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Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.
Type 1 Diabetes (T1D) is an autoimmune disorder in which the own immune system attacks the insulin producing _-cells in the pancreas. Therapy of T1D with anti-CD3 antibodies (aCD3) leads to a blockade of the autoimmune process in animal models and patients resulting in reduced insulin need. Unfortunately, this effect is only temporal and the insulin need increases after a few years. In the first approach, I aimed at a blockade of the cellular re-entry into the islets of Langerhans after aCD3 treatment by neutralising the key chemokine CXCL10, which is important for the T cell migration. In the second approach I tried to block the transmigration of leukocytes trough the endothelial layer into inflamed tissue with an anti-JAM-C antibody (aJAM-C) after aCD3 treatment.
I used the well-established RIP-LCMV-GP mouse model of T1D. As target autoantigen in the _-cells, such mice express the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP). These mice develop T1D within 10 to 14 days only after LCMV-infection. In the combination therapy (CT) I treated diabetic RIP-LCMV-GP mice with 3 5g aCD3 per mouse (3 injections in 3 days) followed by administration of a neutralising anti-CXCL10 (CT) or aJAM-C (CT-J) monoclonal antibody (8 injections of 100 5g per mouse over 2.5 weeks).
CT reverted T1D in RIP-LCMV-GP mice significantly (CT: 67 % reversion; control: 16 % reversion) and with superior efficacy to monotherapies with aCD3 (38 % reversion) and aCXCL10 (36 % reversion).
The CD8 T cells in the spleen have fully regenerated at day 31 after infection. However, the frequency of islet antigen (GP)-specific CD8 T-cells was significantly reduced by 73 % in the spleen after CT compared to isotype control treated mice. In contrast, in aCD3 treated mice the T cells were only reduced by 56 % of the frequency of isotype control treated mice. Flow cytometry and immunohistological examinations demonstrated a marked reduction of CD8 T cells in the pancreas of CT treated mice. Importantly, the number of GP-specific CD8 T cells was reduced dramatically by 78 % in the pancreas of CT treated mice, whereas aCD3 treatment led to a less pronounced reduction of the GP-specific CD8 T cell number (23 %). This reduction of infiltration was long lasting since in the pancreas of CT treated mice the _-cells produce insulin and there were almost no infiltrating T cells present at day 182 post-infection. aCD3 treated mice also showed many insulin producing cells after 182 days post-infection. Nevertheless, their pancreas displayed also some infiltrates around the islets.
In order to confirm my data I treated non-obese diabetic (NOD) mice with CT. In contrast to RIP-LCMV-GP mice, NOD mice develop spontaneous T1D within 15 to 30 weeks after birth, due to a mutation in the CTLA-4 gene. Strikingly CT cured 55 % of diabetic NOD mice, whereas only 30 % showed T1D reversion with aCD3 alone and none reverted after isotype control administration.
The impact of CT on GP-specific T cells (Teff) was stronger in the RIP LCMV-GP than in the NOD model. In contrast, regulatory T cells (Tregs) were induced predominantly in NOD mice rather than in RIP-LCMV-GP mice. However, looking at the Treg/Teff ratio and compared to isotype control antibody treated mice, I found a significant 4-fold increase in the pancreas of CT treated RIP LCMV-GP mice and a 17-fold increase in the PDLN of CT treated NOD mice. In addition, a tendency for an increase in Treg/Teff ratio was obtained in the spleen of CT-treated RIP LCMV-GP as well as NOD mice compared to aCD3 and isotype control antibody treated mice.
In the second combination therapy with neutralising aJAM-C, CT-J (51 % reversion) slightly improved the aCD3 therapy (41 % reversion). However, there was no significant difference between CT-J and aCD3 administration in terms of total CD8 and GP-specific CD8 T cells.
JAM-C also interacts with the integrin receptor macrophage-1 antigen (MAC-1), which is among others expressed by neutrophils. Accordingly, JAM-C could be involved in neutrophil transmigration to the pancreas. Indeed, I found a significant reduction for the infiltrating neutrophils into the pancreas of mice after CT-J compared to aCD3 monotherapy.
In summary the addition of aJAM-C to aCD3 monotherapy showed a small improvement, which was associated with a reduced neutrophil migration into the pancreas. However, JAM C seemed to play only a minor role in T1D development and some other adhesion molecules might be more important. Nevertheless, the combination of aCD3 and aCXCL10 resulted in a significant and long lasting reduction of aggressive T cells in the pancreas in two independent mouse models. Furthermore a protective immune balance was obtained. Since both antibodies are available for as well as tested in humans and the therapy is only for a short period of time after disease onset, this combination therapy might kick-start a novel therapy for T1D.
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.
G protein-coupled receptors (GPCRs) constitute an important class of integral membrane proteins that are involved in several signaling pathways. About 50% of the currently available drugs are targeted against these receptors and high-resolution structures of these receptors will be of immense importance from the perspective of designing specific and potent drugs. However, structure determination of these receptors and of membrane proteins in general, has been a very challenging task till date. A major limitation in the structure determination of these proteins is that they are present in minute amounts in the native tissues and therefore, they must be produced heterologously. Additionally, crystallization of GPCRs is difficult owing to their flexible nature and limited hydrophilic surface area available for crystal contacts. The aim of my Ph.D. thesis work is two fold, first, to address the problem of GPCR crystallization by using a fusion protein complex approach and second, to tailor Rhodobacter sphaeroides as an expression system for the heterologous production of GPCRs. In the first approach, R. sphaeroides was used as an expression system to generate a fusion protein complex of the photosynthetic reaction center (RC) with a GPCR, expecting that such a complex would be easier to crystallize than the receptor alone. The notion behind this approach is that the RC will act as a scaffold in providing surface area to create crystal contacts and at the same time, it will also reduce the flexibility of the receptor, hopefully without perturbing the functionality of the receptor. Based on the computational modelling experiments, two ways to generate a fusion complex were assigned. Long linkers were inserted between the subunits of the RC and the GPCR. The linkers were designed with a possibility of straightforward alteration of their length as they contained a number of restriction enzyme sites. A series of these constructs were designed and expressed in R. sphaeroides deletion strain, which did not possess the chromosomal RC genes. Though most of these fusion constructs could be successfully expressed, as analyzed by western blot, majority of them were not functional in terms of ligand binding of the GPCR component of the fusion complex. Interestingly, one of these constructs, where the M subunit of RC was directly fused to the human angiotensin II type 1a receptor (AT1aR), exhibited significant functional expression. Based on saturation binding analysis using [125I] iodotyrosyl4Sar1Ile8-angiotensin II (an AT1aR subtype specific antagonist), an expression level of 40+5 pmol/mg of total membrane protein was calculated. This expression level corresponds to approximately 0.3 mg of functional receptor per liter culture and it is significantly higher than the AT1aR expression in native tissues. Additionally, the binding affinity of the recombinant receptor for its endogenous ligand angiotensin II was found to be 1±0.1 nM, which is similar to that observed for the AT1aR in native tissues. More interestingly, the RC part of the fusion complex was structurally assembled in other words, properly folded as judged by the presence of the characteristic peaks at 760 nm, 800 nm and 850 nm by absorption spectroscopy. However, a slight change in the intensity of the peak at 800 nm was observed while comparing the spectra of native RC with that in the fusion protein complex. This slight variation might be due to the change in the protein environment. The fusion protein complex RC-AT1aR was functionally solubilized and purified using a decahistidine tag fused at the c-terminus of the AT1aR. Subsequently, the monodispersity and integrity of the complex was confirmed by size exclusion chromatography, which revealed a homogeneous peak. Additionally, it was also possible to solubilize and purify this complex