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The prevalence of food allergies has increased in the westernized countries during the past decades. Clinical manifestations of food allergies involve the skin (e.g. atopic dermatitis), the respiratory tract (e.g. rhinitis, and asthma), the ocular area (e.g. conjunctivitis), the gastrointestinal tract (e.g. food-protein-induced enterocolitis syndrome, food-induced proctocolitis, and eosinophilic gastroenteropathies), and the cardiovascular system (e.g. anaphylaxis). A curative treatment of these diseases has not been established yet. Oral immunotherapy (OIT) has gained attention as a potential therapy for food allergies. Continuous feeding of allergenic diet applied in the model described here mirrors to a certain extent an OIT treatment. It might be therefore useful to investigate efficacy and safety of OIT pre-clinically.
Mouse models have been widely used to analyse novel treatment approaches. Unfortunately, most of them have focussed on IgE-mediated hyperreactivity. Only a limited number of mouse models presenting mixed IgE- and non-IgE-mediated gastrointestinal symptoms and inflammation upon allergen-challenge are available. To study the mechanisms underlying the induction of food-induced gastrointestinal inflammation and subsequent oral tolerance induction, a mouse model of food-induced gastrointestinal allergy was established. BALB/c mice were sensitised with Ovalbumin (OVA) plus ALUM and subsequently challenged by feeding a diet containing egg white (EW diet). During the first seven days on EW diet, OVA-sensitised mice (OVA/ALUM EW mice) developed gastrointestinal symptoms (e.g. weight loss, ruffed fur, soft stool and less mobility) and inflammation in the small intestines accompanied by a strong induction of OVA-specific IgE antibodies and mouse mast cell protease-1 (mMCP-1). Proliferation of CD4+ T cells from spleen of OVA/ALUM EW mice was reduced compared controls. The result indicated that feeding EW diet induced T cell tolerance systemically. In contrast, CD4+ T cells isolated from MLN of OVA/ALUM EW mice showed stronger proliferation upon OVA stimulation in vitro than mice OVA-sensitised but fed a conventional diet, indicating that tolerance was not induced by short-term EW diet. Histological analysis of the small intestinal tissue of OVA/ALUM EW mice revealed strong inflammation present in the duodenum, jejunum and ileum at this time point.
Interestingly, the observed symptoms in OVA/ALUM EW mice resolved spontaneously after 7 days on EW diet, if the feeding was continued. In the next steps the CD4+ T cell-mediated immune response after 28 days continuous EW diet was assessed and revealed that tolerance was induced systemically as well as locally. This was shown by reduced proliferation and cytokine secretion of CD4+ T cells from MLN of OVA/ALUM EW mice after long-term EW diet. However, the inflammation in the jejunum was aggravated instead of resolved at this time point of allergenic diet. Our results suggest that application of OIT in food-allergic patients with gastrointestinal inflammation may need to be reconsidered, since continuous administration of allergenic food may aggravate inflammation in the local tissue. Interestingly, only the jejunum was affected by a worsened condition, whereas duodenum and ileum resolved inflammation. In accordance to the observed jejunal inflammation mMCP-1 levels in the sera were not changed. Allergen-specific IgE levels did not reach baseline level after long-term EW diet, although they were reduced compared to levels in mice after 7 days on EW diet. This result suggests that residual OVA-specific IgE antibodies would promote the jejunal inflammation by sustained activation of mast cells. Furthermore, our results suggest that IL-4 produced by activated Th2 cells could be an effector molecule to induce intestinal inflammation.
The second part of this thesis was aimed at verifying the hypothesis that IgE-mediated mast cell activation is a major effector mechanism in induction of chronic inflammation induced by long-term EW diet. For that mice deficient for FcεRI, a high affinity IgE receptor, were used. These mice were sensitised with OVA and fed EW diet as described for WT mice. Although FcεRI-deficient mice showed an intact Th2 immunity with IgE production, weight loss in the receptor-deficient mice was moderately induced by EW diet compared to WT mice, suggesting that this clinical symptom during the acute phase of allergic response is associated with IgE-mediated mechanisms. Surprisingly, the deficient mice presented comparable intestinal inflammation on day seven of EW diet as WT mice did. However, if EW diet was continued, recovery of intestinal inflammation was observed in FcεRI-deficient mice in contrast to WT mice. These results suggest that the induction of intestinal inflammation is not IgE-dependent. Nevertheless, this does not rule out a potential role of mast cells in the inflammation, because of their IgE-independent activation pathways. It also suggests the involvement of T cell-mediated mechanisms during induction of jejunal inflammation. Interestingly, the aggravated inflammation seen after long-term EW diet in WT mice seems to be IgE-dependent, considering that it was not observed in FcεRI-deficient mice. The elevated number of mast cells in the intestine of WT mice further led to a hypothesis that their continuous activation might be responsible for the chronification of allergic inflammation observed after long-term EW diet. In the context of OIT it further implies that IgE might be a poor prognostic factor for recovery of intestinal inflammation during and after an OIT treatment. In the third part of this thesis regulatory mechanisms employed by the immune system were analysed. Initial results from CD4+ T cells isolated from MLN from OVA/ALUM EW mice showed elevated IL-10 levels in their supernatants after short-term EW diet. IL-10-deficient mice were used to analyse the effect of this immunosuppressive cytokine in the mouse model presented here. However, IL-10-deficient mice tend to develop a strong Th1-dominated immune response. Nevertheless, an accelerated weight loss and slight inflammation of the jejunum was observed after short-term EW diet. Analysis of OVA-specific proliferation and cytokine production CD4+ T cells from Spleen and MLN of IL-10-deficient mice on EW diet suggested that systemic as well as local tolerance was induced after short-term and long-term EW diet feeding, respectively. The result suggests that IL-10 is dispensable for induction of T cell tolerance in our mouse model.
However, the presence of functionally active Tregs was observed during this study in WT mice fed short-term EW diet, suggesting that Tregs might have an important role in regulating the systemic or local immune response. T cell deletion as an alternative immune regulatory mechanism was also observed. Additionally, the efficacy of continuous EW diet (mirroring to a certain extent an OIT treatment) in induction of permanent tolerance was assessed. In OVA-sensitised WT mice continuous allergenic diet was stopped after resolution of clinical symptoms and reintroduced after a defined period on conventional diet. Evaluating the weight development showed that reintroduction of EW diet induced weight loss again, but not as pronounced as seen after short-term EW diet. Also the CD4+ T cell-mediated response was elevated again upon allergen stimulation in vitro. The results suggested that permanent tolerance was not induced in the chosen feeding regime.
The mouse model established and analysed here was used to investigate inflammatory and regulatory mechanisms underlying food-induced gastrointestinal allergy. It presents clinical symptoms and intestinal inflammation (Burggraf et al., 2011). This model is easy to be reproduced in different laboratories, and is useful for testing novel therapy approaches (Schülke et al., 2011; Bohnen et al., 2013). It further provides an opportunity to investigate basic mechanisms underlying OIT. This therapy approach is currently extensively investigated and our mouse model would help to understand the therapeutic mechanism of OIT.
Im Zentralen Nervensystem (ZNS) kommunizieren neuronale Synapsen über eine Kombination von chemischen und elektrischen Signalen, die in ihrer Umgebung eine spezifische Komposition von Ionen benötigen. Um eine strenge Kontrolle des ZNS-Milieus zu gewährleisten, hat sich in Säugetieren eine endotheliale Blut-Hirn-Schranke (BHS) entwickelt. Die BHS limitiert den parazellulären Molekül Transport und wird von den Kapillargefässen des Gehirns gebildet, wobei die physische Barrier von den Tight Junctions (TJs) des vaskulären Endothels generiert wird. Das Gehirnendothel ist Teil einer neurovaskulären Einheit (NVE), zu der auch Perizyten (PZ), Astrozyten (AZ), Mikroglia und Interneurone zählen. Fehlkommunikation oder defekte zelluläre Komponenten in der NVE führen in der Regel zu Störungen in der BHS Funktion und können schwerwiegende neuronale Erkrankungen zur Folge haben.
Vor einigen Jahren haben wir und andere Forschungsgruppen herausgefunden, dass der Wnt/β-Catenin Signalweg essentiell für die Vaskularisierung des Gehirns während der Embryonalentwicklung ist und darüber hinaus auch eine bedeutende Rolle in der Induktion der BHS spielt. Des Weiteren konnte im Zebrafischmodell eine Aktivierung des kanonischen Wnt Signalweges auch im adulten Organismus nachgewiesen werden. Allerdings ist die Quelle der Wnt Wachstumsfaktoren bis dato unbekannt. Der Wnt Signalweg ist eine hoch konservierte und komplexe zelluläre Signalkaskade, die in allen mehrzelligen Organismen vorkommt. Wnt Wachstumsfaktoren sind sekretierte, hydrophobe Signalmoleküle, die sowohl über lange als auch kurze Strecken entweder den β-Catenin-abhängingen („kanonischen“) oder β-Catenin-unabhängingen („nicht-kanonischen“) Wnt Signalweg aktivieren können.
Da die meisten ZNS Erkrankungen mit einem Zusammenbruch der BHS-Funktion assoziiert sind, ist die Forschung bestrebt die Mechanismen, die der Entstehung und Aufrechterhaltung der BHS zugrunde liegen, zu ermitteln und zu verstehen. Das Ziel meiner Doktorarbeit war es herauszufinden, ob AZ Wnts produzieren und ob deren Wirkung auf das Gehirnendothel an der Aufrechterhaltung der BHS beteiligt ist. Zu diesem Zweck, habe ich ein in vitro BHS Kokultivierungs-Modellsystem etabliert das erstmalig ausschliesslich auf der Verwendung von murinen AZ und Gehirnendothelzellen basiert. Zu Beginn der Studie wurden sowohl primäre AZ als auch eine murine Gehirnendothel-zelllinie (MBE) bezüglich ihrer zell-spezifischen Eigenschaften charakterisiert. Dabei konnte belegt werden, dass sowohl die primären AZ als auch die MBE Zelllinie, aufgrund ihrer Proteinexpressionsprofile als repräsentative Vertreter ihres Zelltyps eingestuft werden können. Die darauffolgenden Untersuchungen konnten zeigen, dass primäre AZ über mehrere Passagen hinweg fast alle 19 Wnt Liganden auf mRNA Ebene exprimierten. Ferner konnte in primären Gehirnendothelzellen und zwei Gehirnendothelzelllinien die korrespondierenden Frizzled (FZD) Rezeptoren und low density lipoprotein receptor-related protein (LRP) Korezeptoren nachgewiesen werden. Dieser Befund legte Nahe, dass AZ und Gehirnendothelzellen die basalen Eigenschaften besitzen, um über den Wnt Signalweg miteinander zu kommunizieren. Die Stimulation von pMBEs mit Astrozyten konditioniertem Medium (AKM) induzierte die Hochregulation von Claudin-3 einem bekannten kanonischen Wnt Zielgens. Interessanterweise konnte diese Regulation teilweise durch die Zugabe von dickkopf 1 (Dkk1), einem Wnt/β-Catenin Antagonisten, inhibiert werden.
