Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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The adaptive immune system protects against daily infections and malignant transformation. In this, the translocation of antigenic peptides by the transporter associated with antigen processing (TAP) into the ER lumen is an essential step in the antigen presentation by MHC I molecules. The heterodimeric ATP-binding cassette transporter (ABC) TAP consist of the two halftransporters TAP1 and TAP2. Each monomer contains an N-terminal transmembrane domain (TMD) and a conserved C-terminal nucleotide-binding domain (NBD). Together, the TMDs build the translocation core and the NBDs bind and hydrolyze ATP, energizing the peptide transport. TAP features an asymmetry in the two ATP-binding sites that are built of several conserved motifs. One motif is the D-loop with the consensus sequence SALD. The highly conserved aspartate of the D-loop of TAP1 reaches into the canonic ATP-binding site and contacts the Walker A motif and the H-loop of the opposite NBD, while the Asp of D-loop of TAP2 is part of the non-canonic ATP-binding site.
To examine this ABC transport complex in mechanistic detail, a purification and reconstitution procedure was established with the function of TAP being preserved. The heterodimeric TAP complex was purified via a His10-tag at TAP1 in a 1:1 ratio of the subunits. Nucleotide binding to the purified transporter was elucidated by tryptophan quenching assays and the affinity constants for MgADP and MgATP were determined to be 1.0 μM and 0.7 μM, respectevely. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. Furthermore, TAP was reconstituted into proteoliposomes and the activity was tested by peptide transport and ATP hydrolysis. Interestingly, the kinetic parameters of the transporter in the reconstituted state are comparable to the data gained for TAP in microsomes.
To characterize the functional importance of the D-loop, D-loop mutants of either TAP1 or TAP2 were analyzed. Strikingly, TAP containing a mutated D-loop in TAP1 (D674A) shows an ATP-hydrolysis independent peptide translocation. Accordingly, the MHC I surface expression is similar to the wildtype situation. However, the same mutation in TAP2 (D638A) results in an ATPase dependent peptide transport similar to wildtype, whereas TAP containing mutations in both subunits leads to an inactive transporter. Although all D-loop mutants showed no altered peptide binding activity, the TAP1 mutant is inactive in peptide-stimulated ATPase activity. Strikingly, ATP or ADP binding is strictly required for the peptide translocation. Experiments carried out in proteoliposomes demonstrate that wildtype TAP can export peptides against their gradient when low peptide concentrations are offered. In contrast, the D674A mutant can facilitate peptide translocation along their concentration gradient in the two directions. At high peptide concentrations, TAP is trapped in a transport incompetent state induced by trans-inhibition. In conclusion, a TAP mutant that uncouples solute translocation from ATP hydrolysis was created. Since this passive substrate movement is strictly dependent on binding of ATP or ADP, an active transporter was turned into a “nucleotide-gated facilitator”.
In a cysteine cross-linking approach the conformational changes of TAP during peptide transport and the flexibility of the nucleotide binding domains were examined. Single cysteines were introduced in the D-loops of TAP1 and TAP2. Cross-linking by copper-phenantroline (CuPhe) was possible for all combinations. However, by adding ATP, ADP or peptide to the TAP complex no differences in the cross-linking efficiency were detected. By CuPhe cross-linking TAP was trapped in a conformation, in which the peptide binding site was not accessible. To complete a transport cycle, a flexibility of at least 17.8 Å of the NBDs is needed, since TAP cross-linked by CuPhe (2.0 Å) or bismaleimidoethane (BMOE, 8.0 Å) was transport inactive but when TAP was cross-linked by 1,11-bismaleimido-triethyleneglycol (BM[PEG]3, 17.8 Å) transport activity was preserved.
An exciting in vivo function of ATP-sensitive potassium channels in substantia nigra dopamine neurons Ð Implications for burst firing and novelty coding ÐPhasic burst activity is a key feature of dopamine (DA) midbrain neurons. This particular pattern of excitation of DA neurons occurs via a synaptically triggered transition from low-frequency background spiking to transient high-frequency discharges. Burst-firing mediated phasic DA release is critical for flexible switching of behavioural strategies in response to unexpected rewards, novelty and other salient stimuli. However, the cellular and molecular bases of burst signalling in distinct DA subpopulations of the substantia nigra (SN) or the ventral tegmental area (VTA) are unknown.
DA neuron excitability is controlled by synaptic network inputs, neurotransmitter receptors and ion channels, which generate action potentials and determine frequency and pattern of electrical activity in a complex interplay. ATP-sensitive potassium (K-ATP) channels are widely expressed throughout the brain, where in most cases they are believed to act as metabolically-controlled 'excitation brakes' by matching excitability to cellular energy states. However, their precise physiological in vivo function in DA neurons remains elusive.
To study burst firing and the underlying ionic mechanisms with single cell resolution, in vivo single-unit recordings were combined with juxtacellular neurobiotin labelling as well as immunohistochemical and anatomical identification of individual DA neurons. In vivo recordings were performed in adult isoflurane-anaesthetised wildtype (WT) and global K-ATP channel knockout mice, lacking the pore forming Kir6.2 subunit (Kir6.2-/-). In addition, DA cell-selective functional silencing of K-ATP channel activity in vivo was established using virus-mediated expression of dominant-negative Kir6.2 subunits. Careful control experiments ruled out any significant contributions from nonDA neurons as transduction was effectively limited to SN DA neurons rather than affecting those cells that innervate them. Virus-based K-ATP channel silencing in combination with juxtacellular recording and labelling was achieved to define the electrophysiological phenotype of individually identified, virally-transduced DA neurons in vivo.
Single-unit recordings revealed that K-ATP channels Ð in contrast to their conventional hyperpolarising role Ð in a subpopulation of DA neurons located in the medial SN (m-SN) act as cell-type selective gates for excitatory burst firing in vivo. The percentage of spikes in bursts was threefold reduced in Kir6.2-/- compared to WT mice. Classification of firing patterns based on visual inspection of autocorrelation histograms and on a newly developed spike-train-model confirmed the dramatic shift from phasic burst to tonic single-spike oscillatory firing in Kir6.2-/-. This significant decrease of burstiness was selective for m-SN DA neurons and was not exhibited by DA cells in the lateral SN or VTA. Virus-based K-ATP channel silencing in vivo unequivocally demonstrated that the activity of postsynaptic K-ATP channels was sufficient to disrupt bursting in m-SN DA neuron subtypes. Patch-clamp recordings in brain slices indicated an essential role of K-ATP channels for NMDA-mediated in vitro bursting. In accordance with previous studies in DA midbrain neurons, NMDA receptor stimulation triggered burst-like firing in m-SN DA cells in vitro, but only when K-ATP channels were co-activated in these neurons.
K-ATP channel-gated burst firing in m-SN DA neurons might be functionally relevant in awake, freely moving mice. To explore the behavioural consequences of SN DA neuron subtype-selective K-ATP channel suppression, spontaneous open field (OF) behaviour of mice with bilateral K-ATP silencing across the whole SN (medial + lateral) or in only the lateral SN was tested. Analysis of WT and global Kir6.2-/- mice showed reduced exploratory locomotor activity of Kir6.2-/- in a novel OF environment. Remarkably, K-ATP channel silencing in m-SN DA neurons phenocopied this novelty-exploration deficit, indicating that K-ATP channel-gated burst firing in medial but not lateral SN DA neurons is crucial for WT-like novelty-dependent exploratory behaviour.
In summary, a novel role of K-ATP channels in promoting the excitatory switch from tonic to phasic firing in vivo in a cell-type specific manner was discovered. The present PhD thesis provides several important insights into the pivotal function of K-ATP channels in medial SN DA cells, which project to the dorsomedial striatum, for burst firing and its important consequences for context-dependent exploratory behaviour.
In collaboration with two other research groups transcriptional up-regulation of K-ATP channel and NMDA receptor subunits and high levels of in vivo burst firing were detected in surviving SN DA neurons from Parkinson's disease (PD) patients Ð providing a potential link of K-ATP channel activity to neurodegenerative pathomechanisms of PD. Using high-resolution fMRI imaging another study in humans has recently identified distinct DA midbrain regions that are preferentially activated by either reward or novelty. Taken together, these human data and the results of the present PhD thesis suggest that burst-gating K-ATP channel function in SN DA neurons impacts on phenotypes in disease as well as in health.
The environmental impact of climate change is meanwhile not only discussed in the scientific community but also in the general public. However, little is known about the interaction between climate change and pollutants like pesticides. A combination of multiple stressors (e.g. temperature, pollutants, predators) may lead to severe alterations for organisms such as changes in time of reproduction, reproductive success and growth performance, mortality and geographic distribution. The questions if aquatic organisms tend to react more sensitive towards incidents under climate change conditions remains. Therefore, within the present thesis the aquatic ecotoxicological profile of the fungicide pyrimethanil, as an exemplarily anthropogenic used contaminant, was examined.
A large test battery of ecotoxicological standard tests and supplement bioassays with non-model species was conducted to investigate if species-specific or life stage-specific differences occur or if temperature alteration may change the impact of the fungicide. Two of the most sensitive species (Chironomus riparius and Daphnia magna) were used to investigate the acute and chronic thermal dependence of pyrimethanil effects. The results clearly depict that the ecotoxicity of pyrimethanil at optimal thermal conditions did not depend on the trophic level, but was species-specific. With regard to EC10 values the acute pyrimethanil toxicity on C. riparius increased with higher temperature (6.78 mg L-1 at 14°C and 3.06 mg L-1 at 26°C). The chronic response of D. magna to the NOEC (no observed effect concentration) of the fungicide (0.5 mg L-1) was examined in an experiment which lasted for several generations under three simulated near-natural temperature regimes (‘cold year, today’ (11 to 22.7°C), ‘warm year, today’ (14 to 25.2°C) and ‘warm year, 2080’ (16.5 to 28.1°C)). A pyrimethanil-induced mortality increase was buffered by the strongly related increase of the general reproductive capacity, while population growth was stronger influenced by temperature than by the fungicide. At a further pyrimethanil concentration (LOEC – lowest observed effect concentration: 1 mg L-1), a second generation could not be established by D. magna under all thermal regimes.
Besides daphnids, the midge C. riparius was used for a second multigeneration study. In a bifactorial test design it was tested if climate change conditions alter or affect the impact of a low fungicide concentration on life history and genetic diversity. The NOAEC/2 (half of the no observed adverse effect concentration derived from a standard toxicity test) was used as a low pyrimethanil concentration to which laboratory populations of the midges were chronically exposed under the mentioned temperature scenarios. During the 140-day-multigeneration study, survival, emergence, reproduction, population growth, and genetic diversity of C. riparius were analyzed. The results reveal that high temperatures and pyrimethanil act synergistically on life history parameters of C. riparius. In simulated present-day scenarios, a NOAEC/2 of pyrimethanil provoked only slight to moderate beneficial or adverse effects. In contrast, an exposure to a NOAEC/2 concentration of pyrimethanil at a thermal situation likely for a summer under the future expactations uncovered adverse effects on mortality and population growth rate. In addition, genetic diversity was considerably reduced by pyrimethanil in the ‘warm year, 2080’ scenario, but only slightly under current climatic conditions. The multigeneration studies under near-natural thermal conditions indicate that not only the impact of climate change, but also low concentrations of pesticides may pose a reasonable risk for aquatic invertebrates in the future. This clearly shows that thermal and multigenerational effects should be considered when appraising the ecotoxicity of pesticides and assessing their future risk for the environment.