in the presence of a fluorescein tagged angiotensin II ligand which provides a nice tool to judge the functionality of the AT1aR and integrity of the complex at the same time. The purified RC-AT1aR fusion complex was then subjected to three-dimensional (3-D) crystallization trials and it was possible to obtain reproducible crystals of this complex. The crystals were fluorescent (as the complex was purified in presence of fluorescently labelled angiotensin II) and needle or tetragonal in shape, but produced a powdery diffraction pattern. Further attempts to improve the crystallization condition and to optimize the cryo-conditions are underway. In addition, attempts are also being made to obtain the crystals of this complex with the antagonist (e.g. losartan) bound to the receptor. In view of several limitations in the heterologous expression of GPCRs, as the second part of my Ph.D. thesis, I decided to explore the possibilities of developing a novel expression system based on R. sphaeroides for production of recombinant GPCRs. The notion behind using this host is that lack of inclusion bodies and high concentration of membranes in R. sphaeroides would result in efficient functional overexpression of recombinant membrane proteins. For this purpose, a R. sphaeroides strain, modified by the deletion of the genes encoding the RC and the light harvesting proteins LH1 and LH2, was used. The genes for RC and LHs constitute about 85-90% of total membrane proteins in a R. sphaeroides cell. These membranes are normally housed in special membrane vesicles called intracytoplasmic membranes (ICMs) that can fill almost the entire cell volume under certain growth conditions. Synthesis of a heterologous protein under the control of the moderately strong photosynthetic superoperonic promoter should be coordinated with the synthesis of new membranes to harbour these proteins, thus acting as a natural induction system. Moreover, as most of the native membrane proteins are absent in this deletion strain, heterologously produced protein should not experience a shortage of molecular chaperones for proper folding and insertion. Additionally, the absence of inclusion bodies in this host should enhance the functional and homogenous population of the recombinant proteins. Three human GPCRs, namely the adenosine A2a receptor (A2a), the angiotensin II type 1a receptor (AT1aR) and the bradykinin subtype 2 receptor (B2R) were tested for expression and functionality in this system. Two different constructs were used to determine the optimal position and ribosome-binding site (RBS) in the superoperon for the highest expression level. Of these three receptors, the AT1aR and B2R were successfully produced, while the A2aR failed to express, producing green carotenoid free R. sphaeroides mutants, for unknown reasons. For the recombinant B2R, [3H] bradykinin binding analysis revealed a low functional expression level of 0.7-0.8 pmol/mg of total membrane protein. This expression level corresponds to 0.01 mg functional receptor per liter of culture and is not sufficient for large-scale expression of this receptor. However, for the recombinant AT1aR, [125I] iodotyrosyl4Sar1Ile8- angiotensin II binding analysis revealed an expression level of 12±1 pmol/mg of total membrane protein. This expression level corresponds to approximately 0.1 mg functional receptor per liter culture and this is significantly higher than the AT1aR expression in native tissues. This expression system is still in the nascent stages of development and there are several parameters, which are still to be assessed for the optimal use of this system for the production of GPCRs and other membrane proteins. In conclusion, my Ph.D. work presents a novel fusion protein complex based approach for obtaining crystallizable GPCRs and a novel expression system for producing heterologous GPCRs. It was possible, for the first time, to produce a functional RC-GPCR complex that could easily be crystallized, though further finetuning of the system is required. R. sphaeroides based novel expression system was successfully used to produce functional human GPCRs under the control of a moderately strong photosynthetic superoperonic promoter. This expression system represents a naturally induced system where the expression of a heterologous protein is coordinated with the synthesis of new membranes to harbour the recombinant protein. The fusion protein complex approach and the expression system presented here can hopefully be used as a general method to facilitate the expression and crystallization of other membrane proteins.
The ability of endothelial cells to properly adapt to changes in a dynamic blood perfused environment is essential to maintain the physiological function of the vascular system and of the organs. Epigenetic control of gene expression is believed to be the mechanism controlling cell-fate determination and cell-phenotype maintenance. In the thesis, two JmjC demethylases were screened for their function in endothelial biology. Both of them were proved to play a central role in angiogenesis.
The histone 3 lysine 4 demethylase JARID1B was identified as the most highly expressed demethylase in endothelial cell. Knockdown of JARID1B in human umbilical vein endothelial cells (HUVEC) attenuated cell migration, angiogenic sprouting and tube formation. Jarid1b null mice exhibited attenuated retinal angiogenesis and reduced endothelial sprout outgrowth from aortic segments. Microarray data identified that the antiangiogenic transcription factor HOXA5 was suppressed by JARID1B. Consistently, chromatin immunoprecipitation experiment revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. Hence, as a highly expressed JmjC protein in endothelial cells, JARID1B fundamentally maintains endothelial angiogenic phenotypes perhaps through suppression of HOXA5.
As second enzyme it was identified that the histone plant homeodomain finger protein 8 (PHF8) plays a role in endothelial angiogenic sprouting as well as tube formation and cell migration. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. To characterize the underlying mechanism, E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4, but not E2F1, expression in endothelial cells. Likewise, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Thus, it is proposed that PHF8 maintains endothelial function by controlling E2F4 expression. On the other hand, microarray and subsequent qPCR validation revealed that the expressions of small nuclear RNAs (snRNAs) were regulated by PHF8. Co-immunoprecipitation experiment demonstrated that PHF8 interacts with spliceosome related proteins SNRP70 and SRPK1 as well as snRNA. Indeed, PHF8 contributed to splicing: GLS and VEGF-A displayed alternative splicing in PHF8 depleted cells. In addition, c-FOS introns were showed to be retained after knockdown of PHF8 in endothelial cells. These results demonstrated that, by controlling angiogenic mRNA splicing, PHF8 could affect endothelial properties.
Collectively, the results uncover the important roles of JARID1B and PHF8 in endothelial cells in the control of angiogenesis. Changing histone modifiers appears as an attractive concept for pro- and antiangiogenic therapy. The present work adds JARID1B and PHF8 as novel potential targets to this emerging field.
Die Funktion nukleärer Rezeptoren (NR) beruht auf einem empfindlichen Zusammenspiel zwischen ihren Domänen, Coregulatoren und Liganden. Die meisten Rezeptoren binden die DNA als Homo- oder Heterodimere und transregulieren die Gentranskription in Folge von Ligandenbindung. Klassische Assay-Systeme, die sich auf die Untersuchung der NR-Funktion oder auf die Charakterisierung von Substanzen richten, bilden nur die Coregulator-Rekrutierung zu isolierten NR-Ligandenbindungsdomänen (LBDs) ab und vernachlässigen dabei die NR:NR-Interaktion. Damit klammern sie die NR:NR-Wechselwirkung aus, obwohl die Rekrutierung von Cofaktoren durch allosterischen Crosstalk mit der Oligomerisierung verbunden ist. Dies war die Motivation dafür, Assay-Systeme zu entwickeln, welche die Untersuchung von NR-Interaktionen,
insbesondere der NR-Dimerisierung, und deren Modulation durch verschiedene Arten von Liganden ermöglichen. Im Rahmen dieser Doktorarbeit wird ein vielfältiges modulares Set von Assays für die Untersuchung der NR-Dimerisierung und NR-Coregulator-Rekrutierung vorgestellt und deren Anwendbarkeit auf eine Vielzahl von NRs demonstriert. Die Verwendung einer
rekrutierungsunfähigen RXRα-Variante mit einer mutierten AF-2-Domäne ermöglichte den spezifischen Nachweis der Coaktivatorrekrutierung durch PPARγ im Kontext des Heterodimers mit seinem obligatorischen Dimerpartner RXRα. Außerdem konnte gezeigt werden, dass die Aktivierung der RXRα LBD mit ihrem Agonisten SR11237 zu einer Destabilisierung des RXRα-Homodimers, aber zu einer Förderung der Bildung des Heterodimers mit der PPARγ LBD führte.
