binds on the APX2 promoter was also mapped by the same technique. The direct binding of HsfA2 to the promoter of selected target genes in the Arabidopsis genome was also demonstrated by chromatin immunoprecipitation studies.
Cytochrome b561 (cyt b561) proteins are members of the recently identified eukaryotic ascorbate reducible protein family named CYBASC (CYtochrome B, ASCorbate reducible). CYBASC proteins are di-heme-b-containing membrane proteins that catalyze the transmembrane electron transfer from ascorbate. The function of the CYBASC proteins has been correlated with ascorbate recycling and/or iron facilitation uptake. Therefore, investigations on this family are of great interest as ascorbate is one of the most powerful antioxidants and iron is essential for cell survival both in animals and plants. As the amino acid sequence conservation of animal and plant CYBASC proteins is relatively high, all CYBASC members are proposed to share the same structural motifs. However, no three-dimensional structure of any representative member of the CYBASC family has been determined to date. In the Arabidopsis thaliana (A. thaliana) genome, two complete putative CYBASC open reading frames (ORFs), artb561-a and artb561-b were identified. In this thesis, these two A. thaliana CYBASC ORFs, encoding for Acytb561-A and Acytb561-B proteins respectively, were investigated and obtained main results are listed. 1. A. thaliana CYBASC proteins were heterologously produced in Pichia pastoris and Escherichia coli and purified by a single-step immobilized metal affinity chromatography (IMAC). To facilitate detection and purification, the recombinant A. thaliana CYBASC proteins were produced in both expression systems with the histidine affinity tag. Pure and stable preparations of the cytochromes were obtained via a single-step IMAC in sufficient amounts to perform biochemical characterizations. 2. Detergent solubilized recombinant Acytb561-A and Acytb561-B are dimers. As previously suggested for other CYBASC proteins, analytical gel filtration experiment suggested that both detergent solubilized cytochromes are dimers. 3. Spectroscopic features of Acytb561-B differed from those of previously described bovine chromaffin granule cyt b561. A distinctive feature of the first identified CYBASC protein, the cyt b561 from bovine chromaffin vesicles of adrenal medulla (Bcytb561-CG), is that its differential visible absorbance spectra (visible-spectra) revealed an asymmetric α-band with a maximum at 562 nm and a clear shoulder at 557 nm. This feature was recently used to discriminate CYBASC proteins from not-CYBASC proteins. However, in this thesis, it is shown for the first time that not all CYBASC proteins display in their reduced-minus-oxidized visible-spectra an asymmetric α- band and therefore, this feature can not be used as a discriminating CYBASC characteristic. 4. Ascorbate dependent reduction of the A. thaliana CYBASC proteins is inhibited by diethylpyrocarbonate (DEPC). As previously reported for the Bcytb561-CG, the ascorbatedependent reduction of the A. thaliana CYBASC proteins was inhibited by DEPC treatment. In addition, the ‘ascorbate protectant’ effect against DEPC that was observed on the Bcytb561-CG was also observed on the Acytb561-A and Acytb561-B proteins. Furthermore, as the physiological electron donor of all CYBASC proteins is supposed to be ascorbate, ascorbate-affinity of Acytb561- A and Acytb561-B was monitored and was found to be in the same range of the one of the Bcytb561- CG. 5. A. thaliana CYBASC proteins are Fe3+-chelate reductases. Recently, the Fe3+-chelate reductase activity of various CYBASC proteins was presented. In this thesis, it is shown that also both A. thaliana CYBASC proteins reduced Fe3+-chelates such as Fe3+-EDTA and Fe3+-citrate. Consistently, heme potentiometric reductive-oxidative titration of purified Acytb561-A and Acytb561-B indicated that the midpoint potential of the two heme centres of both cytochromes was lower than the one of those Fe3+-chelates. The values of both heme centre potentials of Acytb561-A and Acytb561-B are also consistent with the observation that both cytochromes were only partially reducible by ascorbate and were fully reduced with the non-physiological reductant Na-dithionite. In summary, this work describes the heterologous production, purification and initial characterizations of two distinct CYBASC proteins from A. thaliana: Acytb561-A and Acytb561-B. Biochemical characterization of these cytochromes showed that the shape of the α-band in the differential spectra is not a discriminating factor for CYBASC proteins but it is likely the DEPC sensitivity and the Fe3+-chelate reductase activity. Establishment of a purification strategy to obtain sufficient amounts of monodispersed and stable A. thaliana CYBASC proteins has also enabled initial screening of three dimensional crystallization conditions which are a prerequisite for a deeper understanding of this new eukaryotic redox enzyme family.
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.
Membrane proteins play vital role in a variety of cellular processes, such as signal transduction, transport and recognition. In turn they are involved in numerous human diseases and currently represent one of the most prevalent drug targets. A comprehensive understanding of the mechanisms mediated by membrane proteins requires information about their structures at near-atomic resolution, although structural studies of membrane proteins remain behind those of soluble proteins. A bottleneck in the study of membrane proteins resides in the difficulties that are encountered during their high-level production in cell based systems. However, many toxic effects attributed to the over production of membrane proteins are eliminated by cell-free expression, as viable host cells are no longer required. Therefore, the objective of this study was to obtain adequate amounts of selected membrane transport proteins for their structural studies using a cell-free expression system. For the establishment of the cell-free system for membrane proteins, the transporters YbgR and YiiP from Salmonella typhimurium LT2, PF0558 and PF1373 from Pyrococcus furiosus, from the cation diffusion family (CDF), BetP from Corynebacterium glutamicum from the betaine/carnitine/choline transporter (BCCT) family and Aq-2030 from Aquifex aeolicus VF5 from the monovalent cation/proton antiporter-2 (CPA2) family were selected. An Escherichia coli S-30 extract based cellfree system was established by generating the best expression constructs of the target proteins, preparing T7 RNA polymerase and an S-30 extract with high translation efficiency. The functionality of the S-30 extract was shown by the cell-free expression of correctly folded Green Fluorescent Protein (GFP). Essential factors of the cell-free system such as the Mg2+ concentration, the bacterial S-30 extract proportion in the reaction mixture and the time-course of cell-free reactions have been optimized. For the cell-free production of membrane proteins in soluble form, the possibility to supplement cell-free reactions with detergents was explored. A wide range of non-ionic or zwitterionic detergents, were found to be compatible with cell-free synthesis, while ionic detergents and non-ionic detergents at high concentrations had an inhibitory effect. Moreover, high concentrations of polyoxyethylene-alkyl-ethers (Brij) detergents were found to have enhancing effect on the production levels as well as on the solubility of cell-free produced proteins. As membrane proteins tend to misfold and aggregate in a membrane-free translation system, the possibility to supplement the cell-free reactions with inner membrane vesicles (IMVs) to obtain correctly folded target transport proteins was explored. All the target proteins were successfully produced in the batch cell-free reactions and were found to be incorporated in the IMVs. A continuous exchange cell-free (CECF) system was established, where consumable substrates (amino acids, nucleotides and energy regenerating compounds) were supplied to the cell-free reaction mixture through a dialysis membrane, which in consequence resulted in high-level production of target proteins compared to the batch system. The osmosensing and osmoregulated sodium-coupled symporter BetP from C. glutamicum was chosen for the large scale production in CECF set-up. The protein is easily produced in E. coli and is functional as assayed by its transport activity, after purification and reconstitution in liposomes. It is therefore possible to compare in-vivo and cell-free production. High-level cell-free production of BetP was achieved in CECF mode in different forms: (i) as precipitate, (ii) as soluble form in detergent, and (iii) incorporated in IMVs. Cell-free production of BetP resulted in the yield of about 0.5 mg of purified BetP from 1 ml of CECF reaction. The yield of purified BetP was increased to 1.6 fold by addition of 1% polyoxyethylene-(20)-cetyl-ether (Brij58) detergent in the reaction mixture. Moreover, the high level cell-free production of BetP (0.5 mg purified BetP/ml reaction mixture) incorporated in IMVs was shown for the first time in this work.However, it was observed that oligomerization of BetP was not efficient in the cell-free system. Factors that can promote the folding of membrane proteins such as lipids and chaperones were investigated. Addition of lipids and molecular chaperone GroE facilitated correct folding of BetP resulting in increased yield and stability of cell-free produced BetP. The results obtained indicate that most of the cell-free produced BetP exists in functional oligomeric form. The possibility of obtaining milligram amounts of BetP, a 12 trans-membrane protein from the cell-free reactions holds promise for structural and functional studies of other membrane proteins. In any case, the strategies adapted in this study should prove extremely valuable for the production of membrane proteins in the E. coli cell-free expression system.
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 ....
1. Halobacillus halophilus akkumuliert zum Ausgleich geringer, extrazellulärer Wasserpotentiale kompatible Solute. Bei Anzuchten in Gegenwart von 0,4 – 1,5 M NaCl wurden Glutamin und Glutamat als die dominierenden kompatiblen Solute identifiziert, während zwischen 2,0 und 3,0 M NaCl Prolin das dominierende Solut darstellt. Außerdem wurde Ectoin als zweites kompatibles Solut gefunden, das spezifisch bei hohen Salzgehalten akumuliert wird. Die Konzentrationen während der exponentiellen Wachstumsphase war jedoch um den Faktor 6 – 7 geringer im Vergleich zu Prolin. 2. Aus Wachstumsexperimenten in Gegenwart unterschiedlicher Anionen war bekannt, dass Glutamat, im Gegensatz zu Gluconat und Nitrat, in der Lage ist, das Wachstum von H. halophilus auch in Abwesenheit von Chlorid zu ermöglichen. Um der Frage nachzugehen, ob die wachstumsfördernde Wirkung von unphysiologisch hohen Glutamat-Konzentrationen im Medium auf die Verwendung von Glutamat als kompatiblem Solut in den Zellen zurückzuführen ist, wurden Gesamtsolutepools von Chlorid-, Nitrat-, Gluconat- und Glutamat-gezogenen Zellen gemessen. In NaCl-gezogenen Zellen zeigte sich Glutamat als dominantes Solut, während Prolin und Glutamin einen geringeren Teil am Gesamtpool ausmachten. In Nitrat-gezogenen Zellen betrug der Gesamtpool nur noch 83% und in Gluconat-gezogenen Zellen nur noch 27% im Vergleich zu Chlorid-gezogenen Zellen. Zellen, die mit Glutamat gezogen wurden, zeigten jedoch eine Gesamtkonzentration an Soluten, die ca. 100% über dem Vergleichswert aus Chlorid-gezogenen Zellen lag. Die Konzentration an Glutamin in den Zellen stieg dabei um 168%, die Konzentration an Glutamat sogar um 299%. Die Prolinkonzentration verringerte sich um 32%. Diese Daten belegen, dass der wachstumsstimulierende Effekt von Glutamat auf die Verwendung als kompatibles Solut zurückzuführen ist. 3. Zur Untersuchung der molekularen Grundlage der Salzadaptation sowie der Abhängigkeit von Chlorid in H. halophilus wurde in Zusammenarbeit mit der Gruppe von Prof. D. Oesterhelt (MPI für Biochemie, Martinsried) die Sequenzierung des Genoms begonnen. Das Projekt ist zur Zeit noch nicht abgeschlossen und befindet sich in der „Lückenschluß-Phase“. Die bisherigen Sequenzdaten konnten dennoch für die in dieser Arbeit beschriebenen Untersuchungen herangezogen werden. Das Genom besitzt eine Größe von ca. 4,1 Mbp mit einem ungefähren GC-Gehalt von 40%. Außerdem wurden 2 Plasmide identifiziert mit einer Größe von 16047 und 3329 bp. 4. Die Schlüsselgene bekannter Biosynthesewege für Glutamin und Glutamat konnten identifiziert werden. Darunter befinden sich zwei Isogene für eine Glutamatdehydrogenase (gdh1 und gdh2), ein Gen für die große Untereinheit einer Glutamatsynthase (gltA), zwei Gene für die kleine Untereinheit einer Glutamat-Synthase (gltB1 und gltB2) und zwei Isogene für eine Glutaminsynthetase (glnA1 und glnA2). glnA1 befindet sich in einem Cluster zusammen mit einem Gen, das für einen Regulator kodiert (glnR), wie er auch aus B. subtilis bekannt ist. Über reverse Transkription von mRNA und anschließender PCR-Analyse konnte gezeigt werden, dass sowohl gltA/gltB1 als auch glnA1/glnR in einem Operon organisiert sind. 5. Wurde die Transkriptmenge der in Punkt 4 erwähnten Biosynthesegene in Zellen quantifiziert, die in Gegenwart unterschiedlicher Salzkonzentrationen (0,4 – 3,0 M NaCl) gezogen wurden, so zeigte sich keine Abhängigkeit von der Salzkonzentration für die Gene gltA, glnA1 und gdh1. Über die Transkriptmengen von gdh2 ließ sich keine abschließende Aussage treffen, da die gefundenen Transkriptmengen sehr gering waren und daher zu sehr großen Varianzen bei der Quantifizierung führten. Eine klare Abhängigkeit der Transkriptmenge von der im Medium zugesetzten Salzkonzentration konnte für glnA2 gezeigt werden. Die glnA2 mRNA-Menge stieg dabei mit steigender Salzkonzentration an und erreichte bei 1,5 – 2.0 M NaCl ein Maximum. Bei diesen Salzkonzentrationen war die Menge an mRNA ca. 4 mal höher als der Vergleichswert bei 0,4 M NaCl. Bei höhern Salzkonzentrationen sank die Menge an Transkript wieder leicht und war dann ca. nur noch 3 mal so hoch wie bei 0,4 M NaCl. 6. Die zelluläre Konzentration der glnA2-Transkripte in Abhängigkeit unterschiedlicher Anionen im Anzuchtmedium wurde untersucht. Die Quantifizierung der glnA2–mRNA ergab eine 2 mal höhere Transkriptmenge in Gegenwart von Chlorid verglichen mit Nitrat oder Gluconat. 7. Es wurde nach Enzymaktivitäten der bekannten Schlüsselenzyme im Glutamat und Glutamin-Biosyntheseweg gesucht. Eine Glutamatdehydrogenase und eine Glutamatsynthase – Aktivität konnte nicht oder nur in vernachlässigbarem Maße nachgewiesen werden. Im Gegensatz dazu konnt eine Glutaminsynthetase – Aktivität eindeutig belegt werden. Diese Aktivität erwies sich abhängig von der Art und der Konzentration des angebotenen Anions im Medium. Maximale Aktivitäten wurden mit NaCl in einer Konzentration von 2,5 – 3,0 M erreicht. Interessanterweise erwies sich die Glutaminsynthetase – Aktivität auch abhängig von der Art des im Testpuffers verwendeten Anions. Hier zeigte sich eine deutliche Stimulierung der Aktivität durch das Anion Chlorid. [Die für diesen Punkt zugrunde liegenden Daten wurden im Rahmen einer von mir mitbetreuten Diplomarbeit von Jasmin F. Sydow erhoben und sind aus Gründen der vollständigen Darstellung des Projektverlaufes mitaufgeführt!] 