In addition to temperature further multiple abiotic and biotic stressors alterate pollutant effects. Moreover, to better discriminate and understand the intrinsic and environmental correlates of changing aquatic ecosystems, it was experimentally unraveled how the effects of a low-dose of pyrimethanil on daphnids becomes modified by different temperatures (15°C, 20°C, 25°C) and in the presence/ absence of predator kairomones of Chaoborus flavicans larvae. The usage of a fractional multifactorial test design provided the possibility to investigate the individual growth, reproduction and population growth rate of Daphnia pulex via different exposure routes to the fungicide pyrimethanil at an environmentally relevant concentration (0.05 mg L-1) - either directly (via the water phase), indirectly (via algae food), dually (via water and food) or for multiple generations (fungicide treated source population).
The number of neonates increased with increasing temperatures. At a temperature of 25°C no significant differences between the individual treatment groups were observed although the growth was overall inhibited due to pyrimethanil. Besides, at 15 and 20°C it is obvious that daphnids which were fed with contaminated algae had the lowest reproduction and growth rate. The obtained results clearly demonstrate that multiple stress factors can modify the response of daphnids to pollutants. The exposure routes of the contaminant are of minor importance, while temperature and the presence of a predator are the dominant factors impacting the reproduction of D. pulex. It can be concluded that low concentrations of pyrimethanil may disturb the zooplankton community at suboptimal temperature conditions, but the effects will become masked if chaoborid larvae are present. Therefore it seems necessary to observe prospectively if the combination of several stress factors like pesticide exposure and suboptimal temperature may influence the life history and sensitivity of several aquatic invertebrates differently.
Besides standard test organisms it is inevitable to conduct test with aquatic invertebrate which are not yet considered regularly in ecotoxicological experiments. For example molluscs represent one of the largest phyla of macroinvertebrates with more than 100.000 species, being ecologically and economically important. Therefore, within the present study embryo, juvenile, half- and full-life cycle toxicity tests with the snail Physella acuta were performed to investigate the impact of pollutants on various life stages. Different concentrations of pyrimethanil (0.06-0.5 or 1.0 mg L-1) assessed at three temperatures (15°C, 20°C, 25°C) revealed that pyrimethanil caused concentration-dependent effects independent of temperature. Interestingly, the ecotoxicity of pyrimethanil was higher at lower temperature for the embryo hatching and F1 reproduction, but its ecotoxicity for the growth of juveniles and the F0 reproduction increased with increasing temperature. More specifically, it could have been observed that especially during the reproduction test high mortality rates occurred at the highest concentration of 1 mg L-1 at all temperatures. Due to high mortality rates no snails were available for the F1 at the highest concentrations (0.5 and 1.0 mg L-1). Compared to the F0, overall more egg masses were produced in the F1, being all fertile and no mortality occurred. For the F1-generation the strongest pyrimethanil effects were detected at 15°C. A comparison of effect concentrations between both generations showed that the F1 is more sensitive than the F0.
These results indicate that an exposure over more than one generation may give a better overview of the impact of xenobiotics. With the establishment of an embryo and reproduction test under different temperatures and various concentrations of pyrimethanil with P. acuta we could successfully show that molluscs can respond more sensitive than model organisms and that both, chemical and thermal stressor strongly influence the behaviour of the pulmonates. It can be concluded that the high susceptibility for the fungicide observed in gastropods clearly demonstrates the complexity of pesticide-temperature interactions and the challenge to draw conclusions for the ecotoxicological risk assessment of pesticides under the impact of global climate change.
Biochemical and functional analysis of the ubiquitin binding properties of the NF-κB regulator NEMO
(2012)
Posttranslationale Modifikationen regulieren wesentliche Eigenschaften von Proteinen, wie z. B. Lokalisation, Konformation, Aktivität, Stabilität und Interaktionsfähigkeit. Eine besondere Form der Proteinmodifikation ist die Ubiquitylierung, bei der das kleine Protein Ubiquitin mit seinem C-Terminus kovalent an ein Substratprotein gebunden wird.
Die am besten untersuchte Funktion der Ubiquitylierung ist die Markierung eines Substrates für den Abbau durch das Proteasom. In den letzten Jahren wurde jedoch entdeckt, dass Ubiquitylierung in vielen Bereichen der Zelle eine wichtige Rolle spielt. Dazu gehören der Transport von Vesikeln, die Reparatur von DNA-Schäden und zelluläre Signalübertragung. Ubiquitin kann verschieden-artige Ketten bilden, indem ein Ubiquitin an eines der sieben Lysine (K6, K11, K27, K29, K33, K48, K63) oder den N-Terminus eines anderen gebunden wird. Diese unterschiedlichen Kettentypen regulieren verschiedene Prozesse. Z. B. dienen K48-verknüpfte Ubiquitinketten als Signal für den proteasomalen Abbau, wohingegen über K63 verknüpfte Ketten hauptsächlich eine Rolle bei Signalübertragungen spielen.
Die meisten Funktionen die durch Ubiquitylierung reguliert werden, werden durch Ubiquitinrezeptoren vermittelt, die eine Ubiquitinbindedomäne (UBD) besitzen. Manche UBDs binden selektiv nur einen Ubiquitinkettentyp und sind somit in der Lage gezielt Prozesse regulieren zu können, indem sie nur durch diesen speziellen Kettentyp aktiviert werden.
Das Protein NEMO ist ein Ubiquitinrezeptor, dessen UBD UBAN selektiv bestimmte Ubiquitinketten bindet. NEMO spielt eine zentrale Rolle bei der Aktivierung der Transkriptionsfaktorfamilie NF-κB, indem es den IKK-Kinasekomplex reguliert. Dieser Kinasekomplex sorgt durch die Phosphorylierung des NF-κB-Inhibitors IκBα für dessen proteasomalen Abbau, wodurch schließlich NF-κB aktiviert wird. Die NF-κB-Aktivierung kann u. a. durch den TNF-Rezeptor (TNFR) induziert werden. Am aktivierten TNFR werden viele Proteine durch verschiedene Ubiquitinketten modifiziert. Bisher wurde angenommen, dass die spezifische Bindung von NEMO an K63-verknüpfte Ubiquitinketten ausschlaggebend für die Aktivierung von IKK ist. Jedoch spielen lineare Ubiquitinketten, die über den N-Terminus verknüpft sind, auch eine wichtige Rolle bei der Aktivierung von NF-κB und die UBAN von NEMO hat eine sehr hohe Affinität zu linearen Ubiquitinketten.
Um die genauen Vorgänge zu verstehen, die zur Aktivierung von NF-κB am TNFR führen, ist es nötig, zu analysieren, welche Proteine mit welchen Ubiquitinketten modifiziert werden und welche Ubiquitinrezeptoren daran binden.
In dieser Studie sollte detailliert untersucht werden, mit welchen Ubiquitin-ketten NEMO bevorzugt interagiert. Dazu wurden in vitro-Bindungsstudien mit bakteriell aufgereinigtem NEMO und verschiedenen Ubiquitinketten durchgeführt. Des Weiteren sollte geprüft werden, wie die Bindung von NEMO an bestimmte Ubiquitinketten die Aktivierung von NF-κB reguliert.
Dabei ergab sich, dass sowohl NEMO in voller Länge, als auch die UBAN, bevorzugt mit linearen Ubiquitinketten interagieren, wohingegen die Interaktion von NEMO mit anderen Ubiquitinketten relativ schwach ist. Ausgehend von einer Kristallstruktur eines Komplexes aus der NEMO-UBAN und linearem di-Ubiquitin, wurden NEMO-Mutanten generiert, die seletkiv die Bindung von NEMO an lineare Ubiquitinketten verhindern, während die schwache Bindung von NEMO an längere K63-verknüpfte Ketten erhalten blieb. Um die Relevanz der Interaktion von NEMO mit linearen Ubiquitinketten für die Aktivierung von NF κB zu überprüfen, wurden diese NEMO-Mutanten dann verwendet um Zellen die kein NEMO exprimieren zu rekonstituieren. Nach Stimulation dieser Zellen mit TNFα wurde NF-κB kaum aktiviert, womit gezeigt werden konnte, dass NEMO gezielt an lineare Ubiquitinketten binden muss, um NF-κB zu aktivieren. Zusätzlich zu seiner Rolle bei der Aktivierung von NF-κB ist NEMO ein wichtiger Inhibitor der durch den TNFR induzierten Apoptose. In dieser Studie wurde gezeigt, dass diese Apoptoseinhibierung abhängig von der Bindung von NEMO an lineare Ubiquitinketten ist, da die Zellen die NEMO-Mutanten exprimierten, die keine linearen Ketten binden können, durch Apoptose starben, währen Wildtyp-Zellen überlebten.
Zusammenfassend konnte in dieser Studie gezeigt werden, dass NEMO bevorzugt und mit vergleichsweise hoher Affinität an lineare Ubiquitinketten bindet und dass diese spezifische Bindung wichtig für die Inhibierung von TNFR-induzierter Apoptose sowie für die Aktivierung von NF-κB ist.
The universal biological energy currency adenosine triphosphate (ATP) is synthesized by the F1Fo-ATP synthase in most living organisms. The overall structure and function of F-type ATPases is conserved in the different organisms. The F1Fo-ATP synthase consist of two domains; the soluble F1 complex has the subunit stoichiometry α3β3γδε and the membrane embedded Fo complex consists of subunits ab2c10-15 in its simplest form found in bacteria. F1 and Fo both function as reversible rotary motors that are connected by a central stalk (γε) and a peripheral stalk (b2δ).
For ATP synthesis, the electrochemical energy formed by a proton or sodium ion gradient is required. The ion translocation across the Fo subcomplex induces torque in the motor part of the enzyme (cnγε), which causes conformational changes in the α3β3 domain leading to ATP synthesis from ADP and inorganic phosphate (Pi) catalyzed in the β-subunits. ATP hydrolysis causes a reverse torque in the Fo subcomplex triggering uphill ion translocation from cytoplasm to periplasm, and the enzyme functions as an ion pump.
The ATP synthesis mechanism is well understood, since several high-resolution structures of F1 are available. In contrast, the ion translocation mechanism across the membrane, mediated by the Fo subcomplex, is not understood in its structural detail.
Subunit a and the c-ring form an ion pathway, but subunit b is needed to form an active ion translocation pathway in both H+- and Na+-dependent systems. Several high-resolution structures of c-rings have provided insights in the ion translocation mechanism. The different ion translocation models based on biochemical, biophysical and structural analysis are in agreement in the fact that ions are translocated through a periplasmic ion access pathway in subunit a to the middle of the membrane and there to the binding site of a c-subunit. After almost a whole rotation of the c-ring the ion returns into the a-c interface, where it can be released to the cytoplasm. In the different models the cytoplasmic access pathway has been proposed to be located in subunit a, at the a-c interface or within the c-ring. The driving force of torque generation has been proposed to be the pH gradient or membrane potential. Several biochemical studies show that a conserved arginine in helix four of subunit a (R226 in Ilyobacter tartaricus or R210 in Escherichia coli)plays a critical role in the ion translocation. The arginine has been proposed to function as an electrostatic separator between the cytoplasmic and periplasmic pathways and as a mediator of the ion exchange into the c-ring ion-binding site.
Structural data of a related enzyme (V1Vo-ATPase from Thermus thermophilus) has provided insight into the helical arrangement of the ion translocating subunits I and Lring (related to subunit a and the c-ring). These structures indicated a small interface between subunit I and the L-ring, and two four-helix bundles in the N-terminal domain of subunit I were proposed to build the periplasmic and cytoplasmic ion pathways. To comprehend the ion-translocation and torque generation mechanism in F1Fo-ATP synthase, structural data of an intact a-c complex is needed.
