Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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Institute
We found that the HMTase G9a, that catalyzes H3K9me2 in euchromatin, plays a key modulatory role in type I IFN expression. This finding raises the possibility of targeted intervention with type I IFN expression by using small synthetic inhibitors of G9a. Given the overall minimal negative effect of G9a-deficiency on differentiated cells, the short-term suppression of G9a could be used to potentiate type I IFN expression during chronic viral diseases such as hepatitis C. Accordingly, pharmacological enhancement of methylation, for example by inhibition of the H3K9me2 specific demethylases, could be potentially used to attenuate type I IFN expression and help to control chronic inflammatory and autoimmune conditions. The mechanism responsible for canvassing the epigenetic profile of type I IFN expressing cells are not known. It is plausible, that similar to neurons, where G9a is targeted to specific loci with the help of noncoding RNAs, IFN expressing cells possess similar mechanisms to target H3K9me2 demethylating enzymes to type I IFN loci, thus keeping these loci accessible for IFN-inducing transcription factors. Identification of non-coding RNAs that may contribute to the establishment of the epigenetic state of IFN producing cells will provide a further opportunity for targeted manipulation of IFN expression.
In my thesis, I describe the collaborative experiments that show the ability of synthetic compounds that interfere with the histone readers to suppress inflammation. Our results present a novel concept for the regulation of inflammatory gene expression. The diversity of histone readers and the combinatorial nature of regulation of gene transcription may provide an opportunity for highly selective interference with disease associated transcriptional programs by interfering with specific readers. In the future we plan to address the therapeutic potential of BET antagonists in autoimmune and chronic inflammatory conditions.In summary, the experiments described in my thesis provide an example of how the understanding of the basic mechanisms of chromatin control of gene expression can facilitate novel therapeutic approaches that target chromatin.
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.
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.
Die im Rahmen dieser Arbeit durchgeführten Untersuchungen führten zu folgenden Ergebnissen:
1. In-silico Analysen von putativen Apoptose-Faktoren im Genom von P. anserina
Es konnten mehrere Gene, die in einer Apoptose-Maschinerie involviert sein könnten, im Genom von P. anserina identifiziert werden. Diese Homologen wurden in zwei Ka-tegorien unterteilt: (i) die nicht-mitochondrialen Proteine PaMCA1, PaMCA2 und PaPARP und (ii) die Homologen des Apoptose-induzierenden Faktors AIF.
2. Einfluss der Metacaspase-Aktivität auf programmierte Zelltodprozesse
Mithilfe von Aktivitätsmessungen konnte eine Arginin-spezifische Aktivität der Meta-caspasen nachgewiesen werden. Diese Metacaspase-Aktivität nimmt in seneszenten Kulturen und nach H2O2-Behandlung signifikant zu. Diese Ergebnisse unterstützen die Hypothese eines programmierten, ROS-induzierten Zelltods im letzten Entwicklungs-stadium des Alternsmodell P. anserina.
3. Die Rolle von AIF-Homologen in der Entwicklung von P. anserina
GFP-Fusionsproteine identifizierten eine mitochondriale Lokalisation der AIF-Homologen PaAIF2, PaAMID2 und PaPRG3. Desweiteren konnte eine altersabhängige PaAIF2-Translokation von den Mitochondrien zum Zellkern gezeigt werden, ähnlich der Apoptose-induzierenden Translokation von humanem AIF. Die Deletion von PaAif2 und PaAmid2 führte zu einer signifikanten Resistenz gegenüber oxidativem Stress und zu einer Verlängerung der Lebensspanne. Diese Befunde weisen auf einen ROS-induzierten, AIF-vermittelten Zelltod hin, der an der Lebensspannen-Kontrolle von P. anserina beteiligt ist.
4. Die Funktion des Proteins PaCYPD bei Seneszenz und programmiertem Zelltod
Membranpotential-Messungen konnten einen Rückgang des mitochondrialen Memb-ranpotentials von 21 % bei den PaCYPD-Überexpressionsstämmen nachweisen. Durch die Behandlung mit dem spezifischen PaCYPD-Inhibitor CSA konnte das Membranpo-tential wieder normalisiert werden. Zusammen mit dem detektierten Verlust von
7 Zusammenfassung
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Cytochrom c in den Mitochondrien der Überexpressionsstämme wird durch diese Studi-en die Vermutung einer PaCYPD-abhängigen Öffnung der mPTP untermauert. Die Pa-PaCypD-Deletion führte zu einer signifikanten Resistenz gegenüber mitochondrial-abhängigem, oxidativem Stress und gegenüber verschiedenen Apoptose-Induktoren. Die Überexpression von PaCypD hingegen führte zu einem beschleunigten Alterungspro-zess (Präseneszenz), einem verschlechterten Resistenzverhalten gegenüber Stress- und Apoptose-Induktoren und zu einer massiven Verkürzung der Lebensspanne. Die Le-bensspanne konnte aber durch die Behandlung mit CSA wieder auf Wildtyp-Niveau verbessert werden. Dies weist auf einen PaCYPD-vermittelte Zelltod hin. Interessan-terweise konnte durch das Wachstum auf CSA-haltigem Medium auch die Lebensspanne des Wildtyps verlängert werden. Um die hier nachgewiesene, lebensver-längernde Wirkung von CSA zu verifizieren, könnte diese Studie leicht auf andere Modellorganismen übertragen werden.
Das humane endogene Retrovirus-K: Grundlagenforschung und Nutzen als Tumor-assoziiertes Antigen
(2011)
Fast die Hälfte des humanen Genoms besteht aus Retroelementen, die während der evolutiven Entwicklung des Menschen im Genom fixiert wurden. Im Wesentlichen kann man diese Retroelemente in DNA-Transposons, LINEs, SINEs und humane endogene Retroviren unterteilen, dabei nehmen die humanen endogenen Retroviren (HERV) 8% des humanen Genoms ein. Bei einer Unterfamilie, der HERV-K Familie, sind bis heute alle offenen Leserahmen erhalten geblieben. Nach heutigem Erkenntnisstand wird eine Expression dieser Elemente in somatischen Zellen jedoch strikt unterdrückt, denn eine Expression von Retroelementen könnte zu Insertionsmutagenesen führen und letztlich dem Organismus erheblichen Schaden zufügen.
Im Gegensatz dazu wird eine reaktivierte Expression von HERV-K häufig in einigen Tumorarten beobachtet: allen voran Keimzelltumore, Melanome und Brustkrebs. Außerdem können in Patienten, die an solchen Tumoren erkranken, häufig HERV-K spezifische Antikörper und mitunter auch gegen HERV-K-gerichtete T-Zellen nachgewiesen werden. Die strikte Unterdrückung der HERV-K Expression in gesunden, somatischen und eine reaktivierte Expression in entarteten Zellen machen HERV-K Proteine daher zu idealen Tumor-assoziierten Antigenen.