8. Wie im Punkt 1 dargelegt, wird Prolin vor allem bei hohen Salzkonzentrationen in H. halophilus - Zellen akkumuliert. Neben der Abhängigkeit von der Salzkonzentration wurde außerdem die Abhängigkeit von der Wachstumsphase untersucht. Die Analyse der Prolinkonzentrationen während verschiedener Wachstumsphasen in Kulturen, die bei 1,0 bzw. 2,5 M NaCl angezogen wurden, zeigte, (i) dass die Prolinkonzentration während der frühen exponentiellen Phase ca. 2,5-fach erhöht war im Vergleich zu Niedrigsalz-Zellen, (ii) dass die Prolinkonzentration beim Übergang von der frühen in die späte exponentielle Phase dramatisch abnahm (um 64% bei 2,5 M NaCl) und dass (iii) in der stationären Phase Prolin praktisch nicht mehr nachzuweisen war. 9. Die Biosynthesegene für die Herstellung von Prolin aus Glutamat konnten im Genom von H. halophilus identifiziert werden. Es handelt sich dabei um ein Cluster von 3 Genen, die für eine putative Pyrrolin-5-carboxylatreductase (proH), eine Glutamat-5-kinase (proJ), und eine Glutamat-5-semialdehyd-dehydrogenase (proA) kodieren. Mittels reverser Transkription von mRNA und anschließenden PCR-Analysen konnte gezeigt werden, dass die drei Gene ein Operon bilden. 10. Eine Quantifizierung der Transkriptmengen der Biosynthesegene proH, proJ und proA mittels quantitativer PCR in Zellen, die bei unterschiedlichen NaCl-Konzentrationen gezogen wurden, zeigte einen deutlichen Zusammenhang zwischen der Salinität des Mediums und der Menge an Transkript. Diese war umso höher, je höher die Salinität des Mediums war. Die maximale Transkriptmenge (6-fach) wurde bei einer Salzkonzentration von 2,5 M NaCl erreicht. Bei noch höherer Salzkonzentration sank die Transkriptmenge auf die ca. 5-fache Menge des Kontrollwertes ab. 11. Um die Regulation und Dynamik der Osmoregulation unabhängig vom Wachstum untersuchen zu können, wurde ein Zellsuspensions-System für H. halophilus etabliert, bei dem eine konzentrierte Zellsuspension direkt von geringen auf hohe Salzkonzentrationen überführt wurde und bei dem die Prozesse der Transkription, Translation und Solut-Biosynthese erhalten blieben. Beispielhaft wurde dieses System an der Produktion von Prolin nach einem Salzschock von 0,8 auf 2,0 M NaCl getestet. Es zeigte sich bei der Analyse, dass sich die Transkriptmengen unmittelbar nach dem Salzschock deutlich erhöhten und bereits nach 1,5 Stunden ein Maximum erreicht wurde. Verglichen mit dem Wert zu Beginn des Versuches waren die Transkriptmengen ca. 13-fach erhöht, sanken im weiteren Verlauf jedoch wieder ab und blieben bei einer 4-fachen Transkriptmenge konstant. Mit der Erhöhung der Transkriptmenge ging auch eine Erhöhung der Prolinkonzentration einher, die ein Maximum von ca. 6 μmol/mg Protein nach 6 Stunden erreichte. Auch diese Konzentration verringerte sich im weiteren Verlauf wieder und erreichte nach 20 Stunden den Ausgangswert. 12. Um den Einfluß diverser Anionen bzw. Osmolyte im Medium auf die Produktion von Prolin zu untersuchen, wurden Zellsuspensionen von H. halophilus einer Erhöhung der Osmolarität von 0,8 M auf 2,0 M unterzogen. Es zeigte sich dabei, dass die maximale Akkumulation von Prolin in Anwesenheit von Chlorid am höchsten war. Nitrat und Glutamat führten zu ähnlichen, aber leicht geringeren maximalen Konzentrationen (92 bzw. 83% des Chloridwertes). Gluconat führte noch zu einer Akkumulation von ca. 51%, während die anderen Osmolyte zu keiner Akkumulation führten. Eine Analyse der Transkriptmengen zeigte jedoch ein völlig anderes Bild. Während Chlorid, Nitrat und Gluconat zu vergleichbaren Anstiegen der Transkripmengen führten, war die maximale Transkriptmenge der Glutamatinkubierten Zellen 3-9 mal höher als in Vergleichszellen mit Chlorid. In anschließenden Titrationsexperimenten mit verschiedenen Glutamatkonzentrationen konnte gezeigt werden, dass eine minimale Konzentration von 0,2 M Glutamat ausreichend ist, um eine 90-fache Steigerung der Transkriptmenge herbeizuführen. 13. Als Antwort auf Hochsalz-Bedingungen akkumuliert H. halophilus neben Prolin auch Ectoin. Die Ectoinkonzentration bei 2,5 M NaCl war ca. 2-3 mal höher als in Zellen, die bei 1,0 M gezogen wurden. Die Bestimmung der intrazellulären Ectoin-Konzentrationen während des Wachstums zeigte außerdem, dass die Produktion von Ectoin wachstumsphasenabhängig ist. Die Konzentration in der stationären Phase war ca. 5-fach höher als in der exponentiellen Phase. Die Entwicklung der Ectoin- Konzentration verhielt sich somit reziprok zur Entwicklung der Prolin-Konzentration während des Wachstums. 14. Es wurde ein Cluster von drei Genen im Genom von H. halophilus identifiziert, deren Genprodukte die Biosynthese von Ectoin aus Aspartatsemialdehyd katalysieren. ectA kodiert dabei für eine putative Diaminobutyrat-Acetyltransferase, ectB für eine putative Diaminobutyrat-2-oxoglutarat-Transaminase und ectC für eine putative Ectoin-Synthase. Mittels reverser Transkription von mRNA und anschließenden PCR-Analysen konnte gezeigt werden, dass die drei Gene ein Operon bilden. 15. Die Transkription der ect-Gene war abhängig von der Salinität des Mediums. Ab 2,0 M stieg die Menge an RNA um das 10-fache an und erreichte bei 3,0 M ein Maximum mit der 23,5-fachen Menge. 16. Nach einem osmotischen Schock stieg die Konzentration an ect-mRNA signifikant und erreichte ein Maximum nach 3 - 4 Stunden. Das Maximum wurde somit 1,5 – 2,5 Stunden später erreicht als bei anderen Genen der Solute-Biosynthese wie etwa gdh1, das für eine Glutamatdehydrogenase, glnA2, das für eine Glutamin-Synthetase oder proH, das für eine Pyrrolin-5-Carboxylase kodiert. Die maximal erreichten Wert lagen 13-fach (ectA), 6,5-fach (ectB) und 3-fach (ectC) über dem Wert vor dem Salzschock. Gegen EctC wurden polyklonale Antikörper generiert. Western-Blot Analysen mit diesem Antikörper zeigten, dass die EctC-Menge nach 4 Stunden um das 2,5-fache stieg, dann aber wieder abfiel auf das 1,6 – 1,7-fache des Ausgangswertes. Der Rückgang an EctC fand keine Entsprechung in der gemessenen Ectoin-Konzentration, welche über einen Zeitraum von 18 Stunden kontinuierlich anstieg. Die maximale Konzentration nach 18 Stunden betrug das ca. 6,3-fache des Ausgangswertes. 17. Wurden H. halophilus Zellen mit anderen Osmolyten außer NaCl geschockt, so ergab sich folgendes Bild der Regulation der Ectoin-Biosynthese: (i) die Transkription der ect-Gene zeigte keine Chlorid-abhängige Regulation. Die maximale Transkriptmenge wurde in Gegenwart von Nitrat erreicht, wohingegen Gluconat zu vergleichbachen mRNA-Mengen führte wie Chlorid. Glutamat führte nur zu schwacher Stimulierung der Transkription. (ii) auf Ebene der Proteinmenge war zu sehen, dass die Menge an EctC nach osmotischem Schock vergleichbar war in Zellen, die mit Chlorid oder Nitrat inkubiert wurden. Gluconat führte nur zu einer 40%-igen Zunahme während andere Osmolyte nahezu wirkungslos auf die Menge an EctC blieben. (iii) die höchste Akkumulation an Ectoin nach einer plötzlichen Erhöhung der Osmolarität wurde erreicht mit Chlorid (6-fache Zunahme) gefolgt von Nitrat (5,6-fache Zunahme). Gluconat führte lediglich zu einer 3,3-fachen und Glutamat nur noch zu einer 2-fachen Steigerung der Ectoinkonzentration. Glutamat hat somit ähnliche Effekte wie Tartrat, Saccharose oder Sulfat. Succinat führte zu keiner Akkumulation und Glycin sogar zu einer deutlichen Abnahme. Die Produktion von Ectoin ist somit hauptsächlich abhängig vom Anion/Osmolyt und nur untergeordnet von der Osmolarität.
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.
Shaped by some of the most dramatic tectonic events of the Cenozoic, the parts of southern and eastern Asia that have become known as the Oriental faunal region comprise vast areas of great geological complexity and ecological diversity. One of the four major groups of terrestrial elapid snakes in this region is the genus Bungarus. These nocturnal and predominantly ophiophagous snakes are widely known as kraits and are an important cause of snakebite mortality throughout their wide range that extends from Afghanistan to Vietnam and eastern China, and south to the Indonesian islands of Java and Bali. Although present on Borneo, kraits have not been found on any island of the Philippines, nor on Lesser Sunda Islands east of Bali. Despite their medical significance and the great importance of Bungarus toxins as tools in neuropharmacology, krait systematics and taxonomy have remained largely unstudied. Twelve species of Bungarus were recognized at the beginning of the present study. Many of these are rare in collections, and most aspects of their biology are unknown. While some species are highly distinct, most kraits are conservative morphologically, rendering molecular methods invaluable for the study of their diversity and biogeography. This study is the first to address the relationships within Bungarus and the historical biogeography of kraits based on molecular evidence. I inferred phylogeographic relationships based on analyses of new nucleotide sequences of the entire mitochondrial cytochrome b gene of 51 kraits and partial NADH dehydrogenase subunit 4 sequences of 40 kraits which I analyzed together with a representative sample of 32 published elapid and non-elapid outgroup taxa using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods. I then used the recovered phylogeny to investigate the evolution of selected morphological characters and, together with collections-based geographical distribution information, in dispersal-vicariance analyses with models of variable taxonomic and biogeographic complexity. The phylogenetic analyses demonstrate that the current taxonomy of kraits does not adequately represent either the relationships or the genetic diversity in this genus. In contrast, I identified monophyletic groups that are congruent with recognized biogeographic units as well as extensive ecomorph evolution and morphologically cryptic speciation. The following additional conclusions are collectively supported by the mitochondrial phylogeny and morphological as well as biochemical synapomorphies: (1) Kraits are monophyletic with respect to the remaining taxa of the Elapidae; (2) Bungarus flaviceps and Bungarus bungaroides form the monophyletic sister clade of a clade formed by B. fasciatus, black-and-white-banded, and uniformly black taxa; (3) the remaining taxa are divisible into two sister clades, the South Asian species (Bungarus sindanus (Bungarus caeruleus, Bungarus ceylonicus)) vs. Himalayan, Burmese, Southeast and East Asian taxa; (4) within the latter, Burmese taxa form the sister clade to Southeast and East Asian taxa; (5) the widespread and medically significant species Bungarus candidus and Bungarus multicinctus are paraphyletic. The results of this study highlight the importance of vicariant geological events and sea level fluctuations for the cladogenesis of kraits. Events of particular importance in the evolution of kraits include the uplift of the Indo-Burman ranges (Arakan-Naga Hills) which separated black-and-white banded kraits in India and Southeast Asia, and the uplift of mountain ranges in Yunnan, China (e.g., the Gaoligong Shan), which coincided with lineage separation in two distantly related clades of kraits. Alternating dispersal and vicariance events due to Pleistocene climatic and sea level changes have caused complex phylogeographic patterns in kraits in Southeast Asia. Zones of contact between closely related evolutionary lineages of the B. candidus complex are identified in Thailand, Vietnam, and southern China (Hainan). Within this complex, two main clades are revealed. One includes populations from the Southeast Asian mainland and is in contact with B. multicinctus in southern China. The other consists of populations from Thailand, southern Vietnam, Java, and Bali. The phylogeny as well as genetic distances suggest a scenario in which a Pleistocene southward dispersal of B. candidus to Sumatra, Java, and Bali during times of low sea levels was temporarily interrupted by vicariant events (rising sea levels, especially flooding of the Malacca Strait between Sumatra and the Malay Peninsula, and of the Bali Strait between Java and Bali). In this context, the close phylogenetic relationship between haplotypes from southern Vietnam and those from Java and Bali suggests that "southern" B. candidus dispersed directly via colonization of the widely receded South Chinese Sea, and not by taking a detour via the Malay Peninsula and Thailand, which were already inhabited by other populations of B. candidus. Using these phylogenetic estimates as the framework for a study on the diversity and evolution of krait venom components, I applied biochemical and molecular genetic approaches to identify and quantify polypeptide and protein toxins in krait venom, focusing on the distribution and molecular evolution of alpha-bungarotoxin, an irreversible competitive antagonist of nicotinic acetylcholine receptors with an exceptionally high applied significance as a receptor probe. I was specifically interested in the medically relevant question of intraspecific and interspecific variability in toxin diversity, and whether receptor-binding postsynaptic toxins evolve at rates different from those of presynaptic neurotoxins like beta-bungarotoxin, which act by destroying the nerve terminal and are believed to exhibit hypervariable functional diversification due to an accelerated mode of molecular evolution. In the context of this question, I isolated and purified the major lethal neurotoxins from B. candidus venoms by sequential steps of liquid chromatography for structural and functional characterization studies. Cloning and sequence analysis of toxin-coding genomic DNAs showed that the gene encoding the alpha-bungarotoxin alanine-31 variant, originally isolated from B. multicinctus venom, is widely present and highly conserved in multiple populations of B. candidus and is expressed as the principal postsynaptic neurotoxin at least in Javan B. candidus. In addition to the widespread presence of genomic DNAs encoding the alpha-bungarotoxin alanine-31 variant, the present study also revealed the partial genes of three novel alpha-bungarotoxin isoforms in addition to the previously known alanine-31 and valine-31 variants, all of which share an invariant exon 3 coding region. While alpha-bungarotoxin is the principal postsynaptic neurotoxin of Taiwanese B. multicinctus and Javan B. candidus, the main postsynaptic neurotoxin of Thai B. candidus both by quantity and lethality was a novel polypeptide of similar toxicity with a mass of 8030 Da and 73 amino acid residues, whose characterization at the genetic and protein levels revealed a novel subgroup of krait neurotoxins, here named alpha-delta-bungarotoxins and represented by four sequences from Bungarus caeruleus and B. candidus. alpha-delta-Bungarotoxins share high sequence homology with alpha-bungarotoxins but the purified, 8030 Da alpha-delta-bungarotoxin-1 exhibits only reversible, low affinity binding to nicotinic receptors and high site-selectivity for the acetylcholine binding site at the alpha-delta-subunit interface of the receptor. These properties render alpha-delta-bungarotoxin not only the first snake long-chain neurotoxin with reversible binding and binding-site selectivity, but also an exciting natural tool with which to address structure-function relationships at the subunit interfaces of the human receptor. The results of comparisons of the number of non-synonymous nucleotide substitutions per nonsynonymous site (dN) to the number of synonymous nucleotide substitutions per synonymous site (dS) strongly suggest that positive selection is acting on exon 2 of the alpha-bungarotoxin and probably also of the alpha-delta-bungarotoxin genes. In addition, the numbers of nucleotide substitutions per site of intron (dI) compared to the dS value of the toxin-coding exon regions provide strong evidence for accelerated molecular evolution in exon 2 of alpha-delta-bungarotoxins —whose value of dI is only one-eighth of the value of dS—whereas the hypothesis of accelerated evolution is rejected for 13 unique genomic DNAs encoding five alpha-bungarotoxin isoforms from B. candidus and B. multicinctus....