Um die physiologische Rolle der Wnt Liganden zu bestimmen, habe ich mir die Eigenschaft des universellen Sekretionsmechanismus der Wachstumsfaktoren, welcher von dem Transmembranprotein evenness interrupted (Evi) abhängig ist, zu Nutze gemacht. Die Verpaarung von Evifl/fl mit hGFAP-Cre Mäusen erlaubt die AZ-spezifische Deletion des Evi Proteins (Evi KO), was zur Folge hat, dass die Astrozyten der Nachkommen keine Wnt Wachstumsfaktoren sekretieren können.
In vitro führte der Verlust von Wnts in AKM zu einer teilweisen Delokalisierung von Junction Proteinen. Während die Kokultivierung mit Evi WT AZ einen straken Anstieg im TEER und reduzierte Permeabilitätsmesswerte induzierten, konnten diese pro-BHS Eigenschaften bei Evi KO AZ nicht beobachtet werden. Diese Ergebnisse zeigten deutlich, dass Wnts sekretiert von AZ den BHS Phenotyp positive beeinflussen, indem sie die Zell-Zell-Verbindung verstärken, was wiederum zu erhöhtem Zellwiderstand und reduzierter transzellulärer Permeabilität führt. Die Analyse des in vivo Phänotyps von Evi KO Mäusen ergab, dass mit fortschreitendem, postnatalem Alter makroskopisch erkennbare zerebrale Blutungen auftraten. Ausserdem konnte ich zeigen, dass eine Subpopulation von Blutgefässen Malformationen aufwies, die mit reduzierter Astrozytenendfuss-Assoziierung einhergingen.
Das Wissen um die Beteiligung des Wnt Signalweges an der Regulation der BHS auch im adulten Organismus kann in Zukunft von wichtiger Bedeutung sein, da es potentielle therapeutische Anwendungen ermöglicht.
Due to recent technical developments, it became evident that the mammalian transcriptome is much more complex than originally expected. Alternative splicing(AS) and the transcription of long non-coding RNAs (lncRNAs) are two phenomenas which have been greatly underestimated in their frequency. Nowadays it is accepted that almost every gene has at least one alternative isoform and the number of lncRNAs exceeds the one of protein-coding genes.
We built user-friendly web interfaces which can process Affymetrix GeneChip Exon 1.0 ST Arrays (exon arrays) and GeneChip Gene 1.0 ST Arrays (gene arrays)for the analysis of alternative splicing events. Results are presented with detailed annotation information and graphics to identify splice events and to facilitate biological validations. Based on two studies using exon arrays, we show how our tools were used to profile genome-wide splicing changes under silencing of Jmjd6 and under hypoxic conditions. Since gene arrays are not intended for AS analysis originally, we demonstrated their applicability by profiling alternative splicing events during embryonic heart development.
To measure lncRNAs expressions with exon arrays, we completely re-annotation all probes and built a lncRNA specific annotation. To demonstrate the applicability of exon arrays in combination with our annotation, we profiled the expression of tens of thousands of lncRNAs. Further, our custom annotation allows for a detailed inspection of lncRNAs and to distinguish between isoforms, as we validated by RTPCR.
To allow for a general usage to the research community, we integrated the annotation in an easy-to-use web interface, which provides various helpful features for the analysis of lncRNAs.
The TolC protein of E. coli is a versatile OMF which is involved in secretion of antibiotics, heavy metal ions, secondary metabolites and proteins. These individual tasks are accomplished by a dynamic formation of different secretion complexes which comprising a plasma membrane transporter, a Membrane Fusion Protein and TolC as the outer membrane channel-tunnel. The TolC-like protein HgdD of the cyanobacterium Anabaena sp. PCC 7120 was previously described as an indispensable OMF involved in formation of the heterocyst-specific glycolipid layer which is needed to sustain the microoxic environment that allows nitrogen fixation in heterocysts of filamentous cyanobacteria. Here I show that HgdD is involved in macrolide antibiotic resistance and ethidium efflux, which is used as a model substrate for cytotoxic compounds and secondary metabolites. It can be shown that ethidium uptake is a passive and porin-dependent process, while multidrug efflux is performed together with the RND efflux pump All3143 (and the MFP All3144). In contrast to HgdD, All3143 can complement the function of its homologue AcrB in E. coli and was suggested to be named anaAcrB. Multidrug efflux is assisted by SmsA and SchE, two secondary transporters of the MFS-type, which facilitate the transport of cytoplasmatic ethidium to the periplasmic space prior to the All3143- and HgdD-dependent efflux. Moreover, it can be demonstrated that SchE and HgdD are involved in secretion of the metal ion-chelating siderophore schizokinin, which functions in iron(III) acquisition. However, a physical interaction of SchE and HgdD is unlikely since SchE does not possess an OMF interacting domain. In addition, both RND efflux pumps All3143 and Alr1656 are needed for the homeostasis of the photosystems during diazotrophic growth. Although a direct involvement in heterocyst development or metabolism cannot be discounted at this stage, it is speculated that both RND transporters are involved in detoxification of reactive nitrogen species, similar to the function of MexF and MdtF of P. aeruginosa and E. coli respectively. In addition to its function in multidrug efflux, HgdD has been shown to be involved in protein secretion. By comparative analysis of the Anabaena sp. wild type and hgdD mutant secretome it was possible to identify eight putative HgdD protein substrates. The localization of four proteins was exemplary demonstrated by secretome isolation and cell fractionation of hemagglutinin-tagged mutant strains. The absence of detectable protein in the hgdD mutant strain suggests a highly efficient secretion system which is quality controlled by proteolysis of mislocalized proteins.
Life-attenuated measles virus (MV) vaccines have revealed their capacity to routinely induce life-long immunity against MV after just a single or two low-dose injections. Moreover, MV vaccines have been shown to be extensively safe and well tolerated, in general. Thus, MV is a prime candidate for a recombinant vaccine platform to protect also against other pathogens after vaccination. For this purpose, foreign genes can be inserted into additional transcription units (ATU) in recombinant MV genomes so that the encoded foreign proteins are co-expressed with MV proteins in infected cells. These so-called bivalent MV should protect against infection by MV or the pathogen, which the encoded foreign protein had been derived from. Bivalent MVs have already been shown to be effective vaccines against e.g. dengue virus or hepatitis B virus infections by inducing humoral and sometimes also cellular immune responses. In most of these studies, soluble or soluble versions of the pathogens' antigens were used for generation of bivalent MVs.
We hypothesized that the form of the antigen expressed by bivalent MVs is crucial for the potency and constitution of the induced immune responses. Therefore, three different forms of an antigen expressed by bivalent MVs were analyzed, here. The model antigen chosen for this purpose has been the envelope protein (Env) of SIVsmmPBj1.9. In its natural mature form, Env is composed of the surface unit gp120 and the transmembrane unit gp41, which stay non-covalently linked after proteolytic processing of the common precursor protein gp160. However, gp120 can be shed by infected cells or virus particles. Therefore, natural gp160 antigen was used as shedding form. Furthermore, stabilized covalently-linked gp160 variants and soluble gp140 variants were used in this thesis. These different antigen forms were inserted either behind the P or behind the H expression cassettes into the MV genome. The respective bivalent MVs were rescued and characterized. Expression of SIVsmmPBj1.9 Env variants by the bivalent MVs was confirmed by immuno blot and in situ immunoperoxidase assays. Replication curves of bivalent MV showed that growth of MVs expressing the different Env variants was slightly delayed by approximately 24 h compared to control viruses.
For immunization of transgenic, MV-susceptible IFNAR-/--CD46Ge mice, which are the current standard to analyze MV vaccines in a small animal model, an optimal dose of 1x105 TCID50 was determined. For the evaluation of humoral immune responses in transgenic mice, two ELISA systems for the detection of total α-MV and α-SIV antibodies and neutralization assays for detection of neutralizing antibodies against MV and SIV in sera of immunized mice were established. Mice immunized with any of the bivalent MVs showed significant humoral immune responses against MV comparable to those elicited by the parental MV vaccine strain without further genetic modifications. Mice immunized with MVvac2-gp140(P) expressing the soluble gp140 variant revealed highest α-SIV titers with a maximal OD of up to 0.4. Second highest levels of α-SIV antibodies were detected in mice that were immunized with the shedding variants or soluble Env in other positions. MVs expressing the stabilized variants induced only very low α-SIV antibody titers. Neutralizing antibodies directed against SIV could be detected in sera of mice immunized with MVs expressing the soluble or shedding variants, but not in sera of mice immunized with MVs expressing the stabilized variants. In sera of control mice immunized with PBS no antibodies could be detected, as expected. Thus, soluble and shedding antigens induced humoral immune responses, whereas stabilized antigens induced only weak humoral immune responses but no neutralizing antibodies. Analysis of cellular immune responses is still ongoing.
Besides Env, further SIV antigens could be tested for their potency to induce humoral as well as cellular immune responses.