Auf Grundlage dieser Untersuchungen wurden, in dieser Arbeit, zwei potentielle Tumorvakzine, basierend auf dem hoch attenuierten Modifizierten Vacciniavirus Ankara (MVA) hergestellt. Durch homologe Rekombination wurde ein HERV-K gag-pro-pol transgenes MVAHKcon und ein HERV-K env transgenes MVAHKEnv hergestellt und charakterisiert. Darüber hinaus wurden die rekombinanten Viren in einem neu etablierten, syngenen Maus-Tumor-Modell untersucht. MVAHKcon immunisierte Mäuse zeigten eine starke humorale Immunantwort und waren in der Lage subkutane, HERV-K Gag positive Tumore fast vollständig zu eliminieren. MVAHKEnv immunisierte Mäuse zeigten dagegen eine moderate humorale Immunantwort und eine starke T-Zellantwort. Nach therapeutischer Immunisierung mit MVAHKEnv konnte im Mausmodell eine signifikante Reduktion an HERV-K Env positiven Lungenmetasten beobachtet werden. Außerdem konnte durch eine prophylaktische Immunisierung mit MVAHKEnv ein vollständiger Schutz der Mäuse vor der Ansiedlung HERV-K Env-exprimierender Tumore erreicht werden. Die hier vorgestellten HERV-K rekombinanten MVA könnten daher der erste Schritt zu einer Immuntherapie gegen reaktivierte Retroelemente in malignen Tumoren darstellen.
MVAHKcon infizierte Zellen produzieren und sekretieren große Mengen HERV-K Virus-ähnlicher Partikel (VLP) somit konnten auch grundlegende Fragestellungen der HERV-K Biologie geklärt
Zusammenfassung
123
werden. Durch die Kombination massenspektrometrischer Analysen und N-terminaler Sequenzierungen konnten, die noch nicht bekannten Schnittstellen der retroviralen Protease im HERV-K Gag Protein identifiziert werden. Zudem wurde eine späte Domäne von HERV-K identifiziert und darüber hinaus Wechselwirkungen von HERV-K VLPs mit zellulären Restriktionsfaktoren wie APOBEC3G und CD317 studiert.
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.
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
The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080 cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.
Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV
infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer”
tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an
HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10
HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral
combination therapy. T cell infusions were tolerated well with no severe side effects. A
significant increase of CD4 counts was observed post infusion. At the end of the one-year
follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified
cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear
follow-up, whereby marking levels correlated with the cell dose. No significant changes of
viral load were observed during the first four months. Four of the seven patients that changed
their antiviral drug regimen thereafter responded with a significant decline in plasma viral load.
In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene
marking and may improve immune competence in HIV-infected patients with advanced disease
and multidrug resistant virus. However, the low level of gene marking and the lack of substantial
long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate
the requirement for new vectors with new strategy.
In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements
were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from
this work provide the basis for the selection of a suitable candidate vector for extensive
preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors
incorporate a number of additional features that are of potential value for gene therapeutic
applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors
and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration
profile. The use of internal promoters to drive expression of the therapeutic transgene maC46
should further improve the safety profile of these new‐generation vectors, while an additional
artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene
expression. The rationale for this is that hematopoietic stem and progenitor cells will be
Summary
98
protected from enhancer‐mediated transactivation effects and also from potential side effects due
to the aberrant expression of maC46 while at the same time the full clinical benefit for the
patients is maintained.
In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors
harboring different regulatory elements were selected based on results from pilot experiments.
The internal promoters used to drive expression of codon optimized maC46 were the PGK
promoter and MPSV promoter. This work focuses on the transgene expression levels in
lymphoid cells and antiviral activity. The issues of long term expression, propensity to
methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first
step the performance of different vectors was evaluated in the human T cell lines. Based on
promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the
MPSV promoter along with intron were selected for in vivo transplantation experiments.
In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells,
along with maintained expression of introduced genes. It was observed that the expression of
these constructs depends strongly on the activation and differentiation status of the targeted T
cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46
can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed
at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no
sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified
lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with
the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed
engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. .
In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of
T cell activation status as related to the desired clinical effect must be addressed. These results
might open the way for a gene therapy targeting mainly or exclusively activated T cells and
could be exploited for immunostimulatory as well as suppressive approaches.
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.
EGFL7 regulates adult neural stem cell maintenance and differentiation by inhibition of Notch1
(2010)
In neurobiology the preexisting dogma on the unchangeability of the adult mammalian brain and its inability to give rise to new neurons has been challenged since the early nineties. Generally, it is now accepted that neurogenesis persists in adults. Progress in developmental and stem cell biology in recent years led to an increasing interest in regeneration-based treatment strategies for damaged tissue of the central nervous system. Thus, the enhancement of the endogenous potential of the brain to repair itself is potentially a feasible therapeutic strategy to treat various types of brain damage. Therefore, it is of great interest to understand the molecular mechanism that regulate adult neurogenesis. One of the prominent pathways suggested to be involved here is the Notch signaling cascade. Previously, it has been shown that various components of Notch signaling are expressed in the stem cell niche of the adult subventricular zone (SVZ) in vivo. Interestingly, a recent study demonstrated that the self-renewal potential of adult neural stem cells (NSCs) isolated from the SVZ depend on Notch signaling in vitro.
Recently, we identified a novel non-canonical Notch ligand termed epidermal growth factor-like domain 7 (EGFL7), which was originally described as a protein secreted by endothelial cells and functionally implicated in cellular responses of the vascular system such as cell migration and blood vessel formation. We were able to show that secreted EGFL7 binds to a region in Notch that is involved in ligand-mediated receptor activation, thus acting as an antagonist of Notch signaling.
The present study identifies neurons of the human and murine brain as a novel source of EGFL7, which suggests functions of EGFL7 in the neural system. Expression analyses by quantitative RT-PCR (qRT-PCR) revealed EGFL7 is down regulated in the adult SVZ, which suggests that endogenous EGFL7 may act as a Notch modulator of NSCs. We assessed the expression of Notch pathway components in adult NSCs isolated from the SVZ of adult mice and demonstrated that inhibition of Notch activity by the γ-secretase inhibitor DAPT reduced the self-renewal potential of NSCs. Accordingly, adenoviral-mediated expression of EGFL7 in vitro decreased Notch-specific signaling and reduced proliferation and self-renewal of NSCs. Conversely, activation of Notch by a constitutive active form of Notch (NICD) rescued the EGFL7-mediated reduction of NSC self-renewal verifying that this effect was directly linked to Notch signaling. Congruent to the reduced proliferation rate measured in vitro, induced expression of EGFL7 in vivo significantly reduced the number of Ki-67 positive cells within the SVZ upon cerebroventricular injection of EGFL7 adenovirus.