Genetic analysis of salt adaptation in Methanosarcina mazei Gö1 : the role of abl, ota and otb genes
(2008)
1. M. mazei ist ein halotolerantes methanogenes Archäon und akkumuliert kompatible Solute als längerfristige Anpassung an erhöhte Osmolarität in der Umgebung. Bei intermediären Salzkonzentrationen (~ 400 mM NaCl) wird vorzugsweise α-Glutamat gebildet und bei höheren Salzkonzentrationen (~ 800 mM NaCl) wird Nε-Acetyl-ß-Lysin zusätzlich zu Alpha-Glutamat synthetisiert. 2. Eine Analyse der intrazellulären Solutezusammensetzung mittels NMR ergab, dass M. mazei Glycin-Betain als Osmolyt akkumulieren kann. Für die Aufnahme von Glycin-Betain konnten zwei putative Glycin-Betain-Transporter in M. mazei identifiziert werden, Ota und Otb. Ota steht für „osmoprotectant transporter A“ und Otb für „osmoprotectant transporter B“. Das Genom von M. mazei wurde, nachdem es vollstänidg sequenziert war, nach Genen durchsucht, die eine Rolle bei der Aufnhame von Glycin-Betain oder anderen kompabtiblen Solute spielen könnten. Dafür wurde die Sequenz eines Substratbindeproteins eines bekannten bakteriellen Glycin-Betain-Transporters, opuAC aus B. subtillis als Referenzsequenz verwendet. Hierbei konnte ein Homolog, otaC, in M. mazei identifiziert werden. otaC ist Teil eines Genclusters, welches für einen ABC-Transporter kodiert. otb wurde bei einer genomweiten Expressionsanalyse zur Salzadaptation von M. mazei identifiziert. Es wurden Gene eines putativen ABC-Transporters identifiziert, die unter Hochsalzbedingungen leicht induziert waren. Es stellte sich heraus, dass es sich hierbei um einen zweiten putativen Glycin-Betain-Transporter handelte. Otb gehört auch zur Familie der ABC-Transporter. Vergleichsanalysen zeigten, dass die beiden Transporter keine große Ähnlichkeit zueinander aufweisen. Die Funktion und Rolle der beiden ABC-Transporter, vor allem von Otb, war zu Beginn dieser Arbeit unklar. 3. Bei Analysen des intrazellulären Solutepools im Wildtyp von M. mazei stellte sich heraus, dass in Anwesenheit von Glycin-Betain die Konzentration von Glutamat und NE- Acetyl-ß-Lysin verringert war. Bei 400 mM NaCl reduzierte Glycin-Betain die Glutamat- Konzentration um 16% und bei 800 mM NaCl um 29%. Besonders deutlich zeigte sich der Einfluß von Glycin-Betain bei der Akkumulation von NE-Acetyl-ß-Lysin. Bei 400 mM NaCl reduzierte Glycin-Betain die Konzentration an NE-Acetyl-ß-Lysin um 60% und bei 800 mM NaCl um 50%. Der Einfluß von Glycin-Betain konnte auf verschiedenen Ebenen in M. mazei beobachten werden. Es konnte gezeigt werden, dass die relative Transkriptimenge von ota unter Hochsalzbedingungen zunimmt. Glycin-Betain reduzierte die Transkription von ota bei verschiedenen Salzkonzentrationen. Die relative Transkriptmenge an mRNA von ota wurde mittels quantitativer real-time PCR (qRT-PCR) quantifiziert und war bis zu 52% reduziert in Zellen, die in Gegenwart von Glycin-Betain gewachsen waren. Die Transkriptmenge von otb war unter den gleichen Bedingungen nicht beeinflusst und zeigte generell keine Zunahme mit der Salinität des Mediums. Des Weiteren konnte ein Effekt von Glycin-Betain auf Ebene der Transportaktivität von Ota gezeigt werden. Hier zeigte sich, dass Zellen, die bei 400 mM NaCl in Gegenwart von Glycin-Betain gezogen waren, eine geringere Transportaktivität aufweisen, als Zellen, die bei 400 mM NaCl ohne Glycin-Betain gewachsen waren. Die Transportaktivität war um 90% geringer. Es muss jedoch berücksichtigt werden, dass es sich bei den Zellen, die ohne Glycin-Betain gewachsen waren, um eine Nettoaufnahme von Glycin-Betain handelte. Im Gegensatz dazu, ist davon auszugehen, dass Zellen, die in Gegenwart von Glycin-Betain gewachsen waren, eine Austaschreaktion zwischen bereits vorhandenem intrazellulärem und extrazellulär angebotenem Glycin-Betain vornehmen. [Die dem letzten Punkt zugrundeliegenden Daten wurden von Silke Schmidt im Rahmen einer Diplomarbeit erhoben, die von mir mitbetreut wurde. Aus Gründen der vollständigen Darstellung des Projektverlaufes werden diese Daten mitaufgeführt.] 4. Zur weiteren Klärung der Rolle und Funktion der beiden putativen Glycin-Betain- Transporter Ota und Otb war es Ziel, Mutantenstudien durchzuführen. Eine Vorraussetzung für die Generierung von Mutanten ist, dass der Organismus auf Agarplatten wächst und Einzelkolonien von einer einzelnen Zelle ausgehend bildet. Dies ist ein wichtiger Punkt bei Methanosarcina spp., die Zellpakete, sogenannte Sarcinen bilden. Deshalb wurde zunächst nach den optimalsten Plattierungsbedingungen gesucht, unter denen M. mazei keine Sarcinen bildet und die Plattierungseffizienz am höchsten war. Die Plattierungseffizienz betrug im Durchschnitt 54%. Für das Einbringen von DNA in die Zellen wurde eine Liposomen-vermittelte Transformation getestet. Ein ähnliches Vorgehen war bereits für Methanosarcina acetivorans beschrieben, konnte bislang aber noch nicht erfolgreich für M. mazei Gö1 und andere Stämme von M. mazei angwendet werden. Erste Schritte zur Anpassung des Transformations-Protokolles beinhalteten das Testen von DOTAP verschiedener Hersteller, sowie die Konzentration an eingesetzter DNA. Das jeweilige Zielgen/Zieloperon, welches deletiert werden sollte, wurde durch eine pac-Kassette ersetzt. Diese kodiert für eine Puromycin-Transacetylase und verleiht dem Organismus Puromycin- Resistenz. Die pac-Kassette wurde von umgebenden Bereichen des Ziellocus flankiert und integrierte mit Hilfe dieser flankierenden Bereiche über doppelt-homologe Rekombination in das Genom. 5. Mit dem oben beschriebenen Verfahren wurden ota::pac- und otb::pac-Mutanten erzeugt und über Southern-Blot Analyse verifiziert. Eine erste Charakterisierung der Mutanten mittels qRT-PCR zeigte, dass auf mRNA-Ebene keine Transkripte von ota in M. mazei ota::pac oder otb in M. mazei otb::pac nachweisbar waren. Zusätzlich konnte auf Proteinebene das Substratbindeprotein OtaC in M. mazei ota::pac und OtbC in M. mazei otb::pac nicht über einen Antikörper gegen das jeweilige Substratbindeprotein nachgewiesen, was die erfolgreiche Deletion bestätigte. Erste phänotypische Charakterisierungen zeigten, dass das Wachstum von M. mazei ota::pac und M. mazei otb::pac unter Hochsalzbedingungen nicht beeinträchtigt und vergleichbar mit dem des Wildtyps war. Auch bei kälteren Wachstumstemperaturen von 22°C wuchsen die Mutanten ohne Phänotyp. 6. Radioaktive Transportstudien mit M. mazei otb::pac zeigten, dass diese Mutante, die noch ein funktionelles Ota besitzt, [14C]Glycin-Betain aufnehmen kann. Es stellte sich heraus, dass diese Mutante eine höhere Transportrate für Glycin-Betain aufwies, als der Wildtyp. Die Aufnahmerate war um einen Faktor 2 höher als beim Wildtyp. Zusätzlich konnten qRT-PCR Analysen zeigen, dass die relative Transkriptmenge an ota in der otb::pac-Mutante um einen Faktor 2 höher war, als im Wildtyp. Umgekehrt konnte dieser Effekt nicht beobachtet werden, d.h. eine erhöhte Transkriptmenge an otb in M. mazei ota::pac. Auf Proteinebene konnte beobachtet werden, dass die intrazelluläre Konzentration an OtaC in der Mutatne leicht höher war als im Wildtyp. Jedoch stellte sich heraus, dass die intrazelluläre Glycin-Betain-Konzentration bei 400 mM NaCl in der Mutante nicht erhöht war verglichen mit Wildtyp, sondern die Konzentrationen gleich waren. Bei höheren Salzkonzentrationen (800 mM NaCl) zeigte sich jedoch ein anderes Bild: die intrazelluläre Glycin-Betain-Konzentration war in der Mutante um 60% erhöht. Dies könnte auf die erhöhte Transportaktivität von M. mazei otb::pac zurückzuführen sein. Die Konzentration anderer kompatibler Solute wie Glutamat und NE-Acetyl-ß-Lysin waren in diesen Zellen bis zu 48% reduziert. In vorherigen Studien konnte gezeigt werden, dass heterolog überproduziertes Ota von M. mazei in E. coli MKH13, eine E. coli-Mutante, die keine Glycin-Betain-Transporter mehr besitzt, die Aufnahme von Glycin-Betain wieder herstellen konnte [die Daten von ota in E. coli MKH13 wurden in der bereits oben erwähnten Diplomarbeit von Silke Schmidt erhoben]. Zur Klärung der Funktion von Otb wurde der gleiche Versuch mit otb in E. coli MKH13 durchgeführt. Jedoch konnte eine heterologe Produktion von Otb aus M. mazei die Aufnahme von Glycin-Betain in E. coli MKH13 nicht wieder herstellen. Hierbei wurde über Western-Blot Analyse sichergestellt, dass Otb tatsächlich in der Membran vorhanden war. Auch Transportstudien mit der Mutante M. mazei ota::pac zeigten, dass diese Mutante kein [14C]Glycin-Betain mehr aufnehmen konnte. Es konnte auch keine Akkumulation von Glycin-Betain mittels NMR in dieser Mutante gemessen werden. Des Weiteren zeigte sich, dass die intrazellulären Konzentrationen an Glutamat und Nε-Acetyl-ß-Lysin bei 400 mM und 800 mM NaCl in der Mutante unbeeinflusst von der Glycin-Betain-Konzentration im Medium waren. Weitere Transportstudien mit M. mazei ota::pac zur Aufnahme von [14C]Cholin zeigten, dass dieses Molekül weder vom Wildtyp, noch von der Mutante aufgenommen wurde. Dieses Ergebnis wurde durch Messung des Solutepools mittels NMR bestätigt. Somit kann ausgeschlossen werden, dass Otb unter den gemessenen Bedingungen weder ein Glycin- Betain-Transporter noch ein Cholin-Transporter in M. mazei ist. Diese Beobachtungen belegen eindeutig, dass Ota der einzige funktionelle Glycin-Betain-Transporter in M. mazei ist, während die Rolle von Otb bislang noch ungeklärt ist. 7. Nε-Acetyl-ß-Lysin, das dominante kompatible Solut in M. mazei bei 800 mM NaCl, wird durch die Enzyme AblA, einer Lysin-2,3-Aminomutase und AblB, einer ß-Lysin- Acetyltransferase synthetisiert. In dieser Arbeit wurde eine Δabl::pac-Mutante generiert, um die Fragen zu klären, ob die beiden Enzyme vom postulierten abl-Operon kodiert werden und wenn ja, welchen Phänotyp eine Nε-Acetyl-ß-Lysin-freier-Mutante bei Salzstress zeigt. NMR-Analysen zeigten, dass in der abl::pac-Mutante kein Nε-Acetyl-ß-Lysin mehr nachweisbar war. Dies belegt, dass die Gene ablA und ablB und deren Genprodukte für die Synthese von NE-Acetyl-ß-Lysin in M. mazei essentiell sind. Unter Hochsalzbedingungen ist das Wachstum von M. mazei abl::pac im Vergleich zum Wildtyp deutlich verlangsamt. Dieses Ergebnis war unerwartet, da eine abl::pac-Mutante von Methanococcus maripaludis unter Hochsalzbedingungen nicht mehr wachsen konnte. Unter Niedrigsalz und bei intermediären Salzkonzentration war das Wachstum von M. mazei abl::pac nicht eingeschränkt und verhielt sich wie der Wildtyp. In Gegenwart von Glycin-Betain akkumulierte die abl::pac-Mutante von M. mazei unter Hochsalzbedingungen 2,4 mal mehr Glycin-Betain als der Wildtyp, um das Defizit im Solutepool auszugleichen und Wachstum bei Hochsalz zu ermöglichen. Dadurch war sie in der Lage, wieder wie der Wildtyp zu wachsen. 8. Der Verlust von NE-Acetyl-ß-Lysin wurde unter Hochsalzbedingungen durch erhöhte Konzentrationen an Glutamat und einem neuen kompatiblen Solut kompensiert. NMRAnalysen zeigten, dass es sich hierbei um Alanin handelte. Bis jetzt wurde die Verwendung von Alanin als kompatibles Solut noch nie beschrieben. Um sicherzustellen, dass Alanin als kompatibles Solut in M. mazei abl::pac dient, wurde die Konzentration bei verschiedenen Salzkonzentrationen gemessen. Die Konzentration an Alanin nahm mit steigender Salzkonzentration zu. Bei 800 mM NaCl war die Konzentration 12 fach erhöht verglichen mit der Konzentration bei 400 mM NaCl. Außerdem redzierte Glycin-Betain die Alanin- Konzentration bei 800 mM NaCl um 58%. Transportexperimente zeigten, dass M. mazei kein Alanin aus dem Medium aufnehmen kann. 9. Erste Analysen möglicher Synthesewege für Alanin zeigten, dass die Alanin- Dehydrogenase nicht auf Transkriptebene unter Hochsalzbedingungen induziert war und somit keine Rolle in der Synthese von Alanin als kompatibles Solut spielen dürfte. Es könnten jedoch Aminotransferasen eine Rolle bei der Biosynthese von Alanin spielen. Des Weiteren sind die Enzyme, die für die Synthese von Glutamat als kompatibles Solut verantwortlich sind, unbekannt. Dies gilt für alle bis jetzt untersuchten Organismen, die Glutamat als kompatibles Solut nutzen. In dieser Arbeit wurde versucht, mit Hilfe der abl::pac-Mutante, die erhöhte Glutamat-Mengen zum Osmoschutz produziert, der Frage nachzugehen, welche Gene/Enzyme eine Rolle spielen könnten bei der Synthese von Glutamat als kompatibles Solut. Dazu wurden unter Hochsalzbedingungen die Transkriptmengen verschiedener Genen, die an der Glutamat-Synthese beteiligt sein könnten, in der Mutante und im Wildtyp untersucht. Hierbei zeigte sich, dass mehrere Gene verschiedener Enzyme unter Hochsalzbedingungen in der Mutante leicht induziert waren. Eines dieser Enzyme ist die Glutaminsynthetase. Dieses Enzym ist für die Umsetzung von Glutamat zu Glutamin unter Verbrauch von ATP verantwortlich. M. mazei besitzt zwei Gene, die für eine putative Gluaminsynthetase kodieren. In M. mazei abl::pac ist unter Hochsalzbedingungen das Gen glnA2 im Vergleich zum Wildtyp (4,03 ± 1,14) leicht induziert (7,63 ± 2,2). Des weiteren konnte in der Mutante eine leichte Induktion von gltB1, gltB2 und gltB3 unter Hochsalz beobachtet werden. Diese Gene kodieren für die einzelnen Domänen einer Glutamatsynthase. Diese ersten Analysen geben einen Hinweis darauf, dass die Synthese von Glutamat als kompatibles Solut über eine gekoppelte Reaktion der Glutaminsynthetase und der Glutamatsynthase verlaufen könnte.