The goal of this work was to obtain structural data of subunit a, most preferably in a complex with the c-ring or additionally with subunit b. Therefore, a new purification procedure for the I. tartaricus Fo-subcomplex, heterologously expressed in E. coli cells, was established. The purified Fo was characterized biochemically and by Laserinduced liquid bead ion desorption mass spectrometry (LILBID-MS). These analyses showed that pure and completely assembled Fo containing all its subunits in the correct stoichiometry (ab2c11) was obtained. The purified Fo complex was stable at 4°C for several months and at room temperature in the presence of lipids for several weeks. A lipid analysis was performed by thin-layer chromatography (TLC) to investigate the qualitative lipid composition of I. tartaricus whole lipid extract and various I. tartaricus F1Fo isolates. The whole lipid extract contained PC, PG and PE lipids and probably cardiolipin. PC, PG and PE lipids were bound to wild type I. tartaricus F1Fo, whereas recombinant I. tartaricus F1Fo did not have any bound lipids, but was able to bind the synthetic lipids POPC and POPG if they were provided during the purification.
For subsequent structural studies the purified Fo was subjected to two-dimensional (2D) crystallization trials. Vesicles and sheets tightly packed with protein and crystals with a rare plane group for I. tartaricus c11 (p121) were obtained. The c-ring was visible in the CCD images, and immunogold-labeling revealed the presence of the His-tagged a-subunit in the reconstituted vesicles. Furthermore, atomic force microscopy (AFM) imaging showed protein densities next to the c-rings, which protruded less from the membrane (0.4±0.1 nm) than the c-ring (0.7±0.1 nm). These protein densities presumably belonged to subunit a.
Cryo-electronmicroscopy (cryo-EM) was used to collect data of the p121 crystals and a merged projection density map was calculated to 7.0 Å resolution. The unit cell of the crystals (81 × 252 Å) contained two asymmetric units with three c-rings in each and next to the c11-rings new prominent densities were visible. In each extra density up to 7 transmembrane helices were visible, belonging to the stator subunit a and/or subunit b. To elucidate whether there are conserved elements in the three extra densities non-crystallographic averaging was applied using a single-particle approach.
Six possible arrangements for the c-rings and the extra densities were identified and used for the averaging. The extra densities were enhanced only in one of the possible arrangements. The average showed a four-helix bundle and a fifth helix in close proximity to the c-ring. Two more helices were present in each position but their position was ambivalent. The data obtained in this work provides the first insight in the helical arrangement in the a-c interface of F1Fo-ATP synthase.
Im Rahmen dieser Arbeit konnte die Bindeeigenschaft des synaptischen Vesikelproteins SV31 zu den divalenten Metallionen Zn2+, Ni2+ sowie Cu2+ nachgewiesen und reproduziert werden. Die Bindung an Zn2+ wurde dabei sowohl in vitro an der Sepharosesäule als auch in vivo in NGF-differenzierten PC12-Zellen bestätigt (3.2.1 - 3.2.3). In einer Kollaboration mit dem Max-Planck-Institut für Biophysik wurde des Weiteren eine mögliche Zinktransportfunktion von SV31 untersucht. Dafür wurde die Ladungstranslokation durch myc-SV31-enthaltene CHO-Zellmembranen nach Zinkzugabe gemessen (3.2.5). Weiterhin konnte durch subzelluläre Fraktionierung von PC12-Zellen ein Verteilungsmuster des neuen Proteins in Mikrosomen unterschiedlicher Dichte dokumentiert werden. Durch die andauernde Expression von SV31-RFP in stabil transfizierten PC12-Zellen kommt es außerdem zur Beeinflussung des Expressionsmusters zahlreicher Markerproteine und damit einhergehend zu einer Dichteverschiebung somatischer Organellen (3.3.1 - 3.3.3). Kolokalisationsstudien von SV31 mit Markerproteinen zahlreicher Zellorganellen ergaben partielle Fluoreszenzüberlagerungen mit synaptischen Vesikelproteinen sowie eine Anreicherung von SV31 in Nähe der Plasmamembran. In diesem Zusammenhang zeigt sich ebenfalls eine Übereinstimmung der Lokalisation von SV31 mit den SNAREProteinen SNAP25 und Syntaxin1A (3.4.1 - 3.4.3). Die Ergebnisse der vorliegenden Arbeit erweitern nicht nur das Wissen um die funktionellen Eigenschaften von SV31, sie geben auch Anlass zum Nachdenken über mögliche Interaktionspartner des neuen Vesikelproteins. Die Fähigkeit zur Zinkbindung und -akkumulation auf präsynaptischer Seite rückt SV31, im Hinblick auf neurodegenerative Erkrankungen wie Alzheimer und Parkinson, auch in einen medizinisch relevanten Kontext. Durch Deduktion der hier aufgezeigten Ergebnisse entsteht ein erweitertes Verständnis der Relevanz von SV31 als funktionelle, zinkbindende Einheit im Rahmen der synaptischen Transmission.
BMPs control postnatal dendrite growth and complexity in sympathetic neurons / von Afsaneh Majdazari
(2012)
The vertebrate nervous system is a complex network of billions of neurons connected by dendrites and axons, integrated to functional circuits and areas/organs in the central and peripheral nervous system. The cells of the nervous system origin from common progenitors, which take on different cell fates based on intrinsic and extrinsic factors. These factors determine general neuronal traits, but also the morphology and the type of connections made to other cells. Mechanisms underlying axonal and dendritic growth are well described in contrast to the initiation of neurite growth, which remains to be fully elucidated, especially concerning dendrite formation. Recently BMPs have been identified as candidate dendrite inducing factors in sympathetic, cortical and hippocampal neurons. Here we focus on the in vivo role of BMPs on dendrite growth in sympathetic neurons as their development and differentiation processes have been analyzed in detail.
The importance of RNA in molecular and cell biology has long been underestimated. Besides transmitting genetic information, studies of recent years have revealed crucial tasks of RNA especially in gene regulation. Riboswitches are natural RNA-based genetic switches and known only for ten years. They directly sense small-molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within recent years, artificial riboswitches have been developed that operate according to user-defined demands. Hence, they represent powerful tools for synthetic biology.
This study focused on the development of engineered catalytic riboswitches for conditional gene expression in eukaryotes. A self-cleaving hammerhead ribozyme was linked to a tetracycline binding aptamer in order to regulate ribozyme cleavage allosterically with tetracycline. By integrating such a hybrid molecule into a gene of interest, mRNA cleavage and thereby gene expression is controllable in a ligand dependent manner. The linking domain between ribozyme and aptamer was randomised. Tetracycline inducible ribozymes were isolated after eleven cycles of in vitro selection (SELEX). 80% of the analysed ribozymes show cleavage that strongly depends on tetracycline. In the presence of 1 μM tetracycline, their cleavage rates are comparable to that of the parental hammerhead ribozyme. In the absence of tetracycline, cleavage rates are inhibited up to 333-fold. The allosteric ribozymes bind tetracycline with similar affinity and specificity as the parental aptamer. Ribozyme cleavage is fully induced within minutes after addition of tetracycline. Interestingly, the isolated linker domains exhibit structural consensus motives rather than consensus sequences.
When transferred to yeast, three switches reduced reporter gene expression by 30 - 60% in the presence of tetracycline; none of them controlled gene expression in mammalian cells. In vitro selected molecules do not necessarily retain their characteristics when applied in a cellular context. Therefore, high throughput screening and selection systems have been developed in mammalian cells. The screening system is based on two fluorescent reporter proteins (GFP and mCherry). 1152 individual constructs of the selected ribozyme pool were tested, but none of them reduced reporter gene expression significantly in the presence of tetracycline. The selection system employs a fusion peptide encoding two selection markers (Hygromycin B phosphotransferase and HSV thymidine kinase) facilitating both negative and positive selection. 6.5 x 104 individual constructs of the selected ribozyme pool are currently under investigation.
Das Burkitt Lymphom ist ein aggressives B-Zelllymphom, das in tropischen Regionen Afrikas und in Neu Guinea endemisch auftritt und vor allem bei Kindern vorkommt. Die sporadische Form des Burkitt Lymphoms tritt weltweit in geringerer Häufigkeit auf und betrifft alle Altersschichten. In nahezu allen endemischen Fällen ist das Epstein-Barr Virus in den Tumorzellen nachweisbar, jedoch nur in ca. 20 % der sporadischen Fälle. Der Beitrag von EBV zur Entstehung EBV-positiver Burkitt Lymphome ist seit über 50 Jahren EBV-Forschung ungeklärt. Im Jahr 2004 wurden im Genom des Epstein-Barr Virus eine Reihe von microRNAs entdeckt, die potentiell für die Pathogenese des EBV-positiven Burkitt Lymphoms relevant sein könnten. Da die Expression der viralen microRNAs seither für das Burkitt Lymphom nur unvollständig beschrieben worden sind, wurden sie in dieser Arbeit systematisch analysiert und dadurch ein vollständiges Expressionsprofil erstellt. Es konnte dabei keine Unterscheidung zwischen endemischen und sporadischen Fällen erreicht werden, jedoch wurden hierbei erstmals Fälle identifiziert, die trotz nachgewiesener EBV-Assoziation keine viralen microRNAs enthielten. Neben den viralen microRNAs könnten im Burkitt Lymphom auch die zellulären microRNAs für die Tumorentstehung von Bedeutung sein. Deshalb wurde in dieser Arbeit auch die Expression der zellulären microRNAs aus Burkitt Lymphom-Biopsien charakterisiert. Durch hierarchisches „Clustering“ bildeten sich drei Gruppen, die hauptsächlich durch An- und Abwesenheit von zwei microRNAs (miR21 und miR92a) definiert wurden, denen onkogenes Potential zugeschrieben wird. Die Expressionsmuster der einzelnen Gruppen weisen auf zelluläre Mechanismen der Pathogenese des Burkitt Lymphoms hin.
Die genetische Charakteristik des Burkitt Lymphoms ist eine Chromosomentranslokation, welche das Protoonkogen c MYC unter die Kontrolle von regulatorischen Elementen der Immunglobulingene bringt. Durch die somit erhöhte Transkription von c-MYC entfaltet das Genprodukt sein onkogenes Potential. Mutationen im offenen Leserahmen können dieses Potential zusätzlich verstärken. Da c MYC ein pleiotroper Transkriptionsfaktor ist und somit auf eine ganze Reihe zellulärer Prozesse Einfluss hat, bewirkt die Translokation massive Veränderungen in der Zelle. Vorangegangene Untersuchungen der Arbeitsgruppe zeigten, dass die antivirale Interferonantwort durch hohe c MYC-Expression unterdrückt wird. Diese Beobachtung liefert eine mögliche Erklärung für die Immunevasion von Burkitt Lymphom-Zellen, trotz Anwesenheit des EBV-Genoms. In Zelllinien, die aus Burkitt Lymphom-Biopsien generiert wurden, konnte gezeigt werden, dass EBV eine Interferoninduktion auslöst, die durch c-MYC unterdrückt wird. In dieser Arbeit konnte auch gezeigt werden, dass Epstein-Barr-virale Nukleinsäureprodukte durch den zytosolischen Rezeptor RIG-I Interferon induzieren, dieser aber durch die hohe c-MYC-Expression transkriptionell gehemmt wird. Neben RIG-I wurden weitere Rezeptoren und Mediatoren der Interferoninduktionskaskade identifiziert, die ebenfalls transkriptionell von c-MYC unterdrückt werden. Diese Ergebnisse stützen die Hypothese, dass c-MYC durch Unterdrückung der angeborenen Immunität die Immunevasion von Burkitt Lymphom-Zellen ermöglicht.