Ein zentrales Ergebnis war das Phänomen, dass der Einbau von PPARγ in das Heterodimer zu einem erheblichen Anstieg an Affinität gegenüber Coaktivatoren führt, auch in Abwesenheit von Liganden. Somit fördert die RXRα-Aktivierung die Coaktivator-Rekrutierung von PPARγ indirekt durch eine Verschiebung der Oligomerisierungspräferenz von RXRα in Richtung des Heterodimers. Zusätzlich wurde die Wirkung von Tetrac, einem nicht-klassischen Schilddrüsenhormon, auf PPARγ und RXRα untersucht und dessen Aktivierungsvermögen gegenüber beiden Rezeptoren mit einer deutlich vervielfachten Wirkung auf das Heterodimer demonstriert. Mit Hilfe des neu etablierten Cofaktor-Rekrutierungsscreens konnte die Dynamik
zwischen dem Nurr1 NR und 29 kanonischen Coregulatoren, von denen einige ligandenabhängig hohe Affinitäten zum Rezeptor aufwiesen, beleuchtet werden. Diese Interaktionen wurden
bidirektional durch eine Reihe von strukturell unterschiedlichen nicht-steroidalen Antirheumatika moduliert, die auch die Affinitäten sowohl des Nurr1-Homodimers als auch des Heterodimers mit der RXRα LBD beeinflussen konnten. Die Nurr1-Dimere zeigten zudem auch eine hohe Empfindlichkeit gegenüber dem Endocannabinoid Anandamid. Zusätzlich zu PPARγ, RXRα und Nurr1 wurden erste Schritte zur Untersuchung der TLX NR-Funktion unternommen. Unter Anwendung der entwickelten Assays konnte die Heterodimerbildung der TLX und der RXRα LBD
beschrieben und die ligandenabhängige Rekrutierung des Corepressors SMRT beobachtet werden.
Zusammenfassend beschreibt diese Arbeit einen Satz von Werkzeugen für die Untersuchung von ligandenabhängiger NR-Coregulator-Interaktion und Oligomerisierung. Auf diese Weise trug sie zu einer umfassenderen Identifizierung und Charakterisierung von NR-Liganden bei und stellt eine valide Basis für die weitere Assayentwicklung und Ligandendesign dar.
Tumor development usually follows predictable paths where tumor cells acquire common characteristics and features known as the hallmarks of cancer. Recently, additional characteristics have been added to these hallmarks since solid tumors are composed of a very heterogeneous population of transformed, formerly normal tissue cells and stromal cells, e.g. immune cells and fibroblasts. Compelling evidence suggests that stromal cells and tumor cells maintain a symbiotic relationship to build up the tumor microenvironment and to fuel tumor growth. In cancer therapies, common features of tumors such as unrestricted cell growth, suppression of immunological responses, and the ability to form new blood vessels (angiogenesis) have emerged as the main targets of interest. The lipid mediator prostaglandin E2 (PGE2) is known to promote all these features and thus, is connected to cancer progression in general. Its synthesis is triggered in response to stress factors or during inflammation. Inducible PGE2 production relies on the enzymes cyclooxygenase 2 (COX-2) and microsomal prostanglandin E synthase 1 (mPGES-1), which are simultaneously expressed in response to a variety of different stimuli and are functionally coupled. Inhibition of COX-2 with non-steroidal antiinflammatory drugs (NSAIDs) for cancer treatment is, however, limited by cardiovascular risks, since selective COX-2 inhibition disrupts the prostacyclin/thromboxane balance. Therefore targeting mPGES-1 downstream of COX-2 for PGE2 inhibition was evaluated in this work in different steps of carcinogenesis. Knockdown of mPGES-1 in DU145 prostate cancer cells revealed that the mPGES-1 status did not affect growth of monolayer tumor cells, but significantly impaired 3D growth of multi-cellular tumor spheroids (MCTS). Spheroid formation induced COX-2 in DU145 and other prostate cancer spheroids. High levels of PGE2 were detected in supernatants of DU145 MCTS as opposed to monolayer DU145 cells. Pharmacological inhibition of COX-2 and mPGES-1 confirmed the pivotal role of PGE2 for DU145 MCTS growth. Besides promoting spheroid growth, MCTS-derived PGE2 also inhibited cytotoxic T lymphocyte (CTL) activation. When investigating the mechanisms of COX-2 induction during spheroid formation, the typical tumor microenvironmental factors such as glucose deprivation, hypoxia or tumor cell apoptosis failed to enhance COX-2. Interestingly, when interfering with apoptosis in DU145 spheroids, the pan-caspase inhibitor Z-VAD-FMK triggered a Summary 12 shift towards necrosis, thus enhancing COX-2 expression. Coculturing viable DU145 monolayer cells with isolated heat-shocked-treated necrotic DU145 cells, but not with necrotic cell supernatants, induced COX-2 and PGE2, confirming the impact of necrosis for MCTS growth and CTL inhibition. As mentioned, in vivo tumors are very heterogenous mixtures of tumor cells and stromal cells e.g. immune cells. Hence, the interaction of the immune system with tumors was investigated in further experiments. When coculturing MCF-7 breast cancer spheroids with human peripheral blood mononuclear cells (PBMCs), only low levels of PGE2 were detected, since MCF-7 cells did not upregulate COX-2 during spheroid formation and did not induce PGE2 production by PBMCs. Under inflammatory conditions, by adding the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) to cocultures, PGE2 production was triggered, spheroid sizes were reduced, and numbers of high levels of granzyme B expressing (GrBhi) CTLs were increased, while CD80 expression by tumor-associated phagocytes was also elevated. Inhibition of CD80 but not CD86 diminished numbers of GrBhi CTLs and attenuated spheroid lysis. To determine the role of ctivation-induced PGE2 production, use of the COX-2 inhibitor celecoxib and the experimental mPGES-1 inhibitor C3 further increased CD80 expression. Addition of PGE2, the prostaglandin E2 (EP2) receptor agonist butaprost, and the phosphodiesterase 4 (PDE4) inhibitor rolipram reduced LPS/C3-triggered CD80 expression, confirming the impact of COX- 2/mPGES-1-derived PGE2 on shaping phagocyte phenotypes in an EP2/cAMP-dependent manner. In a spontaneous breast cancer model (MMTV-PyMT), mPGES-1-deficiency significantly delayed tumor growth in mice, confirming an overall protumorigenic role of mPGES-1 in breast cancer development in vivo. However in tumors of mPGES-1-/- mice, tumor-infiltrating phagocytes expressed low levels of CD80 similar to their wildtype counterparts. These data suggest that the immunosuppressive microenvironment does not allow for immunostimulatory effects by mPGES-1 inhibition without an activating stimulus. Evidences in this study recommend the application of mPGES-1 inhibitors for treating cancer diseases, since mPGES-1 promotes tumor growth in multiple steps of carcinogenesis, ranging from well-characterized effects of tumor cell growth to immune suppression of CTL activity and phagocyte polarization. Regarding the latter, blunting PGE2 during immune activation may limit the tumor-favoring features of inflammation and improve the efficiency of TLR4 based immune therapies.