Besides being used as a vaccine platform, recombinant MVs are evaluated as future agent for cancer therapy due to their significant inherent tumor-lytic, so-called oncolytic activity. Currently, the anti-tumoral activity of MV is analyzed in clinical phase I trials. MV strains with high fusion activity are used as oncolytic agents. The fusion protein F of MV strain NSe is highly fusogenic, in contrast to e.g. F of MVwt323, a clone of the pathogenic strain IC-B. Sequence analysis of these two proteins identified one coding nucleotide difference at aa 94 in the F2 domain: a valine (V) in FNSe and a methionine (M) in Fwt323. To evaluate impact of this difference, residues at aa 94 were exchanged. After transient-transfection of MV F and H expression plasmids in receptor-positive cells, V94 in the F2 subunit of FNSe or Fwt323 led to about 6-fold higher fusion activity compared to F proteins with M94. The co-expressed H protein (HNSe or Hwt323) did not influence fusion activity, indicating that the receptor (CD46 or SLAM) bound by H does not quantitatively affect the F proteins' activation. Analysis of F and H showed that formation and transport of MV glycoprotein complexes are not altered by substitution in aa 94 of FNSe or Fwt323.
Furthermore, recombinant MVNSe, MVNSe-F-M94, MVwt323, or MVwt323-F-V94 were rescued. Viral replication revealed slightly higher titers for recombinant MVs expressing M94 in F after 96 h of replication, compared to MVs expressing V94. MVs expressing V94 in F2 showed 2.5-fold higher fusion activity on CD46- and SLAM-positive Vero-hSLAM cells and 2-fold higher fusion activity on B95a cells expressing only SLAM compared to MVs expressing F with M94. Fusion activity of recombinant MVs can thus be modulated by substituting a single aa. V94 in the F protein results in highly fusion active MVs with possibly increased direct cytotoxicity in infected tumors, whereas M94 in F could be associated with decreased fusion activity for therapies, where higher virus titers are required.
Retroviral vectors are powerful tools in clinical gene therapy as they integrate permanently into the target cell genome and thus guarantee long-term expression of transgenes. Therefore, they belong to the most frequently used application platforms in clinical gene therapy involving a broad range of different target cells and tissues. However, stable genomic integration of retroviral vectors can be oncogenic, as reported in several animal models and in clinical trials. In particular, γ-retroviral vectors, which derive from naturally mutagenic γ-retroviruses, integrate semirandomly into the host genome with regard to the target sequence, but have a preference for regions of active transcription and regulatory elements of transcriptionally active genes. The integration can result in overexpression of adjacent genes or disruption of ‘target’ gene expression. Moreover, γ-retroviral integration can cause modified transcripts and proteins through alternative or aberrant splicing or through premature termination of transcription.
Initially, the event of insertional mutagenesis and subsequent induction of leukemia by the genotoxicity of a γ-retroviral vector was described in a mouse model after genetic modification of hematopoietic stem cells (HSCs). Vector-related activation and overexpression of the oncogene ecotropic viral integration site-1 (Evi1) fostered clonal outgrowth and leukemogenesis. Additional genotoxic events of γ-retroviral vectors were observed in clinical HSC gene therapy trials for X-linked severe combined immune deficiency (SCID-X1), chronic granulomatous disease (X-CGD), and Wiskott-Aldrich Syndrome (WAS). But, genotoxicity induced by γ-retroviral vectors has never been described in clinical gene therapy trials involving adoptive transfer of genetically modified mature T lymphocytes. This fact is surprising, since T cells are long-lived and have a high capacity of self-renewal.
In a previous study, the susceptibility towards oncogenic transformation of mature T cells and HSCs after genetic modification was compared. It could be demonstrated that T-cell receptor (TCR)-polyclonal mature T cells are far less prone to transformation after γ-retroviral transfer of (proto-)oncogenes in vivo than HSCs. Additional experiments revealed that TCR-oligoclonal (OT-I and P14) mature T cells are transformable in the same setting and give rise to mature T-cell lymphomas (MTCLs).
In the present thesis, the susceptibility of mature T cells towards insertional mutagenesis was investigated. Within the first part of the thesis, retroviral integration sites (RISs) from 33 murine MTCLs were retrieved and subsequently analyzed in terms of integration pattern, detection of common integration sites (CIS) and gene ontology (GO). As these bioinformatic results demonstrated that insertional mutagenesis most likely contributed to mature T-cell lymphomagenesis, the susceptibility of mature T cells was directly assessed in a mouse model. Therefore, murine TCR-oligoclonal OT-I T cells were transduced with an enhanced green fluorescent protein (EGFP) encoding γ-retroviral vector and gene-modified T cells were transplanted into RAG1-/- mice. After 16 months, including one round of serial transplantation, a case of MTCL emerged. Tumor cells were characterized by CD3, CD8, TCR and ICOS expression. Integration site analysis via ligation-mediated polymerase chain reaction (LM-PCR) revealed a proviral insertion in the Janus kinase 1 (Jak1) gene. Subsequent overexpression of Jak1 could be demonstrated on transcriptional and protein level. Furthermore, T-cell lymphoma cells were characterized by an activated Jak/STAT-pathway as signal transducer and activator of transcription 3 (STAT3) was highly phosphorylated. The overexpression of Jak1 was causally implicated in tumor growth promotion as specific pharmacological inhibition of Jak1 using Ruxolitinib significantly prolonged survival of mice transplanted with these Jak1-activated tumor cells. A concluding systematic metaanalysis of available gene expression data on human mature T-cell lymphomas/leukemias confirmed the relevance of Jak/STAT overexpression in sporadic human T-cell tumorigenesis.
This was the first reported case of an insertional mutagenesis event in mature T cells in vivo. Thus, the results obtained in this thesis underline the importance of long-term monitoring of genetically modified T cells in vivo and the evaluation of vector toxicology and safety in T-cell based gene therapies. In particular, the transduction of T cells with a recombinant TCR or CAR (chimeric antigen receptor) bears a risk enhancement, as normal T-cell homeostasis is perturbed besides the general risk of insertional mutagenesis.
Eine Infektion mit dem Hepatitis B Virus (HBV) kann bei 5-10 % der infizierten Erwachsenen und 70-90 % der infizierten Kinder chronisch verlaufen. Trotz einer verfügbaren Impfung gegen die Erkrankung sind heute nach Angaben der WHO weltweit etwa 350 Mio. Menschen chronisch HBV-infiziert [Lupberger and Hildt, 2007, Hollinger and Liang, 2001]. In 5-10 % der Fälle führt eine chronische Infektion zu einer Leberfibrose und Zirrhose, welche letztlich zur Ausbildung eines hepatozellulären Karzinoms (HCC) führen kann. HCCs sind die dritthäufigste karzinomassoziierte Todesursache weltweit [Blum, 2005]. Um Therapien gegen eine HBV-Infektion und das damit erhöhte Risiko einer HCC-Entstehung entwickeln zu können, müssen die einzelnen Schritte des HBV-Replikationszyklus verstanden sein. Wesentliche Schritte der frühen Infektionsphase, insbesondere der Rezeptor bzw. Rezeptorkomplex, welcher den Zelleintritt des Virus vermittelt sowie der Transport des Virusgenoms in den Zellkern, sind bisher noch unklar. Auch der Exportprozess und die Freisetzung der Viruspartikel ist bisher noch nicht im Detail verstanden. Es ist jedoch bekannt, dass die Viruspartikel unter Nutzung der zellulären ESCRT (endosomal sorting complex required for transport)-Maschinerie aus der Zelle freigesetzt werden [Lambert et al., 2007]. Auf der Suche nach Faktoren, die in diese Vorgänge involviert sind, konnte in dieser Arbeit das vesikeltransportassoziierte Protein α-Taxilin identifiziert werden. Der Einfluss von HBV auf die α-Taxilin-Bildung und seine mögliche Beteiligung am viralen Export wurden dabei näher charakterisiert. In HBV-positiven Zellen konnte in vivo und in vitro eine signifikante Steigerung der α-Taxilin-Expression nachgewiesen werden. Diese wird hierbei durch die HBV-Proteine HBx und LHBs über den Raf/Mek/Erk-Signalweg induziert [Glatzel, 2011]. Mithilfe von knockdown-Experimenten konnte beobachtet werden, dass α-Taxilin für den Export der Viruspartikel, nicht aber für den Export subviraler Partikel (SVPs) essentiell ist. Der Export der Virionen findet hierbei über das ESCRT-System statt. Den HBV-Strukturproteinen fehlen jedoch die für die Interaktion mit dem ESCRT-System essentiellen late-Domänen. Die Proteinstruktur von α-Taxilin dagegen weist diese late-Domänen auf. In dieser Arbeit konnte diese interaktionsvermittelnde Funktion von α-Taxilin zwischen dem Virus und dem ESCRT-System charakterisiert werden. Über eine Interaktion von α-Taxilin mit dem viralen LHBs-Protein auf der einen Seite und der tsg101-Komponente des ESCRT-I-Komplexes auf der anderen Seite agiert α-Taxilin als eine Art Linker zwischen dem ESCRT-System und HBV.
Darüber hinaus wurde Annexin A5 als zellulärer Interaktionspartner für α-Taxilin identifiziert [Röttger, 2011]. Es dirigiert α-Taxilin in einer Art shuttle-Funktion auf die Zellmembran suszeptibler Zellen und bindet es an deren Zelloberfläche. Diese Exposition von α-Taxilin nimmt während der Dedifferenzierung in Korrelation mit dem Suszeptibilitätsverlust primärer Hepatozyten ab. Eine Maskierung von α-Taxilin durch eine vorherige Inkubation der Zellen mit α-Taxilin-spezifischen Antikörpern konnte die Bindung und die Aufnahme der Viren inhibieren. Überexpressionsstudien bestätigten die essentielle Rezeptorfunktion von α-Taxilin. Die verstärkte Produktion von α-Taxilin führte zur Suszeptibilität der Zellen. Auch die Speziesspezifität der Bindung zwischen humanem α-Taxilin und HBV konnte in einem Co-Immunpräzipitationsexperiment mit den rezeptorbindenden Domänen von HBV, WHV und DHBV identifiziert werden.
In der vorliegenden Arbeit konnte somit zum ersten Mal eine Rezeptorfunktion von α-Taxilin bei der Aufnahme von HBV in die Wirtszelle nachgewiesen werden. Darüber hinaus schreiben die in dieser Arbeit gemachten Beobachtungen α-Taxilin eine essentielle Funktion für die Vermittlung des ESCRT-abhängigen Exports der Virionen aus der Zelle zu. Die hierbei gewonnen Erkenntnisse sind von hoher Relevanz für die weitere Erforschung der HBV-assoziierten Pathogenese und die Etablierung eines in vivo Infektions-Modells.
Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a rare inherited childhood neurodegenerative disease that is caused by a mutation in the gene CLN3. The function of the protein produced by the gene has remained elusive, and therefore the disease mechanism of JNCL is as of yet unknown. The disease is fatal, and no cure is currently available. We believe that simvastatin shows promise as a possible treatment. Simvastatin is well tolerated in children, and as currently no other viable, less invasive treatment for JNCL exists, at least pilot-scale clinical trials for this new off-label use of simvastatin are warranted.
The protein CLN3 has been indicated to have several different subcellular localizations and functions, but conclusive evidence about its role in cellular metabolism is lacking. It is also unclear why the mutation causes the distinct phenotype of the JNCL disease. In order to bring lucidity to the issue, we set out to identify metabolic pathways related to the phenotype of JNCL by using Multi-Epitope Ligand Cartography (MELC) and the related field of toponomics. Toponomic methods are required to process the massive amount of data generated by the MELC runs in order to extract information from them.
Our disease model of choice was the CLN3Δex7/8 knock-in mouse. To separate cause from effect, we compared embryonal wild type and mutant mouse brains to their adult counterparts. The first analyses revealed progressively abnormal Combinatorial Molecular Patterns (CMPs, an unit of toponomic data) related to cholera toxin/ganglioside 1 (Ctx/GM1), which is a membrane microdomain marker.
Cholesterol is an essential part of microdomains, so we utilized filipin staining to see if there were actual changes in cholesterol concentration and localization between healthy and diseased animals. After the disturbance in cholesterol metabolism was verified, we investigated the metabolic pathway that synthesizes cholesterol, the mevalonate pathway. Simvastatin is a drug that specifically down-regulates the mevalonate pathway. Fish oil affects lipid homeostasis and has some effects similar to those of simvastatin, and both of these drugs have previously been studied for their effects on neurodegenerative diseases. After treatment of mice with these drugs, highperformance liquid chromatography (HPLC) measurements on the brain homogenate showed a decrease in levels of farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), products of the mevalonate pathway, confirming the effect of these drugs on the brains of the animals. Analyses of motor function of the mice further supported the notion that simvastatin had a positive effect on the condition of the diseased animals.
CMP analyses from the simvastatin treated mice showed a rescue of the Ctx/GM1 CMPs, suggesting at least a partial restoration of membrane microdomain homeostasis. Filipin staining revealed reversion of the apparent cholesterol depletion in the adult mutant mouse hippocampus by simvastatin. Interestingly, an additional effect of the treatment was found: simvastatin also affected glutamate receptor homeostasis, especially as regarding to N-methyl-D-aspartate (NMDA) and alphaamino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors. This finding suggested that excitotoxicity could be a part of the disease process, and pointed towards glutamate receptors as possible therapy targets. This is in line with previous studies that have shown that attenuation of AMPA receptors and L voltage-dependent channels improve the phenotype of a JNCL mouse and cell model, respectively.
Simvastatin mediates many of its effects via downregulation of the mevalonate pathway products, such as isoprenoids and cholesterol. However, simvastatin also has multiple pleiotropic effects that include suppression of excitotoxicity and granting neuroprotection. It is apparent that simvastatin treatment has a positive effect on JNCL mice, but if its effects are mediated via cholesterol (and membrane microdomains), isoprenoids (and isoprenylated proteins) or via a fully cholesterol independent mechanism remains to be solved.
In this study we have shown that with the MELC method and toponomics it is possible to approach rare diseases with confounded disease mechanisms with a hypothesis-free approach, to identify possible drug targets, and to monitor the effects of the drugs on treated individuals. This should open up a new avenue in the research of the many diseases that so far have avoided all attempts at discerning their nature.
Cell-cell adhesion is an essential process during the development of multicellular organisms. It is based on various cellular junctions and ensures a tight contact between neighboring cells, enabling interactive exchanges necessary for morphological and functional differentiation and maintaining the homeostasis of healthy tissue organization. Two important types of cell-cell adhesions are the adherens junction (AJ) and the desmosome which link the actin cytoskeleton and intermediate filaments to cadherin-based adhesion sites. The core of these structures is composed of single-span transmembrane proteins of the cadherin superfamily which include, among other members, the classical cadherins, e.g. E-cadherin, as well as the desmosomal cadherins, e.g. desmoglein-3. The cytoplasmic domains of the desmosomal and classical cadherins enable interactions with proteins of the catenin family. Classical cadherins preferentially associate with β-catenin and p120-catenin, whereas desmosomal cadherins bind to γ-catenin and plakophilins. Intriguingly, γ-catenin, also known as plakoglobin, is so far the only protein known to be present both in the AJ and the desmosome.
In this study, we showed that the two homologous, membrane raft-associated proteins flotillin-1 and flotillin-2 associate with core proteins of the AJ and the desmosome in vitro and in vivo. In confluent human, non-malignant epithelial MCF10A cells and human skin cryosections, flotillin-2 colocalized with E-cadherin, desmoglein-3 and γ-catenin at cell-cell contact sites, whereas flotillin-1 showed barely any overlap with these proteins. In addition, we detected a colocalization of both flotillins with the actin-binding protein α-actinin in membrane ruffles in subconfluent and at cell-cell contact sites in confluent MCF10A cells as well as in human skin cryosections. The interaction with α-actinin was later shown to be flotillin-1 dependent by performing indirect GST pulldown experiments with purified α-actinin-1-GST in MCF10A cell lysates.
Since flotillin-2 strongly colocalized with cell-cell junctions, this suggested that flotillins might be found in complex with cell adhesion proteins. Thus, we performed coimmunoprecipitation experiments in murine skin lysates and various cell lines of epithelial origin, such as human breast cancer MCF7 cells, human keratinocyte HaCaT cells and primary mouse keratinocytes. These experiments demonstrated that flotillins, especially flotillin-2, coprecipitated with E-cadherin, desmosomal cadherins and γ-catenin in relation to the respective cell type and the maturation status of these cell-cell adhesion structures. However, since γ-catenin is so far the only protein known to be present in the AJ and the desmosome, we further assumed that the complex formation of flotillins with cell adhesion structures is mediated by γ-catenin. For this, we performed indirect GST pulldown experiments in MCF10A cell lysates with bacterially expressed, purified flotillin-1-GST, flotillin-2-GST and γ-catenin-GST and were able to verify the complex formation of adhesion proteins and flotillins in vitro. To further test if the interaction of γ-catenin and flotillins is a direct one, we used purified flotillin-1-GST or flotillin-2-GST and γ-catenin-MBP fusion proteins. Both flotillins directly interacted with γ-catenin in this in vitro assay. In addition, mapping of the interaction domains in γ-catenin by using GST fusion proteins carrying different parts of γ-catenin suggested that flotillins bind to a discontinuous γ-catenin binding domain which consists of a Major determinant around ARM domains 6-12, most likely with a major contribution of the ARM domain 7, and possibly including the NT part of γ-catenin.
To study the effect of flotillin depletion on cell-cell adhesion, we generated stable MCF10A cell lines in which flotillins were knocked down by means of lentiviral shRNAs. Staining of E-cadherin and γ-catenin in these cells showed that the localization at the cell-cell borders was significantly altered after flotillin-2 depletion, which pointed to a role for flotillin-2 in the formation of cell-cell adhesion structures in epithelial cells. Furthermore, isolation of detergent resistant membranes (DRMs) from these cells demonstrated that upon depletion of flotillin-2, a significant amount of E-cadherin and γ-catenin shifted into raft fractions. On the contrary, no change was detected in flotillin-1 knockdown cells. These observations point to a functional role of flotillin-2 in the regulation of raft association of cell-cell adhesion proteins. To gain more insight into the in vivo relevance of our findings, we next studied the function of flotillins in the skin of Flot2-/- knockout mice. Analysis of lysates prepared from the skin of one year old female animals revealed an increased expression of E-cadherin, desmoglein-1 and γ-catenin but not β-catenin, implicating that specific adhesion proteins are upregulated in flotillin-2 knockout skin.
Since flotillins are tightly associated with membrane microdomains we next studied the interaction of flotillin-2 with membrane cholesterol. Using the photoreactive cholesterol analog azocholestanol, we were able to show that flotillin-2 and cholesterol directly interacted. In addition, previous studies speculated that flotillin-2 interacts with cholesterol via two putative cholesterol recognition/interaction amino acid consensus (CRAC) motifs. Analysis of the flotillin-2 sequence revealed that flotillin-2 actually contains four putative CRAC motifs. However, using various flotillin-2 CRAC mutant GFP fusion proteins, we were able to show that none of the putative CRAC motifs is functional, which suggested that flotillin-2 interacts with membrane cholesterol, e.g., via posttranslational modifications, such as myristoylation and palmitoylation which were previously shown to be essential for membrane association of flotillin proteins.
Ribosome biogenesis is best understood in the yeast Saccharomyces cerevisiae. In human or mammalian ribosome biogenesis, it has been shown that basic principles are conserved to yeast, but additional features have been reported. Our understanding about the interplay between proteins and RNA in human ribosome biogenesis is far from complete.
The present study focused on the analysis of the human ribosome biogenesis co-factors PWP2, EMG1 and Exportin 5 (XPO5) to understand the degree of conservation of ribosome biogenesis. The proteins were characterized in respect to their localization and interaction partners. For the early 90S co-factor, PWP2, it was possible to pull down and identify the human UTP-B complex with MALDI mass spectrometry. Besides the orthologues of the members of this complex known in yeast (TBL3, WDR3, WDR36, UTP6, UTP18), the human UTP-B complex is not only conserved from yeast to humans, but contains also additional components, like the DEAD-box RNA helicase DDX21, which lacks a yeast orthologue. DDX21 was localized to the nucleus, assembled to the native UTP-B complex and co-precipitated also with other UTP-B complex members, presumably extending the functions of this complex in ribosome biogenesis.