Die 5-Lipoxygenase (5-LO) ist eines der Schlüsselenzyme der Leukotrienbiosynthese. Sie katalysiert zunächst die Umsetzung der freigesetzten Arachidonsäure(AA) zu 5-Hydroperoxyeicosatetraensäure (5-HpETE), in einem zweiten Reaktionsschritt wandelt sie diese in Leukotrien A4 (LTA4) um. Leukotriene sind potente Entzündungsmediatoren und spielen eine wichtige Rolle bei entzündlichen und allergischen Reaktionen. Außerdem wird die Beteiligung an verschiedenen Krebsarten kontrovers diskutiert.
Sie besteht aus 673AS, ist 78 kDa schwer und gliedert sich wie alle bisher bekannten Lipoxygenasen in eine N-terminale C2-ähnliche, regulatorische Domäne(AS 1–114) (C2ld), die für die Membran- und Calciumbindung sowie die Interaktion mit dem Coactosin-like Protein (CLP) verantwortlich ist, und in eine C-terminale, katalytische Domäne (AS 121–673), die das Nicht-Häm-gebundene Eisen im aktiven Zentrum trägt. Ein weiteres Strukturmerkmal sind zwei ATP-Bindungsregionen, eine befindet sich in der C2ld (AS 73–83), die andere auf der katalytischen Domäne (AS 193–209), das molare Verhältnis von 5-LO zu ATP konnte dabei auf 1:1 festgelegt werden [167].
Bereits 1982 wurde in einer Veröffentlichung von Parker et al. beschrieben, dass 5-LO aus Rattenzellen in Gegenwart von Calcium auf einer Gelfiltration dimerisieren kann [204], 2008 schließlich wurde von Aleem et al. publiziert, dass humane 12-LO aus Thrombozyten Dimere bilden kann [219]. Somit konnte es möglich sein, dass auch die humane 5-LO zur Dimerisierung fähig ist.
Zunächst wurde aufgereinigtes Enzym mit nativer Gelelektrophorese und anschließender Coomassiefärbung oder Western Blot untersucht, dabei konnten mehrere Banden pro Bahn detektiert werden. Um dieses Phänomen weiter zu untersuchen, wurde im Anschluss eine Gelfiltration etabliert; da die C2ld der 5-LO recht hydrophob ist, war es nötig, 0,5% T20 zum Elutionspuffer PBS/EDTA zuzusetzen, da das Enzym ansonsten unspezifisch mit dem Säulenmaterial interagiert und für seine Größe zu spät eluiert hätte. In Anwesenheit von T20 eluierte 5-LO in zwei getrennten Peaks, die exakt zu den vorher mit Referenzproteinen bestimmten Elutionsvolumina des Monomers und Dimers passten. Weiter wurde getestet, ob niedermolekulare Substanzen einen Einfluss auf das Dimerisierungsverhalten haben, allerdings konnte weder durch Ca2+noch durch ATP eine Verstärkung der Dimerisierung beobachtet werden. Dahingegen konnte, nach Vorinkubation mit GSH und Diamid, das alleinige Monomer auf der Gelfiltration nachgewiesen werden, nach Vorinkubation nur mit Diamid, lag das gesamte Protein ausschließlich als Dimer vor. Durch Gelelektrophorese mit oder ohne Zusatz von ß-Mercaptoethanol und LILBID-MS konnte die Ausbildung von intermolekularen Disulfidbrücken bestätigt werden. Ein Bindungsassay mit radioaktivem 35S-GSH konnte die kovalente Bindung des GSH an die 5-LO bestätigen. Quantifizierungsstudien mit Ellmans Reagens zeigten, dass mindestens eins der Oberflächencysteine mit GSH modifiziert wurde. Die von der Gelfiltration erhaltenen Fraktionen wurden auf enzymatische Aktivität getestet und in allen 5-LO-haltigen Fraktionen konnte Aktivität gefunden werden. Leider war es nicht möglich, eine Aussage darüber zu treffen, ob das Mono- oder das Dimer aktiver war. Es liegt offenbar in einem Fließgleichgewicht vor, da erneute Injektion des Monomerpeaks im bekannten Elutionsprofil aus zwei Peaks resultierte. Außerdem führt die Anwesenheit von 0,5% T20 während des Aktivitätstests zu einer Hemmung des Enzyms und weniger detektierbaren 5-LO-Produkten; es fiel vor allem auf, dass so gut wie keinerlei trans- und epitrans-LTB4, die nicht-enzymatischen Zerfallprodukte der 5-HpETE, nachzuweisen waren. Betrachtet man die Struktur der 5-LO, so findet man zehn Cysteine an der Oberfläche; die Cysteine 159, 300, 416 und 418 liegen dabei in einem Interface. Mutiert man diese Cysteine zu Serinen, so verschwindet der Dimer-induzierende Effekt des Diamids, wohingegen die Mutante weiterhin glutathionylierbar bleibt. Interessanterweise zeigt diese Mutante auch eine wesentlich weniger ausgeprägte Hemmung durch T20. Um eine Aussage treffen zu können, ob auch 5-LO aus humanen Zellen Dimere bilden kann, wurde 5-LO-haltiger S100 aus polymorphkernigen Leukozyten (PMNL) untersucht. Dabei konnte mit Western Blot und einem Aktivitätsnachweis gezeigt werden, dass die 5-LO in einem breiten Bereich von der Gelfiltration eluiert. Das deutet darauf hin, dass sie in PMNL ebenfalls dimerisiert vorliegen kann. In Gegenwart von Ca2+kam es zu einer Verschiebung der 5-LO zu höhermolekularen Gewichten, wobei dieses Phänomen nicht bei S100 aus transformierten E.coli auftrat, was auf einen gerichteten Komplex nach Calciuminduktion in PMNL hindeutet.
Außerdem wurde im Rahmen dieser Arbeit der Bindemodus von Sulindac an die 5-LO mittels Crosslinking untersucht. Dabei konnte gezeigt werden, dass konzentrationsabhängig der einfache Komplex aus 5-LO und CLP abnimmt, dafür aber ein hochmolekularer Komplex, der beide Enzyme enthält, entsteht. Weder das Prodrug Sulindac noch der weitere Metabolit Sulindacsulfon oder andere Inhibitoren, die ebenfalls an der C2ld angreifen sollen, zeigten diesen Effekt. Leider konnte nicht weiter geklärt werden, was diesen Effekt verursacht, allerdings liegt die Vermutung nahe, dass es zu einer Aggregation kommt. Weitere Untersuchungen könnten wichtige Hinweise auf das Design von neuen Arzneistoffen bringen, um selektivere und damit nebenwirkungsärmere Inhibitoren zu finden.
Chemical contamination of the environment and thus of aquatic ecosystems is steadily increasing. Whenever environmental pollutants enter a water body, they affect not only the water, but also the sediment. Substances that bind to sediment particles can be stored for a long time, whereby sediments act as sinks for some contaminants. Therefore, sediment
assessments often more accurately describe the contamination of a water body than investigations of the water itself. Among environmental chemicals, endocrine disrupting compounds (EDCs) have gained more and more attention in recent years. Since they interfere with endocrine systems and may disturb reproduction, they endanger the survival of populations or even species. Hazardous substances enter the aquatic environment by different pathways, with sewage treatment plants (STPs) belonging to the most important contamination sources.The main objective of this work is a comprehensive sediment assessment of predominantly small surface waters in the German federal state of Hesse. The 50 study sites, located in 44 different creeks and small rivers, are situated in the densely populated and economically important Frankfurt/Rhine-Main area, as well as in rural and less urbanized regions.
Chemical analytical data, provided by the Hessian Agency for the Environment and Geology (HLUG), indicated different contamination levels of the study sites. In order to investigate the general toxicity of the sediment samples, the oligochaete Lumbriculus variegatus and the midge Chironomus riparius were exposed to whole sediments and apical endpoints regarding biomass, survival, and reproduction were determined. In further experiments, special attention was paid to the contamination with endocrine active compounds. For this purpose, the reproductive success of the New Zealand mudsnail Potamopyrgus antipodarum was analyzed after exposure to whole sediments. Additionally, a yeast-based reporter gene assay was applied with sediment eluates to assess the estrogenic and androgenic activity of the samples. Biotest results were compared with chemical analysis data to investigate whether the test organisms reflect the measured pollution of the study sites and if the observed effects can be explained by chemical contamination.
Five study sites, all located less than 1 km downstream of a STP discharger, were selected for further investigations based on the results of the sediment monitoring. The sediments from these sites were conspicuous due to their general toxic and/or estrogenic activity. In order to investigate whether the observed effects can be ascribed to the effluents, an active biomonitoring study was conducted with the mudsnail P. antipodarum and the zebra mussel Dreissena polymorpha, exposed at study sites located up- and downstream of the discharger.
In addition to endocrine activity, genotoxic effects were investigated using the comet assay and the micronucleus assay. Endocrine activity was examined based on the reproductive output of P. antipodarum and the content of vitellogenin-like proteins in D. polymorpha. Yeast-based reporter gene assays were used to estimate the endocrine potential (estrogen, anti-estrogen, anti-androgen, dioxin-like) of sediment and water samples.
22% of the 50 sediments showed ecologically relevant effects in the biotests with L. variegatus and C. riparius. Only one sediment caused a relevant effect on both test organisms, while the other ten positively tested sediments affected either L. variegatus or C. riparius, probably due to differences in inter-species sensitivities. This suggests that a combination of different biotests is necessary for a comprehensive evaluation of sediment toxicity. 78% of the sediments caused a significantly increased number of embryos in P. antipodarum, which could be ascribed to estrogenic contamination of the sediment samples. An increase in the number of embryos by 60%, as observed in this study, and an associated increase in population size may result in the displacement of other, less competitive species.
In the in vitro tests, 66% of the sediments showed estrogenic activity and 68% showed androgenic activity. Maximum observed values were 40.9 ng EEQ/kg sediment (EEQ = estradiol equivalent) for estrogenic and 93.4 ng TEQ/kg sediment (TEQ = testosterone equivalent) for androgenic activity. Natural and synthetic hormones as well as alkylphenols were the major contributors to the total estrogenicity of environmental samples in several other studies, and are likely responsible for a large part of the estrogenic activity in this case as well. Similarly, androgenic activity is mainly due to natural steroids and their metabolites.
Bioassay results reflect the analytically measured contamination levels at the study sites only very infrequently. This can be ascribed to the occurrence of integrated effects of chemical mixtures present in the sediments. Additionally, effects of substances not included in the analytical program or of substances present in concentrations below the detection limit of the chemical analytical investigations as well as varying bioavailabilities might be relevant. The fact that a large part of the observed effects cannot be explained by the chemical contamination demonstrates the need for effect studies in ecotoxicological sediment assessments.
In order to identify possible causes for the effects observed in the sediment monitoring, e.g. contamination sources, the area types (urban fabrics, arable lands, pasturages, etc.) of the catchment areas belonging to the study sites were analyzed. No significant differences were found between the area profiles of the sampling sites with and without effects in the biotests.
The results indicate that the contamination responsible for the observed effects can be ascribed to different sources. Furthermore, study sites whose sediments exerted significant effects in biotests were located in anthropogenic as well as in predominantly natural areas. The active biomonitoring study at STPs revealed genotoxic and endocrine effects only sporadically.
However, in the in vitro tests considerable endocrine activities of sediment and water samples were determined. No conclusive picture emerges as to whether the observed effects occur more frequently downstream of the dischargers, and thus could be attributed to a contamination by sewage. This indicates that contamination sources other than STP dischargers, for example agricultural runoff, may contribute to the observed effects. Weaker effects and biological activities downstream of a discharger compared to an upstream site might be ascribed to a dilution effect by the effluents. A comparison of the measured in vitro estrogenicity with exposure studies described in the literature shows that adverse effects in aquatic organisms can be expected at the EEQ concentrations determined in the present study.