Macrophages in the tumor microenvironment respond to complex cytokine signals. How these responses shape the phenotype of tumor-associated macrophages (TAMs) is incompletely understood. Here we explored how cytokines of the tumor milieu, interleukin (IL)-6 and IL-4, interact to influence target gene expression in primary human monocyte-derived macrophages (hMDMs). We show that dual stimulation with IL-4 and IL-6 synergistically modified gene expression. Among the synergistically induced genes are several targets with known pro-tumorigenic properties, such as CC-chemokine ligand 18 (CCL18), transforming growth factor alpha (TGFA) or CD274 (programmed cell death 1 ligand 1 (PD-L1)). We found that transcription factors of the signal transducer and activator of transcription (STAT) family, STAT3 and STAT6 bind regulatory regions of synergistically induced genes in close vicinity. STAT3 and STAT6 co-binding further induces the basic leucine zipper ATF-like transcription factor (BATF), which participates in synergistic induction of target gene expression. Functional analyses revealed increased MCF-7 and MDA-MB 231 tumor cell motility in response to conditioned media from co-treated hMDMs compared to cells incubated with media from single cytokine-treated hMDMs. Flow cytometric analysis of T cell populations upon co-culture with hMDMs polarized by different cytokines indicated that dual stimulation promoted immunosuppressive properties of hMDMs in a PD-L1-dependent manner. Analysis of clinical data revealed increased expression of BATF together with TAM markers in tumor stroma of breast cancer patients as compared to normal breast tissue stroma. Collectively, our findings suggest that IL-4 and IL-6 cooperate to alter the human macrophage transcriptome, endowing hMDMs with pro-tumorigenic properties.
Diese Zusammenfassung ist in zwei Abschnitte gegliedert. Im Abschnitt 6.1. wird die physiologische Bedeutung der Glutamatrezeptoren (GluR) und ihr biologischer Hintergrund kurz erklärt. Am Ende dieses Abschnitts wird der Stand der Strukturanalyse des GluR-B Ionenkanals zu Beginn des Projektes zusammengefasst. Im nachfolgenden Abschnitt 6.2. sind die wesentlichen Ergebnisse der hier vorgelegten Arbeit zusammengefasst. 6.1. Die Bedeutung von Glutamatrezeptoren - Stand der Strukturanalyse zum Beginn dieser Arbeit Die Kommunikation zwischen Nervenzellen erfolgt vorwiegend an hochspezialisierten Kontaktstellen den chemischen Synapsen. Der enge Raum zwischen sendender und empfangender Nervenzelle wird auch als synaptischer Spalt bezeichnet. Der Prozess der synaptischen Übertragung beruht auf der präsynaptischen Freisetzung von chemischen Botenstoffen, sogenannten Neurotransmittern in den synaptischen Spalt. Die Aminosäure L- Glutamat (Glu) ist der wichtigste erregende Neurotransmitter im menschlichen Gehirn und Rückenmark. Dementsprechend bedeutend ist die Rolle der ionotropen Glutamatrezeptoren (iGluRs), die sie bei der elektrochemischen Erregungsübertragung am synaptischen Spalt spielen (Seeburg, 1993), (Hollmann and Heinemann, 1994), (Dingledine et al., 1999). Die Freisetzung von Neurotransmittern wird durch ein elektrisches Signal (Aktionspotential) ausgelöst, das sich entlang der Nervenfaser, dem Axon, bis zur Nervenendigung, der Synapse, fortpflanzt. Nach der Freisetzung diffundieren die Neurotransmitter durch den synaptischen Spalt und binden an sogenannte Rezeptoren. Ionotrope Glutamatrezeptoren sind Ionenkanäle, die in die Membran der nachgeschalteten (postsynaptischen) Nervenzelle eingebaut sind. Sie zählen deshalb zu den Membranproteinen. Als ligandgesteuerte kationenselektive Ionenkanäle machen Glutamatrezeptoren (GluRs) die postsynaptische Membran nach Aktivierung durch Ligandbindung für bestimmte Kationen durchlässig. Der Einstrom von Ionen bewirkt eine Änderung des Membranpotentials. Die Stärke der synaptischen Übertragung ist lebenslang modulierbar; die sogennante synaptische Plastizität wird als eine entscheidende Grundlage für die Erklärung von Lernen und Gedächtnis angesehen. Drei synthetische Agonisten aktivieren die GluRs selektiv und wurden deshalb für die Klassifizierung der ionotropen Glutamatrezeptoren herangezogen. Bei den Agonisten handelt es sich um -Amino-3-hydroxy-5-methyl-4-isoxazol-4-propionat (AMPA), Kainat and N- Methyl-D-Aspartat (NMDA). Die ersten beiden Subtypen werden auch als non-NMDA- Rezeptoren zusammengefasst. Die Aktivierung und Desensitivierung der non-NMDA Rezeptoren ist schneller als die der NMDA-Rezeptoren. Aus molekularbiologischer Sicht (siehe Kapitel 1.3.2.) zeigen die drei Klassen der ionotropen Glutamatrezeptoren eine beträchliche Diversität. So gibt es vier verschiedene Unterheiten vom AMPA-Subtyp, nämlich GluR-A, GluR-B, GluR-C und GluR-B. In dieser Arbeit steht die Strukturanalyse eines aus GluR-B Untereinheiten bestehenden AMPA-Rezeptors im Vordergrund. (Die weitere Unterteilung der NMDA- und Kainatrezeptoren kann dem Kapitel 1.3.2. auf Seite 6 entnommen werden.) Bestimmte Abschnitte der Aminosäurensequenz von Glutamatrezeptoren sind durch hydrophobe Bereiche gekennzeichnet ((M1-M4) in Abbildung 6.1.A (A.)). Das durch verschiedene Untersuchungen etablierte Modell der Glutamatrezeptor-Topologie zeigt 3 Transmembrandomänen (M1, M3 und M4) und eine Membranschleife (M2) (Hollmann et al., 1994), (Kuner et al., 1996). Der Aminoterminus ist extrazellulär, der Carboxyterminus hingegen intrazellulär. Daraus ergibt sich die in Abbildung 6.1.A (B.) abgebildete Topologie (Paas, 1998). S1 und S2 kennzeichnen die Ligandbindungsdomäne. Glutamatrezeptoren (GluR) sind Oligomere, die sich mit grosser Wahrscheinlichkeit aus vier Untereinheiten (Rosenmund et al., 1998), (Ayalon and Stern-Bach, 2001) zusammensetzen (siehe Kapitel 1.3.3.). Die Zusammenlagerung verschiedener Untereinheiten zu einem funktionellen Kanal setzt voraus, dass die Untereinheiten zum gleichen Subtyp gehören, d.h. AMPA Untereinheiten können nur mit anderen AMPA Untereinheiten einen Ionenkanal bilden. Das gleiche gilt für die Zusammensetzung von NMDA und Kainat-Rezeptoren. Das Modell eines tetrameren Glutamatrezeptors ist im Bild C. der Abbildung 6.1.A zu sehen. Die Bestimmung der Quartärstruktur eines vollständigen Glutamatrezeptors ist bislang nicht veröffentlicht. Die strukturelle Analyse von Proteinen erfordert die Isolierung von reinem und funktionellem Protein. Im Vergleich zu den meisten löslichen Proteinen erfordert die Isolierung von Membranproteinen oft besonderer Optimierung. Falls das Vorkommen des Proteins in natürlichem Gewebe gering ist, so kann die strukturelle Analyse durch rekombinante Expression in einem geeigneten Wirtsorganismus zugänglich gemacht werden. Die Isolierung von Milligramm-Mengen eines rekombinanten homomeren GluR-B Rezeptors aus dem entsprechenden Baculovirusexpressionssystem (Keinänen et al., 1994) wurde in unserem Labor etabliert (Safferling et al., 2001) und wurde im ersten Jahr dieses Projektes fortgeführt. Durch zonale Ultrazentrifugation konnte gezeigt werden, dass die molekulare Masse des GluR-B Proteinkomplexes ca. 495 kD beträgt. Dieser Wert liegt in der Nähe des theoretischen Molekulargewichts