5-LO is the key enzyme in the biosynthesis of proinflammatory leukotrienes. It catalyses the conversion of arachidonic acid to the hydroperoxy intermediate 5(S)-hydroperoxy-6- trans-8,11,14-cis-eicosatetraenoic acid (5-HpETE). In a second step 5-LO catalyses a dehydration reaction forming the unstable epoxide intermediate 5(S)-trans-5,6-oxido-7,9- trans-11,14-cis-eicosatetraenoic acid (leukotriene A4 , LTA4). The 5-LO gene is subjected to versatile regulation mechanisms. Apart from regulation by DNA-methylation and histone acetylation / deacetylation 5-LO gene expression can be regulated by the differentiation inducers calcitriol (1,25-dihydroxyvitamin D3) and transforming growth factor beta (TGFβ) 5-LO gene expression. In the myeloid cell lines Mono Mac 6 (MM6) and HL-60, differentiation with both agents caused a prominent upregulation of 5-LO mRNA level, of 5-LO protein expression and of 5-LO activity. Treatment with calcitriol alone already has an impact on 5-LO gene expression which is additionally potentiated by TGFβ treatment. Previous nuclear run-off analysis and reporter gene analysis could not associate the 5-LO promoter with the induction of 5-LO mRNA expression mediated by calcitriol and TGFβ. Inclusion of the 5-LO coding sequence (cds) and inclusion of the 5-LO cds plus the last four introns of the gene (J to M) in the 5-LO promoter construct pN10 led to an enhanced reporter gene activity. The inductions were dependent on vitamin D receptor (VDR) and retinoid x receptor (RXR) cotransfection. Therefore the work was concentrated on identifying elements outside the 5-LO promoter region which contribute to the calcitriol / TGFβ effect on 5-LO mRNA expression. Insertion of the LTA4 hydrolase coding sequence – a coding sequence of similar size - instead of the 5-LO cds led to a loss of the calcitriol / TGFβ effect (pN10LTA4Hcds 1-fold induction). Therewith, it was proven that the presence of the 5-LO cds is crucial for the upregulating effect of calcitriol / TGFβ on 5-LO mRNA level. Cloning of the SV40 promoter instead of pN10 upstream of the 5-LO cds still showed inducibility by treatment with the inducers which argues for a promoter unspecific effect. Insertion of the 5-LO cds in a promoterless basic vector (pGL3cds) displayed same inductions by calcitriol / TGFβ treatment as the 5-LO promoter 5-LO cds construct (pN10cds). Thus, the effect of the inducers is not dependent on the 5-LO promoter under the in vitro conditions of the reporter gene assay. Hence, further cloning was done with promoterless constructs. Through 5-LO cds deletion constructs a positive regulating region in exon 10 to 14 was discovered. To adapt the natural gene context the last four introns (J-M) of the 5-LO gene were inserted in a promoterless construct containing exon 10 to 14 (pGL3cdsΔABInJM). 5end deletion constructs of it revealed putative vitamin D responsive elements (VDREs) in exon 12 and intron M. Mutation of the putative VDREs led to a reduced calcitriol effect –more prominent when the putative VDRE in intron M was mutated (reduction of 40%). Moreover another putative VDRE in exon 10 with an adjacent SMAD binding element (SBE) was detected. SMAD proteins are effector proteins of TGFβ signalling. Gelshift experiments demonstrated in vitro binding of the VDR-RXR heterodimer to those three putative VDREs. By chromatin immunoprecipitation (ChIP) assay in vivo binding of VDR and RXR was shown to the VDRE in the region of exon 10, exon 12 and intron M. 8h and 24h incubation with calcitriol / TGFβ resulted in enhanced expression of VDR in each of the examined regions. The VDR is able to bind to the VDRE without its ligand, whereas this goes along with corepressor recruitment and thus the VDR has a repressive effect on transcription. Histone H4 acetylation was increased when MM6 cells were treated for 8h or 24h with calcitriol or the combination of calcitriol / TGFβ. This finding implies that at that point of time corepressors associated with the VDR are replaced by coactivators. It seems convincing that 5-LO transcription is mainly promoted by calcitriol alone which leads to a more accessible chromatin structure. Previous data indicated that calcitriol and TGFβ upregulate 5-LO RNA maturation and 5- LO transcript elongation. Thus several elongation markers were investigated by ChIP analysis: Histone H3 lysine 36 (H3K36) trimethylation and H4K20 monomethylation were detected in the analysed regions in exon 10, exon 12 and intron M. In region exon 10 the H3K36 trimethylation status was enhanced after 24h calcitriol or calcitriol / TGFβ treatment. An increased H4K20 monomethylation status in all regions was observed when MM6 cells were treated for 24h with calcitriol / TGFβ. 24h treatment with both agents also enhanced the recruitment of the elongation form of RNA polymerase II, which is phosphorylated at serine 2 of the carboxyterminal domain, to the investigated regions. These findings prove the positive regulating role for calcitriol and TGFβ on 5-LO transcript elongation. A putative mechanism of the effect of calcitriol and TGFβ on 5-LO RNA maturation might be the elevated phosphorylation of serine 2 of the RNA Polymerase II which is known to be followed by recruiting polyadenylating factors.
Plastids are complex plant organelles fulfilling essential physiological functions, such as photosynthesis and amino acid metabolism. The majority of proteins required for these functions are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursors, which are subsequently translocated across the outer and inner membrane of the organelle. Their targeting to the organelle is ensured by a so called transit peptide, which is specifically recognized by GTP-dependent receptors Toc159 and Toc34 at the cytosolic side of outer envelope. They cooperatively regulate the insertion of the precursor protein into the channel protein Toc75, thereby initiating the translocation process. Toc34 is regarded as the primary receptor, while Toc159 probably provides the driving force for the insertion. Precursor transfer is achieved by the physical interaction between both receptors in the GTP loaded state. One translocon unit, also called the Toc core complex, is formed by four molecules Toc34, four molecules Toc75 and one molecule Toc159. In the GDP-loaded state, Toc34 preferably forms homodimers, whose physiological function was investigated in the presented study. It could be shown that the dissociation of GDP and therefore the nucleotide exchange are inhibited by the homodimeric state of Toc34. Dissociation of the homodimer is induced by the recognition of a precursor protein, which renders the binding of GTP and subsequent interaction with Toc159 possible. Thus, the homodimeric conformation could reflect an inactive state of the translocon, preventing GTP consumption in the absence of a precursor protein. Both homodimerization as well as heterodimerization of the receptor are regulated by phosphorylation, which could be demonstrated by in vitro and in vivo approaches using atToc33 from Arabidopsis thaliana as a model system. Since the phosphorylated form of Toc34 cannot be assembled with the Toc core complex, it can be concluded that the interactions between GTPase domains not only regulate the transfer of precursor proteins, but also warrant the integrity of the translocon.
The reggie protein family consists of two homologous members, reggie-1 and reggie-2, also termed flotillin-2 and flotillin-1, respectively, that are ubiquitously expressed and evolutionarily well conserved, suggesting an important but so far ill-defined function. In various cell types, both reggies have been found to be constitutively associated with lipid rafts by means of acylation modifications and oligomerization. Lipid rafts are glycosphingolipid- and cholesterol-rich membrane microdomains which have been implicated in several cellular processes including membrane transport and signal transduction through growth factor receptors. However, the molecular details of these processes are still poorly understood. With the observation that reggies colocalize with activated glycosylphosphatidylinositolanchored proteins (GPI-APs) and Fyn kinase in rafts, a role for these proteins in signaling events has been suggested. In agreement with that, we have previously shown that reggie-1 becomes multiply tyrosine phosphorylated by Src kinases in response to epidermal growth factor (EGF) stimulation, pointing to a function for reggie-1 in growth factor signaling. Furthermore, overexpression of reggie-1 enhances spreading on fibronectin substrate in a tyrosine-dependent manner, thus revealing a role for reggie-1 in regulation of actin cytoskeleton through growth factor receptors. Due to the similarity shared by reggie proteins at amino acid level and to their ability to form hetero-oligomeric complexes, the first aim of this study was to analyze the putative tyrosine phosphorylation of reggie-2 in growth factor stimulated cells. Similarly to reggie-1, reggie-2 was found to be multiply tyrosine phosphorylated by Src kinase and to exist in a molecular complex with Src, with the degree of co-immunoprecipitation dependent on the activity of Src. Recent studies from us have also shown that administration of EGF results in the endocytosis of reggie-1 from the plasma membrane into endosomes, which is in line with a proposed role for reggies in membrane trafficking processes. In order to characterize in detail the endocytic mechanism that mediates the uptake of reggie-1, the dependency of reggie-1 endocytosis on clathrin and dynamin was investigated by means of overexpressing a variant form of Eps15 or a dominant negative form of dynamin-2. In either case the translocation of reggie-1 into endosomes in response to EGF was not affected, and this, together with the results that reggie-1 colocalized with cholera toxin (CTX) but not with transferrin receptor (TfnR) during EGF signaling, indicates that reggie-1 is taken up by means of a dynaminindependent, raft-mediated pathway. These findings are very well in line with recent data showing the pathway of entry into cells of reggie-2 as a raft-mediated endocytic pathway. The endocytosis of reggie-2 in response to EGF was also analyzed in this study. Similarly to reggie-1, in growth factor stimulated cells reggie-2 underwent a translocation from the plasma membrane to endosomes where the two reggies were found to colocalize with each other, suggesting that epidermal growth factor signaling might trigger the endocytosis of reggie oligomers. In addition, colocalization with both the late endosomal marker LAMP3/CD63 and epidermal growth factor receptor (EGFR) was detected, again indicating a function for reggies in signal transduction through growth factor receptors. EGFR has been reported to localize in rafts but, although this association is thought to be functional during EGF stimulation, how segregation of EGFR into rafts modulates its endocytosis and signaling is still under debate. Since reggie oligomers have recently been suggested to define a raft subtype, a further aim of this study was to investigate whether the depletion of reggies by means of small interfering RNA could interfere with the signaling and the trafficking through EGFR. Knockdown of reggie-2 resulted in an altered tyrosine phosphorylation of EGFR in response to EGF, while the degree of ubiquitination was not affected. Less efficient phosphorylation of tyrosine residues, especially of those which are docking sites for Grb2 and Shc, led in turn to an impaired activation of p38 and ERK1/2 MAPKs. Depletion of reggie-2 did not affect the early trafficking of activated EGFRs, with receptors being endocytosed and delivered to late endosomes as efficiently as in control cells. This would be in line with the normal degree of ubiquitination observed for EGFR, as ubiquitin moieties have been proposed to represent sorting tags that ensure receptor endocytosis into early endosomes and its proper intracellular trafficking. On the contrary, after prolonged EGF stimulation, depletion of reggie-2 resulted in a decreased downregulation of both receptor-bound ligand and EGFR, and in their accumulation in intracellular vesicles, thus pointing to a role for reggie-2 in the degradative pathway. Taken all together, these data ndicate that the association of EGFR with reggie-microdomains is likely to be important for proper receptor trafficking and signaling.