This phenomenon was also observed for the 90S co-factor EMG1, an RNA methyltransferase, whose mutant form causes the Bowen-Conradi syndrome, if aspartic acid is mutated to glycine at position 86. This study revealed that the mutant, EMG1-D86G, clearly lost its nucleolar localization and co-precipitated to histones for unknown reasons.
A participation of the nuclear export receptor XPO5 in human ribosome biogenesis was shown in this study. Pulldown analysis, sucrose density gradients and UV crosslinking and analysis of cDNAs of XPO5 revealed the involvement of XPO5 in pre-60S subunit maturation. Moreover, besides the known pre-miRNAs and tRNAs as substrates for nuclear export, XPO5 crosslinked to snoRNAs. XPO5 was further demonstrated to interact with the miRNA Let-7a, which has an important regulatory function for MYC, a transcription factor required for ribosome biogenesis.
All results support a role of these proteins in human ribosome biogenesis and therefore it seems that the biogenesis of ribosomes in human cells requires additional components, like DDX21 and XPO5.
The biogenesis and function of photosynthetically active chloroplasts relies on the import of thousands of nuclear encoded proteins via the coordinated actions of two multiprotein translocon machineries in the outer and inner envelope membrane. Trafficking of preproteins across the soluble compartment of InterMembrane Space (IMS) is currently envisioned to be facilitated by an IMS complex composed of outer envelope proteins Toc64 and Toc12, a soluble IMS component, Tic22 and an IMS-localized Hsp70. Among them, currently Tic22 is the only component that stands undisputed in terms of its existence. Having two closely related homologs in A. thaliana, their biochemical and functional characterization was still lacking. A critical analysis of Tic22 knockout mutants displayed growth phenotype reminiscent of ppi1, the mutant of Toc33. However, both the genes have similar expression patterns with no clear preference for photosynthetic or nonphotosynthetic tissues, which explained the absence of a detectable phenotype in single mutants. In addition, transgenic complementation study with either of the homolog affirmed the identical localization of both proteins in the IMS which characterizes the two homologs as functionally redundant. Based on the pale-yellow phenotype exhibited by the double mutant plants, an attempt to analyze the import capacity of a stromal substrate in the double mutant revealed threefold reduction when compared to wild-type acknowledging the essential role of Tic22 in the import mechanism. Initially, Tic22 was identified together with another protein, Tic20, which has been heavily discussed as a protein conducting channel in the inner membrane. Despite being characterized, in A. thaliana, two out of four homologs of Tic20 are differentially localized with one being additionally localized in mitochondria and the other, exclusively residing in the thylakoids.
According to in silico analysis, for all the Tic20 proteins, a four-helix transmembrane topology was predicted. Accordingly, its topology was mapped by employing the recently established selfassembling GFP-based in vivo experiments. Astonishingly, the expression of one of the inner envelope localized Tic20 homolog enforces inner membrane proliferation affecting the shape and organization of the membrane. Therefore this study focuses on analyzing the effects of high envelope protein concentrations on membrane structures, which together with the existing results, an imbalance in the lipid to protein ratio and a possible role of signaling pathway regulating membrane biogenesis is discussed.
ß1-integrins are essential for angiogenesis but the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. BRAG2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of BRAG2 in EC and angiogenesis and the underlying molecular mechanisms remains unclear. siRNA-mediated BRAG2-silencing reduced EC angiogenic sprouting and migration. BRAG2-siRNA-transfection differentially affected a5ß1- and aVß3-integrin function: specifically, BRAG2-silencing increased focal/fibrillar adhesions and EC adhesion on ß1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the aVß3-integrin-ligand, vitronectin. Consistent with these results, BRAG2-silencing enhanced surface expression of a5ß1-integrin, while reducing surface expression of aVß3-integrin. Mechanistically, BRAG2 mediated recycling of aVß3-integrins and endocytosis of ß1-integrins and specifically of the active/matrix bound a5ß1-integrin present in fibrillar/focal adhesions (FA), suggesting that BRAG2 contributes to the disassembly of FA via ß1-integrin-endocytosis. Arf5 and Arf6 are promoting downstream of BRAG2 angiogenic sprouting, ß1-integrin-endocytosis and the regulation of FA. In vivo silencing of the BRAG2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitral injection of plasmids containing BRAG2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveals that BRAG2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating ß1-integrin internalization and associates for the first time the process of ß1-integrin endocytosis with angiogenesis.
Der programmierte Zelltod (Apoptose) ist ein wichtiger Mechanismus zur Eliminierung von beschädigtem Gewebe und entarteten Zellen. Die Deregulierung der Apoptose führt zu zahlreichen Erkrankungen wie neuro-degenerativen Störungen und Krebs. Insbesondere in Tumoren wird der programmierte Zelltod mit Hilfe von hochregulierten, anti-apoptotischen Proteinen umgangen und es entstehen Resistenzen gegen Chemotherapien. Um innovative therapeutische Ansätze zu finden, wurden in diesem Projekt mit Hilfe eines Hefe-Survival-Screens neue, potentiell anti-apoptotische Proteine im Pankreaskarzinom identifiziert. Von den insgesamt 38 identifizierten Genprodukten wurden zwei für eine weiterführende Analyse ausgewählt.
Eins der näher untersuchten Proteine ist die Pyruvoyl-tetrahydrobiopterin-Synthase (PTS), ein wichtiges Enzym für die Biosynthese von Tetrahydrobiopterin (BH4). BH4 ist ein Kofaktor, der von mehreren Enzymen der Zelle für ihre Funktionen benötigt wird. In Zellkultur-Experimenten konnte gezeigt werden, dass eine Überexpression von PTS die Zellen vor Apoptose schützen kann, während eine Herunterregulation durch genetischen knockdown die Zellen gegenüber Apoptose-Stimuli sensibilisiert und ihr Wachstum beeinträchtigt. In Xenograft-Experimenten mit NOD/SCID-Mäusen konnte zudem gezeigt werden, dass Tumore mit einem PTS-Knockdown signifikant langsamer wachsen als die der Kontrollgruppe. Zusammengenommen deuten diese Ergebnisse auf eine Rolle von PTS bei der Apoptose-Regulation und beim Tumorwachstum hin, was das Protein zu einem attraktiven Target für die Krebstherapie macht.
Als zweites wurde ein Protein analysiert, das eine Untereinheit des respiratorischen Komplex I bildet: NDUFB5 (NADH-Dehydrogenase 1 beta Subcomplex, 5). Das besondere an diesem Protein sind die verschiedenen Isoformen, die durch alternatives Splicing zustandekommen. Eine Isoform, der die Exone 2 und 3 fehlen, wurde im Hefe-Survival-Screen identifiziert. Bei Überexpression in Zelllinien konnte sie im Gegensatz zum Volllänge-Protein die Apoptoserate reduzieren. Und auch Ergebnisse aus Versuchen mit Isoformen-spezifischem knockdown deuten an, dass hauptsächlich die verkürzte Isoform sNDUFB5 für die Regulation von Apoptose und Proliferation verantwortlich ist. Diese Beobachtungen konnten mit denselben Zellen im Xenograft-Tiermodell jedoch nicht bestätigt werden. Die Ursachen dafür blieben unklar. Zusätzlich wurden immunhistochemische Analysen von Pankreaskarzinomen und normalem Pankreasgewebe durchgeführt. Sie ergaben, dass die kurze Isoform sNDUFB5 im Tumor stark überexpremiert ist, während die Expression des Volllänge-Proteins in normalem und Tumorgewebe ähnlich hoch ausfällt. Dieser Befund macht NDUFB5 zu einem interessanten therapeutischen Target.
Die näher untersuchten Kandidaten-Gene zeigen beide Potential als neue Angriffspunkte für eine molekulare Krebstherapie. Andere in dem Hefe-Survival-Screen identifizierte Proteine wurden bereits als anti-apoptotisch und/oder in Krebszellen überexprimiert beschrieben. Diese Ergebnisse demonstrieren, dass ein funktionelles, Hefe-basiertes Screeningsystem geeignet ist, neue bisher unbekannte Proteine mit anti-apoptotischer Funktion zu identifizieren. Auch zeigen die Befunde, dass bereits bekannte Proteine weitere bisher unbekannte Funktionen wie z.B. die Inhibition von Apoptose aufweisen können. Basierend auf solchen mehrfachen Proteinfunktionen lassen sich weitere therapeutische Möglichkeiten ableiten.
In dieser Arbeit wurde die physiologische Funktion der Klasse I Methyltransferase Rrp8 bei der Ribosomen-Biogenese der Hefe Saccharomyces cerevisiae untersucht. Ziel war es, die Bedeutung des Proteins für die rRNA-Prozessierungsschritte besser zu verstehen und das Substratmolekül zu identifizieren, das durch die katalytische Aktivität von Rrp8p modifiziert wird.
In einer rrp8-ΔC Mutante, bei der die für die C-terminale Methyltransferase-Domäne codierende Sequenz deletiert vorlag, konnte eine leichte Mengenreduktion der 40S Untereinheit gefunden werden, was für eine Beteiligung von Rrp8p an der Biogenese der kleinen Untereinheit sprach. Unter Anwendung eines artifiziellen Tetrazyklin-Aptamer-Systems, das die Regulation der Expression eines spezifischen Gens erlaubt, wurde eine bereits vorher bekannte synthetische Interaktion mit der essentiellen 90SKomponente Nep1p bestätigt. Mit Hilfe dieses Expressionssystems konnte auch für eine reduzierte Expression von Nop14p, einem Interaktionspartner des Nep1-Proteins, eine synthetisch kranke Beziehung mit rrp8-ΔC festgestellt werden. Zusammen mit der Untersuchung des Sedimentationsverhaltens eines markierten Rrp8-Proteins und bekannten Daten aus der Literatur wiesen die genetischen Analysen darauf hin, dass Rrp8p neben dem Einfluss auf späte Reifungsschritte des 90S prä-Ribosoms auch für die frühen Reifungsschritte der 60S Untereinheit wichtig ist. Weitere Interaktionen mit Faktoren, die an der Translation beteiligt sind (TIF4631, DOM34) und die Messung der Translationsaktivität zeigten, dass der Ausfall von Rrp8p nicht nur die Biogenese verzögert, sondern gleichfalls die Funktionsfähigkeit des Ribosoms beeinflusst.