eines tetrameren Ionenkanals, dessen Molmasse sich aus vier GluR-B Untereinheiten (104 kD) und einer Detergenzmizelle von ca. 63-97 kD zusammensetzt (Safferling et al., 2001). Die elektronenmikroskopische Analyse des Proteinkomplexes von W. Tichelaar aus unserer Gruppe erfolgte 1999 durch Negativfärbung. Für die Strukturanalyse mit Hilfe der Software IMAGIC wurden 10 000 Proteinteilchen selektiert. Das Ergebnis der Bildrekonstruktion ist in der folgenden Abbildung 6.1.B gezeigt. Die projezierten Dimensionen des Models entsprechen einem Molekül mit den Dimensionen 17 nm × 11 nm × 14 nm. Das Model zeigt keine ausgezeichnete Symmetrie, die auf die Stöchiometrie des GluR hinweisen könnte. Das Molekül zeigt mit Färbemittel gefüllte Vertiefungen und innere Strukturen, die vielleicht an der Ionenleitung beteiligt sind. 6.2. Funktionelle und strukturelle Charakterisierung des GluR-B Ionenkanals In der Fortsetzung des oben beschriebenen Projektes wurden für die rekombinante Expression desselben Rezeptors (GluR-B homomer) stabil transformierte Insektenzellen eingesetzt. Dazu wurde die für die GluR-B Untereinheit kodierende und in Plasmiden enthaltene DNA in Insektenzellen transformiert (siehe APPENDIX A.2.2.). Im Vergleich zu dieser auf Dauerhaftigkeit angelegten Integration der Rezeptor DNA wird die Proteinexpression beim Baculovirusexpressionssystem durch Infektion mit rekombinanten Baculoviren initiiert. Der Vergleich zeigte, dass die mit Baculoviren erzielten Ausbeuten bei GluR-B etwa doppelt so hoch waren als bei stabil transformierten Zellen. Allerdings fallen bei stabil transformierten Zellen die eventuellen Nachteile der viralen Belastung auf die zellulären Sekretionsprozesse weg. Im Verlauf der elektronenmikroskopischen Analyse von baculoviral erzeugtem GluR-B Protein hat sich gezeigt, dass Proteine viralen Ursprungs unter Umständen selbst doppelt aufgereinigte GluR-B Proben verunreinigen können (siehe APPENDIX A.2.1.). Dieser Punkt ist bei einer Einzelbildverarbeitung von grosser Relevanz, falls die virusspezifischen Proteinverunreinigungen eine ähnliche Grösse haben wie das eigentliche Zielprotein. Das Hauptziel dieser Arbeit war es, das Potenzial stabil transformierter Insektenzellen für die Expression von homomeren GluR-B Ionenkanälen zu bewerten und dabei die Stöchiometrie der Untereinheiten in diesem Ionenkanal aufzuklären. Zu diesem Zweck wurden biochemische und elektronenmikrosopische Techniken eingesetzt. Zur Isolierung des GluR-B Ionenkanals aus stabil transformierten Insektenzellen wurde das bestehende Aufreinigungsprotokoll für die Affinitätchromatographie an immobilisierten Metallionen (IMAC) (Safferling et al., 2001) optimiert, indem das Chargenverfahren durch das Durchflussverfahren ersetzt wurde (zur genaueren Erklärung der Optimierung siehe RESULTS 4.1.2.). Abbildung 6.C zeigt ein silbergefärbtes Gel mit den Eluaten der IMAC und Eluaten der abschliessenden Affinitätschromatographie mit immobilisiertem M1-Antikörper. Die auf den Bahnen 5-8 aufgetragen GluR-B Proben wurden auch für die Einzelteilchenanalyse mittels Elektronenmikroskopie verwendet. Die Ligandbindungsaktivität von GluR-B wurde durch Filterbindungsexperimente mit dem Radioliganden [3H]-AMPA vor und nach der Isolierung aus den Membranfragmenten bestimmt. Die KD-Werte sind für beide Proben ähnlich gross. Der Bmax-Werte ist für die aufgereinigte Probe wie erwartet sehr viel (mehr als 200×) höher. Die Ergebnisse der Ligandbindungsexperimente sind im Kapitel 4.2.1 tabellarisch zusammengefasst. Die oligomere Struktur des isolierten Ionenkanals wurde durch Quervernetzungsexperimente (Cross-linking) und Einzelteilchenanalyse von negativ gefärbten Proteinmolekülen bewertet. Die Quervernetzungsexerimente selbst erbrachten kein eindeutiges Ergebnis im Hinblick auf oligomere Struktur des komplett zusammengesetzten Rezeptors. Kontrollexperimente mit dem Lysat vom Rattenhippocampus zeigten, dass mit DTSSP ein geeigneter Cross-Linker verwendet wurde (siehe RESULTS 4.3.2.). Neben einem aus 4 Banden bestehenden Muster (siehe RESULTS 4.3.1.) lieferten die Quervernetzungsexperimente mit isoliertem GluR-B aber einen deutlichen Hinweis auf die Stabilität von dimeren GluR-B Strukturen, die im Einklang mit einer jüngst veröffentlichten Arbeit stehen (Ayalon and Stern-Bach, 2001). Diese Veröffentlichung liefert zusätzliche (Armstrong et al., 1998) Hinweise auf die Bedeutung von Dimeren in der Glutamatrezeptorstruktur und postuliert, dass sich ein kompletter Glutamaterezeptor aus einem Dimer-Paar zusmmensetzt, wobei die Dimere zuerst gebildet werden. Die nachfolgende Abbildung 6.2.B zeigt negativ gefärbte GluR-B Ionenkanäle bei einer 46000× Vergrösserung. Die Aufnahme stammt von einem Philips EM 400 Elektronenmikroskop. Für die 3D Rekonstruktion wurden 500 der in Abbildung 6.2.B gezeigten Rezeptormoleküle ausgewählt. Dieser relativ kleine Datensatz besteht aus GluR-B Ionenkanälen deren Präservierung in Uranylacetat als besonderes vielversprechend eingeschätzt wurde. Dieser positive Effekt wurde auf die Verwendung frisch von einer Wasseroberfläche aufgefischter Kohlefilme zurückgeführt (siehe RESULTS 4.4.3.3.). Während der Klassifizierung dieses Datensatzes fiel auf, dass die beim Band-Pass-Filtern für die niedrigen Frequenzen gesetzten Cut-offs einen deutlichen Einfluss auf die erste Klassifizierung der unterschiedlichen zweidimensionalen Ansichten des Proteinkomplexes haben (siehe RESULTS 4.4.3.4.). Aus diesem Grund wurde der gleiche Datensatz mit 5 verschiedenen low-frequency cut-offs (LFCO) gefiltert (siehe Table 4.4.3.4.) und getrennt klassifiziert. Von den 5 resultierenden Klassifikationen wurden 3 (LFCO 0,005, 0,03 und 0,05) für die weiterführende 3D Rekonstruktion ausgewählt. Die Evaluierung der resultiernden 3D Modelle ergab, dass der mit einem LFCO von 0,03 gefilterte Datensatz eine Klassifikationen erlaubte, die zu einem 3D Modell (Modell GluR-BII/a siehe RESULTS Figure 4.4.3.4.H) führte, das im Vergleich zu den beiden anderen Rekonstruktionen konsistenter war. Am stärksten spricht für dieses Modell die Übereinstimmung der Input-Projektionen mit den Reprojektionen der 3D Rekonstruktion (siehe siehe RESULTS Figure 4.4.3.4.H). Zur Verfeinerung des Modells GluR-BII/a wurden die beiden Projektionen mit der höchsten Standardabweichung vom Klassendurchschnitt (class average) eliminiert. Die verbleibenden 11 Projektionen bildeten die Input-Projektionen für die Berechung eines verfeinerten Modells, GluR-BII/b, das auf einer neuen Zuordnung der Euler-Winkel beruht. Das Ergebnis dieser Berechung ist in der nachfolgenden Abbildung gezeigt. Das Modell in Abbildung 6.2.C zeigt einen zentralen Kanal und hat die Dimensionen 18 nm × 14 nm × 11 nm. Die Stöchiometrie der Untereinheiten ist aus dem Modell, das mit grosser Wahrscheinlichkeit einen komplett zusammengesetzten GluR darstellt, nicht ablesbar. Ebensowenig zeigt das Modell eine eindeutig vierzählige oder fünfzählige Symmetrie. Allerdings ist die erkennbare zweizählige Symmetrie im Einklang mit dem vorgeschlagenen Pair-of-Dimer Modell (Ayalon and Stern-Bach, 2001), das auf eine teramere Struktur des oligomeren