Ataxin-2 is a novel protein, within which the unstable expansion of a polyglutamine domain can cause Spinocerebellar Ataxia type 2 (SCA2), a neurodegenerative disease which belongs to the group of polyglutamine disorders. SCA2 is characterised by a progressive loss of neurons that first affects the cerebellum and brain stem and then may extend to other areas of the brain, like substantia nigra, motoneurons and thalamus. Several lines of research have attempted to determine therole of ataxin-2 in its normal and mutant version. Different animal models and cell culture approaches to study ataxin-2 function implicated ataxin-2 in RNA processing, embryonic development, apoptosis and cytoskeleton. However, the function of ataxin-2 still remains unclear. In this thesis, a protein interaction approach was chosen as an alternative to gain insights into the cellular function of ataxin-2. Full-length ataxin-2 was used as bait in a yeast two-hybrid screen of human adult brain cDNA. Among five candidate interactor proteins identified, two were the endophilins A1 and A3, proteins involved in vesicle endocytosis. Co-immunoprecipitation studies confirmed the association of these proteins in an endogenous complex of mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilins A1/A3. Ataxin-2 and endophilins A1/A3 colocalised at the endoplasmic reticulum as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in untransfected rat hippocampal neurons. In mouse brain, associations of ataxin-2 with endocytic proteins such as the adaptor CIN85, the ubiquitin ligase c-Cbl and also GRB2, in the last case by means of a SH3 domain array chip, were also demonstrated. GST pull-down assays showed ataxin-2 to interact directly with the SH3 domains A and C of CIN85, the C-terminal SH3 domain of GRB2, and the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR) by reducing the EGFR internalisation after EGF stimulation. Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.
The growth of blood vessels is crucial for organ growth in the embryo and repair of wounded tissues in the adult. An imbalance in this process contributes to numerous malignant, inflammatory, ischemic, infectious and immune disorders (Ferrara et al., 2003). Postnatal neovascularization occurs through the recruitment of progenitor cells and angiogenesis. Integrins are heterodimeric cell surface molecules and are the main receptors for extracellular matrix proteins. Regulation of integrin activation is crucial during embryonic development and during adult life. Dysregulation of integrin activity leads to severe diseases. In this study, we have demonstrated that Rap1, a small GTPase regulating integrin activity, and its GEF Epac1 are expressed in both EPC and endothelial cells. Moreover, the pharmacological activator of Epac activates the small GTPase Rap1 in progenitor cells. In parallel the angiogenic growth factors VEGF and bFGF activate Rap1 in endothelial cells. In addition, the regulation of Rap1 activity in EPC and in endothelial cells plays an important role in the regulation of migration and adhesion to matrix proteins, by regulating the activity of different integrins, a mechanism known as integrin inside‐out signaling. Furthermore, regulation of Rap1 activity affects probably indirectly through outside‐in signaling of integrins the activity of several and crucial proteins such PKB/Akt and focal adhesion kinase in endothelial cells. In line with these results, we have demonstrated that Rap1 activity affect angiogenesis, homing of EPC to ischemic tissues and thereby postnatal neovascularization. The understanding how Rap1 regulates integrin activity in endothelial cells is still not completely clear, for example we have demonstrated that the known effectors of Rap1 mediating the increase of integrin activity in T and B cells, such as RAPL and RIAM are, respectively, either not increasing integrin activity or not expressed in endothelial cells. We aim to find the effector of Rap1 promoting integrin activity in endothelial cells and how RAPL regulates integrin functions and angiogenesis. Moreover data from us and others using genetic models and generation of Rap1a or Rap1b deficient mice or deficient for Rap1a and Rap1b led to embryonic lethality suggesting that Rap1 is a key node protein during embryonic development. The development of conditionnal Rap1a/b endothelial/pericytes restricted deficient mice will help us to decipher more precisely the role of Rap1 during vascular development and angiogenesis.
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.
In this thesis I have investigated the regulation of eicosanoid synthesizing-enzymes by cannabinoid receptor agonists. Rat renal mesangial cells were used as a model system. I could show that all three (CB1, CB2, and GPR55) cannabinoid receptors are expressed on the mRNA level in rat renal mesangial cells – but with differing expression profiles. The CB1 and GPR55 receptors are expressed in comparable amounts, whereas the CB2 receptor is considerably less expressed than the CB1 and the GPR55 receptors. Furthermore I could show that stimulation of renal mesangial cells with CB1 receptor agonists, such as R(+)MA or ACEA, increased IL-1β-induced cPLA2, sPLA2-IIa, and COX2 protein and mRNA expression which subsequently led to an enhanced IL-1β-induced PGE2 formation. Additionally, the IL-1β- induced sPLA2-IIa promoter activity was also increased by CB1 receptor stimulation. Besides the modulated expression of the eicosanoid synthesizing enzymes, I could show that CB1 agonists also led to an increase of IL-1β-induced iNOS expression and subsequent NO formation. In contrast, stimulation with CB2 selective agonists led to a decrease in IL-1β- induced sPLA2-IIa protein expression and PGE2 formation. Accordingly, the IL-1β-induced sPLA2-IIa promoter activity was also reduced by CB2 receptor agonists. IL-1β-induced iNOS expression and subsequent NO formation were not influenced by CB2 recptor activation. Matching the results I obtained with CB1 receptor agonists on IL-1β-induced PGE2 formation, I could observe an increased cPLA2 protein and mRNA expression with a subsequent increase in IL-1β-induced PGE2 formation by GPR55 stimulation. Stimulation with THC, an unselective CB agonist, increased the IL-1β-induced sPLA2-IIa protein expression and subsequently led to an enhanced IL-1β-induced PGE2 formation. Subjecting the cells to higher THC concentrations surprisingly led to a reduction of the IL-1b-induced sPLA2-IIa protein expression and PGE2 formation. A possible explanation may be the differential expression of the three CB receptors. At low concentrations THC may predominantly activate CB1 and GPR55 and with increasing concentration CB2 receptors may also be activated, slightly reversing the enhancing effect. Moreover, I could show that the CB1 receptor stimulation mediated phosphorylation and hence the activation of ERK1/2 MAPK. Additionally to ERK1/2, there was also a phosphorylation and activation of NFkB observed by CB1 receptor stimulation. In my thesis I could show for the first time that PPARα was activated by IL-1β in rMC. The IL-1β-induced PPARα promoter activity was completely inhibited by addition of the CB2 receptor agonist, JWH015. These findings were confirmed by inhibition of the IL-1β-induced PGE2 formation by a PPARα antagonist (MK-886). In summary, I could show that activation of CB1 receptors in our system led to a worsening of an inflammatory condition, whereas activation of the CB2 receptors led to the complete opposite; namely a reduction of the inflammatory response by reducing the sPLA2-IIa expression and PGE2 formation. GPR55 activation did not display any alteration of inflammatory conditions, since the classical inflammatory pathway was not influenced.
RNA interference (RNAi) is triggered by recognition of double-stranded RNA (dsRNA), and elicits the silencing of gene(s) complementary to the dsRNA sequence. RNAi is thought to have emerged as a way of safeguarding the genome against mobile genetic elements and viral infection, thus maintaining genomic integrity. dsRNA is first processed into small interfering RNAs (siRNA) by the enzyme Dicer. siRNAs are ~21 to 25 -nt long, and contain a signature 5’ phosphate group and a two nucleotide long 3’ overhang (Bernstein et al., 2001). The siRNA is then loaded into the RNA-induced si-lencing complex (RISC), of which Argonaute is the primary catalytic component (Liu et al., 2004). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (Khvorova et al., 2003; Schwarz et al., 2003). Argonaute cleaves the passen-ger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (Gregory et al., 2005; Matranga et al., 2005; Rand et al., 2005). This single-stranded guide strand siRNA bound to Argonaute is able to recognize target mRNA in a sequence-specific manner, and cleaves the mRNA. Argonaute 2 in complex with single-stranded siRNA is sufficient for mRNA recognition and cleavage, thus forming a minimal RISC (Rivas et al., 2005). miRNAs, endogenously expressed small RNA genes which typically contain mismatches and non-Watson-Crick base pairing, are processed by this general pathway, although typically modulate gene expression by translational repression as opposed to cleavage of their target mRNA. The number of Argonaute genes is highly variable between species, ranging from one in S. pombe to twenty-seven in C. elegans. Earlier crystal structures of Argonaute apoen-zymes show the architecture of Argonaute to be a multidomain protein composed of N terminal, PAZ, MID, and PIWI domains (Song et al., 2004; Yuan et al., 2005). These multi-domain proteins are present in both prokaryotic and eukaryotic organisms. The role of Argonaute proteins in prokaryotes is still unknown, but based similarity to eu-karyotic Argonautes, they may also be involved in nucleic acid-directed regulatory pathways. These proteins have served as excellent models for learning about the struc-ture and function of this family of proteins. RNAi has found a widespread application for the simple yet effective knockdown of genes of interest. The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conforma-tional changes during the cycle of mRNA recognition by RISC (Filipowicz, 2005; Tom-ari and Zamore, 2005). Nevertheless, it remains unclear how the multi-domain ar-rangement of Argonaute recognizes and distinguishes between single-stranded and dou-ble-stranded oligonucleotides, which correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand / mRNA duplex. The Argonaute protein from Aquifex aeolicus was cloned, expressed, crystallized and solved by molecular replacement. Relative to earlier Argonaute structures, a 24° reorientation of the PAZ domain in this structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. A 5.5-ns molecular dynamics simulation of Argonaute showed a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute was monitored by total internal reflection fluorescence spectroscopy (TIRFS). The experi-ments showed biphasic kinetics indicative of large conformational changes, and re-vealed a hotspot of binding energy corresponding to the first 9 nucleotides, the so-called “seed region” most crucial for sequence-specific target recognition. As RNAi may have evolved as a way of safeguarding the genome viral infection, it is not surprising that viruses have evolved different strategies to suppress the host RNAi response in the form of viral suppressor protein. (Hock and Meister, 2008; Lecellier and Voinnet, 2004; Rashid et al., 2007; Song et al., 2004; Vastenhouw and Plasterk, 2004). These viral suppressors are widespread, having been identified in a number of different viral families. Not surprisingly, they generally share little sequence homology with one another, although they appear to exist as oligomers built upon a ~ 100-200 amino acid protomer. Tomato aspermy virus, a member of the Cucumoviruses, encodes for protein 2B (TAV 2B, 95 a.a., ~11.3 kDa) that acts as an RNAi suppressor. Intriguingly, a similar genomic arrangement is seen in RNAi suppressors in the Nodaviruses, a family of viruses that can infect both plants and animals, such as Flock house virus b2 (FHV b2). The 2B and b2 proteins are both derived from a frameshifted ORF within the RNA polymerase gene (Chao et al., 2005). In spite of this genomic similarity, the 2B and b2 proteins share little sequence identity, and it is not well understood how the Cucumovirus 2B proteins suppress RNAi. To address how TAV 2B suppresses RNAi, the oligonucleotide-binding properties of TAV 2B were studied. TAV 2B shows a preference for double-stranded RNA oligonucleotides corresponding to siRNAs and miRNAs, and also binds to single-stranded RNA oligonucleotides. A stretch of positively charged residues between amino acids 20-30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B into a primarily helical structure. These studies sug-gest that suppression of RNAi by TAV 2B may occur by targeting different stages of the RNAi pathway. TAV 2B falls under the category of more general RNAi suppres-sors, with potentially multiple targets for suppression.