Die in dieser Arbeit durchgeführte phänotypische Analyse einer rrp8-ΔC tc-GAR1 Doppelmutante unterstützte die Vermutung, dass Rrp8p auch frühe Reifungsschritte der 60S Untereinheit beeinflusst. Mit einem in vitro Experiment konnte die Bindung von SAM an Rrp8p gezeigt werden und RP-HPLC Analysen der 25S rRNA verdeutlichten, dass Rrp8p neben dem Einfluss auf die Prozessierungsstelle A2 für die m1A645 Modifikation in Helix 25.1 verantwortlich ist. Die phänotypische Untersuchung einer von P. Kötter und S. Lamberth angefertigten rRNA Mutante (A645U) zeigte, dass die Sequenzveränderung innerhalb der Helix 25.1 der 25S rRNA, die zugleich zum Verlust der Modifikation führt, eine deutliche Auswirkung auf das Zellwachstum und auf das Polysomenprofil hat. Ähnliche Polysomenprofile wurden in den Mutanten rrp8-G209R und rrp8-G209A beobachtet, die ein punktmutiertes Rrp8-Protein exprimieren. Eine reduzierte SAM-Bindungsaktivität des mutierten Proteins führte ebenfalls zu einer reduzierten Menge an m1A645 modifizierter 25S rRNA. Eine im Unterschied zur rrp8-ΔC Mutante auftretende Reduktion der 60S Untereinheit in den Punktmutanten spricht für einen bisher noch unbekannten Einfluss von Rrp8p auf die Biogenese der 60S Untereinheit.
In Zusammenarbeit mit S. Sharma durchgeführte 2D-DIGE Experimente und quantitative Messungen von Transkriptmengen zeigten, dass im Vergleich zu einem Wildtyp-Stamm in einer rrp8-ΔC Mutante einige glykolytische Enzyme in geringerem Maße exprimiert werden, was in Zusammenhang mit einer in höheren Eukaryoten bekannten nukleolären Stressantwort gebracht werden kann. Dies verdeutlicht die komplexe Wechselwirkung zwischen der Ribosomenfunktion und dem Energiemetabolismus.
Nervous system development requires a sequence of processes such as neuronal migration, the development of dendrites and dendritic spines and the formation of synapses. The extracellular matrix protein Reelin plays an important role in these processes, Reelin regulates for example the migration of neurons from proliferative zones to their target positions in the brain. As a consequence, layered structures are formed in the neocortex, the hippocampus and cerebellum (Lambert de Rouvroit et al., 1999). Reelin exerts its functions by binding to two transmembrane receptors, apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR). This binding causes phosphorylation of the intracellular adapter protein Disabled-1 (Dab1) (D’Arcangelo et al., 1999) via activation of Src-family kinases (SFKs) (Bock and Herz, 2003), leading to cytoskeletal reorganization which enables cell migration and morphological changes (Lambert de Rouvroit and Goffinet, 2001). Since ApoER2 and VLDLR do not possess intrinsic kinase activity to activate SFKs, the existence of a co-receptor was suggested. EphrinBs are transmembrane ligands for Eph receptors and have signaling capabilities required for axon guidance (Cowan et al., 2004), dendritic spine maturation (Segura et al., 2007) and synaptic plasticity (Essmann et al., 2008; Grunwald et al., 2004). As stimulation of cultured cortical neurons with soluble EphB receptors causes recruitment of SFKs to ephrinB-containing membrane patches and SFK activation (Palmer et al., 2002), we investigated whether ephrinB ligands would be the missing co-receptors in the Reelin signaling pathway functioning during neuronal migration, dendritic spine maturation and synaptic plasticity. We found that the extracellular part of ephrinBs directly binds to Reelin and that ephrinBs interact with Dab1, phospho-Dab1, ApoER2 and VLDLR. EphrinB3 is localized in the same neurons as ApoER2 and Dab1 in the cortex and hippocampus, and in the cerebellum ephrinB2 is detected in neurons that express Dab1. To investigate the requirement of ephrinBs for neuronal migration, triple knockout mice lacking all ephrinB ligands were analyzed. The cortical layering of ephrinB1, B2, B3 knockout brains is inverted, showing the outside-in pattern typical for the reeler cortex. The hippocampus and cerebellum of triple knockout mice also exhibit reeler-like malformations, although less penetrant than the cortical defects. Dab1 phosphorylation is impaired in mice lacking ephrinB3 and this effect is strongly enhanced in neurons lacking all ephrin ligands. Moreover, activation of ephrinB3 reverse signaling induces Dab1phosphorylation in reeler primary neurons. In agreement with an important regulatory function of ephrinBs in Reelin signaling, activation of ephrinB3 reverse signaling is even able to rescue reeler defects in cortical layering in organotypic slice cultures. In summary, all these results identify ephrinBs as co-receptors for Reelin signaling, playing essential roles in neuronal migration during the development of cortex, hippocampus and cerebellum (Sentürk et al., 2011).
Durch RNAinterferenz (RNAi) läßt sich die Expression eines beliebigen Gens spezifisch unterdrücken. Dafür müssen in das Zytoplasma kurze, doppelsträngige RNA Moleküle (siRNA bzw. shRNA) eingebracht werden, die teilweise komplementäre Sequenzen zu dem Zielgen aufweisen. Um siRNAs mit einer hohen Effizienz und Kopienzahl in die Zielzelle einzubringen, wurden Transfersysteme unterschiedlicher Art entwickelt. Nicht-virale Transfersysteme können nur einen transienten Effekt auslösen - ein Umstand, der für Langzeitstudien eine mehrfache Transfektion bedingt. Zur Lösung dieses Problems wurden retrovirale Vektorsysteme entwickelt, die durch Integration der shRNA-Expressionskassette in das zelluläre Genom eine stabile Unterdrückung eines Zielgens erreichen können. Insbesondere für präklinische Studien in vivo ist jedoch ein System mit erhöhter Transferrate wünschenswert, um in möglichst vielen Zielzellen einen RNAi-Effekt zu bewirken. Sliva et al. konnten zeigen, dass das Murine Leukämie Virus (MLV) theoretisch diese Anforderung erfüllt. Dafür wurde eine shRNA-Expressionskassette in das Virusgenom eingefügt und in vitro ein RNAi-Effekt nachgewiesen. In der vorliegenden Arbeit wurde dieses System nun durch die Verwendung von microRNA-adaptierten shRNAs (shRNAmir) verbessert. In mehreren Publikationen wurde bestätigt, dass shRNAs, die endogenen microRNAs nachempfunden sind, eine höhere Effizienz und niedrigere Toxizität aufweisen. Zunächst wurde die für die genetische Stabilität optimale Orientierung der shRNAmir-Expressionskassette bestimmt. Das Konstrukt in reverser Orientierung wies eine Deletion in der shRNAmir Promotersequenz auf, die wahrscheinlich durch Interferenz mit dem 5’LTR Promoter entstanden ist. Mit dem genetisch stabilen Viruskonstrukt wurden Experimente zur Reduktion der Expression von Markergenen durchgeführt, um die Effizienz der RNAi-Aktivität leicht zu quantifizieren. Dafür wurden humane Fibrosarkom (HT1080) Zellen infiziert, die eGFP oder Luziferase stabil exprimieren.
Mit eGFP- und Luziferase-spezifischen shRNAmir-Expressionskassetten konnte nach Infektion eine Herunterregulation von eGFP auf etwa 20 % und für Luziferase auf unter 10% beobachtet werden. Das Kontrollvirus, das eine unspezifische shRNAmir kodiert, hatte keinen Einfluss auf die Expression beider Markerproteine. Die Kinetik mit der die Markerproteine herrunterreguliert wurden, war abhängig von der Virusdosis. Die Virusdosis hatte aber keinen Einfluß auf die Stärke des RNAi-Effekts, der nach Infektion aller Zellen festgestellt werden konnte. Dieses Ergebnis entspricht der Erwartung an ein replikatives Transfersystems, das je nach applizierter Virusdosis unterschiedlich schnell RNAi in der Zellkultur ausbreitet und induziert. Die Anwendbarkeit dieses RNAi-Transfersystems auch für endogene Gene wurde mit MMP14-spezifischen shRNAmirs gezeigt. Nach Infektion von HT1080 Zellen mit den entsprechenden Viren in HT1080 Zellen konnte eine verringerte Menge an MMP14 mRNA und Protein nachgewiesen werden. Dies konnte funktionell durch eine verringerte Menge an intermediärem MMP2 und durch eine reduzierte Invasivität bestätigt werden. Zudem war die Fähigkeit dieser Zellen subkutane Tumore zu bilden stark eingeschränkt.
Um die Anwendbarkeit dieses Systems für in vivo Applikationen zu zeigen, wurde in Mäuse, die Luziferase-exprimierenden Tumoren trugen, MLV-shLuc oder das Kontrollvirus systemisch appliziert. 21 Tage nach Virusgabe konnte in den Tumoren von MLV-shLuc infizierten Mäusen eine Abnahme der Luziferaseaktivität auf 15 % nachgewiesen werden. Auch in Mäusen, die systemisch applizierte Tumorzellen erhielten, konnte eine Tendenz von RNAi-vermittelter Luziferase-Reduktion beobachtet werden.
Damit wurde in dieser Arbeit ein neuartiges RNAi-Transfersystem geschaffen, das in der Lage ist, auch in vivo einen starken und lang andauernden RNAi-Effekt auszulösen. Die Einzigartigkeit besteht in der Kombination von shRNAmir und Replikations-kompetenten Retroviren. Dadurch konnte eine erweiterte Transferrate von shRNAmir in Tumorzellen erreicht werden, so dass nun Genfunktionsstudien mit sehr hoher Aussagekraft möglich sind.
In the absence of apparent mutations, alteration of gene expression patterns represents the key mechanism by which normal cells evolve to cancer cells.