Expression analyses in the developing brain showed single EGFL7-positive cells within the marginal zone of the neocortex as measured by in situ hybridization. These cells might be Cajal-Retzius cells, specialized neurons, which specifically express Reelin, which is a protein of the extracellular matrix known to control neuronal migration and differentiation. Interstingly, we could show that Reelin and EGFL7 are expressed in a subtype of neurons of the adult mouse cortex. This implied an interaction of both proteins and was verified by co-immunoprecipitation assays, suggesting an additional role for EGFL7 in neuronal maintenance. QRT-PCR based expression analyses in vitro comparing differentiated and non-differentiated NSCs displayed an increase in EGFL7 expression during the differentiation process, which was paralled by reduced Notch signaling. NSCs differentiated on coverslips coated with EGFL7 differentiated into all three cell types - neurons, oligodendrocytes and astrocytes. EGFL7 favored the formation of neurons as compared to control comparable to the effect of the Notch-inhibitor DAPT. Furthermore, additional oligodendrocytes were formed. These cells displayed a mature morphology with distinct sprouts and branches in contrast to the small and round oligodendrocytes that formed on control coverslips, which resembled us of precursor cells. Neurons and oligodendrocytes were formed at the expense of astrocytes. Congruently to the effect observed in vitro, adenoviral-based expression of EGFL7 in the SVZ yielded a slight induction of neuronal differentiation in vivo. Taken together, these results show for the first time a previously unrecognized role for EGFL7 in the brain by modulation of the Notch pathway in adult NSCs, which changes the proliferation and differentiation potential of adult NSCs in vitro and in vivo.
Protein translocation across the chloroplast membrane is mediated by molecular machinery composed of protein complexes termed the TOC/TIC (the outer/inner envelope chloroplasts translocases). This translocation process is regulated by metabolic energy in form of GTP and ATP and is influenced by the lipid composition of the membrane. The ability to study the function of a single complex “TOC” in vitro using purified protein or purified chloroplast outer envelope vesicles has been instrumental for our understanding of the mechanism underlying this process.
Indeed, the TOC complex has been purified by previously established procedures. However its functional and structural analyses are impaired by the limited yield of purified protein. Therefore, protocols for native TOC complex purification are described here. The complex isolation is achieved by direct biochemical treatment of biological membrane hosting this complex or by tandem affinity purification of modified protein complex components from generated transgenic plants.
Furthermore, in this thesis, radioactive based in vitro import assays are described, namely those that allow monitoring translocation activity across the outer envelope of chloroplast. Based on the analysis of knock-out plants and isolated complexes it was previously suggested that lipid dependence of protein translocation might exist. Thus, the question was raised whether the lipid composition of the membrane has a direct influence on the behavior and functionality of the TOC translocon, or whether additional components of the chloroplast membrane account for the observed effect in vivo. To answer this question, a technique for vesicle fusion was developed. The principal aim was to explore the effect of an exchange of the lipid environment surrounding the complex translocon. This method helped to demonstrate that the SQDG and PI act stimulatory on the translocation across the outer envelope of chloroplast, whereas DGDG exhibits an inhibitory effect on TOC complex functionality.
Sponges are one of the major components of benthic communities and are considered to be a
key role organism in marine ecosystems. In addition to their importance in terms of
biodiversity, sponges are becoming increasingly attractive to the industry, as they themselves
or associated symbionts, produce various kinds of secondary metabolites of pharmaceutical
properties. Some of them have already been clinically applied.
The taxonomic characters of Porifera are limited to only a few morphological and
histological characters. In addition, sponges of the same species often show a wide
morphological variability, whereas the latter depends on different ecological parameters such
as water depth and current conditions. Thus, the taxonomic classification of sponges often
becomes a scientific challenge.
The fauna of the Yellow Sea rates among the least studied worldwide. At the same time,
according to the UN Atlas of the Ocean, the Yellow Sea is one of the most intensively
exploited marine areas in the world. This is not least due to the dense human population living
in the entire catchment area of the Yellow Sea region. In order to compile medium- and longterm
conclusions about the anthropogenic impact on biota of the Yellow Sea, the knowledge
of species and their distribution is of crucial importance, as these data form the baseline for all
future conservation efforts.
Until now the sponge fauna of the Chinese Yellow Sea is insufficiently investigated.
Thus, there is only one publication on sponges from this region that has been released
hitherto. This paper is dealing with only a view species. However, there is no reference
concerning the present location of the voucher material, on which this publication is based on.
Consequently, no scientific collection on Porifera from the Chinese part of the Yellow Sea
exists to date.
In order to compile a documentation of the recent sponge community of the Chinese
Yellow Sea, 12 study sites along the coast of the Liaoning Peninsula, China, Northeast
Yellow Sea, were investigated with focus on sponge distribution. The corresponding habitats
were characterized in regard to their topographical features, abiotic parameters, and common
composition of benthic megafaunal and macroalgal assemblages.
Due to the lack of comparable studies, a comprehensive literature research on sponges of the
shallow Northwest Pacific Ocean was required. As a result the first compilation of
publications is presented, dealing with sponges from shallow depths of the northwestern
Pacific Ocean.
Abstract
2
In the course of this study, 31 sponge species in total were recorded, which are scientifically
processed. With the exception of four all specimens were determined to species- level.
Twelve out of the total number of species are new to science and are described and classified
according to the recent taxonomic system of the phylum Porifera.
The results of this study indicate considerable differences in species composition between
investigated sites. It is shown that physical factors (particularly current regime, sedimentation,
seasonally related variations in temperatures), as well the availability of suitable substrates are
directly related to the diversity and abundance of investigated sponge communities. In this
context possible adaptation strategies of the corresponding sponges were discussed in detail.
Two sponge species, Clathria (Clathria) asodes and Antho (Acarnia) lithophoenix, formerly
known exclusively from the northeastern Pacific Ocean, are now recorded from the Northwest
Pacific Ocean for the first time. Furthermore, Penares hongdoensis, Clathria (Clathria)
hongdoensis and Celtodoryx girardae were synonymized with Penares cortius, Clathria
(Clathria) acanthostyli, and Celtodoryx ciocalyptoides respectively. Moreover, the occurrence
of eight sponge species, which were known from previous records from the Yellow Sea, could
be confirmed.
As a result of this study the Asian origin of a sponge species that is invasive to the French and
Dutch coasts of the Northeast Atlantic Ocean since the 1990s could be established. Moreover,
it is demonstrated that Celtodoryx girardae from the northeastern Atlantic is in fact
conspecific with Cornulum ciocalyptoides described by Burton (1935) from the Posiet Bay,
Sea of Japan. Apart from taxonomic remarks, variations between populations from both
oceans are examined and discussed thoroughly in regard to possible ecological implications.
The community of documented sponges shows overlapping with the one from the Sea of
Japan. According to the results it is assumed that the endemic degree of the sponges from the
Chinese Yellow Sea is rather low to moderate.
The material obtained in the course of this study was integrated in the collection of the
Senckenbergischen Naturforschenden Sammlungen. Therefore, it is the first scientific
collection of sponges from the Chinese Yellow Sea that can be consulted as a basis for all
further studies on sponges of this region.
The present study is the only investigation of sponges from Dalian and adjacent waters before
the spill occurred in the Dalian harbour in July 2010. Therefore, it provides an essential
baseline needed to assess the impact of the oil spill on benthic communities.