Ionenkanals schliessen lässt. Die Ergebnisse dieser Arbeit zeigen, dass stabil transifzierte Insektenzellen eine durchaus geeignete Quelle für GluR-B Ionenkanäle sind. Nachteilig sind die geringen Ausbeuten. Allerdings kann durch weitere Selektion der Zellen die GluR Expression noch gesteigert werden (siehe APPENDIX A.2.2.). Bei höheren GluR-B Ausbeuten könnte zukünftig auch die Detektion des Rezeptors in vitrifizierten Proben in Verbindung mit Kryo-Elektronen- mikroskopie und auch die 2D-Kristallisation gelingen. Die während dieses Projekts gemachten Kristallisationsexperimente (siehe APPENDIX A.3.) und Kryo-Experimente mit GluR-B Protein aus dem Baculovirusexpressionssystem (siehe RESULTS 4.4.1. und 4.4.2.) ergaben negative Ergebnisse. Das Potential der Kryo-Methode konnte allerdings in Kontrollexperimenten mit Tabak-Mosaik-Virus (TMV) gezeigt werden. Kryo-Daten von GluR-B würden die Berechnung eines genaueren Strukurmodells erlauben. Die Reprojektionen des hier besprochenen Strukturmodells GluR-BII/b aus der Abbildung 6.2.C könnten als Referenzen für das Alignment der vitrifizierten GluR Ionenkanäle dienen. Für das langfristige Ziel der Rekonstituition des Rezeptors in Liposomen sollte die Delipidierung des Membranproteins während der Aufreinigung möglichst reduziert werden. Hier erscheinen zwei Ansätze sinnvoll. Die Aufreinigung des Proteins in einem Schritt durch die Erweiterung des tags am Carboxyterminus von nur 6 auf 10 Histidin-Reste. Ausserdem gibt es Hinweise, dass die Anwesenheit von Lipiden während der Aufreinigung für seine Rekonstituierbarkeit förderlich ist (Huganir and Racker, 1982).
Functional expression of recombinant N-methyl-D-aspartate (NMDA) receptors in eukaryotic cell lines
(2000)
Proliferation and apoptosis are fundamental cellular processes that are important for the development and homeostasis of multi-cellular organisms. Deregulation of these processes plays an important role in tumor formation. Often, genes that control homeostasis by regulating proliferation and apoptosis are mutated or improperly expressed in tumors. In this project, the physiological and pathological functions of FUSE Binding Protein 1 (FBP1) were studied to elucidate the involvement of this gene in the context of embryonic development and tumorigenesis. Two reasons led to the hypothesis that FBP1 might be relevant in this context. FBP1 was isolated in the group of PD Dr. Martin Zörnig using a functional yeast survival screen for the identification of anti-apoptotic genes involved in tumorigenesis, and the anti-apoptotic function of FBP1 was confirmed in the human colon carcinoma cell line RKO. In addition, FBP1 had been published to function as a transcriptional regulator that activates expression of the proto-oncogene c-myc. This gene stimulates cell proliferation and is overexpressed in many tumors. Analysis of FBP1 expression by immunhistochemistry in normal and tumor tissue samples revealed frequent and significant overexpression of FBP1 in Hepatocellular Carcinoma (HCC). To study the functional relevance of FBP1 activity for this tumor type, apoptosis and proliferation of the HCC cell line Hep3B were studied in dependence of FBP1 expression. Downregulation of FBP1 by lentiviral expression of FBP1-specific short hairpin RNA (shRNA) reduced proliferation and increased sensitivity to apoptosis. Subcutaneous injection of FBP1-deficient Hep3B cells into immunodeficient NOD/SCID mice demonstrated that tumor growth was strongly decreased in comparison to control cells. mRNA expression studies by quantitative real time PCR showed reduced mRNA levels of the pro-apoptotic genes Bik, Noxa, TRAIL and TNF-􀀁 in the absence of FBP1. In addition, the cell cycle inhibitors p21 and p15 were repressed by FBP1 while Cyclin D2 expression was decreased in the absence of FBP1. Surprisingly, expression of c-myc was not altered by FBP1 downregulation, indicating a different mechanism of c-myc regulation in HCC cells. These results demonstrate that overexpression of FBP1 inhibits apoptosis and stimulates proliferation in HCC cells by regulating the transcription of relevant target genes. Therefore, FBP1 might represent a promising therapeutic target for the treatment of HCC. For analysis of the physiological function of FBP1, a gene trap mouse model was established. In these mice, the gene trap vector pT1􀀂geo is inserted in intron 19 of the FBP1 locus, leading to the expression of a fusion protein consisting of a truncated FBP1 (lacking the last 62 amino acids), 􀀁-Galactosidase and Neomycin Phosphotransferase. Luciferase reporter assays demonstrated that the fusion protein was not capable of activating the c-myc promoter and even showed a dominant negative effect. Thus, this gene trap mouse serves as a functional FBP1 knockout model. Phenotyping of the FBP1 gene trap mice showed that homozygous mutation of FBP1 resulted in embryonic lethality at late stages of embryonic development (E15.5-E16.5). Heterozygous mice were viable, but born at lower frequencies, indicating a gene dosage- or a dominant negative effect of the FBP1 fusion protein. The cellular effects of FBP1 inactivation were tested in mouse embryonic fibroblasts isolated from FBP1 gene trap mice. While proliferation was reduced in the absence of wildtype FBP1, apoptosis was not affected. Expression analysis showed that in homozygous MEFs p15 and p21 transcripts were upregulated, while decreased cmyc mRNA levels were measured. Closer inspection of homozygous gene trap embryos revealed an anemic phenotype that appeared most pronounced around embryonic day 15.5. Analysis of fetal livers, the main site of hematopoiesis at this stage of development, showed a strongly reduced total cell number in homozygous embryos. Evaluation of the different hematopoietic cell lineages did not reveal significant changes in particular differentiated cell types. Instead, all cell lineages seemed to be affected equally by FBP1 inactivation. In contrast, analysis of hematopoietic progenitor cell populations showed an increased percentage of multipotent progenitor cells (MPPs) and a strongly reduced number of long-term hematopoietic stem cells (LT-HSCs). Functional analysis of MPPs by in vitro colony formation assays demonstrated that the FBP1-mutant cells possess a normal colony formation potential while their expansion capacity was reduced. Competitive transplantation of lineage negative fetal liver cells into irradiated recipient mice resulted in reduced engraftment of liverderived progenitor cells from homozygous FBP1 gene trap mice. However, stable engraftment was observed over a period of 12 weeks, demonstrating that the FBP1-deficient LT-HSCs are in principle capable of long-term repopulation. These results demonstrate that FBP1 exerts an essential function during definitive hematopoiesis. It can be speculated that FBP1 influences proliferation, apoptosis and possibly also stem cell self-renewal through the regulation of specific target genes within the hematopoietic progenitor cells. Alternatively, extrinsic effects caused by the absence of FBP1 activity could impair the function of the progenitor cells.