Gene expression is tightly regulated by posttranscriptional processes. Within this context, RNA-binding proteins (RBPs) represent fundamental factors, since they control mechanisms, such as mRNA-stabilization, -translation and -degradation. Human antigen R (HuR) was among the first RBPs that have been directly associated to carcinogenesis. HuR modulates the stability and translation of mRNAs which encode proteins facilitating various ‘hallmarks of cancer’, namely proliferation, evasion of growth suppression, angiogenesis, cell death resistance, invasion and metastasis. Furthermore, it is well established that tumor-promoting inflammation contributes to tumorigenesis. In this process, monocytes are attracted to the site of the tumor and educated towards a tumor-promoting macrophage phenotype. While HuR has been extensively studied in various tumor cell types, little is known about HuR in hepatocellular carcinoma (HCC). Thus, the aim of my work was to characterize the contribution of HuR to the development of cancer characteristics in HCC. I was particularly interested to investigate if HuR facilitates tumor-promoting inflammation, since a role for HuR has not been described in this context. To this end, I depleted HuR in HepG2 cells (HuR k/d) and used a co-culture model of HepG2 tumor spheroids and infiltrating monocytes to study the impact of HuR on the tumor microenvironment. I could show that depletion of HuR resulted in the reduction of cell numbers. Additionally, the expression of proliferation marker KI-67 and proto-oncogene c-Myc was reduced, supporting a proliferative role of HuR. Furthermore, exposure to cytotoxic staurosporine elevated apoptosis in HuR k/d cells compared to control cells. Concomitantly, the expression of the anti-apoptotic mediator B-cell lymphoma protein-2 (Bcl-2) was markedly reduced in the HuR k/d cells, pointing to an involvement of HuR in cell survival processes.
Accordingly, a pro-survival function of HuR was also observed in tumor spheroids, since HuR k/d spheroids exhibited a larger necrotic core region at earlier time points and showed elevated numbers of dead cells compared to control (Ctr.) spheroids. Interestingly, HuR k/d spheroids isplayed reduced numbers of infiltrated macrophages, suggesting that HuR contributes to a tumor-promoting, inflammatory microenvironment by recruiting monocytes/macrophages to the tumor site. Aiming at identifying HuR-regulated factors responsible for the recruitment of monocytes, I found reduced levels of the chemokine interleukin 8 (IL-8) in supernatants of HuR k/d spheroids, supporting a critical involvement of HuR in the chemoattraction of monocytes. Analyzing supernatants of co-cultures of macrophages and HuR k/d or Ctr. spheroids revealed additional differences in chemokine secretion patterns. Interestingly, protein levels of many chemokines were elevated in co-cultures of HuR k/d spheroids compared to control co-cultures. Albeit enhanced chemokine secretion was observed, less monocytes are recruited into HuR k/d spheroids, further underlining the necessity of HuR in cancer related monocyte/macrophage attraction and infiltration. Differences between chemokine profiles of mono- and co-cultured spheroids could be attributable to changes in spheroid-derived chemokines as a result of the crosstalk with the immune cells. Provided the chemokines originate from monocytes/macrophages, the different secretion patterns suggest that HuR contributes to the modulation of the functional phenotype of infiltrated macrophages, since the tumorenvironment is critically involved in the shaping of macrophage phenotypes. Regions of low-oxygen (hypoxia) represent another critical feature of tumors. Therefore, I next analyzed the impact of HuR on the hypoxic response. Loss of HuR attenuated hypoxia-inducible factor (HIF) 2α expression after exposure to hypoxia, while HIF-1α protein levels remained unaltered. Considering previous results of our group, showing that HIF-2α depletion (HIF-2α k/d) resulted in the enhanced expression of HIF-1α protein, I aimed to determine the involvement of HuR in the compensatory upregulation of HIF-1α protein in HIF-2α k/d cells. I could demonstrate that not only total HuR protein levels, but specifically cytoplasmic HuR was elevated in HIF-2α depleted cells pointing to enhanced HuR activity. Silencing HuR in HIF-2α deficient cells attenuated enhanced HIF-1α protein expression, thus confirming a direct role of HuR in the compensatory upregulation of HIF-1α. This as also reflected on HIF-1α target gene expression. I further investigated the mechanism underlying the compensatory HIF-1α expression in HIF-2α deficient cells. Analyzing HIF-1α mRNA expression, I excluded enhanced HIF1-α transcription and stability to account for elevated HIF-1α expression in HIF-2α k/d cells. HIF-1α promoter activity assays confirmed the mRNA data. Furthermore, HIF-1α protein half-life was not elevated in HIF-2α k/d cells compared to control cells, indicating that HIF-1α protein stability is not altered in HIF-2α k/d cells. Analysis of the association of HIF-1α with the translational machinery using polysomal fractionation finally revealed an increased istribution of HIF-1α mRNA in the heavier polysomal fractions in HIF-2α k/d cells compared to control cells. Since augmented ribosome occupancy is an indicator for more efficient translation, I propose enhanced HIF-1α translation as underlying principle of the compensatory increase in HIF-1α protein levels in HIF-2α k/d cells. In summary, my results demonstrate that HuR is critical for the development of cancer characteristics in HCC. Future work analyzing the impact of HuR on tumor-promoting inflammation, specifically macrophage attraction and activation could provide new trategies to inhibit macrophage-driven tumor progression. Furthermore, I provide evidence that HuR contributes to the hypoxic response by regulating the expression of HIF-1α and HIF-2α. Targeting single HIF-isoforms for tumor therapy should be carefully considered, because of their compensatory regulation when one α-subunit is depleted. Thus, therapeutic strategies targeting factors such as HuR that control both α-subunits and at the same time prevent compensation might be more promising.
In this thesis the integral membrane protein diacylglycerol kinase (DAGK) from E.coli is investigated with solid-state NMR. The aim is to gain an insight into the enzyme’s mechanism through integration of kinetic, structural and dynamic data. The biological function of DAGK is the transfer of the γ-phosphate group from Mg*ATP to diacylglycerol (DAG) building phosphatidic acid (PA)[6] as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases[12]. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found[13]. In its physiological form DAGK is a homo-trimer with nine transmembrane helices, three catalytic centers and a size of 39.6 kDa.
First, the set-up of a real-time 31P MAS NMR experiment is shown. This experiment allows measuring in real-time the simultaneous ATP hydrolysis in the aqueous phase and lipid substrate phos-phorylation in the membrane phase with atomic resolution under magic angle spinning[56]. After fast transfer of the sample into the NMR spectrometer the enzymatic reaction is started with a temperature jump. This approach of real-time MAS NMR in a dual-phase system was demonstrated for the lipid substrate analogs dioleoyl- (DOG) and dibutyrylglycerol (DBG), with a C8 and C4 aliphatic chain, respectively. The combination of 31P direct and cross polarization functions as a dynamic filter. In the 31P direct polarized experiment nuclei in both phases are detected, while in the 31P cross polar-ized experiment, only nuclei in the membrane phase are detected. Rates for substrate turnover, i.e. degradation of γP-, βP, αP-ATP and build-up of βP-, αP-ADP, free phosphate as side reaction, and PA are obtained, which reveal a Michaelis-Menten behavior with regard to Mg*ATP and DBG. Here Mg*ATP and DBG follow a random-equilibrium model, where every substrate can bind indepen-dently from the other substrate. Analyses of the peak integrals from educts and products of the enzymatic reaction, revealed the stoichiometry of the reaction: 1.5 ATP molecules are used to phos-phorylate one DBG molecule. The excess of ATP is attributed to the basal ATPase activity. Further-more, experiments with ATPγS, usually regarded as a non-hydrolysable ATP-analog, where carried out. Surprisingly, DAGK hydrolyzes ATPγS and also transfers the thio-phosphate group to the lipid acceptor DBG, which points to a certain degree of plasticity in the active center. A phosphorylated enzyme intermediate was not detected. These results suggest the building of a ternary complex of Mg*ATP, DBG and DAGK performing a direct-phosphoryl transfer reaction, without passing through a phosphorylated enzyme intermediate. Experiments with the transition state analog ortho-vanadate (Vi) showed a decoupling of the ATP hydrolysis activity from lipid substrate phosphorylation. This indicates a specific transfer site for the γ-phosphate group from ATP to DAG, which can be blocked by Vi.
A general disadvantage of NMR spectroscopy compared to other spectroscopic methods is its inherent low sensitivity. One possible starting point for the improvement of signal-to-noise per unit time is the reduction of the spin-lattice relaxation time of protons[209]. Usually 95 % of the experi-mental time is required for the relaxation of the 1H to equilibrium. The addition of paramagnetic species can be used to reduce the 1H T1[233]. In a comprehensive study four different paramagnetic agents were tested: Cu2+-EDTA, Cu2+-EDTA-tag, Gd3+-TTAHA and Gd3+-DOTA. The titration of these paramagnetic complexes showed the principle feasibility of this approach, but differences between the tested species exist. The most promising complex is Gd3+-DOTA which, at a concentration of 2 mM, causes a 10-time improvement of signal-to-noise ratio per unit time. This allowed measuring 2D 13C-13C correlation spectra of proteoliposomes in one tenth of the usual required experimental time (i.e. 10 hours vs. 4 days) with good signal-to-noise.
For the investigation of structural or dynamic changes in the protein upon substrate interaction with MAS NMR, the spectral properties CP efficiency and resolution of the DAGK in liposomes needed to be improved. The most critical step during sample preparation is the reconstitution of the membrane protein from detergent micelles into a membrane of synthetic lipids under detergent removal. For this procedure the important criteria are enzymatic activity, measured in a coupled ATPase assay[55], and homogeneity of the proteoliposomes, which was tested e.g. on a discontinuous sucrose step gradient. Therefore an extensive study was carried out, in which different detergents, lipids and lipid mixtures, techniques for detergent removal and different protein-to-lipid ratios were tested. A direct correlation between high ATPase activity and good resolution was not found. Moreover, active DAGK in a mixture of DMPC and cholesterol, which emulates the membrane features of a membrane containing DAG, showed the best CP efficiency and resolution.