Strukturelle Organisation und Mobilisierung des Primaten-spezifischen Non-LTR-Retrotransposons SVA
(2011)
SVA-Elemente repraesentieren die juengste Familie der Non-LTR-Retrotransposons,
welche das humane Genom fortwaehrend modifizieren. SVA-Elemente zeichnen sich
durch ihre Organisation aus zusammengesetzten repetitiven Elementen aus. Um
Rueckschluesse auf den Assemblierungsprozess, der zur gegenwaertigen Organisation der
SVA-Elemente fuehrte, und ueber transkriptionelle Regulation dieser Elemente zu ziehen,
wurden Unterschiede in der Struktur der 116 SVA-Elemente, die auf humanem
Chromosom 19 lokalisiert sind, detailliert untersucht.
SVA-Elemente konnten in sieben unterschiedliche Strukturvarianten eingeteilt werden,
einschliesslich neuer Varianten wie SVA2, 3`-verkuerzte Elemente und Elemente mit 5`-
flankierenden Transduktionen. Ich habe auch eine extrem erfolgreiche human-spezifische
5`-Transduktionsgruppe identifiziert, SVA_F1, die trotz ihres jungen evolutionaeren Alters
ca. 32% aller Mitglieder der SVA-Subfamilie SVA_F umfasst. Die transkriptionelle
Kontrolle einer retrotransponierten und 5`-verkuerzten SVA_F-Kopie durch den Promotor
des MAST2-Gens diente als urspruengliches Source-Element dieser umfangreichen 5`-
Transduktionsgruppe, die mindestens 84 Elemente einschliesst. Die zusaetzlichen 5`-
sowie 3`-Transduktionsereignisse der vollstaendigen Alu-Sequenzen bei Mitgliedern der
SVA_F1-Transduktionsgruppe 4 weisen auf ihre wichtige Rolle in der erfolgreichen
Expansion im humanen Genom hin. Diese nachtraeglich erworbenen Alu-Sequenzen
machen SVA_F1-Familienmitglieder offensichtlich zum besseren Substrat fuer die Trans-
Mobilisierung durch die L1-Proteinmaschinerie. Die unterschiedlichen konsekutiven 5`-
Tansduktionsereignisse der SVA_F1-Familienmitglieder deuten auf transkriptionelle
Kontrolle ihrer Source-Elemente durch eine Vielzahl externer zellulaerer Promotoren hin,
die im Laufe der Evolution in Keimzellen aktiv waren. Ausserdem zeigt die Existenz von
5`-Transduktionen, dass SVA-Elemente sich die 5`-flankierenden Sequenzen aneignen
koennen. Die Daten zeigen auch, dass SVA-vermittelte 5-Tansduktionsereignisse
alternatives RNA-Spleissen an putativen Spleissstellen involvieren. Aus der EST-
Datenbankanalyse ist ersichtlich, dass Mitglieder der SVA_F1-Subfamilie auch
gegenwaertig transkribiert werden.
SVA-Elemente sind hoch aktiv im humanen Genom, aber der Mechanismus ihrer
Retrotransposition wurde bislang nicht aufgeklaert. Vorangehende Analysen genomischer
SVA-Kopien liessen auf eine L1-vermittelte Mobilisierung schliessen; allerdings wurde
der experimentelle Beweis dieser Hypothese bislang nicht geliefert. Mit Hilfe der
Zellkultur-basierten Trans-Mobilisierungsassays wurde in dieser Arbeit zum ersten Mal
experimentell bewiesen, dass SVA-Elemente tatsaechlich durch die L1-kodierten Proteine
in trans mobilisiert werden. Zu diesem Zweck wurden HeLa-Zellen mit einem
vollstaendigen oder mit einem 5`-verkuerzten SVA-Retrotranspositionsreporterkonstrukt
sowie mit einem L1-Expressionsplasmid bzw. Leervektor kotransfiziert und dann die
jeweiligen Raten der SVA-Retrotransposition anhand Neo-resistenter Kolonien, die
mindestens ein de novo-Retrotranspositionsereignis widerspiegeln, bestimmt. Die
Experimente zeigen, dass die Entstehung der Neo-resistenten Kolonien von der
Koexpression L1-kodierter Proteine abhaengig ist. Ich konnte auch zeigen, dass das
vollstaendige SVA-Testkonstrukt - im Gegensatz zum 5`-verkuerzten SVA-Konstrukt -
mit einer signifikant hoeheren Retrotranspositionsrate als die Kontrollkonstrukte, die zur
Generierung der prozessierten Pseudogenformation eingesetzt wurden, trans-mobilisiert
wird. Die Ergebnisse der Trans-Mobilisierungsassays belegen, dass SVA-Elemente ein
bevorzugtes Substrat fuer die L1-Proteinmaschinerie darstellen, und ihre 5`-Region
einschliesslich der Alu-homologen Sequenz fuer die hohe Retrotranspositionsrate essentiell
ist. Die elf analysierten SVA de novo-Integrationsereignisse weisen Merkmale der L1-
vermittelten Retrotransposition auf, wie Poly(A)-Enden, L1-EN-spezifische Konsensus-
Zielsequenz (NNAUNA), Zielsequenz-Verdoppelungen (TSDs), Mikrohomologien und
zusaetzliche Guanosin-Nukleotide am 5`-UEbergang.
Zusammenfassend demonstrieren die Ergebnisse dieser Studien, dass ein signifikanter Teil
der Mitglieder der human-spezifischen SVA-Subfamilie aus transkriptioneller Kontrolle
ihrer Source-Elemente durch externe Promotoren hervorgeht. Durch die in dieser Arbeit
durchgefuehrten in silico-Analysen wurde auch gezeigt, dass SVA-vermittelte 5`-
Transduktionsereignisse zur strukturellen Vielfalt der SVA-Elemente fuehren, und eine
neue Art von genomischen Umstrukturierungen darstellen, die zur Plastizitaet des
humanen Genoms beitragen. Ausserdem bestaetigen die Ergebnisse der Trans-
Mobilisierungsassays die Hypothese, dass SVA-Elemente tatsaechlich durch die L1-
kodierte Proteinmaschinerie trans-mobilisiert werden. Dabei sind Module am 5`-Ende der
SVA-Elemente fuer diesen Prozess hoechst relevant.
Die Ergebnisse der Dualen-Luciferase-Reportergen-Assays unterstuetzen die Hypothese,
dass innerhalb der SINE-R-Sequenz von SVA H19_27 cis-aktive Elemente vorhanden
sind, die auf aehnliche Weise wie die cis-aktiven Elemente innerhalb der 5`LTR von
HERV-K reguliert werden.