Reggie-1 (flotillin-2) and reggie-2 (flotillin-1) are membrane microdomain proteins which are associated with the membrane by means of acylation. They influence different cellular signaling processes, such as neuronal, T-cell and insulin signaling. Upon stimulation of the EGF receptor, reggie-1 becomes phosphorylated and undergoes tyrosine 163 dependent translocation from the plasma membrane to endosomal compartments. In addition, reggie-1 was shown to influence actindependent processes. Reggie-2 has been demonstrated to affect caveolin- and clathrin-independent endocytosis. Both proteins form homo- and hetero-oligomers, but the function of these oligomers has remained elusive. Moreover, it has not been clarified if functions of reggie-1 are also influenced by reggie-2 and vice versa. The first aim of the study was to further investigate the interplay and the heterooligomerization of reggie proteins and their functional effects. Both reggie proteins were individually depleted by means of siRNA. In different siRNA systems and various cell lines, reggie-1 depleted cells showed reduced protein amounts of reggie-1 and reggie-2, but reggie-2 knock down cells still expressed reggie-1 protein. The decrease of reggie-2 in reggie-1 depleted cells was only detected at protein but not at mRNA level. Furthermore, reggie-2 expression could be rescued by expression of siRNA resistant wild type reggie-1-EGFP constructs, but not by the soluble myristoylation mutant G2A. This mutant was also not able to associate with endogenous reggie-1 or reggie-2, which demonstrates that membrane association of reggie-1 is necessary for hetero-oligomerization. In addition, fluorescence microscopy studies and membrane fractionations showed that correct localization of overexpressed reggie-2 was dependent on co-overexpressed reggie-1. Thus, hetero-oligomerization is crucial for membrane association of reggie-2 and for its protein stability or protein expression. Moreover, the binding of reggie-2 to reggie-1 required tyrosine 163 of reggie-1 which was previously shown to be important for endosomal translocation of reggie-1. Since reggie-2 was implicated to function in clathrin- and caveolin-independent endocytosis pathways, the effect of reggie-2 depletion on reggie-1 endocytosis was investigated. Indeed, reggie-1 was dependent on reggie-2 for endosomal localization and EGF-induced endocytosis. By FRET-FLIM analysis it could be shown that reggie heterooligomers are dynamic in size or conformation upon EGF stimulation. Thus, it can be concluded that reggie proteins are interdependent in different aspects, such as protein stability or expression, membrane association and subcellular localization. In addition, these results demonstrate that the hetero-oligomers are dynamic and reggie proteins influence each other in terms of function. A further aim was the characterization of reggie-1 and reggie-2 function in actindependent processes, where so far only reggie-1 was known to play a role. Depletion of either of the proteins reduced cell migration, cell spreading and the number of focal adhesions in steady state cells. Thus, also reggie-2 affects actin-dependent processes. Further investigation of the focal adhesions during cell spreading revealed that depletion of reggie-1 displayed different effects as compared to reggie-2 knock down. Reggie-1 depleted cells had elongated cell-matrix-adhesions and showed reduced activation of FAK and ERK2. On the other hand, depletion of reggie-2 resulted in a restricted localization of focal adhesion at the periphery of the cell and decreased ERK2 phosphorylation, but it did not affect FAK autophosphorylation. Hence, reggie proteins influence the regulation of cell-matrix-adhesions differently. A link between reggie proteins and focal adhesions is the actin cross-linking protein -actinin. The interaction of -actinin with reggie-1 could be verified by means of co-immunoprecipitations and FRET-FLIM analysis. Reggie-1 binds -actinin especially in membrane ruffles and in other locations where actin remodeling takes place. Moreover, -actinin showed a different localization pattern during cell spreading in reggie-1 depleted cells, as compared to the control cells. These results provide further insights into the function of both reggie proteins. Their interplay and hetero-oligomerization was shown to be crucial for their role in endocytosis. In addition, both reggie proteins influence actin-dependent processes and differentially affect focal adhesion regulation.
The respiratory chain is composed of protein complexes residing in the inner mitochondrial membrane of eukaryotes or in the cytoplasmic membrane of prokaryotes. This cellular energy converter transforms a redox potential stored in low potential substrates into an electrochemical potential across the respective membrane. Typical respiratory chains contain the complexes I, II, III and IV named according to their sequence in the respiratory chain reaction. Electrons of low potential substrates enter at complex I or II and are passed via complex III to complex IV where they are transferred to oxygen. The transport of electrons between the complexes is mediated by small electron shuttles like quinol or cytochrome c. Two different models describe their exchange either by (1) random collision of freely diffusible electron shuttles and membrane protein complexes or (2) arrangement of the complexes in supercomplexes enabling direct channeling of electron shuttles. In the Gram positive bacterium Corynebacterium glutamicum, the complex III to complex IV electron shuttle cytochrome c is not diffusible but a covalently bound part of the diheme cytochrome subunit QcrC of complex III. Therefore, the complexes III and IV have to form a supercomplex for electron transduction. The aim of this thesis was to purify and characterise this obligatory supercomplex III/IV of C. glutamicum. To gain sufficient biomass of C. glutamicum as starting material for purification, a phosphate buffered minimal medium was developed that enabled yield of total 120 g wet cell mass (38 g dry mass) in 12 L (6×2 L) shaking cultures. The determined conversion factor of glucose into biomass was 0.46 g/g indicating an intact respiratory chain. The yield was increased by bioreactor cultivation to ~690 g wet cell mass (~220 g dry mass) in ~10 L culture volume. A previously described homologous expression system was applied that produces the complex IV subunit CtaD with a fused Strep-tag II to facilitate purification. Affinity purifications using the Strep-tag II affinity to Strep-Tactin resin yielded a mixture of complexes and supercomplexes. Two supercomplex III/IV versions named supercomplex A and B and free complex IV were identified in this mixture by size exclusion chromatography, redox difference spectroscopy and two dimensional polyacrylamide gel electrophoresis including blue native polyacrylamide electrophoresis. The here presented downscaled blue native polyacrylamide electrophoresis method with analysis times of ~1 h enabled efficient screening of factors influencing the stability of supercomplex III/IV. The screening resulted that the integrity of supercomplex III/IV is preserved by using neutral detergents at minimal detergent to protein ratios for solubilisation and low detergent concentrations for purification and storage slightly above the required critical micellar concentration. Furthermore, pH <=7.5 is required for stability of supercomplex III/IV. Large biomass yields enabled upscaling of supercomplex III/IV affinity purification. Application of the identified stability conditions resulted in affinity purified samples free of supercomplex B. The major component supercomplex A was efficiently separated from residual free complex IV by preparative size exclusion chromatography. Concentration of purified supercomplex A by ultracentrifugation resulted in integrity of the supercomplex for several days at 4 °C. Purified supercomplex A contains ten different previously described subunits. The heme content of supercomplex A relative to the protein mass is heme A: 6.0 μmol/g, heme B: 6.5 μmol/g, and heme C: 5.8 μmol/g determined by redox difference spectroscopy and biochemical protein quantification. This indicates an equimolar ratio of complex III and complex IV in supercomplex A. Supercomplex A has quinol oxidase activity that is inhibited by stigmatellin or sodium azide. The turnover number of transferred electrons per complex III monomer is 148 s−1 at 25° C. The homogeneity and stability of the prepared supercomplex A enabled the growth of threedimensional crystals of up to 0.1 mm in length. Their composition of supercomplex A was verified by redox difference spectroscopy of intact crystals and blue native polyacrylamide electrophoresis of dissolved crystals. The crystals diffracted X-rays corresponding to a resolution of ~10 Å. Electron microscopy of negative stained samples revealed the uniform shape of purified supercomplex A particles with dimensions of 22 × 9 nm in the view plane. Combined heme quantification, size determination, determined activity, symmetry considerations, and particle shape indicate that supercomplex A has a central dimer of complex III and two monomers of complex IV on opposite sides. This conformation is functionally reasonable because it provides each complex III monomer with one complex IV monomer as electron acceptor. Therefore, the stoichiometry of supercomplex A is most likely III2IV2. The sensitivity of supercomplex A to detergents indicated a role of phospholipids in its stability. Therefore, a method for phospholipid identification and quantification was developed that is suitable for detergent solubilised crude and purified membrane protein samples. The analysis combines separation of phospholipid classes according to their head group by normal phase high performance liquid chromatography with evaporative light scattering detection. Calibration with external standard allows quantification of phospholipid amount in the range of 0.25-12 μg. The method is verified by analysing the phospholipid content of the well characterised complex III of Saccharomyces cerevisiae. The reduction of its phospholipid content during its purification steps is monitored. The complex III sample purified to crystallisation quality contains the phospholipid content that was also observed in previously reported structures determined by X-ray crystallography. Purified stable supercomplex A from C. glutamicum revealed a large content of bound phospholipids. The main differences between intact supercomplex A and a mixture of potentially disintegrated smaller complexes is that intact supercomplex A has a doubled phosphatidic acid content and an increased phosphatidyl glycerol content. The importance of the small anionic phosphatidic acid for mediation of contacts between complexes in a supercomplex is discussed. The total phospholipid content of stable supercomplex A is sufficient for a complete belt surrounding the supercomplex in the membrane plane. This indicates that also all essential internal phospholipid binding positions are occupied and potentially stabilise supercomplex A.
Mitochondria are dynamic organelles indispensible for viability of eukaryotic cells. Diffusion of proteins in mitochondrial membranes is a prerequisite for the correct functionality of the organelles. However, its study is made complicated due to the nontrivial geometry, small size and positional instability of the organelle, restricting the usability of regular experimental methods and theoretical understanding of acquired data. Therefore, here the molecular transport along the main mitochondrial axis was investigated using highly accurate computational methods combining them with traditional experimental approaches. Using recently reported electron microscopic tomography data concerning the constitution of mitochondria [Fre02], a lattice model of the inner mitochondrial membrane (IM) reproducing its structure in great details was built up. With Monte Carlo (MC) simulations of particle dynamics on this model, it was found that the membrane geometry induces nonlinear effects in the motion of molecules along the mitochondrial axis, which in turn lead to a transient violation of the 2nd Fick?s equation. We show that mere curvature of the IM resulting from the presence of cristae is sufficient for the emergence of transient anomalous diffusion (TAD) in the membrane. The MC calculations have enabled an accurate estimation of regularities in the extent of deviations from the normal regime, therefore allowing us to propose non-homogenous power law as a suitable generalization of the current approach to the analysis of experimental data for the transient dynamics. The general cause of TAD resulting from the membrane curvature alone, without any involvement of specific inter-particle interactions prompted us to predict the similar dynamical effect also for other curved cellular membranes, be it diffusion in endoplasmic reticulum or in plasma membrane of cells possessing dense microvilli. The data indicate that the geometry-induced anomalous diffusion should be easily detectable with current experimental methods, but only in the restricted range of time scales corresponding to high temporal resolution. Until now, experimental measurements of molecular diffusion in biological membranes indiscriminately assumed either pure normal or pure anomalous diffusion schemes for the analysis of data acquired in very wide range of temporal resolutions, which often lead to ambiguities in the interpretation of diffusion parameters. The MC calculations have clearly illustrated the necessity for a more subtle treatment of experimental conditions: the assumption of pure Gaussian diffusion model is justified only if the applied temporal resolution is sufficiently low (as is often the case when using scanning techniques exemplified further); otherwise, the transient regime should be tested for by means of the non-homogenous power function. In the second part of the study the Fluorescence Recovery after Photobleaching (FRAP) with the laser scanning microscope is introduced as a method of choice for studying protein mobility within mitochondrial membranes. The conventional FRAP methodology [Axe76] was extended to enable its application for the determination of confined diffusion with conventional laser scanning microscopes which allowed us to communicate for the first time the direct measurement of protein diffusion in mitochondrial membranes of living cells. This is achieved through adaptation of FRAP data analysis to account for the spatial dimensions of the organelle and the spatiotemporal pattern of light pulses induced by the microscope. The experimental circumstances existing during the particular measurement session are computationally recreated and this way the best suited values of diffusion parameters are found. The method is validated experimentally for four FP-tagged mitochondrial membrane proteins: the IM OxPhos complexes F1F0 ATPase and cytochrome c oxidase and for Tom7 and hFis1 - components of the mitochondrial protein import and fission machineries respectively localized in the outer membrane. We find that for all proteins simple normal diffusion is not a sufficient description. In the inner membrane, diffusion coefficient of F1F0 ATPase expressed in HeLa cell line is found to be 0.2 ?m2/s, with more than 1/3 of the protein molecules being immobilized, while cytochrome c oxidase (in CEF primary cells) demonstrated a similar diffusivity pattern (0.4 ?m2/s, 30% immobile). In the outer membrane, the D (0.7 ?m2/s) and immobile fraction (7-8%) of GFP-Tom7 and GFP-hFis1 (both in HeLa cells) are identical, which designates a substantial difference in comparison to the IM protein mobility. Diffusion coefficients of mitochondrial membrane proteins studied here lay in the intermediate region between those measured in artificial bilayers and in plasma membranes. Protein crowding and intermolecular interactions will be among the major causes responsible for the detected slowdown of diffusion.