The assignment of the protein backbone and amino acid side chains the first mandatory step towards the investigation of structural and dynamical features influencing and defining the enzymatic mechanism by MAS NMR. As the assignment procedure is very time consuming for a total protein, a special labeling scheme for DAGK was developed, which allows assigning most of the protein areas presumably involved in enzyme catalysis. The assignment of DAGK with solution NMR[132] was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs[335], two consecutive amino acids which only appear once in the amino acid sequence. These unique pairs served as anchor points. Five different multinuclear MAS NMR experiments (DARR, NCO, NCA, NCACX, NCOCX) were required for the sequential assignment. It was possible to assign 35 % of the total amino acid sequence with one sample and 8 experiments acquired at 850 MHz. The secondary structure analysis showed subtle differences to the DAGK assignment with solution NMR[132], which can be attributed to the different environment in lipid bilayers and detergent micelles.
Data about structural and dynamical changes under substrate interaction can reveal details about the enzymatic mechanism. Therefore changes in chemical shift in 2D heteronuclear correlation experiments in the apo-state and under substrate saturated conditions with the substrates Mg*AMP-PNP, a non-hydrolysable ATP-analog, DOG, a mixture of Mg*AMP-PNP and DOG as well as inhibited by Vi were recorded. The most significant peak changes were observed at the interface membrane-cytoplasm as well as the the N-terminal amphipathic helix. The residues revealing chemical shift perturbations correlate with conserved residues or such residues, for which importance for catalysis and/or folding could be shown in mutation studies[8]. Especially noticeable were the changes at the amino acids Asn 72, Lys 64, His 87, Tyr 86 and Asp 95.
Beside changes of the chemical shift, changes of line width or signal doubling were observable. These changes can point to a correlation with dynamic reorientations in the μs-ms time regime, which are most relevant for enzymatic processes. The protein backbone dynamics in the apo-state as well as saturated with the substrates or inhibited with Vi were investigated with a 15N-CODEX experiment, which is based on the reorientation of the CSA tensor upon dynamical changes[350]. Specific effects of the different substrates or analogs on the protein backbone dynamic were revealed complementing the structural data and the chemical shift perturbation experiments.
Plants absorb sunlight via photosynthetic pigments and convert light energy intochemical energy in the process of photosynthesis. These pigments are mainly bound to antenna protein complexes that funnel the excitation energy to the photosynthetic reaction centres. The peripheral antenna of plant photosystem II (PSII) consists of the major light-harvesting complex of PSII (LHC-II) and the minor LHCs CP29, CP26 and CP24. Light intensity can change frequently and plants need to adapt to high-light conditions in order to avoid photodamage. When more photons are absorbed than can be utilised by the photosynthetic machinery, excessive excitation energy is dissipated as heat by short-term adaptation processes collectively known as non-photochemical quenching (NPQ). A decrease in PSII antenna chlorophyll (Chl) fluorescence yield and a reduction in the average Chl fluorescence lifetime are associated with NPQ. The main component of NPQ is the so-called energy-dependent quenching (qE), and it is triggered by the rapid drop in thylakoid lumenal pH resulting from the plant’s photosynthetic activity. This process is thought to take place at the PSII antenna complexes, which therefore not only capture and transfer light energy but are also involved in balancing the energy flow. The decrease in lumenal pH acivates the enzyme violaxanthin de-epoxidase (VDE), which converts the xanthophyll violaxanthin (Vio) into zeaxanthin (Zea) in the xanthophyll cycle. In addition, the PSII subunit PsbS was discovered to be essential for qE by screening qE-deficient Arabidopsis thaliana mutants. This membrane protein is considered a member of the LHC superfamily, which also includes LHC-II and the minor LHCs. Previous studies on PsbS isolated either from native source or refolded in vitro have produced inconsistent results on its pigment binding capacity. Interestingly, a pH-dependent change in the quaternary structure of PsbS under high light conditions has been reported. This observed dimer-tomonomer transition very likely follows the protonation of lumenal glutamates upon the drop in pH and is accompanied by a change in PSII supercomplex localisation. PsbS dimers are preferentially found in association with the PSII core, whereas PsbS monomers co-localise with LHC-II.Despite the identification of !pH, Zea and PsbS as key players in qE, both the nature of the quencher(s) as well as the underlying molecular mechanism leading to excess energy dissipation still remain unknown. Several models have been put forward to explain the reversible switch in the antenna from an energy-transmitting to a quenched state. Proposals include a simple pigment exchange of Vio for Zea, and aggregation or an internal conformational change of LHC-II. Charge transfer (CT)quenching in the minor LHCs or quenching by carotenoid dark state (Car S1)-Chl interactions have also been suggested. However, none of these qE models has so far been capable of accommodating all the physiological observations and available experimental data. Most importantly, the function of PsbS remains an enigma. A recent qE model suggested that monomerisation of PsbS enables the protein to transiently bind a carotenoid and form a quenching unit with a Chl of a PSII LHC. In view of the various proposed qE mechanisms, this thesis aimed at understanding the interplay of the different qE components and the contribution of the PSII subunits LHC-II, the minor LHCs and PsbS to qE. The initial approach was to investigate the properties of the PSII subunits in the most simple in vitro model system, namely in detergent solution. For this purpose, LHC-II was isolated either from native source or refolded from recombinantly produced protein. Investigation of the minor LHCs and PsbS required heterologous expression and refolding. In addition, experiments were performed on aggregated LHC-II. Aggregates of LHC-II have been used as a popular model system for qE because they exhibit highly quenched Chl fluorescence. At the final stage of this doctoral work, a more sophisticated model system to approximate the thylakoid membrane was developed by reconstitution of the PSII subunits LHC-II and PsbS into liposomes. This system not only allowed for investigation of these membrane proteins in their native environment, but also for mimicking the xanthophyll cycle by distribution of Zea within the membrane as well as !pH by outside buffer exchange. The role of Zea in qE was first investigated with detergent solubilised antenna proteins. The requirement of this xanthophyll for qE is well-known, but the specific contribution to the molecular quenching mechansim is unclear. Previous work had shown that replacement of Vio for Zea in LHC-II was not sufficient to induce Chl fluorescence quenching in Zea-LHC-II, as suggested by the so-called molecular gearshift mechanism. However, by means of selective two-photon excitation spectroscopy, an increase in electronic interactions between Car S1 and Chls was observed for LHC-II upon lowering the pH of the detergent buffer. Electronic Car S1-Chl coupling became even stronger when Zea-LHC-II was probed. The extent of Car S1-Chl coupling correlated directly with the extent of Chl fluorescence quenching, in a similar way as observed previously in live plants under high-light conditions. However, very similar results were obtained with LHC-II aggregates. This implied that the increase in electronic interactions and fluorescence quenching was independent of Zea and low pH. Further experiments on aggregates of LHC-II Chl mutants indicated that the targeted pigments were also not essential for the observed effects. It is proposed that the same molecular mechanism causes an increase in electronic Car S1-Chl interactions and Chl fluorescence quenching in Zea-LHC-II at low pH as well as in aggregated LHC-II. Most likely, surface exposed pigments form random quenching centres in both cases. On the other hand, it was possible that Zea could act as a direct quencher of excess excitation energy in the minor LHCs. However, enrichment of refolded CP29, CP26 and CP24 with Zea did not lead to a change in the Chl excited state lifetime. Formation of a carotenoid radical cation, previously implied in CT quenching, was also not observed, although artificial generation of such a radical cation was principally possible as shown for CP29. During the course of this work, a study reporting the formation of Zea radical cations in minor LHCs was published. Therefore, Zea-enriched minor LHCs were again investigated on the experimental apparatus used in the reported study. Indeed, the presence of at least one carotenoid radical cation for each minor complex was detected. It is suggested that either the preparation method of incubating the refolded minor LHCs with Zea in contrast to refolding the complexes with only Zea and lutein causes the observed differences or that the observed spectral radical cation signatures are due to experimental artifacts. While the experiments with LHC-II and the minor LHCs gave useful insights into the putative qE mechanism, the quencher site and the mode of action of Zea could still not be unambiguously identified. Most importantly, these studies could not explain the function of the qE keyplayer PsbS. Therefore, the focus of the work was shifted to PsbS protein production, purification and characterisation. In view of inconsistent reports on the pigment binding capacity of this PSII subunit, refolding trials with and without photosynthetic pigments were conducted. The formation of a specific pigmentprotein complex typical for other LHCs was not observed and neither was the earlier reported “activation” of Zea for qE by binding to this protein. Nevertheless, PsbS refolded without pigments displayed secondary structure content in agreement with previous studies, indicating pigment-independent folding. Reconstitution of pigmentfree, refolded PsbS into liposomes confirmed that the protein is stable in the absence of pigments. Zea distributed in PsbS-containing liposomes also showed no spectral alteration that would indicate its “activation”. With the ability to reconstitute PsbS, it was then possible to proceed to modelling qE in a proteoliposome system. For this purpose, PsbS was co-reconstituted with LHC-II, which has been reported to interact with PsbS. One-photon excitation (OPE) and two-photon excitation (TPE) spectroscopy measurements were performed on LHC-II- and LHC-II/PsbS-containing liposomes. This enabled both quantification of Chl fluorescence quenching as well as determination of the extent of electronic Car S1-Chl interactions. The effect of Zea was investigated by incorporating it in the proteoliposome membrane. It was shown that Zea alone was not able to induce significant Chl fluorescence quenching when only LHC-II was present. However, when LHC-II and PsbS were co-reconstituted, pronounced Chl fluorescence quenching and an increase in electronic Car S1-Chl interactions were observed and both effects were enhanced when Zea was present. Western blot analysis indicated the presence of a LHC-II/PsbS-heterodimer in these proteoliposomes. In addition to the OPE and TPE measurements, the average Chl fluorescence lifetime was determined in detergent-free buffer at neutral pH and directly after buffer exchange to low pH. No significant changes in the average lifetime were observed for LHC-II proteoliposomes when either Zea was present or after exchange for low pH buffer. This indicated that Zea alone cannot act as a direct quencher, which concurs with the OPE measurements. Moreover, the complex was also properly reconstituted as no aggregation or significant Chl fluorescence quenching were observed. The average lifetime was not significantly affected in LHC-II/PsbS-proteoliposomes, independent of Zea or pH. However, a shortlived component in the presence of a long-lived component was not resolvable with the time resolution of the fluorescence lifetime apparatus.
Implications for qE model systems and the in vivo quenching mechanism are discussed based on the experiments in detergent solution, on LHC-II aggregates and with the proteoliposome model system.