Ausserdem wurde in dieser Arbeit die Existenz interner reguatorischer Sequenzen
innerhalb der SVA-Sequenz bestaetigt. Mit Hilfe der Dualen-Luciferase-Reportergen-
Assays konnte zum ersten Mal gezeigt werden, dass SVA-Elemente cis-aktive Elemente
enthalten, die hauptsaechlich in der SINE-R-Region lokalisiert sind. Diese cis-aktiven
Elemente werden auf aehnliche Weise wie die cis-aktiven Elemente innerhalb der 5`LTR
von HERV-K reguliert. Die starke transkriptionelle Aktivitaet des vollstaendigen SVA-
Testelements und des L1RP-Promotors in den Teratokarzinom-Zelllinien bekraeftigen die
Annahme, dass haeufige SVA-Mobilisierung in Keimzellen durch die gleichzeitig
hochregulierte SVA- und L1-Transkription bedingt sein koennte.
Es konnte gezeigt werden, dass SVA-Elemente cis-aktive Elemente enthalten, die
hauptsaechlich in der SINE-R-Region lokalisiert sind, und auf aehnliche Weise wie die cis-
aktiven Elemente innerhalb der 5`LTR von HERV-K reguliert werden. Die starke
transkriptionelle Aktivitaet des vollstaendigen SVA-Testelements und des L1RP-Promotors
in Teratokarzinom-Zelllinien bestaetigen die Annahme, dass haeufige SVA-
Retrotransposition in Keimzellen durch die gleichzeitig hochregulierte SVA- und L1-
Transkription bedingt sein koennte.
NOSTRIN belongs to the recently defined F-BAR protein family. F-BAR proteins are
multi-domain proteins, which serve as adaptors between plasma membrane and
cytoskeleton components in processes such as membrane protrusion formation,
endocytosis and migration. NOSTRIN encompasses a F-BAR domain at the N-terminus,
which mediates membrane association, followed by a HR1 motif and an intermediate
domain (ID) domain in the middle, and a SH3 domain at the C-terminus. The domain
architecture and ability to form oligomers enable NOSTRIN to coordinate several
interaction partners namely dynamin, caveolin, N-WASP and endothelial nitric oxide
synthase (eNOS) in the process of eNOS trafficking. In this context NOSTRIN was
originally identified and hence termed eNOS traffick inducer. NOSTRIN is expressed in
vascularized tissues (e.g. liver and lung) and in primary endothelial cells.
Aims of the present work were (1) to investigate if NOSTRIN is involved in other
processes besides eNOS trafficking, (2) to analyse the function of NOSTRIN in vivo
through knockdown of NOSTRIN in developing zebrafish and (3) to study the
consequences of the loss of NOSTRIN on signal transduction in a primary cell culture
model derived from NOSTRIN knockout mice.
To study the possible involvement of NOSTRIN in other processes besides eNOS
trafficking a yeast two-hybrid screen was performed in which fibroblast growth factor
receptor 1 (FGFR1) was identified as a putative novel interaction partner of NOSTRIN. In
a series of yeast two-hybrid, pulldown and co-immunoprecipitation experiments the
interaction between NOSTRIN and FGFR1 was confirmed to occur between
endogenously expressed proteins and determined to be direct and to depend on the ID
domain of NOSTRIN and the 130 C-terminal amino acid residues of FGFR1. FGFR1 is
activated by binding of fibroblast growth factors (FGFs) and induces several different
signal transduction pathways (e.g. MAPK and Akt pathway). Overexpression of
NOSTRIN in HeLa cells specifically enhanced FGF2-dependent MAPK activation.
Accordingly, depletion of NOSTRIN attenuated FGF2-dependent MAPK activation and
did not affect FGF2-induced Akt activation.
In summary, NOSTRIN has been identified as a novel interaction partner of FGFR1
involved in FGF2-dependent signal transduction.
The morpholino oligonucleotide-mediated knockdown of NOSTRIN in developing
zebrafish caused vascular leakage and irregular vascular patterning e.g. a loss of the
proper trajectory of intersegmental vessel and interruptions of the dorsal longitudinal
anastomotic vessel. The vascular phenotype was consistent upon use of two different
morpholinos and could be rescued in a dose dependent manner by the injection of
zebrafish NOSTRIN mRNA. Detailed analysis involving confocal and time lapse
microscopy in zebrafish with endothelial specific expression of EGFP revealed that the
knockdown of NOSTRIN impacts in vivo on the migration and morphology of endothelial
tip cells and leads to a reduction of filopodia number and length.
Additionally a NOSTRIN knockout mouse was generated. The analysis of FGFR1 signal
transduction in primary mouse lung endothelial cells (MLECs) from NOSTRIN knockout
and wild type mice revealed that FGF2-dependent MAPK activation was attenuated in
MLECs isolated from NOSTRIN knockout mice when compared to MLECs isolated from
wild type mice. The effect of NOSTRIN on FGF2-dependent signal transduction seems to
be specific, since VEGF-induced MAPK activation was not affected in NOSTRIN
knockout MLECs. The importance of NOSTRIN for FGF2 signal transduction in vivo is
demonstrated by the greatly impaired angiogenic response to FGF2 in NOSTRIN
knockout mice in matrigel plug assay. In a detailed biochemical analysis it was
discovered that NOSTRIN interacts with the activated small GTPase Rac1 and that
overexpression of NOSTRIN enhances Rac1 activation. Furthermore, the interactions of
NOSTRIN with both Rac1 and its GEF Sos1 are required for NOSTRIN-mediated
activation of Rac1. In accordance, activation of Rac1 was not detected upon FGF2
stimulation in NOSTRIN knockout MLECs.
In conclusion, the present work describes a novel function of the F-BAR protein
NOSTRIN in FGFR1 signal transduction. Data presented in this work demonstrate that
NOSTRIN is required for the assembly of a complex consisting of FGFR1, Sos1 and
Rac1 and subsequently for the FGF2-dependent activation of Rac1 in endothelial cells.
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.
In der vorliegenden Dissertation stand die Aufklärung der Funktion und Regulation von p21 in der Mitose im Mittelpunkt. p21 ist als Cdk-Inhibitor und Schlüsselregulator bekannt, der in viele fundamentale zelluläre Prozesse involviert ist: Zellzyklusregulation, Apoptose, Seneszenz, Zellmigration und Dynamik des Zytoskeletts, Transkription, Differenzierung sowie DNA-Reparatur, aber auch in die Umprogrammierung induzierter pluripotenter Stammzellen (Besson et al. 2008; Abbas und Dutta 2009; Jung et al. 2010).
Die unkontrollierte Proliferation von Zellen ist mit der Tumorgenese assoziiert und wird unter anderem durch die Fehlregulation von p21, aber auch durch die wichtigen mitotischen Kinasen Cdk1, im Komplex mit ihrer regulatorischen Untereinheit Cyclin B1, sowie Plk1 bedingt. Zudem ist das Fehlen von p21 oder die Fehllokalisation in das Zytoplasma mit einer schlechteren Prognose für den Patienten und Chemotherapie-Resistenz von Tumoren verbunden (Abukhdeir und Park 2008). Aufgrund der zunehmenden Inzidenz und Mortalität von Krebserkrankungen ist es daher von besonderem klinischem Interesse, die molekularen Ursachen für die Entstehung maligner Tumorerkrankungen aufzuklären. Bislang existieren kaum Studien über welche molekularen Mechanismen die Funktionen von p21, dem wichtigsten Cdk-Inhibitor, der zum Beispiel durch die Anwendung niedermolekularer Inhibitoren wie BI 2536, das sich bereits in klinischen Phase II Studien befindet (Strebhardt 2010), beeinflusst wird, während der Mitose reguliert werden.