Amphibians of Malawi : an analysis of their richness and community diversity in a changing landscape
(2009)
This study summarizes the state of the knowledge of the amphibian diversity in Malawi highlighting the possible threats impending on this fauna correlated with human encroachment and land use change. New data about diversity, distribution and ecology have been gathered, whereas the old ones have been summarised, reviewed and commented. In order to put in context the responses of the amphibian communities to land use change, the main environmental characteristics of the country at a broad space and time scale have been explored. Furthermore, the original habitats and vegetation have been described, and their status in the present day Malawi discussed. In the same way, an overview of the actual state of the knowledge about the Malawian amphibians has been provided, and their ability to act as surrogate of environmental integrity in Sub-Saharan Africa commented on the basis of the available studies. Afterwards, the results of the study of the selected areas and samples have been analysed within this newly generated context. Different field and laboratory methods were applied for the quantitative analysis of the richness and diversity of the communities. Opportunistic search was used to detect species richness, whereas the visual encounter survey was applied to detect the relative abundance of species. Several indices of diversity and similarity, and extrapolations by means of true richness estimators were used for the analysis of the alpha and beta diversities. Additional information were gathered by means of pitfall traps with drift fence, and by the recording of the advertisement calls. Supplementary methods were applied for the analysis of the taxonomic composition of the collected material. In Malawi 84 amphibian species are recorded, two of which still undescribed (Leptopelis sp. and Phrynobatrachus sp.). Three further species need to be confirmed and might be possibly present too: Amietia viridireticulata, Hemisus guineensis, and Hyperolius minutissimus. Additionally, other unrecognised cryptic species — at least one — are present within the Hyperolius nasutus complex. Most of the species belong to the order Anura (82 species; 97.6%), whereas only two species belong to the Gymnophiona (2.4%). Anurans are divided into 12 families and 23 genera, whereas the two caecilians species into one family (Caecilidae) and two genera. The more diverse family is the Hyperoliidae (21 species, 25%) followed by the families Ptychadenidae (13 species, 15%), Arthroleptidae (11 species, 13%), Phrynobatrachidae (10 species, 12%), and Bufonidae and Pyxicephalidae (9 species, 11% respectively). The remaining high family diversity (seven families, Caecilidae included) is contrasted by a low number of species (11 species in total, 14%). Based on the available distribution data, the value of species richness of the anuran communities in Malawi is comprised between 5‒45 species. In average 16.8 ± 9.0 species (N=80) are to be found, 75% of the sites have less than 21 species, and only two sites have more than 25 species. Four hot spots of amphibian diversity were identified: the Nyika Plateau (24 species), Mangochi-Malombe (25 species), Zomba Plateau (32 species) and the Mulanje Massif (45 species). In the studied areas a mean of 14.7 ± 1.6 species was observed and extrapolations by means of the true richness estimators were in good agreement with this result. Among the studied areas the richest was Palm Forest Reserve (17 species), followed by Kaningina Forest Reserve (16 species) and Vinthukutu F. R., and Vwaza W. R (15 species). The poorest area was the Misuku Mountains with 12 species only and a slightly different ranking was generated by the true richness estimators. The mean of the species present in the samples was 4.8 ± 2.1 species, considerably less than the true species richness detected in the respective areas. Basing on the ranking generated by the K-dominance plot the most diverse samples were Palm F. R. and Misuku, whereas the less diverse were Kaningina F. R. and Fort Lister, confirmed by the values of the diversity indices. The main finding of this study was the observation of the lack of a clear match between environmental degradation and amphibian diversity, and the crucial importance of temporary water bodies for the preservation of the amphibian diversity. In fact, despite most of the original habitat formerly present in Malawi have been destroyed and replaced by cultivations, the amphibian communities of different areas showed a comparable diversity at both family and species richness level, and no evident match between environmental degradation and amphibian diversity was recognisable. Differences in species richness could mostly be explained by natural factors such the elevation gradient and the presence of temporary water bodies. However, it was not possible to exclude that the communities have changed during historical time and the shift in species composition already occurred together with the modification of their relative frequencies. Most of the species showed a remarkable ecological plasticity and several species were found in a variety of both natural and altered habitats. The classification of the Malawian amphibians on the basis of ecological guilds based on the available natural history data showed the preponderance (76%) of generalist pond breeders. As a consequence, most of these amphibians possessed a scarce capacity to act as surrogates of habitat integrity. Based on the result of this study the farm bush landscape with traditional agriculture practices bears a great potential to support amphibian diversity in terms of species richness, representing a compromise between local economic development and conservation. Furthermore, the results of this study indicate the outstanding importance of the southern-east region of Malawi for the conservation of the country’s amphibians.
5-lipoxygenase (5-LO) is the key enzyme in the formation of inflammatory leukotrienes, which are mediators of inflammation and allergy. The 5-LO catalyses the oxidation of arachidonic acid to 5-HPETE and subsequently to LTA4. The leukotrienes are involved in the development and maintenance of inflammatory diseases, like asthma and allergic rhinitis. Additionally, 5-LO is overexpressed in some cancer types, although its relevance is still not fully understood. 5-LO expressing cells are B- lymphocytes and cells of myeloid origin like monocytes, macrophages and granulocytes. The 5-LO promoter lacks a TATA or CCAT box and covers two CpG islands. These are characteristics of a housekeeping gene, but as the 5-LO is not expressed ubiquitiously, the expression of the 5-LO is tightly regulated. Epigenetic mechanisms were known to be involved in the control of the 5-LO expression. The HDAC inhibitor TsA significantly induced the transcriptional activity of the 5-LO promoter in reporter gene assays as well as on 5-LO mRNA transcript level in MM6 cells. The GC-boxes GC4 and GC5 in the proximal 5-LO promoter were identified to be essential for the TsA effect, as deletion of these element led to an attenuated TsA effect in reporter gene assay. Recruitment of the transcription factors Sp1 and Sp3 and the RNA polymerase II to the 5-LO promoter was detectable after TsA treatment in MM6 cells by chromatin immunoprecipitation assays (ChIP), while the acetylation status of histone H4 remained unchanged. Likewise it is known that DNA methylation leads to silencing of 5-LO expression in-vitro and in-vivo. The 5-LO promoter is densely methylated in the cell line U937, but unmethylated in HL-60 cells and - elucidated in this study - also in MM6 cells. Reporter gene assays with in-vitro methylated 5-LO promoter containing plasmids revealed that the frequency of methylated CpGs is directly proportional to reduction of 5-LO promoter activity. Incubation of U937 cells with 5-AdC, an inhibitor of DNA methyltransferases, was able to reactivate 5-LO transcription and to demethylate CpG dinucleotides. In the first part of this study the mechanism of TsA induced promoter activation was further investigated. I elucidated the mechanism of Sp1 and Sp3 recruitment to the 5-LO promoter after TsA treatment. Immnoprecipitation assay was used to detect a transcription factor complex containing Sp1 or Sp3 interacting with HDAC proteins, which might change its composition after TsA treatment. Besides the posttranslational modifications of the transcription factors Sp1 and Sp3 after TsA treatment were investigated, potentially causing an increased interaction of the proteins with the 5-LO promoter. Both aspects and their response in HDAC inhibition have been described. TsA did not affect the composition of the Sp1/HDAC1/HDAC2 complex. Sp3 was not located in a complex with the HDAC enzymes. Acetylation of Sp1 and Sp3 was detectable, but no change occurred after TsA treatment. Since neither release of the transcription factors off a complex, nor alterations in posttranslational modifications of Sp1 and Sp3 are the reason for the increased Sp1 and Sp3 binding to the 5-LO promoter, I elucidated alterations in the chromatin structure. The acetylation status of the histone proteins H3 and H4, as well as the chromatin marks H3K4me3, representing active chromatin, and H3K9me, representative for repressive state, were investigated. Additionally, the time course of the TsA effect was determined on 5-LO mRNA level using real-time PCR. The acetylation status of the histone proteins on the 5-LO core promoter correlated with the basal 5-LO mRNA transcript expression in MM6, HL-60 and U937 cells. The highest 5-LO mRNA level was detectable in MM6 cells, followed by HL-60 cells. The lowest 5-LO mRNA level was detected in 5-LO promoter methylated U937 cells. The order of the basal 5-LO mRNA expression of the three cell lines correlates with the basal acetylation status of histone proteins H3 and H4. In MM6 cells the highest basal levels in acH3 and acH4 were detected, followed by HL-60 and U937 cells. Moreover, the data obtained in U937 cells revealed that the correlation between DNA methylation and histone hypoacetylation is alike on the 5-LO promoter. TsA treatment induced the 5-LO mRNA level in the three cell lines with different intensity: 5-LO mRNA level in MM6 cells was induced 11-fold, in HL-60 cells 6- fold and in U937 cells 4- fold. The histone acetylation and methylation levels on the 5-LO promoter after TsA incubation were investigated. No increase in acH3 and acH4, but in H3K4me3 was detectable in MM6 cells by ChIP assay. HL-60 cells showed an increase in acH3 and acH4 as well as in H3K4me3. H3K9me was only detectable in untreated U937 cells, but disappeared after TsA treatment, while acH3, acH4 and H3K4me3 increased constantly after TsA treatme nt. A strong correlation between the histone modifications and the time course of the mRNA expression was detectable in all three cell lines. The combination of the posttranslational modifications acH3, acH4 and H3K4me3 led to a fast effect in transcriptional activation and the maxima of acH3 and acH4 were usually associated with the maximum in 5-LO mRNA transcript level. An increase in H3K4me3 alone, as detected in MM6 cells, led to continuous increase in the 5-LO mRNA expression with a late maximum. Additionally, we detected a slight overall decrease in 5-LO promoter methylation in U937 cells after TsA treatment. This fact taken together with the observed histone modifications could explain the 4- fold response in 5-LO mRNA level to TsA treatment of the methylated cell line U937. Another aim of the present study was to identify the specific HDAC enzymes involved in the 5-LO promoter regulation. Reporter gene assays and real-time PCR with selective HDAC inhibitors revealed that HDACs of class I are involved in 5-LO promoter regulation, namely HDAC 1, 2 and 3. The influence of each of the enzymes seemed to depend on the cell type, as inhibition of HDACs 2, 3 strongly induced 5-LO promoter activity in reporter gene assay in HeLa cells, whereas in MM6 cells HDACs 1 and 2, 3 seemed to be responsible for the 5-LO promoter regulation, measured as 5-LO mRNA level. The HDACs of class IIa and class III are not involved in the regulation of 5-LO mRNA expression. The second part of this study investigated the influence of MBD proteins on the methylated 5-LO promoter and the 5-LO mRNA expression. ChIP assays revealed MBD1, 2 and MeCP2 protein binding to the proximal 5-LO promoter in U937 cells. MBD1 was detectable on the 5-LO promoter in unmethylated HL-60 cells, while no MBD protein was located on the 5-LO promoter in MM6 cells. To elucidate the functional role of the MBD proteins, stable knocked down of MBD proteins was established in U937 cells. 5-LO mRNA transcript level was determined in the knock down clones by real-time PCR. The 5-LO transcript level was increased in all knock down samples. MBD2 knock down clones showed the highest effect in activating 5-LO with a 3- and 4.4-fold increase in the 5-LO mRNA level, followed by MBD1 (3.5- fold) and MeCP2 (2.5-fold) knock down clones. A combined participation of these three enzymes in the corepression of the methylated 5-LO promoter is indicated. Taken together, the data reveal that epigenetic mechanisms are strongly involved in the regulation of 5-LO transcription and might function as a crucial control mechanism of 5-LO expression.
Delthyridoid spiriferids are characterized by a global abundance and fast evolution during Silurian and Devonian, and, therefore, are used as important biostratigraphical and palaeobiogeographical tools. In this work, delthyridoid brachiopod faunas from different regions of today’s world, resp., of different palaeobiogeographical units, are compared side-by-side to investigate their phylogenetic relationships and to improve, in a second step, the palaeobiogeography from Late Silurian to Early Eifelian time. A new systematics of Delthyridoidae is established which is more complicated than hitherto assumed. The results of this study are mainly based on direct comparison of articulated and isolated brachiopod shells, external and internal moulds, as well as latex casts and serial sections. The computer supported cladistic analyses have turned out not to be useful due to different kinds of preservation resulting in an incomplete matrix which is insufficient for reliable cladograms. A further problem in terms of cladistical analyses are various convergences during the evolution of spiriferids. Many characters evolved independently from each other at different times in each lineage so that autapomorphies are hardly or not at all recognizable. As a result, families and genera are only definable by a combination of characters rather than by a single or a few autapomorphies. As a new method, 3D reconstruction from serial sections is introduced which made it possible for the first time to compare directly mouldic and shelly material. Preliminary results are presented herein. Statistical analyses of measurements taken from new taxa are made but regarded as a descriptive argument rather than a deciding factor for taxonmy due to incomplete preservation and/or tectonic deformation. Brachiopods, especially type material, from collections of different institutions and museums are studied as well as personal material, whenever possible collected from topotype outcrops. Emended diagnoses, if necessary, from family to species level are given. During this work several new taxa have been erected: 7 new families: Australospiriferidae, Murchisonispiriferidae, Orientospiriferidae, Otospiriferidae, Patriaspiriferidae, Rostrospiriferidae, and Trigonospiriferidae; 6 new genera, 1 of these in open nomenclature: Cyclopterospirifer, Hallispirifer, Parlinispirifer, Murchisonispirifer, Shujiapingensispirifer, and gen. nov. B; and 3 new species: Patriaspirifer merriami, Patriaspirifer johnsoni, and Murchisonispirifer feldmani; 1 taxon is defined as nomen novum: Orientospirifer nakaolingensis wani. In the framework of this project, 2 families: Filispiriferidae and Multispiriferidae; 1 subfamily: Multiplicatispiriferinae, 6 genera, 1 of them in open nomenclature: Frequentispirifer, Leonispirifer, Multiplicatispirifer, Ovetensispirifer, Turcispirifer, and Gen. A; and 9 new species, 3 of them in open nomenclature: Filispirifer hamadae, Leonispirifer leonensis, Multiplicatispirifer foumzguidensis, Oventensispirifer novascotianus, Quiringites arensentiae, Turcispirifer turciae, Multiplicatispirifer cf. foumzguidensis, Quiringites cf. arensentiae, and ?Turcispirifer sp. A which have already been established are also described in this work. The brachiopod faunas studied consist of externally very similar spiriferids which have been identified as same genera, species, or even subspecies in earlier times. These forms are considered as 6 distinct morphotypes Howellella-, Arduspirifer-, Acrospirifer-, Euryspirifer-, Paraspirifer-, and Multiplicatispirifer-like morphotypes, which are briefly introduced. The new systematics is characterized by different clades, the European/North African delthyridoid spiriferid clade, the North American delthyridoid spiriferid clade, the Asian delthyridoid spiriferid clade, the Malvinokaffric delthyridoid spiriferid clade, and the delthyridoid multiplicated spiriferid clade. Each of them is described in a cladistic and in a phylogenetic way. Their phylogenetic relationship sheds new light on palaeobiogeographical interpretations for the different stages from Late Silurian to early Middle Devonian time. A tendency for increasing endemicity is seen until the end of the Early Emsian, which is interrupted by short term regional faunal exchange within a province or within a realm, followed by a loss of endemicity resulting in global distribution of brachiopod genera until the end of Givetian time. The Old World Realm is re-defined due to the lack of phylogenetic relationship between its faunas and subdivided into the European Realm, consisting of the Gondwanan and Avalonian provinces, and the Asian Realm, consisting of the Siberian, Sino, and Mongolian provinces. A reconstruction of Lower Devonian palaeobiographical map is introduced.