The results of the sediment monitoring and the STP study revealed a widespread endocrine pollution of small surface waters in Hesse. The fact that the bioassay results only rarely reflect study site contamination as determined by chemical analysis demonstrates the need for effect studies in comprehensive sediment assessments. In some cases STP dischargers increased, in other cases they decreased the observed in vivo effects and in vitro activity of environmental samples. Transferring the results obtained in laboratory studies to the field, adverse effects on aquatic ecosystems can be expected. The study illustrates the need for restrictive measures that contribute to the removal or reduction of environmental pollutants.
For the identification of substances that have so far not been linked to adverse effects on the environment, methods such as effect-directed analyses (EDA) or toxicity identification evaluation (TIE) should be increasingly applied in future studies. Furthermore, bioassays for the assessment of endocrine activity should be implemented in standardized monitoring programs.
The midbrain DA system comprising dopamine (DA) neurons of the substantia nigra (SN) and the ventral tegmental area (VTA) is involved in various brain functions, including voluntary movement and the encoding and prediction of behaviorally relevant stimuli. In Parkinsonʼs disease (PD), a progressive degeneration of particularly vulnerable SN DA neurons causes a progressive DA depletion of striatal projection sites. As a consequence, motor symptoms such as tremor, hypokinesia and rigidity appear once about 50 % to 70 % of SN DA neurons have been lost. Under physiological conditions, SN DA neurons can encode behaviorally salient events and coordinated movements through tonic and phasic activity and correlated striatal DA release. Burst-activity mediates a phasic, supralinear rise of striatal DA levels and allows to activate coordinated movements via modulation of corticostriatal signals.
In the present dissertation project, pathophysiological adaptations of surviving SN DA neurons after a partial degeneration of the nigrostiatal system have been studied using a 6-hydroxydopamine mouse model of PD. Combining in vivo retrograde tracing techniques with in vitro whole-cell patch-clamp recordings, multifluorescent immunolabeling and confocal microscopy allowed an unambiguous correlation of electrophysiological phenotypes, anatomical positions and neurochemical phenotypes of recorded neurons on a single-cell level. In vitro, neuronal activity of SN DA neurons is characterized by spontaneous, slow pacemaker activity of 1 to 10 Hz and a high degree of spike-timing precision. In vitro current-clamp recordings of surviving SN DA neurons using acute brain slice preparations after a partial, PD-like degeneration of the nigrostriatal DA system showed a significant perturbation of spontaneous pacemaker activity, mirrored by a decreased spike-timing precision compared to controls. Selective pharmacology and whole-cell voltage-clamp recordings served to identify calciumactivated SK channels as molecular effectors of a perturbated pacemaker activity of surviving SN DA neurons. SK channels and have been shown to critically contribute to the spike-timing precision of SN DA neurons. Consistently, in vitro current-clamp recordings after pharmacological blockade of SK channels in vitro caused a significant decrease of spike-timing precision, occluding previously observed differences between surviving SN DA neurons and controls.In addition to in vitro patch-clamp recordings, extracellular single-unit recordings in anaesthetized animals in vivo served to study surviving SN DA neurons embedded in an intact neuronal network after a partial, PD-like degeneration of the nigrostriatal DA system. Combining in vivo single-unit recordings, juxtacellular neurobiotin labeling and multifluorescent immunohistochemistry allowed to directly correlate electrophysiological and neurochemical phenotypes as well as anatomical positions on a single-cell level. In vivo, surviving SN DA neurons showed a significant decrease of spike-timing precision as reflected by an increased irregularity and an augmented burst activity compared to controls.
The present dissertation project provided a unique combination of a neurotoxicological PD mouse model, retrograde tracing techniques and in vitro as well as in vivo electrophysiologiy, allowing to unambiguously correlate electrophysiological adaptations, projection-specific anatomical positions and neurochemical phenotypes of SN DA neurons after a partial degeneration of the nigrostriatal system. Surviving SN DA neurons exhibited a significant deficit of SK channel activity after a partial degeneration of the nigrostriatal DA system. In consequence of a diminished SK channel activity observed in vitro, surviving SN DA neurons exhibited and enhanced burst activity in vivo, providing a plausible mechanism to compensate a striatal DA depletion.
Synaptic plasticity is the basis for information storage, learning and memory and is achieved by modulation of the synaptic transmission. The amount of active AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid) receptors at the synapse determines the transmission properties, therefore the regulation of AMPA receptor trafficking affects the synaptic strength. The protein GRIP (glutamate receptor interacting protein) binds to AMPA receptors and is one of the important regulators of AMPA receptor stability at the synapse (Dong et al., 1997; Osten et al., 2000). Previous studies have shown that the ablation of ephrinB2 or ephrinB3 in the nervous system leads to severe defects in hippocampal LTP (long term potentiation) and LTD (long term depression) (Grunwald et al., 2004). We found that ephrinB2 ligands play an important role in the stabilization of AMPA receptors at the cellular membrane (Essmann et al., 2008). Treating cultured hippocampal neurons with AMPA resulted in a robust AMPA receptor internalization, which could be inhibited by simultaneous ephrinB2 activation with soluble EphB4-Fc fusion proteins. Conditional hippocampal ephrinB2 knock-out (KO) neurons showed enhanced constitutive internalization of AMPA receptors. Interaction and interference experiments revealed that ephrinB ligands and AMPA receptors are bridged by GRIP. This interaction is regulated by phosphorylation of a single serine residue in close proximity to the C-terminal PDZ protein target site in ephrinB ligands (Essmann et al., 2008). To investigate the in vivo relevance of this previously undescribed feature of ephrinB reverse signaling, we generated ephrinB2 S-9>A knock-in mice, where the serine at position -9 was replaced by an alanine to prevent phosphorylation. The mutated ephrinB2 of this mouse line was expressed and able to form clusters following stimulation with the preclustered receptor EphB4-Fc. Surface ephrinB2 cluster size and cluster number was slightly smaller in comparison to wild type (WT) mice. Analyzing AMPA receptor internalization, we oserved an increased basal GluR2 endocytosis in cultured hippocampal neurons of ephrinB2 S-9>A mice. Dendrite and spine morphology was similar in pyramidal CA1 neurons of brain slices from adult ephrinB2 S-9>A and WT mice, suggesting a redundancy between the different ephrinB familily members.
Apart from regulating AMPA receptor stability at the synapse, GRIP1 also has an important role in the secretory pathway to deliver cargo proteins along microtubules to dendrites and synapses (Setou et al., 2002). Proteins involved in synaptic transmission and plasticity, as well as lipids required for the outgrowth and remodeling of dendrites and axons have to be transported. We showed in our laboratory with a directed proteomic analysis using the tandem affinity purification-mass spectrometry methodology (Angrand et al., 2006) and with immunoprecipitation assays with brain lysates that the small regulatory protein 14-3-3 interacts with GRIP1. Further immunoprecipitation assays with lysates from HeLa cells transfected with various parts and sequence mutants of GRIP1 revealed that threonine 956 in the linker region L2 between PDZ6 and PDZ7 of GRIP1 is necessary for the interaction with 14-3-3. GRIP1 has been postulated to influence dendritic arborization and maintenance in hippocampal neurons in culture due to defective kinesin-dependent transport along microtubules (Hoogenraad et al 2005). In order to address the role of the association of GRIP1 and 14-3-3 in dendritogenesis, we transfected rat hippocampal neurons with GRIP1-WT and GRIP1 mutants and performed Sholl analysis to evaluate dendritic arborization defects. We could observe striking increased formation and growth of dendrites in developing neurons as well as in mature neurons overexpressing GRIP1-WT. However, overexpression of GRIP1-T956A, where the threonine 956 was replaced by an alanine to prevent phosphorylation, did not show enhanced dendritogenesis, indicating a role for threonine 956 phosphorylation in dendrite branching. To investigate the importance of the interaction between GRIP1 and 14-3-3 in vivo, we generated transgenic mouse lines with a GRIP1-T956A transgene or a GRIP1-WT transgene as control. These mice were crossed with heterozygous GRIP1 mice and by further breedings we obtained some surviver mice carrying either the wild type or the mutated GRIP1 transgene in the usually embryonic lethal GRIP1-KO background (Bladt et al., 2002; Takamiya et al., 2004). In embryonic day (E) 14.5 cultured hippocampal GRIP1-KO neurons we could observe reduced dendritic growth. We also showed reduced GluR2 staining on the dendritic surface in cultured hippocampal neurons from GRIP1-KO and GRIP1-KO neurons containing the GRIP1-T956A transgene. GRIP1-KO neurons containing the GRIP1-WT transgene showed a similar surface GluR2 signal intensity as WT neurons. Reduced surface GluR2 staining in GRIP1-KO neurons and GRIP1-KO neurons with the GRIP1-T956A transgene might be a consequence of defective kinesin-dependent transport of GluR2 to dendrites, indicating an important role of threonine 956 phosphorylation of GRIP1 for GluR2 trafficking.
The tumor suppressor programmed cell death 4 (Pdcd4) exerts its function by inhibiting protein translation initiation. Specifically, it displaces the scaffold protein eukaryotic initiation factor 4G (eIF4G) from its binding to the eukaryotic initiation factor 4A (eIF4A). Thereby, Pdcd4 inhibits the helicase activity of eIF4A, which is necessary for the unwinding of highly structured 5’ untranslated regions (UTRs) of messenger RNAs (mRNAs) often found in oncogenes like c-myc to make them accessible for the translation machinery and subsequent protein production. Overexpression of Pdcd4 inhibits tumorigenesis in vitro and in vivo and inversely, Pdcd4 knockout mice show enhanced tumor formation. In line, Pdcd4 is lost in various tumor types and proposed as prognostic factor in colon carcinomas. Unlike most other tumor suppressors that are rendered nonfunctional by mutations (e.g., p53), Pdcd4 loss is not attributable to mutational inactivation. It is regulated via translational repression by microRNAs and increased degradation of the protein under tumor promoting, inflammatory conditions and mitogens. Specifically, proteasomal degradation of Pdcd4 is controlled by p70 S6 Kinase (p70S6K)-mediated phosphorylation in its degron sequence (serines 67, 71 and 76). Stimulation of the PI3K-AKT-mTOR pathway by growth factors, hormones and cytokines initiates p70S6K activity. Phosphorylated Pdcd4 is subsequently recognized by the E3 ubiquitin ligase beta-transducin repeats-containing protein (β-TrCP) and marked with a polyubiquitin tail to be detected by the 26S proteasome for degradation. β-TrCP represents the substrate specific recognition subunit of the ubiquitin ligase complex responsible for protein-protein interaction with Pdcd4 as substrate for ubiquitin transfer and subsequent proteasomal disassembly.
The first part of the present work aimed at identifying novel stabilizers of the tumor suppressor Pdcd4 in a high throughput screen (HTS). As assay design, a fragment of Pdcd4 from amino acid 39 to 91, containing the phosphorylation sensitive degron sequence, was fused to a luciferase reporter gene construct. Stable expression of this Pdcd4(39-91)luciferase (Pdcd4(39-91)luc) fusion protein in HEK 293 cells served as read-out for the Pdcd4 protein amount to be detected in a high throughput compatible cell-based assay. Loss of Pdcd4(39-91)luc was induced by treatment with 12-O-
tetradecanoylphorbol-13-acetate (TPA), a phorbolester, which activates the PI3K signaling cascade leading to degradation of Pdcd4. The cut-off for hit definition was set at >50% activity in rescuing the Pdcd4(39-91)luc signal from TPA-induced degradation. Activity was calculated relative to the difference of DMSO- and TPA-treated cells (ΔDMSO-TPA = RLUDMSO-RLUTPA). Initial screening of a protein kinase inhibitor library (PKI) revealed hit substances expected to show Pdcd4 stabilizing activity by inhibition of kinases involved in Pdcd4 downregulation, e.g., the mTOR inhibitor rapamycin, the PI3K inhibitors wortmannin and LY294002 and the PKC inhibitors GF 109203X and Ro 31-8220.