In der vorliegenden Dissertation wurde daher die physiologische Rolle des Cdk-Inhibitors bzw. Regulators p21 während der Mitose untersucht und mit der Kinaseaktivität von Cdk1/Cyclin B1, wie auch Plk1 korreliert. Es konnte gezeigt werden, dass p21 während der Mitose stark exprimiert wird und dass mitotisches p21 in verschiedenen Krebszelllinien unabhängig von dem p53-Status in einer phosphorylierten Form vorkommt, welche mit der Aktivität von Cdk1 und weniger mit der von Cdk2 assoziiert ist. Durch Untersuchungen der isogenen HCT116-Zelllinien mit und ohne p21 wurde aufgezeigt, wie wichtig p21 für den ordnungsgemäßen Ablauf der Mitose ist. Ohne p21 sind sowohl die Anaphase wie auch die Zytokinese verlängert, die Zellen ordnen die Chromosomen fehlerhaft in der Metaphaseplatte an (congression Fehler), besitzen weitaus mehr lagging Chromosomen und fast 20 % der Zellen weisen im Versuchsverlauf Polyploidie auf. Durch den Verlust des Cdk-Regulators p21 kommt es zur Fehlregulation von Cdk1 und seiner Substrate (wie MCAK) und es treten die oben beschriebenen Probleme auf.
Weiterhin phosphoryliert Cdk1/Cyclin B1 p21 an Ser-130 in vitro und ex vivo in der frühen Phase der Mitose, der Prophase bzw. Prometaphase. Die nicht phosphorylierbare p21 Form S130A befindet sich hauptsächlich im Zellkern und führt zu vermehrtem Auftreten von congression Fehlern, während die S130D-Mutante, die die Phosphorylierung durch Cdk1 vortäuscht, schneller degradiert wird und zudem den Phänotyp der HCT116 p21-/- Zellen verstärkt. Zellen, die S130D exprimieren, benötigen mehr Zeit für das Durchlaufen der Mitose. Hier ist vor allem die Metaphase stark verlängert, aber auch Anaphase und Zytokinese. Dies führt zu congression Fehlern und zu Polyploidie. Diese Ergebnisse bestätigen, wie wichtig die zeitlich korrekte Phosphorylierung von p21 und die dadurch vermittelte Aktivierung von Cdk1/Cyclin B1 ist.
Darüber hinaus stabilisiert die Suppression von Plk1 das p21 Protein, was darauf hinweist, dass die Degradation von p21 während der Prometaphase von Plk1 kontrolliert wird. Dies wird von der Tatsache unterstützt, dass Ser-114, wie auch Ser116 von Plk1 in vitro phosphoryliert wird. Die Deregulation von p21 durch Plk1, SS114/116AA bzw. SS114/116DD induziert Chromosomenfehler, wodurch die molekularen Mechanismen, warum fehlreguliertes Plk1 die Tumorgenese fördert, hervorgehoben werden.
Nach Abschluss der bisherigen Untersuchungen steht fest, dass man sich von der starren Rolle von p21 als Tumorsuppressor und Akteur während der G1/S-Phase lösen muss. Der Cdk-Inhibitor p21 trägt entscheidend zur mitotischen Progression bei, vor allem bedingt durch die zeitlich ordnungsgemäße Inaktivierung bzw. Aktivierung von Cdk1/Cyclin B1, der Kinase, die wiederum zahlreiche für die Mitose essentielle Proteine reguliert. In Zukunft muss zum besseren Verständnis der Rolle von p21 in der Mitose die genaue Abfolge der Ereignisse unter Einbeziehung der Degradationsmechanismen eingehender untersucht werden.
Im Zuge des hessischen Wiederansiedlungsprojektes für die Europäische Sumpfschildkröte (Emys orbicularis) konnten in den Jahren 2002-2007 insgesamt 79 juvenile Sumpfschildkröten an vier Standorten (NSG „Hölle von Rockenberg“, NSG „Reinheimer Teich“, NSG „Nachtweid von Dauernheim“ und NSG „Nidderauen von Stockheim“) in Hessen ausgewildert werden. Ziel der vorliegenden Untersuchung war es, hinreichende Kenntnisse zur Biologie, Physiologie und Ökologie der ausgesetzten Jungtiere zu erhalten. Die generellen Erfolgsaussichten eines solchen Langzeitprojektes, das Verhalten der ausgesetzten Jungtiere und deren Gesundheitszustand standen hierbei im Vordergrund der Arbeit. Anhand verschiedener Methoden (direkte Beobachtung, Radiotelemetrie, Fang-Wiederfang) wurde die Ansiedlung über einen Zeitraum von fünf Jahren überwacht. Radiotelemetrische Studien sowie eine direkte Beobachtung erbrachten hierbei Informationen zur Raum- und Habitatnutzung der Jungtiere und den daraus resultierenden Lebensraumansprüchen. Durch dieses Wissen konnten weitere, sowie bereits bestehende, Wiederansiedlungsgebiete in dieser Region durch individuelle Maßnahmen optimiert werden. Des Weiteren konnte mit temperatursensitiven Messmethoden ein Überblick über das tägliche und saisonale Körpertemperaturspektrum der Art in Hessen ermittelt werden. Vor der Wiederansiedlung sowie im weiteren Verlauf der Untersuchung wurden in den jeweiligen Gebieten diverse Biotopsmaßnahmen durchgeführt. So z.B. das Ausbringen von zusätzlichen Sonnenmöglichkeiten in Form von Stämmen, das Anlegen von permanenten und temporären Flachwasserteichen, verschiedenste Biotopspflegemaßnahmen (Entschlammen, Entbuschen) und die Offenhaltung potentieller Eiablageplätze. Diese Maßnahmen zeigten im Laufe der Untersuchung nicht nur einen positiven Effekt für die Zielart Emys orbicularis, sondern auch für weitere, bestandsbedrohte Begleitarten (z.B. die Wechselkröte Bufo viridis). Die ausgesetzten Jungtiere hatten ein mittleres Alter von 3,44 ± 1,29 Jahren (2-6 Jahre) bei einer mittlere Masse von 197,1 ± 110,2 g (66-619 g) und einer mittleren Carapaxlänge von 9,91 ± 1,77 cm (6,71-14,87 cm). Alle Tiere wurden vor dem Aussetzen durch einen Mikrotransponder und eine individuelle Farbmarkierung auf dem Panzer gekennzeichnet.