The aim of the study was to investigate the role of the CX3C chemokine FKN in the role of platelet adhesion. The presence of the FKN receptor CX3CR1 in platelets is demonstrated and G-protein dependent activation of platelets with soluble FKN results in the increased adhesion of platelets to collagen and fibrinogen under flow 228 and adhesion of leucocytes to firmly attached platelets 231. Whether membrane-bound FKN is capable to promote the direct adhesion of platelets in flowing blood analogue to leucocytes was completely unknown. The adhesion mechanisms of FKN in mediating the adhesion of leucocytes under flow are well characterised and represent a novel unique mechanism of leucocyte capture and firm adhesion: FKN is responsible for immediate arrest of flowing CX3CR1 expressing leucocytes without the participation of additional adhesion receptors and ligands. This is in contrast to the classical leucocyte adhesion pathways, which are multistep processes involving leucocyte arrest, rolling and subsequent cell activation prior to firm arrest. In leucocytes, the FKN – CX3CR1 axis is sufficient to allow rapid arrest of leucocytes at low shear flow conditions 67, 101, 115, 122, 261. The set of data from this study demonstrates that immobilised FKN was capable to mediate the adhesion of platelets under low shear conditions, whereas there was no interaction in the absence of shear flow. In the presence of vWf in the adhesion matrix, FKN mediated the potent increased adhesion of platelets. This was in parts due to the activation of flowing platelets via CX3CR1 and the augmented translocation of platelets on FKN via the vWf receptor GPIbα. With respect to platelet activation, the function of endothelial FKN was comparable to leucocytes: in both cell types, the FKN dependent activation is mediated by its cognate receptor CX3CR1. This is in contrast to the adhesive capacity: in leucocytes, FKN dependent adhesion is mediated by CX3CR1, whereas in platelets, the adhesive capacity was mostly mediated by the vWf receptor GPIbα with only minor contribution from CX3CR1. In platelets, activation and adhesion by FKN were mediated by two distinct receptors, whereas in leucocytes, CX3CR1 is solely responsible for FKN dependent activation and adhesion. The presented results point out to a role of platelets in early stage of atherosclerosis. The in vivo expression of both, FKN and vWf is regulated by TNF-α, which is released in early stages of inflammation. The presence of vWf and FKN in the endothelial lining of blood vessels during these conditions is sufficient to initiate the capturing and translocation of platelets on the tunica interna. The rolling of platelets on the endothelium can induce endothelial damage and inflammation of the vessel, which might advance to the generation of clinically significant atherosclerotic plaques and fibrous atheroma.
A generic drug product (World Health Organization (WHO) terminology: multisource product) is usually marketed and manufactured after the expiry date of the innovator’s patent. Generic drugs are less expensive than the innovator products because generic manufacturers do not have to amortize the investment costs of research, development, marketing, and promotion. Multisource products must contain the same active pharmaceutical ingredients (APIs) as the original formulation and have to be shown to be interchangeable with the original formulation. Multisource products have to be shown bioequivalent to the innovator counterpart with respect to pharmacokinetic and pharmacodynamic properties. Multisource products are therefore identical in dose, strength, route of administration, safety, efficacy, and intended use. Bioequivalence can be demonstrated by in vitro dissolution, pharmacokinetic, pharmacodynamic or clinical studies. Since 2000, the U.S. Food and Drug Administration (FDA) allows the approval of certain multisource products solely on the basis of in vitro studies, i.e. by waiving in vivo studies in humans (“Biowaiver”), based on the Biopharmaceutics Classification Scheme (BCS). The BCS characterizes APIs by their solubility and permeability in the gastrointestinal tract (GIT). The different BCS Classes I-IV (Class I: high solubility, high permeability; Class II: low solubility, high permeability; Class III: high solubility, low permeability and Class IV: low solubility, low permeability) result from all possible combinations of high and low solubility with high and low permeability. Since the adoption of the BCS by the FDA in 1995, the BCS criteria have been under continuous development. In 2006, the WHO has released the most recent bioequivalence guidance including relaxed criteria for bioequivalence studies based on modified BCS criteria. According to this guidance, APIs belonging to the BCS classes I – and under defined conditions - II and III – are eligible for a biowaiver-based approval. The principal objective of this work was to characterize the first-line anti tuberculosis APIs, isoniazid, pyrazinamide, ethambutol dihydrochloride and rifampicin, according to their physicochemical, biopharmaceutical, pharmacokinetic and pharmacological properties and to classify them according to the BCS. Ethambutol dihydrochloride and isoniazid were classified as borderline BCS class I/III APIs. Pyrazinamide was classified as a BCS class III and rifampicin as a BCS class II API. Based on the BCS classification and the additional criteria defined in the WHO bioequivalence guidance, the possibility of biowaiver-based approval for immediate release (immediate release) solid oral dosage forms containing the first-line antituberculosis drugs was evaluated. A biowaiver-based approval with defined constraints was recommended for immediate release solid oral dosage forms containing isoniazid (interaction with reducing sugars), pyrazinamide and ethambutol dihydrochloride (relative narrow therapeutic index). Rifampicin was classified as a BCS class II API, and it was concluded that rifampicin containing solid oral immediate release drug products as well as Scale-Up and Post-Approval Changes (SUPAC) changes should not be approved by a biowaiver on the following basis: (i) its solubility and dissolution are highly variable due to polymorphism and instability, (ii) concomitant intake of food and antacids reduces its absorption and bioavailability, (iii) no in vitro predictive dissolution test has been found which correlates to in vivo absorption and (iv) several publications reporting cases of non-bioequivalent and bioinequivalent rifampicin products have been located in the literature. Thus, it is recommended that bioequivalence of rifampicin containing solid oral immediate release drug products should be established by in vivo pharmacokinetic studies in humans. This risk-benefit benefit assessment of a biowaiver-based approval was presented as a poster at the American Association of Pharmaceutical Scientists (AAPS) 2005 and subsequently published as “Biowaiver Monographs” in the Journal of Pharmaceutical Sciences. Based on the assessment of the dissolution properties of the antituberculosis drugs for a biowaiver approval, quality control dissolution methodologies for the International Pharmacopoeia (Pharm. Int.) were developed, presented at the WHO expert meeting and adopted in the Pharm. Int. (http://www.who.int/medicines/publications/pharmprep/OMS_TRS_948.pdf). Additionally, preliminary biowaiver recommendations were also developed for four firstline antimalarial drugs listed on the WHO Essential Medicines List (EML): Quinine, as both the hydrochloride and sulphate, and proguanil hydrochloride were classified as borderline BCS class I/III APIs. Since quinine is a narrow therapeutic index drug and many cases of non-bioequivalence have been reported in the literature, a biowaiverbased approval was not recommended. For solid oral immediate release dosage forms containing proguanil a biowaiver-based approval was recommended under the condition that they dissolve very rapidly. Primaquine phosphate was classified as a BCS class I API. Therefore, a biowaiver-based approval was recommended for immediate release solid oral dosage forms containing primaquine phosphate. Mefloquine hydrochloride was classified as a basic, BCS class IV/II API, making it ineligible for the biowaiver. Additionally, reports of non-bioequivalence and a narrow therapeutic index were found in the scientific literature. Consequently, bioequivalence of solid oral immediate release dosage forms containing mefloquine hydrochloride should be established by in vivo pharmacokinetic studies. The results for quinine hydrochloride and sulphate, proguanil hydrochloride, primaquine diphosphate and mefloquine hydrochloride were presented as a poster at the Pharmaceutical Sciences World Congress (PSWC) 2007 and published as a WHO Collaborating Center Report in June 2006. The aim of this project was to collect, evaluate, generate and publish relevant information for a biowaiver-based approval of essential medicines in order to provide a summary to local regulatory authorities. This information complements the selected list of essential medicines by providing information about the biopharmaceutical properties and pharmaceutical quality of solid oral immediate release dosage forms containing these APIs. The aim of the biowaiver project, inspired by the WHO and brought in life by the International Pharmaceutical Federation (FIP), is to enable access to essential medicines in standardized quality at an affordable price. In this work, a significant contribution to this aim in the form of four biowaiver monographs for the antituberculosis drugs and several reports on the antimalarials has been achieved.
Höhere Eukaryoten stellen ein Ensemble von Zellen dar, die in Kompartimente unterteilt sind. Somit sind intra- und interzelluläre Transportprozesse entscheidend für das Überleben dieser Zellverbände. In meiner Arbeit habe ich Evolution und Struktur von Translokationskomplexen untersucht, um einige Aspekte dieser komplexen Systeme zu untersuchen. Eingangs befassten wir uns mit Rezeptorsystemen am Beispiel des Proteintransports. Mittels phylogenetischer Analysen fanden wir heraus, dass Pex5 nicht der Urahn der anderen untersuchten 3-TPR-Domänen ist, obwohl Pex5 in allen eukaryotischen Organismen vorkommt. Ein Vergleich der 3-TPR-Domänen mit der restlichen Sequenz des Rezeptorproteins ergab, dass die 3-TPR-Domänen eine langsamere Evolutionsgeschwindigkeit aufweisen, was für eine Evolutionseinschränkung durch Interaktionspartner spricht. Sec72 ist möglicherweise aus einer TPR1 (Hop) Domäne entstanden und eine Funktion als Hsp70-erkennende Komponente des Sec-Komplexes für den post-translationalen Import kann daraus abgeleitet werden. „Recycling“ von 3-TPR-Domänen anderer Proteine konnten wir durch unsere phylogenetische Analyse auch für die zweite 3-TPR-Domäne von Tom34 nachweisen, die mit CYP40/FKBP51/52 clustert. Darüber hinaus war es uns möglich, die plastidär bzw. mitochondriell lokalisierten Formen von Toc64 phylogenetisch zu unterscheiden. Durch Erzeugung von Homologiemodellen konnten organellspezifische Aminosäuren strukturell eingeordnet werden. Dabei stellten wir fest, dass sich fast alle Positionen, die sich in der Aminosäurekomposition unterscheiden, auf der konvexen Seite der 3-TPR-Domäne befinden. Molekulardynamische Simulationen zeigten zudem deutliche Veränderung der Hauptbewegungen der 3-TPR-Domänen nach Komplexierung mit dem Hsp90-C-Terminus. Bei Bindung des Liganden werden intramolekulare Wasserstoffbrücken sowohl auf der konvexen als auch konkaven Seite der 3-TPR-Domäne „umgeschaltet“. Diese Erkenntnisse führen zu zwei Hypothesen: 1.) die Organellspezifität der Rezeptoren wird durch die Interaktion mit anderen Komplexpartnern garantiert und 2.) die Änderungen des Wasserstoffbrückennetzwerkes auf der konvexen Seite nach Hsp90-Bindung führen zur Ausbildung der Bindungsstelle für die andere Komplexkomponente. Beide Hypothesen erklären die experimentellen Beobachtungen bezüglich der Rezeptoren und warum keine phylogenetischen Hinweise für die Existenz von Vorstufenprotein-spezifischen Hsp70/90-Proteinen gefunden werden konnten. Nach dem Rezeptor haben wir uns mit dem Translokationsprozess befasst. Wir konnten phylogenetisch zeigen, dass sich Omp85 aus Proteobakterien im Vergleich zu Cyanobakterien und Eukaryoten insbesondere durch andersartige POTRA Domänen auszeichnet und fanden zwei konservierte Motive in der Porenregion. Zudem konnten wir im Heterokontophyten P. tricornutum ein vollständiges Omp85 identifizieren (bipartite Signalsequenz, 2 POTRAs, Pore mit langen Schleifen). Die Aminosäuresequenz weicht teils deutlich von den bekannten Omp85-Proteinen ab, was die Entdeckung erschwerte. Wir haben damit geklärt, dass auch im Translokationsapparat von komplexen Plastiden ein b-Fassprotein der Omp85 Familie die Kerneinheit bildet. Ebenfalls zu den Protein-transportierenden b-Fassproteinen gehört TolC, das aber im Gegensatz zu Omp85 auch andere Substanzen, wie zum Beispiel Siderophore transportiert. Alr2887 ist das einzige TolC-ähnliche Protein aus Anabaena sp. PCC7120. Vergleichende Phänotypuntersuchungen weisen auf eine Interaktion eines ABC-Transporters (DevBCA Operon) mit Alr2887 hin. Die Distanz zwischen äußerer Membran und Plasmamembran ist in Anabaena doppelt so groß wie in E. coli. Entsprechend fanden wir im Adapterprotein DevB eine stark verlängerte dimere Doppelwendel, die das von TolC gebildete a-Fass im Periplasma bis hin zum ABC-Transporter in der Plasmamembran theoretisch fortsetzen kann. Da verschiedenste in Anabaena existierende ABC-Transporter TolC als Abflusskanal benötigen, nehmen wir an, dass Alr2887 ein Rundumtalent in Bezug auf die zu transportierenden Substrate darstellt. Dieses ist auch aufgrund der basalen Einordnung im phylogenetischen Baum zu vermuten; es könnte somit auch in den „Multi-Drug-Efflux“ involviert sein. Nicht nur ABC-Transporter, auch TonB-abhängige Transporter stehen in funktionellem Zusammenhang mit TolC. Wir haben Aminosäuresequenzen von ~4600 TBDTs aus Gram-negativen Bakterien und Cyanobakterien zusammengetragen und nach ihrer paarweisen Ähnlichkeit geclustert. Anhand experimentell charakterisierter TBDTs mit bekannten Substraten und TBDTs mit vorhergesagten Substraten konnten wir sehr vielen Clustern ein Substrat zuordnen, das die in ihnen zusammengefassten TBDTs aller Wahrscheinlichkeit nach importieren. Wir konnten ferner feststellen, dass es noch eine Menge weiterer Cluster mit unbekannten Substratspezifitäten gibt und unsere Analysen stimulieren somit die Arbeiten an diesem System im Allgemeinen und in Cyanobakterien im Besonderen.