The Molecular Targets Laboratory (MTL) of the National Cancer Institute (NCI) in Frederick, USA, hosts one of the largest collections of crude natural product extracts as well as a big substance libraries from pure synthetic sources. Screening of over 15 000 pure compounds and over 135 000 natural product extracts identified 46 pure and 42 extract hits as Pdcd4 stabilizers. For nine synthetic and six natural product derived compounds (after bioassay-guided fractionation), dose-dependent activities for recovering the TPA-induced Pdcd4(39-91)luc loss defined IC50s in the low micromolar range. Most importantly, these compounds were confirmed to stabilize endogenous Pdcd4 protein levels from forced degradation as well. This result proved the assay design to be highly representative for endogenous cellular mechanisms regulating Pdcd4 protein stability. The next step was to stratify the hit substances according to their likely mechanism of action to be located either up- or downstream of the p70S6K-mediated phosphorylation of Pdcd4. Therefore, phosphorylation of S6, as proto-typical p70S6K target, was analyzed and uncovered two natural derived compounds to influence p70S6K activity. Four substances did not affect p70S6K phosphorylation activity and were therefore considered to stabilize Pdcd4 by acting downstream, i.e. on the β-TrCP-mediated proteasomal degradation.
In the second part of this work, one of these compounds, namely the sesquiterpene lactone erioflorin, isolated by bioassay-guided fraction from the active extract of Eriophyllum lanatum, Asteraceae, was further characterized in detail with respect to its molecular mechanism of action. Erioflorin dose-dependently protected both Pdcd4(39-91)luc and endogenous Pdcd4 protein from TPA-induced degradation with IC50s of 1.28 and 2.64 μM, respectively. Pdcd4 stabilizing activity was maximal at 5 μM erioflorin. Up to this concentration, erioflorin was verified not to inhibit p70S6K activity. In addition, it was observed that erioflorin rescued Pdcd4(39-91)luc from both, wild type and constitutively active p70S6K-mediated downregulation. Only wild type p70S6K was inhibitable by the mTOR inhibitor rapamycin which served as an upstream acting control. To study the next section of Pdcd4 regulation, i.e. recognition by the E3 ubiquitin ligase β-TrCP, Pdcd4(39-91)luc and endogenous Pdcd4 were immunoprecipitated from whole cell extracts with the corresponding antibodies. In this key experiment, treatment with TPA increased overexpressed β-TrCP binding to both and this coimmunoprecipitation could be strongly reduced by erioflorin treatment. This result strongly pointed to an inhibitory mechanism of the β-TrCP specific binding to Pdcd4 by erioflorin. In addition, erioflorin disrupted the binding of in vitro transcribed/translated β-TrCP to Pdcd4 in an in vitro interaction assay to exclude nonspecific intracellular signals. Furthermore, polyubiquitination of Pdcd4 was decreased by erioflorin treatment as well. To clarify questions regarding specificity of erioflorin for the E3 ubiquitin ligase β-TrCP, stability of another important β-TrCP target was explored, i.e. the tumor suppressor inhibitor of kappa B alpha (IκBα). Indeed, the tumor necrosis factor alpha (TNFα)-mediated loss of IκBα could be prevented by erioflorin cotreatment. On the other hand, the E3 ubiquitin ligase von Hippel Lindau protein (pVHL) was left unaffected as its target hypoxia inducible factor 1 alpha (HIF-1α) could not be stabilized from oxygen-dependent degradation by erioflorin treatment. These results argued strongly for erioflorin being a specific inhibitor of β-TrCP-mediated protein degradation. Functional consequences of erioflorin treatment were investigated by observing its influence on the transcriptional activities of the transformation marker activator protein 1 (AP-1, an indirect downstream target of Pdcd4) and nuclear factor κB (NF-κB which is directly inhibited by IκBα). Indeed, erioflorin showed significant inhibition of AP-1 and NF-κB reporter constructs at 5 μM, a concentration for which an impact on cell viability was excluded. Finally to characterize the significance of erioflorin in a cell-based tumorigenesis assay, the highly invasive colon carcinoma cell line RKO was tested in a two dimensional migration assay. Erioflorin was discovered to significantly lower cell migration in a wound closure assay.
In conclusion, development of a high throughput compatible cell-based reporter assay successfully identified novel substances from pure synthetic and natural product derived background as potent stabilizers of the tumor suppressor Pdcd4. In addition, this work aimed at elucidating the detailed mechanism of action of the sesquiterpene lactone erioflorin from Eriophyllum lanatum, Asteraceae. Erioflorin was discovered to inhibit the E3 ubiquitin ligase β-TrCP, thereby preventing protein degradation of tumor suppressors like Pdcd4 and IκBα. This may offer the possibility to more specifically target protein degradation and generate less adverse side effects by blocking a particular E3 ubiquitin ligase compared to general proteasome inhibition.
The long sought molecular function of membrane raft-associated flotillin proteins is slowly becoming resolved, partially owing to the increasing knowledge about their interaction partners. Being ubiquitously expressed and evolutionarily highly conserved, flotillins carry out important cellular functions, one of which is the regulation of signal transduction pathways. This study shows that the signaling adaptor protein fibroblast growth factor receptor substrate 2 (FRS2) directly interacts both in vivo and in vitro with flotillin-1 (flot-1). FRS2 is an important docking protein of many receptor tyrosine kinases. It regulates downstream signaling by forming molecular complexes with other adaptor proteins and tyrosine phosphatases, and seems to be a critical mediator of sustained extracellular signal regulated kinase (ERK) activity. Flot-1 has also been implicated in the regulation of ERK activity upon EGF and FGF stimuli. Furthermore, flot-1 forms signalosomes with EGFR and the downstream components of the MAP kinase pathway. The newly discovered interaction between FRS2 and flot-1 was shown to be mediated by the phosphotyrosine binding (PTB) domain and, to a lesser extent, the C-terminus (CT) of FRS2 and by the C-terminus of flot-1. Flot-1 coprecipitated together with FRS2 from murine tissues and cell lysates, demonstrating that this interaction also takes place in vivo. Interestingly, flot-2, which shows a high homology to flot-1 and forms stable oligomeric complexes with it, does not appear to directly interact with FRS2. Novel insights into the functional role of the interaction between flot-1 and FRS2 were provided by the results showing that depletion of flot-1 affects the cellular localization of FRS2. In hepatocytes stably depleted of flot-1, FRS2 appeared to be more soluble. Furthermore, upon pervanadate stimulation of the cells, a small fraction of FRS2 was recruited into detergent resistant membranes, but the recruitment did not take place in the absence of flot-1. Triggered by the same stimulus, a fraction of FRS2 was translocated to the nucleus independently of flot-1. Overexpression of FRS2 has previously been shown to result in increased ERK activation. However, in cells depleted of flot-1, FRS2 was not able to compensate for the compromised ERK activation after EGF or FGF stimulation. This might imply that FRS2 and flot-1 are functionally interconnected and that FRS2 resides upstream of flot-1. Taken together, the results presented here indicate that this complex may be involved in the control of signaling downstream of receptor tyrosine kinases and is important for ensuring a proper signaling response. In the absence of flot-1, increased Tyr phosphorylation of FRS2 was observed. It is known that Tyr and Thr phosphorylation of FRS2 are reciprocally regulated. Since ERK is a known executor of the FRS2 Thr phosphorylation, and ERK activity was shown to be severely diminished upon flot-1 depletion, the increased Tyr phosphorylation of FRS2 was in agreement with this and might be a direct consequence of a decreased ERK activity upon flot-1 depletion. FRS2 owes its name to the major and the first described function of this protein as a substrate for FGFR. PTB domain of FRS2 was published to constitutively bind the juxtamembrane domain of FGFR. In this study, the PTB domain was mapped to be involved in the constitutive interaction with flot-1 and the competition was shown to exist between flot-1 and FGFR1 for binding to FRS2. Another novel interaction partner of FRS2 was discovered in the present study. Cbl-associated protein (CAP) is an adaptor protein with three SH3 domains and it plays a role during insulin signaling by recruiting the signaling complex to lipid rafts. CAP was previously shown to interact with flot-1 via the SoHo domain, and this interaction was found to be crucial for the lipid raft recruitment of other signaling components. Both the PTB domain and CT of FRS2 were found to mediate the interaction with CAP, whereas in CAP, the SoHo domain, together with the third SH3 domain, seems to bind to FRS2. SH3 domains mediate the assembly of specific protein complexes by binding to proline rich sequences, several of which are present in FRS2. Due to overlapping interaction domains, FRS2 and flot-1 competed for the binding to CAP. However, the interaction with neither CAP nor flot-1 was necessary for the observed nuclear translocation of FRS2. Since CAP is expressed as several tissue- and developmental stage-specific isoforms, a further aim of this study was to analyze the expression of its isoforms in mouse embryonic fibroblasts (MEFs). Many new isoforms were discovered here which have not been described in the literature so far. They all contain the SoHo domain and three SH3 domains, but differ among themselves by the presence and length of a proline-rich region that preceeds the SoHo domain and by a novel 20-amino acid (AA) stretch between the second and the third SH3 domain. The length of the proline-rich region turned out to be an important factor determining the strength of the interaction with FRS2. The interaction was found to be weakened by the increasing length of this region. The new isoforms possessing the 20-AA stretch are specifically expressed in murine muscular tissues, with the highest level in the heart. During adipogenesis, we observed a shift in the abundance of the isoforms, in that only the isoforms without the insertion were shown to be upregulated on mRNA level. However, during myogenesis, preferentially expressed isoforms were those with the insertion. The collected data implicate that isoforms with the 20-AA insertion might be more ubiquitous in nondifferentiated/embryonic cells and that the observed "isoform-switch" might be dependent on the cell fate and differentiation state.
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Here, the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD) was investigated. In the present study, the identification of two truncated transcripts and four novel 5-LO splice variants containing premature termination codons (PTC) was reported. The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
RT-PCR analysis of different cell types revealed the existence of a large number of 5-LO splice variants. The most interesting splice variants were observed in BL41-E95A cells, which give a raise to novel 5-LO protein isoforms. This leads to the hypothesis of a novel regulatory mechanism in which the dimerization of 5-LO with 5-LO isoforms might regulate the 5-LO activity.