Mit Hilfe der Radiotelemetrie wurden die Aufenthaltsbereiche sowie das Wanderverhalten der Sumpfschildkröten in den Gebieten NSG „Hölle von Rockenberg“, NSG „Reinheimer Teich“ und NSG „Nachtweid von Dauernheim“ dokumentiert. Zusätzlich zu den herkömmlichen Radiotelemetriesendern konnten temperatursensitive Radiotelemetriesender verwendet werden, die sowohl über den Standort des Tieres als auch seine momentane Körpertemperatur Auskunft gaben. Es wurden hierbei insgesamt 34 Telemetriesender (9 ohne, 25 mit Temperaturfunktion) für eine Besenderung von 27 Individuen verwendet. Das entspricht einer Besenderungsquote von 42,3 %. Die durchschnittliche Masse der Sumpfschildkröten bei der Erstbesenderung betrug 238,6 ± 68,2 g bei einer Carapaxlänge von 10,80 ± 1,07 cm. Um einen Überblick über das Körpertemperaturspektrum der Art in Hessen zu erhalten, wurden temperatursensitive Radiotelemetriesender und ergänzend Temperaturdatenlogger (iButtons®) verwendet. Es zeigten sich sowohl individuelle als auch stark saisonal geprägte Temperaturmuster. Erwartungsgemäß konnten die höchsten Körpertemperaturen im Sommer (bis zu 44 °C) und die niedrigsten im Winter (bis zu -0,8 °C) dokumentiert werden. Der bevorzugte Temperaturbereich von Emys orbicularis wird aufgrund der vorliegenden Daten bei 25-32 °C vermutet. Die Aktivitätsperiode von Emys orbicularis in Hessen lässt sich von Mitte/Ende März bis Mitte Oktober, mit einem Aktivitätsmaximum in den Monaten Mai und Juni, angeben. Eine Erhöhung bzw. Erniedrigung der Körpertemperatur wurde durch Verhaltensweisen, wie Sonnenbaden oder das Aufsuchen von Wasser, erreicht. Sonnenbadende Sumpfschildkröten konnten in der Zeit von 07:15 bis 19:45 Uhr direkt beobachtet werden, die Hauptsonnenaktivität lag zwischen 09:00-15:00 Uhr. Hierbei konnte eine Tagesrhythmik des Sonnenbadens dokumentiert werden, die im Normalfall einen einphasigen Verlauf hatte. Die Schildkröten erschienen im Laufe des Vormittags auf dem Sonnenplatz und verblieben dort, unterbrochen von kurzzeitigem Aufsuchen des Wassers, bis in den Nachmittag hinein. Die bevorzugten Aufenthaltsbereiche und Strukturen unterlagen sowohl saisonalen als auch individuellen Präferenzen. Die Tiere nutzten zu 69,9 % Baumstämme als Sonnenplatz. Es wurden sowohl größere als auch kleinere Gewässer als Aufenthaltsbereich genutzt. Charakteristisch waren hier vor allem ausgedehnte Unterwasser- und Schwimmblatt-Gesellschaften in den Randbereichen, wie z.B. die „Untergetauchte Laichkrautgesellschaft“.
Die Störanfälligkeit der beobachteten Sumpfschildkröten war sehr stark individuell ausgeprägt. Es konnte aber auch beobachtet werden, dass Tiere, die gerade das Wasser verließen, und noch nicht getrocknet waren, schneller auf eine Störung reagierten, als Tiere, die schon vollständig getrocknet waren. Es ist anzunehmen, dass dieses Verhalten mit der Thermoregulation der Tiere in Zusammenhang steht. Die besenderten Tiere verblieben in den Aussetzgebieten und es konnten nur geringe Wanderbewegungen (bis max. 250 m) notiert werden. Der hierbei ermittelte Aktionsraum („home range“) variierte sowohl individuell als auch in den einzelnen Gebieten. So konnte in dem kleineren Gebiet „Hölle von Rockenberg“ (13 ha) eine zehnmal geringere Aktionsraumgröße notiert werden als in dem weitaus größeren Gebiet „Reinheimer Teich“ (75 ha). Sie betrug in Rockenberg 0,21 ± 0,03 ha (0,19 bis 0,32 ha) und in Reinheim 2,00 ± 1,58 ha (0,24 ha bis 4,71 ha). In der Phase der Überwinterung konnte keinerlei Mortalität dokumentiert werden. Als Überwinterungsplatz nutzten die Tiere in der Regel schilfbewachsene Uferbereiche mit einer Wassertiefe von 50 cm. Im Gebiet „Hölle von Rockenberg“ konnten einige Tiere mehrmalig in der Überwinterung beobachtet werden und es zeigten sich hierbei individuelle Standortpräferenzen. Die Überwinterungsplätze wurden zu 50 % wiederholt aufgesucht. In den Wintermonaten Dezember-Februar betrug die mittlere Körpertemperatur 3,09 ± 1,59 °C. Die verwendeten Fangmethoden (Sonnenfalle, Reusenfalle, Handfang) konnten nur im Gebiet „Hölle von Rockenberg“ erfolgreich eingesetzt werden. Im Gebiet „Reinheimer Teich“ konnte nur einmalig eine Schildkröte wiedergefangen werden. Die wiederangesiedelten Tiere nahmen sowohl an Masse als auch an Größe zu. Die durchschnittliche Massenzunahme betrug im Gebiet „Hölle von Rockenberg“ 40,33 ± 7,20 g (29,38-48,40 g) bei einem Wachstum des Carapax von 0,61 ± 0,08 cm (0,52-0,75 cm). Es konnten keinerlei Krankheiten oder Verhaltensauffälligkeiten dokumentiert werden. Im gesamten Untersuchungszeitraum wurde nur einmalig der Verlust eines Tieres detektiert.
Diese Massen- und Größenzunahmen sowie die Überlebensrate sprechen für die verwendeten Aufzuchtsmethoden und die ausgewählten Wiederansiedlungsgebiete. Es zeigt, dass sich die Methode des „headstarting“ bei Emys orbicularis sehr gut eignet und kann somit für solche Wiederansiedlungsprojekte durchaus empfohlen werden. Eine Stützung der Bestände durch das Auswildern „headstarted“ Emys orbicularis (aufgezogenen unter den hier vorgestellten Bedingungen) wird daher für weitere Projekte empfohlen Basierend auf den Ergebnissen der vorliegenden Arbeit lassen sich die Lebensraumansprüche der Art in Hessen benennen. Ausgehend von diesen speziellen Ansprüchen lässt sich ein optimales Emys orbicularis Habitat (für nördliche Vertreter der Art) definieren.
Zusammenfassende Grundlagen eines idealen Emys orbicularis Habitats:
- begleitendes (natürliches) Fließgewässer
- ausreichend Kleingewässer, auch als Trittsteine zum Fließgewässer
- mind. ein großes Hauptgewässer (mind. 2000 m2) mit einer Tiefe von mind. 1,50 m
- flach abfallendes Gewässerprofil mit sich schnell erwärmenden Flachwasserzonen
- ausreichend Wasser-/Unterwasservegetation
- ausreichend, ganztägig besonnte Sonnenplätze in Form von ins Wasser ragenden Stämmen und Ästen
- breiter, südexponierter Schilfgürtel zur Überwinterung
- geringer Fischbesatz zur Sicherung der Nahrungsgrundlage (Konkurrenz)
- möglichst keine fremdländischen Schildkröten (Konkurrenz)
- südexponierte, xerotherme offene Hanglage mit Magerrasencharakter zur Eiablage (im Bedarfsfall sichergestellt durch Beweidung und/oder Mahd) - keinerlei oder nur eingeschränkte freizeitliche Nutzung in Randbereichen - keine Zerschneidung der Gewässer und Wanderwege durch Straßen.