In dieser Studie haben wir die Modulation von Arachidonsäure (AA)-Stoffwechselwegen während einer Wurminfektionen mit dem Fadenwurm Heligmosomoides polygyrus bakeri (Hpb) als angeborene regulatorische Strategie zur Modulation der Typ-2-Entzündung untersucht. Wir zeigten, dass Hpb in frühen Stadien der Infektion (Tag 7) die Produktion von regulatorischen Prostaglandinen (PGE2 und 6-keto PGF1-α, ein Abbauprodukt von PGI2) und COX-Metaboliten (12-HHT und TXB2) fördert, jedoch die Sekretion von entzündungsfördernden Mediatoren PGD2 und LTs (LTB4, cysLTs) unterdrückt. Die Hpb-gesteuerte Regulierung des AA-Stoffwechsels könnte eine Strategie zur Immunsuppression/ Immunevasion dieses Parasiten darstellen, die darauf abzielt, die vom Wirt ausgelösten Immunantworten des Typs-2 zu unterbinden und sowohl die Infiltration und Rekrutierung von Granulozyten als auch die Schleimproduktion zu begrenzen und auf diese Weise das Abtöten bzw. Ausscheiden der Larven zu verhindern.
Als Schwerpunkt der Arbeit, konnten wir ebenso zeigen, dass ein Larvenextrakt aus Heligmosomoides polygyrus bakeri (HpbE) den AA Stoffwechsel in myeloiden Zellen wie Makrophagen und Granulozyten moduliert, indem die Synthese von 5-LOX in Richtung COX-Metaboliten verschoben wird. Die Behandlung von murinen und humanen Makrophagen mit HpbE induzierte die Synthese von regulatorischen Prostaglandinen (PGE2) und Prostaglandinen, die an der Wundheilung und Blutgerinnung beteiligt sind (12-HHT, TXB2), wohingegen die Produktion von entzündungsfördernden Lipidmediatoren (LTs, PGD2) unterdrückt wurde. Weiter induzierte HpbE in humanen und murinen Makrophagen die Synthese der Typ-2 hemmenden Mediatoren IL-10 und IL-1β und modulierte die Produktion von Zytokinen, die an der Regulierung von M2-Polarisierung und der Typ-2-Entzündung (IL-12, IL-28, IL-27 und TNF-α) in humanen Makrophagen beteiligt sind. Ähnlich zu der HpbE-vermittelten Eicosanoid-Umprogrammierung in Makrophagen, veränderte HpbE den AA-Stoffwechsel humaner Granulozyten und zeigt eine Verschiebung von LOX- in Richtung COX-Metabolismus. Außerdem kann HpbE direkt auf humane Granulozyten wirken und die Chemotaxis von Granulozyten effizienter hemmen als zur Asthmabehandlung verwendete Standardarzneimittel, indem es die Expression von LT synthetisierenden Enzymen (LTA4H und LTC4S) verringert und die Expression von chemotaktischen Rezeptoren (CCR3 und CRTH2) herunterreguliert.
Darüber hinaus, konnten wir die Mechanismen identifizieren, die der HpbE-gesteuerten Eicosanoid-Umprogrammierung in Makrophagen zugrunde liegen. Hpb Produkte induzierten die Aktivierung von p38 MAPK, welche COX und die Transkriptionsfaktoren HIF-1α und NFκβ aktiviert und die Produktion von Prostaglandinen (PGE2 and TXB2) sowie der Typ-2 unterdrückenden Zytokine IL-10 and IL-1β fördert. Der der Induktion des COX-Signalwegs zugrunde liegende Upstream-Mechanismus umfasste mehrere PPRs (TLR2, Dectin-1/2). Diese Rezeptoren waren allerdings nicht an der HpbE-gesteuerten Induktion von IL-10 beteiligt. Die Mechanismen der Modulation des 5-LOX-Signalweges muss noch in zukünftigen Studien weiter erforscht werden.
Das therapeutische Potential von HpbE oder HpbE-behandelten Makrophagen wurde in einem Maus Model mit HDM-induzierter allergischer Atemwegsentzündung in vivo gezeigt. Eine intranasale Behandlung mit HpbE vor HDM-Sensibilisierung und -Provokation führte zu einer Umprogrammierung des AA-Stoffwechsels und verhinderte die Allergie-induzierte Eosinophilie, Zellinfiltration, Atemwegsentzündung und Schleimproduktion. Die Modulation der Typ-2-Entzündung durch HpbE wurde vor allem durch COX-2-Metabolite vermittelt, die von HpbE-stimulierten Makrophagen freigesetzt wurden. Dies zeigte sich insbesondere darin, dass der Transfer von HpbE-stimulierten Wildtyp- aber nicht COX-2-defizienten Makrophagen vor Provokation die Granulozyten Rekrutierung und Typ-2-Entzündung während der HDM-induzierten Allergie in vivo abschwächte.
Mittels eines Maus Models für die allergische Atemwegsentzündung in unterschiedlichen Altersstufen (Neugeboren, Jungtier und Erwachsen) zeigte dieses Forschungsprojekt, dass das Alter der Sensibilisierung eine Schlüsselrolle bei der Produktion von LTs, der Expression von LT-Synthese Enzymen sowie von Faktoren, die zu strukturellen Veränderungen in den Atemwegen führen, spielt. Hier haben wir auch festgestellt, dass der Mechanismus hinter der LT-Produktion und dem Atemwegs-Remodeling im Epithel von ausgewachsenen sensibilisierten Mäusen die Aktivierung der Faktoren sPLA2X, TGM2 und Wnt5a beinhaltet.
Des Weiteren zeigte unsere Studie, dass eine Wechselwirkung zwischen entzündetem Atemwegsepithel und Alveolar-ähnlichen Makrophagen die Synthese von LTs fördern kann. Der vorgeschlagene Mechanismus startet mit der Sekretion von Wnt5a durch das entzündete Atemwegsepithel, welches die Expression von TGM2 in Makrophagen aktiviert und die Produktion von entzündungsfördernden LTs induziert, wodurch die Rolle der Makrophagen in entzündeten Atemwegen bei Erwachsenen weiter unterstützt wird. Die Relevanz der entdeckten Kaskade konnte auch in Geweben von Patienten mit chronischer Rhinosinusitis und Nasenpolypen (CRSwNP) bestätigt werden. Hohe Konzentrationen von LT Enzymen (5-LO, LTC4S LTA4H), sPLA2-X, TGM2 und Wnt5a wurden in humanen Nasenpolyp Geweben beobachtet, und hohe Konzentrationen von CysLTs wurden in Nasenpolyp Sekreten dieser Patienten gemessen. Dies lässt vermuten, dass die Expression von Atemwegs Remodeling-Faktoren, LT-Synthese Enzymen und die LT Synthese steroidresistent sind. Daher könnte diese entzündliche Kaskade ein alternatives therapeutisches Ziel für die Behandlung von Asthma darstellen, speziell bei Patienten mit steroidresistenten Formen von Atemwegsentzündungen.