The 5-LO protein expression was reduced on translational level in UPF1 knock down cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry based proteomics study was started to identify compartment specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis demonstrated that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~ 21%) but not in the soluble fraction (< 1%). Western blot data confirmed the trend of the proteomics analysis. This data suggest that UPF1 is a critical gene expression regulator in a compartment specific way. During differentiation by TGFβ and calcitriol the majority of UPF1 regulated proteins was adjusted to normal level. It appears that that not only the NMD mechanism alters its composition during differentiation. Also the gene expression regulation on translational level by UPF1 seems to be also cell differentiation dependent. An interesting group of UPF1 target genes represent the downregulated proteins. qRT-PCR analysis of randomly chosen genes revealed no effect on mRNA expression upon UPF1 knockdown, suggesting that UPF1 positively influences the translation of these genes. Computational sequence analysis identified a conserved C-rich sequence which might be a hnRNP E2-binding site. hnRNP E2 has been characterized as a translational repressor in myeloid cells. Western blot analysis revealed a differentiation independent up regulation of hnRNP E2 by UPF1 knockdown. Additionally, microRNA-328 (miR-328) has been described as an RNA decoy modulating hnRNP E2 regulation. Due to this, stem loop qRT-PCR showed an up regulation of miR-328 in TGFβ and calcitriol differentiated MM6 cells. Based on this data we suggest a model in which downregulation of UPF1 increases hnRNP E2 expression, leading to translation inhibition. During differentiation, miRNA-328 is upregulated thereby competing with hnRNP E2 leading to an efficient translation
Respiration is one of the key processes of energy transduction used by the cell. It consists of two components: electron transfer and ATP production. The electron transfer chain converts the energy released from several biochemical redox reactions into an electrochemical proton gradient across membranes. This stored energy is used as the driving force for the production of ATP by the ATP synthase. The mitochondrial electron transfer chain contains four major protein complexes called complexes I-IV, with counting starting at the lower side of the redox potentials. It has been discussed for a long time how these protein complexes are organized in the membranes. Do they diffuse freely in the membrane? Alternatively, do they form a supercomplex built up of several neighboring complexes? The evidence supporting the free diffusion mode is that both electron transfer intermediates (cytochrome c and quinone) behave as “pool”. However, respiratory supercomplexes have been detected in membranes from bacteria, fungi, yeast, plant and animal during the last decade, and sometimes the respiratory complexes are only stable inside a supercomplex. Therefore, the idea of supercomplex formation has become more popular. The argument that the supercomplex arises from solubilization and is a detergent artifact could be rejected because: 1) supercomplexes can be isolated from many organisms in an active form; 2) supercomplexes have been proven to stabilize the individual complexes in some cases; 3) supercomplexes can be very stable after chromatographic isolation in some cases....
Menschliche Aktivitäten beeinflussen beinahe alle Bereiche des Lebens auf der Erde (MEA 2005a; UNEP 2007). Die Zerstörung und Veränderung natürlicher Lebensräume sind als Hauptursache für den weltweiten Biodiversitätsverlust identifiziert (Harrison and Bruna 1999; Dale et al. 2000; Foley et al. 2005; MEA 2005a). Zusammen mit dem Klimawandel wird die Landnutzungsveränderung daher als einflussreichster Aspekt anthropogen verursachten globalen Wandels betrachtet (MEA 2005a). Landnutzungsveränderung schließt sowohl die Umwandlung natürlicher Habitate in Agrarland oder Siedlungen als auch die Landnutzungsintensivierung in bereits kultivierten Landschaften mit ein. Diese Veränderungen haben weitreichende Konsequenzen für die Artenvielfalt und resultieren häufig in dem Verlust von Arten mit zunehmender Intensität der Landnutzung (Scholes and Biggs 2005).
Biodiversität und Ökosysteme stellen viele verschiedene Funktionen zur Verfügung, wie z. B. die Sauerstoffproduktion, die Reinigung von Wasser und die Bestäubung von Nutzpflanzen.
Einige dieser Funktionen sind hilfreich, andere wichtig und wieder andere notwendig für das menschliche Wohlergehen (MEA 2005b; UNEP 2007). Mittlerweile sind Ökosystemfunktionen und die vielen Nutzen, die sie erbringen, zu einem zentralen Thema der interdisziplinären Forschung von Sozialwissenschaften und Naturwissenschaften geworden (Barkmann et al. 2008 und darin enthaltene Referenzen). Dadurch bedingt ist es zu einiger Verwirrung bezüglich der verwendeten Begriffe der "Ökosystemfunktion" (engl. "ecosystem function") und dem der "Ökosystemdienstleistung" (engl. "ecosystem service") gekommen (deGroot et al. 2002). Da der Fokus meiner Arbeit auf grundlegenden Funktionen von Ökosystemen liegt, verwende ich im Folgenden den Begriff der Ökosystemfunktion.
Für viele Ökosystemfunktionen ist noch sehr unzureichend bekannt, wie diese von externen Störungen beeinflusst werden (Kremen and Ostfeld 2005; Balvanera et al. 2006). Ökosystemfunktionen werden selten von nur einer einzigen Art aufrechterhalten, sondern meist von einer ganzen Reihe unterschiedlicher taxonomischer Gruppen – alle mit ihren ganz eigenen Ansprüchen. Diese Arten, wie auch deren intra- und interspezifischen Interaktionen, können durchaus nterschiedlich auf die gleiche Störungsquelle oder Störungsintensität reagieren. Dies kann Vorhersagen zum Verhalten von Ökosystemfunktionen extrem erschweren. ...
Plastids are complex organelles that fulfil numerous essential cellular functions, such as
photosynthesis, amino acid and fatty acid synthesis. he majority of proteins required for
these functions are encoded in the nuclear genome and synthesised on cytosolic ribosomes as
precursors, which are posttranslationally transported to and imported into the organelle by
concerted actions of translocons in the outer and inner chloroplast membrane. For most
preproteins, targeting to the organelle is ensured by a specific import signal, a so called
transit peptide, which is specifically recognised by receptors at the chloroplastês surface. A transit peptide is generally defined as essential and sufficient for precursor targeting to and
translocation into chloroplasts, (however, an analysis of the ability of transit peptides to drive translocation of tightly folded passenger domain revealed that the transit peptide is not
always sufficient for the translocation event. A critical signal length requirement of amino
acids has been determined in vivo and in vitro. In the case of shorter transit peptide, the
succeeding portion of the mature domain provides an extension of an unfolded polypeptide
stretch required for successful translocation. The analysis of the unfolding mode of a folded
model passenger during translocation links the observed transit peptide length requirement
to the action of an energising unit present in the intermembrane space of chloroplasts.
The likely candidate for this energising unit space is putative imsHsp70, previously hypothesised to function in translocation of precursor proteins across the outer membrane. However, as the identity of this protein has up to now remained unknown, its existence has
been a matter of debate. The present study focuses on the isolation and characterisation of
imsHsp70 at the molecular level. Mass spectrometry analyses and in vivo localisation studies
demonstrate that while no specific imsHsp70 exists, multiple cytosolic Hsp70 isoforms are
targeted to the intermembrane space, but not to the stroma of chloroplasts. Thus, a so far unrecognised mode of dual targeting to chloroplasts and cytosol is most likely to ensure the
allocation of (sp s into the intermembrane space.
The canonical Wnt pathway, also known as Wnt/β-‐catenin pathway, comprises a network of proteins which control diverse developmental and adult processes in all metazoan organisms. The binding of canonical Wnt ligands to a cell surface receptor complex, consisting of frizzled family members and low density lipoprotein receptor-‐ related protein 5 or 6 co‐receptors, triggers a signaling cascade which results in a β-catenin-‐mediated transcriptional activation of different target genes, implicated in cellular proliferation, apoptosis, migration and differentiation. A couple of years ago, several groups including us, iden2fied transient activation of the canonical Wnt-pathway in endothelial cells (ECs) of the developing central nervous system (CNS). In this context, Wnt/β-‐catenin signaling could be demonstrated to be crucial for brain angio genesis as well as for the establishment of the blood-brain barrier (BBB) phenotype in the newly formed vessels.
Gliomas, in particular the glioblastoma (GBM), belong to the group of highly vascularized solid tumors which gain their vascularization due to an angiogenic switch occurring during tumor progression. Interestingly, nuclear localized β-‐catenin could be exclusively detected in the activated endothelium of induced rat gliomas and of human GBM, suggesting a so far unknown and not further characterized involvement of the canonical Wnt pathway in pathological angiogenesis. In order to systematically decipher the precise role of endothelial Wnt/β-‐catenin signaling in tumor angiogenesis, I established
murine GL261 glioma cell lines overexpressing either Wnt1 or Dickkopf (Dkk) 1 in a doxycycline-‐dependent manner, an activator and potent inhibitor of Wnt/β-‐catenin signaling, respectively. In subcutaneous and intracranial transplantations, tumor-derived Wnt1 reduced, while Dkk1 increased GL261 tumor growth without affecting in vitro proliferation, cell cycle or cell death of the established cell lines. Nowadays, it is well accepted that solid tumors are dependent on vascular support allowing them to grow beyond a certain size. In my work I could show that tumor-‐derived Wnt1 targets the tumor vasculature by increasing endothelial Wnt/β-‐catenin signaling, which reduced tumor vessel density and resulted in a more quiescent tumor vasculature. Furthermore, Wnt1-‐expression mediated tight association of smooth muscle cells (SMCs) and pericytes to the tumor endothelium, a phenotype which is unusual for tumor vessels and a described hallmark of tumor vessel normalization. In contrast, inhibition of endothelial Wnt/β-‐catenin signaling by Dkk1 mediated an opposing effect, characterized by endothelial hyper-proliferation and a tumor vasculature with a rough basal lamina distribution and loosely anached mural cells, indicative of a strong angiogenic activity. The described vascular effects in Wnt1-expressing GL261 tumors could be verified by subcutaneous transplantations of a rat glioma cell line constitutively expressing Wnt1. Furthermore, an applied in vivo MatrigelTM plug assay uncovered the reduction in vessel density upon Wnt1 simulation to be tumor cell independent, suggesting an EC-‐autonomous effect. This hypothesis was confirmed by subcutaneous transplantations of parental GL261 cells into mice with genetically generated endothelial β-‐catenin gain-of-function (GOF). The derived GOF tumor from this experiment comprised a quiescent and normalized tumor vasculature and phenocopied the vascular effects observed in Wnt1-expressing tumors.
Our previous work provided evidence that Wnt/β-‐catenin signaling contributes to the BBB phenotype of the developing CNS through the transcriptional regulation of the tight junction protein claudin-‐3. Furthermore, the coverage of pericytes to brain vessels has been described to correlate with BBB integrity. In agreement with these publications, vessels of intracranial Wnt1-‐expressing GL261 tumors retained or regained barrier properties, indicated by a reduced leakage of the tracer Evans blue and endogenous mouse immunoglobulin G and increased junctional localiza2on of the tight junction proteins claudin-‐3, -‐5 and zonula occludens-‐1.
Overall, we detected sustained endothelial Wnt/β-‐catenin signaling to induce a quiescent and normalized tumor vascularization. Interestingly, the Notch signaling pathway has been shown to inhibit the angiogenic tip cell and to promote the quiescent stalk cell phenotype via its ligand Delta-like ligand 4 (Dll4) and the receptors Notch1 and 4. Mechanistically, my work demonstrated for the first time that overactivation of endothelial Wnt/β-‐catenin signaling reactivated expression of Dll4 in the tumor endothelium, which could be shown in vitro to increase Notch signaling and to favor a stalk cell-like gene signature. Furthermore, we uncovered the platelet-derived growth factor subunit B (pdgm) as a novel transcriptional target of Wnt/β-catenin signaling in ECs. Hence endothelial-‐derived PDGF-‐B is known to promote the recruitment of mural cells, the upregulation of this factor might explain the increased SMC/pericyte coverage observed in the tumor vasculature upon sustained endothelial Wnt/β-‐catenin signaling which additionally might promote a cycle of vascular normalization.
Taken together, my work reveals several vascular effects, being mediated by reinforced endothelial Wnt/β-‐catenin signaling during tumor angiogenesis. While a moderate level of canonical Wnt signaling, observed in vessels of human astrocytomas and murine control tumors, is considered to be associated with tumor angiogenesis, dominant activation of this pathway in ECs is shown to limit angiogenesis and to promote a quiescent and normalized tumor vasculature with increased barrier properties. Furthermore, my work discovers pdgm as a novel target of canonical Wnt signaling in ECs.