Die vorliegende Arbeit bestätigt den bisher getätigten Bemühungen zum Schutz der Europäischen Sumpfschildkröte gute Erfolgsaussichten für eine weitere Etablierung der Art in Hessen. Da die untersuchten Tiere noch nicht geschlechtsreif waren und somit noch keine Reproduktion im Freiland dokumentiert werden konnte, lässt sich der langfristige Erfolg noch nicht abschließend beurteilen. Aufgrund der bisher getätigten Untersuchungen lässt sich ein Reproduktionserfolg aber in den nächsten Jahren vermuten. Ein wichtiger Schritt zum Schutz der Europäischen Sumpfschildkröte in Hessen ist mit dem hier vorgestellten Projekt und den verwendeten Aufzuchts- und Wiederansiedlungsmethoden getan.
Parvulustat ist ein kleines Protein (8,2 kDA), das aus dem Bodenbakterium Streptomyces
parvulus sekretiert wird. Es ist ein saures Polypeptid mit einem isoelektrischen Punkt von 4,49
und inhibiert spezifisch α-Amylasen (Ki = 9 x 10-12).
In der vorliegenden Arbeit sollen neben der Isotopenmarkierung des Parvulustats PL-Mutanten
mittels PCR-Mutagenese und DNA Rekombinationstechnick hergestellt werden.
Zur Charakterisierung und Strukturbestimmung der Mutanten werden größere Mengen des
jeweiligen Proteins benötigt, wofür wiederum ein effektives Expressionssystem bereitstehen
muß. Ein Expressionssystem besteht aus einem Wirtsorganismus der seinen Protein-Biosynthese-
Apparat zur Verfügung stellt und einem Vektor (Plasmid) der das Zielgen trägt. Im Falle des
Parvulustats und seiner Mutanten bietet sich der gut charakterisierte Stamm Streptomyces
lividans TK 24 (Hopwood et al, 1985) als Wirtsorganismus an. Der Inhibitor wird in ihm als
Vorläuferprotein mit Signalpeptid als Transportsignal (Leader) gebildet. Da dieses Bakterium
keine äußere Membran besitzt, wird der Inhibitor direkt ins Kulturmedium ausgeschleust. Dabei
wird das Signalpeptid durch die Signalpeptidase abgespalten.
Das Strukturgen wird zuerst im Klonierungsplasmid pSH09 in E. coli vervielfältigt, bevor es mit
den Enzymen EcoRI und SpeI geschnitten wird, um in den Expressionsvektor pSH017 integriert
zu werden. Dieser Shuttlevektor trägt genauso die gewünschten Restriktionsschnittstellen, SpeI
und EcoRI, die die paßgenaue Ligation des rekombinanten Strukturgens mit den funktionellen
Elementen erlauben.
Nach erfolgtem Einbau des Zielgens in das neue Vektorkonstrukt kann dieser Expressionsvektor
leicht in den Wirtsorganismus eingebracht werden und sich dort vermehren. Nach Klonierung der
mutierten Parvulustatsgene in den Expressionsvektor werden die damit transformierten Zellen auf
R2YE-Platten aufgebracht und bei einer Temperatur zwischen 20 und 28°C je nach PL-Mutanten
inkubiert. Zur Selektion wird Thiostrepton verwendet. Nach Transformation und Vermehrung
geeigneter Wirtszellen wird das klonierte Gen transkribiert. Durch anschließende Translation der
gebildeten mRNA in der Wirtszelle entsteht das gewünschte Protein in großen Mengen. Zur
Überprüfung der Aktivität der einzelnen Mutanten steht der qualitative α-Amylase-Plattentest zur
Verfügung, wobei die aktiven Mutanten einen blauen Hemmhof bilden (siehe Abbildung 24). Der
blaue Jod-Stärke-Komplex weist, bei gleichzeitigem Vorhandensein von α-Amylase, auf nicht
abgebaute Stärke und damit indirekt auf die Produktion des α-Amylase-Inhibitors hin.
Da das Parvulustat von den Bakterien ins Medium abgegeben wird, besteht die Abtrennung der
Zellen durch Zentrifugation des Kulturmediums. Es folgen Ammoniumsulfatfällung des
Überstandes, Auftragung auf PAGE und präparative HPLC, nach der reines, einheitliches Protein
isoliert werden konnte.
The NS5B protein of the hepatitis C virus (HCV) is a RNA-dependent RNA polymerase, which is the key enzyme for viral replication. It is recognized as one of the promising targets for antiviral intervention within the new HCV treatment approach of direct-acting antivirals (DAA). However, several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GT). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. The work submitted here gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1 - 6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a > 2a, 3a, 4a, 5a > 1a. Furthermore, the optimized assay conditions for GT profiling were confirmed by testing the inhibitory activity of four known prototype NNIs, each addressing one of the four NNI binding sites. Additionally, a novel NNI chemotype - identified by screening - is described, the substituted N-phenyl-benzenesulphonamides (SPBS). This inhibitor class showed reversible inhibition of NS5B from HCV 1b Con1 with IC50 values up to 39 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M, it was assumed that the SPBS inhibitors bound to the thumb site II as it has been described for the carboxy thiophene inhibitors. The postulated binding site was consequently confirmed by analysing a provided co-crystal structure of NS5B in complex with a SPBS analogue. Notably, the two SPBS analogues SPBS-1 and SPBS-2 reported here revealed significant differences in addressing the NH-group of the main chain Y477 by hydrogen-bonds, watermediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the H475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using the previously optimized biochemical primer-dependent transcription assay, inhibitory activity of the SPBS could be demonstrated against polymerases from HCV GTs 1a and 1b whereas the inhibitor class failed to inhibit any of the non-GT 1 polymerases. Furthermore, initial antiviral activity for SPBS was demonstrated against the subgenomic replicons of HCV GTs 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types. Finally, concerning a possible future treatment without PEG-IFN α or ribavirin, the SPBS analogues were found to display additive to synergistic effects in combination with the benzothiadiazine, the benzofuran and the indole - representative inhibitors for the binding sites palm I, palm II and thumb I, repectively - in the biochemical assay. Within the same binding site as the SPBS, the reference compound hydroxydihydropyranone displayed additive interactions only with the benzothiadiazine (palm I) in the biochemical assay as well as in cell culture. Hence it could be concluded that, having characterized one individual NNI, no universal predication is possible concerning the combinatory behaviour of NNIs binding to the same binding site. As synergistic, antagonistic or additive interactions are inhibitor-dependent (not binding sitedependent) each novel NNI has to be characterized individually in one-to-one combinations.