The work presented in this dissertation therefore not only uncovers the role of endothelial Wnt/β-‐catenin signaling in tumor angiogenesis but additionally reveals this pathway to be a novel modulator in pathological vessel development which might proof to be a valuable therapeutic target for anti-angiogenic and edema glioma therapy.
Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.
Typ I Interferone sind bekannt für die durch sie vermittelten immunaktivierenden bzw. antiviralen Effekte. Nach ihrer Induktion, im Rahmen der angeborenen Immunantwort, vermitteln Interferone nicht nur einen systemischen anti-viralen Status, sondern können auch wichtige Effektormechanismen der adaptiven Immunität dahingehend beeinflussen, dass sie diese verstärken bzw. ermöglichen. Im Allgemeinen kann diese Eigenschaft als pro-inflammatorische Aktivität der Interferone bezeichnet werden. Allerdings gehört es ebenfalls zu den Eigenschaften der Interferone eine Verminderung der adaptiven Immunität bewirken zu können, was als anti-inflammatorische Aktivität verstanden werden kann. Insgesamt kann man die durch Interferone induzierten Effekte also als ambivalent bezeichnen.
Die Leber als Immunorgan besitzt, ähnlich wie die Interferone, eine zentrale Rolle in der Immunität und sollte in ihrer Funktion als Vermittler zwischen Immunaktivierung und Immuntoleranz nicht unterschätzt werden. Die Aufgaben der Leber können ebenfalls als ambivalent bezeichnet werden, da sie zum einen eine unnötige Aktivierung des Immunsystems verhindern muss um eine Schädigung der Leberzellen zu vermeiden (Immuntoleranz). Zum anderen muss auch in der Leber eine Immunaktivierung stattfinden können, um den Schutz vor Pathogenen zu gewährleisten.
In einem Leberschadenmodell, das künstliche Doppelstrang-RNA (poly(I:C)) zur Induktion von Typ I Interferonen verwendet, sollen im Rahmen der vorliegenden Arbeit Immunmodulationen, insbesondere in der Leber, untersucht werden. Hierbei liegt das Hauptaugenmerk auf den Interferon-vermittelten Effekten, die eine Schädigung der Leber verhindern.
Werden Interferonrezeptor-defiziente Tiere (IFNAR-/-) intraperitoneal mit poly(I:C) behandelt kann eine ausgeprägte Schädigung der Leber sowie Hepatitis in diesen Tieren beobachtet werden. Wildtyp (WT) Mäuse zeigen hingegen keinerlei Schädigungen der Leber, was für einen protektiven bzw. anti-inflammatorischen Effekt spricht, der über den IFNAR und damit über Typ I Interferone vermittelt wird. Unter Verwendung von Mäusen, die eine selektive Deletion des IFNAR auf bestimmten Immunzellen tragen (alle anderen Zellen der Maus exprimieren jedoch weiterhin den IFNAR), konnte der Immunzelltyp ermittelt werden, der beim IFNAR-vermittelten Schutz der Leber eine Schlüsselrolle übernimmt. Aus diesen Experimenten wird deutlich, dass es myeloide Zellen sind, die über den IFNAR durch Typ I Interferone stimuliert werden müssen, um im poly(I:C)-induzierten Leberschadenmodell einen Schutz der Leber zu bewirken. Ergänzend dazu konnte gezeigt werden, dass CD11b- und F4/80-doppelt positive Makrophagen nach poly(I:C)-Behandlung in die Leber von WT Mäusen infiltrieren. Zudem wurde in Experimenten mit Interferon-Reporter Mäusen deutlich, dass diese infiltrierenden Makrophagen über den IFNAR durch Typ I Interferone stimuliert sind. Nach poly(I:C)-Behandlung konnte gezeigt werden, dass Leber-infiltrierende Zellen in WT Mäusen anti-inflammatorischen Interleukin-1 Rezeptor Antagonisten (IL-1RA) sekretieren. In Abwesenheit eines funktionalen Interferonsystems hingegen (in IFNAR-/- Mäusen) konnte eine gestörte IL-1beta- und IL-1RA-Balance festgestellt werden. Für diese Zytokine, die sich gegenseitig regulieren, indem der anti-inflammatorische IL-1RA mit dem pro-inflammatorischen IL-1beta um die Bindung an den IL-1 Rezeptor konkurriert, konnte gezeigt werden, dass ihre Expression in der Leber Interferon-abhängig reguliert wird. In IFNAR-/- Mäusen und in Mäusen, deren IFNAR selektiv auf myeloiden Zellen deletiert war, konnte keine IL-1RA-Expression durch infiltrierende Zellen detektiert werden. Da in diesen Tieren nach poly(I:C)-Behandlung massive Leberschäden beobachtet wurden, kann vermutet werden, dass das Vorhandensein des anti-inflammatorischen IL-1RA unerlässlich für den Schutz der Leber ist.
Abschließend kann zusammengefasst werden, dass die Interferon-vermittelten Effekte, die eine Schädigung der Leber verhindern, zum einen auf der Stimulation und Rekrutierung von Makrophagen beruhen. Zum anderen beruhen diese Effekte auf der Induktion des anti-inflammatorischen Zytokins IL-1RA, und der dadurch blockierten Wirkung des pro-inflammatorischen IL-1beta.
Durch diese Ergebnisse werden neue Einblicke in die Interferon-vermittelte Hemmung von Virus- und Autoimmun-induzierten Erkrankungen der Leber ermöglicht. Genutzt werden könnten diese für die Optimierung IFN-basierter Therapien. Beispielsweise kann durch die gezielte Induktion anti-inflammatorischer Zytokine über IFNAR-induzierte Signalwege oder die direkte Gabe anti-inflammatorischer Zytokine (z.B. IL-1RA) eine Therapie entwickelt werden, die neben den vorteilhaften Eigenschaften der Zytokine eine verbesserte Aktivierung von Immunzellen ermöglicht.
Sowohl die Gifte der Kegel- (Conidae) als auch die der Schraubenschnecken (Terebridae) enthalten eine Vielzahl pharmakologisch aktiver Peptide. Vor allem die Conopeptide bzw. Conotoxine aus den Giften der Kegelschnecken werden aufgrund ihrer Selektivität für Ionenkanäle und Rezeptoren seit langem als Werkzeuge in der neuropharmakologischen Forschung eingesetzt. Hier rücken gerade neuronale nikotinische Acetylcholinrezeptoren immer mehr in den Fokus der medizinischen Forschung, da sie vermutlich an der Entwicklung neurodegenerativer Erkrankungen wie Alzheimer, Parkinson, Demenz, Schizophrenie und Epilepsie beteiligt sind. Ziel dieser Dissertation war es daher, neue Inhibitoren in den Giften Kegelschnecken (Conidae) und der Schraubenschnecken (Terebridae) für nikotinische Acetylcholinrezeptoren, vor allem der neuronalen Subtypen, zu identifizieren. Es erfolgte die:
1. Identifizierung neuer αD-Conotoxine
Aus den Giften von Conus capitaneus und C. mustelinus konnten zwei native αDConotoxine (αD-CAP und αD-MUS) isoliert und charakterisiert werden. Beide Toxine sind Homodimere mit Molekulargewichten von 11 kDa und inhibieren nikotinische ACh-Rezeptoren. Sie blockieren die Subtypen α7>α3β2>α4β2, wobei sich αD-MUS als potenter als αD-CAP erweist (IC50-Werte von αD-MUS: α7 0,12 nM, α3β2 1,08 nM, α4β2 4,5 nM; IC50-Werte von αD-CAP α7 0,25 nM, α3β2 2,8 nM, α4β2 28,6 nM). Hingegen haben die αD-Conotoxine auf die Rezeptorsubtypen α3β4, α4β4 und α1β1γδ keinen hemmenden Einfluss. Zusätzlich konnten drei weitere αD-Conotoxine mit Hilfe der cDNA von C. vexillum und C. betulinus identifiziert werden. Eine Besonderheit hierbei war, dass innerhalb der Familie der αD-Conotoxine zwei unterschiedliche Signalsequenzen vorkommen und somit diese Sequenzen nicht stark konserviert sind.
2. Charakterisierung des α-Conotoxins SI aus dem Gift von C. striatus
Im Gift der Kegelschnecke Conus striatus wurde ein Peptid mit inhibierender Wirkung an α7-Rezeptoren nachgewiesen. Molekulare Masse (1.352,5 Da) und Aminosäuresequenz entsprachen dem α-Conotoxin SI, das als Antagonist muskulärer nACh-Rezeptoren bekannt ist. Da die Ergebnisse mehrere Jahre zurück lagen und bisher keine Analysen im Oozytenexpressions-System durchgeführt wurden, wurdeeine mögliche Aktivität sowohl an neuronalen als auch an muskulären nACh-Rezeptoren vermutet. Voltage Clamp-Messungen bestätigten die spezifische Wirkung am Muskeltyp, wodurch die Aktivität am α7-Rezeptorsubtyp einem anderen Conopeptid, zugewiesen werden muss, das als Beiprodukt isoliert wurde.
3. Identifizierung neuer Conotoxine der A-Superfamilie Mit molekularbiologischen Methoden unter Nutzung von cDNA-Bibliotheken gelang es, 27 Conotoxine (17 neue und 10 bekannte) aus der A-Superfamilie zu identifizieren: drei α- und zwei κA-Conotoxine aus Conus striatus, zwei α-Conotoxine aus C. betulinus, zwei α- und zwei κA-Conotoxine aus C. carinatus, drei α-Conotoxine aus C. catus, drei α- und zwei κA-Conotoxine aus C. circumcisus, ein α-Conotoxin aus. C. geographus, zwei aus C. imperialis, jeweils eines aus C. lividus, C. quercinus, C. sponsalis sowie zwei aus C. terebra Die Vielzahl der identifizierten α-Conotoxine belegt die hohe Diversität dieser Toxine in den Giften der Kegelschecken. Anhand von Vergleichen mit bereits bekannten Toxinen werden die möglichen Wirkungsweisen einiger neuer α-Conotoxine diskutiert. Für einen Teil der α-Conotoxine wurden 3D-Strukturmodelle erstellt, die Einblicke in die Bindung der Toxine an den Rezeptor geben können.
4. Untersuchung der Gifte der Terebridae
Die inhibierende Aktivität einiger Gifte (Terebra consobrina, T. argus, Myurella affinis, Acus felina, A. chlorata, A. maculata und Hastulopsis pertusa) an nACh-Rezeptoren (α7, α3β2, α4β2, α3β4, α4β4 und α1β1γδ) wurde erstmals nachgewiesen. An Kalium- und Natriumkanälen zeigten die Giftextrakte keine Wirkung. Die Giftextrakte von Myurella affinis und Acus maculata waren am potentesten und blockierten alle untersuchten nACh-Rezeptoren. Dies ist besonders ungewöhnlich, da diese Terebriden-Arten nach der Literatur (Puillandre & Holford, 2010) keinen Giftapparat besitzen sollen. Eine weitere Auffälligkeit aller Terebriden-Giftextrakte war neben der Selektivität für a7-Rezeptorsubtypen, eine hohe Aktivität gegenüber α4-enthaltenden Rezeptoren. In den Giften und mit Hilfe von cDNA-Bibliotheken von Kegelschnecken konnte eine Vielzahl neuer Inhibitoren für neuronale nikotinische Acetylcholinrezeptoren identifiziert werden. Sie zeigen ein breites Wirkungsspektrum, das die unterschiedlichsten nAChRSubtypen einschließt, was ihre Verwendung als pharmaklogische Werkzeuge begrenzt. Hingegen zeigen die Gifte der Schraubenschnecken ein Selektivitätsspektrum, das die Analyse ihrer Peptide als vielversprechend erscheinen lässt.