Basierend auf den möglichen therapeutischen Anwendungen von HpbE haben wir begonnen, an der Charakterisierung der im HpbE vorhandenen immunmodulatorischen Wirkstoffe zu arbeiten. Glutamatdehydrogenase (GDH) und Ferritin wurden als potenzielle immunmodulatorische Komponenten von HpbE identifiziert. Es ist jedoch weitere Arbeit erforderlich, um diese in HpbE vorhandenen Proteine rekombinant herzustellen und den Wirkungsmechanismus im Bezug auf die Typ-2-Entzündung weiter aufzuklären.
G protein-coupled receptors (GPCRs) play regulatory roles in many different physiological processes and they represent one of the most important class of drug targets. However, due to the lack of three-dimensional structures, structure based drug design has not been possible. The major bottleneck in getting three-dimensional crystal structure of GPCRs is to obtain milligram quantities of pure, homogenous and stable protein. Therefore, during my Ph.D. thesis, I focused on expression, characterization and isolation of three GPCRs namely human bradykinin receptor subtype 2 (B2R), human angiotensin II receptor subtype 1 (AT1aR), and human neuromedin U receptor subtype 2 (NmU2R). These receptors were heterologously produced in three different expression systems (i.e. Pichia pastoris, insect cells and mammalian cells), biochemically characterized and subsequently solubilized and purified for structural studies The human bradykinin receptor subtype 2 (B2R) is constitutively expressed in a variety of cells, including endothelial cells, vascular smooth muscle cells and cardiomyocytes. Activation of B2R is important in pathogenesis of inflammation, pain, tissue injury and cardioprotective mechanisms. During this study, recombinant B2R was produced in methylotrophic yeast Pichia pastoris (3.5 pmol/mg), insect cells (10 pmol/mg) and mammalian cells (60 pmol/mg). The recombinant receptor was characterized in terms of [3H] bradykinin binding, G protein coupling, localization, and glycosylation. Subsequently, it was solubilized and purified using affinity chromatography. Homogeneity and stability of purified B2R was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from BHK cells and Sf9 cells, which were used for three-dimensional crystallization attempts. The second receptor, which I worked on, is human angiotensin II receptor subtype 1 (AT1aR). AT1aR is distributed in smooth muscle cells, liver, kidney, heart, lung and testis. Activation of AT1aR is implicated in the regulation of blood pressure, hypertension and cardiovascular diseases. Recombinant AT1aR was produced at high levels in Pichia pastoris (167 pmol/mg), while at moderate levels in insect cells (29 pmol/mg) and mammalian cells (32 pmol/mg). The recombinant receptor was characterized in terms of [3H] angiotensin II binding, localization, and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Homogeneity and stability of purified AT1aR was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from Pichia pastoris, which were used for threedimensional crystallization attempts. In addition to B2R and AT1aR, I also attempted to produce and isolate the human neuromedin U receptor subtype 2 (NmU2R), which was deorphanized recently. It is found in highest abundance in the central nervous system, particularly the medulla oblongata, spinal cord and thalamus. The distribution of this receptor suggests its regulatory role in sensory transmission and modulation. During this study, recombinant NmU2R was produced in Pichia pastoris (6 pmol/mg) and BHK cells (9 pmol/mg). Recombinant receptor was characterized with regard to [125I] NmU binding, localization and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Due to its low expression level, further expression optimization is required in order to obtain milligram amounts for structural studies. The long-term goal of this study was to obtain three-dimensional crystal structure of recombinant GPCRs. However, 3-dimensional crystallization of human recombinant membrane proteins still remains a difficult task. On the other hand, recent advances in the solid-state NMR spectroscopy offer ample opportunities to study receptor-ligand systems, provided milligram quantities of purified receptor are available. Therefore, in parallel to 3-dimensional crystallization trials, purified B2R was also used for solid-state NMR analysis in order to investigate the receptor bound conformation of bradykinin. Preliminary results are promising and indicate significant structural changes in bradykinin upon binding to B2R. Further experiments are ongoing and will hopefully result in the structure of receptor bound bradykinin. One of the challenges in GPCR crystallization is the small hydrophilic surface area that is available to make crystal contacts. One possibility to overcome this problem can be the reconstitution of a GPCR complex with an interacting protein for cocrystallization. For this purpose, I coexpressed B2R and AT1aR, which form a stable heterodimer complex, in BHK cells. I could successfully isolate the heterodimer complex by using two-step affinity purification. Unfortunately, this complex was not stable over time and disassociates within three days of purification. However, during coexpression of B2R and AT1aR in BHK cells, I observed that B2R was localized in the plasma membrane in coexpressing cells while it was retained intracellularly when expressed alone. This coexpression of AT1aR with B2R resulted in a four-fold increase in [3H] bradykinin binding sites on the cell surface. In addition, these two receptors were cointernalized in response to their individual specific ligands. Interestingly, colocalization of B2R and AT1aR was also found in human foreskin fibroblasts (which endogenously express both receptors), in line with the possibility that heterodimerization may be required for surface localization of B2R in native tissues as well. This is the first report where surface localization of a peptide GPCR is triggered by a distantly related peptide GPCR. These data support the hypothesis that heterodimerization may be a prerequisite for cell surface localization of some GPCRs. A second approach that I followed to stabilize the purified B2R was to reconstitute the B2R-β-arrestin complex. β-arrestin is a cytosolic protein that participates in agonist mediated desensitization of GPCRs and therefore dampens the cellular responses initiated by the activation of GPCRs. I tried to reconstitute B2R-β-arrestin complex in vitro by mixing purified B2R and purified β-arrestin. But, no interaction of these two proteins was observed in the pull-down assays. However, a C-terminal mutant of B2R (where a part of the C-terminus of the B2R is exchanged with that of the vasopressin receptor) was found to interact with β-arrestin in vitro as revealed by pull-down assays. In conclusion, this work establishes the production, characterization and isolation of three recombinant human GPCRs. Recombinant receptors were produced in milligram amounts and therefore, pave the way for structural analysis. The heterodimer complex of B2R-AT1aR and B2R-β-arrestin complex can be of great help during crystallization. In addition, it was also found for the first time that the surface localization of a peptide GPCR can be triggered by heterodimerization with a distantly related peptide GPCR.
During the last years, chemopreventive activity of NSAIDs against a great variety of tumors was highly investigated. COX-2 seemingly plays a major part in tumorigensis and tumor development, underlined by several studies in animals and humans. At first, NSAIDs were thought to accomplish chemoprevention by inhibition of COX-2 as their so far known mode of action comprises unselective inhbition of COX-enzymes. However, further studies revealed COX-independent mechanisms. Sulindac is known as a well established drug used to treat inflammation and pain exerting the most prominent chemopreventive action, mainly in colorectal cancer or FAP and can be classified into the group of NSAIDs inhibting both COX-isoformes. As interference with the AA metabolism is evident, it was speculated whether Ssi has targets other than COX-enzymes providing evidence and explanation of its beneficial side effect profile and its ability to reduce tumor growth. 5-LO is another master enzyme in the AA cascade which produces inflammatory lipid mediators (LTs) upon stimulation in inflamed tissues. The present work should answer the question if Ssi targets the 5-LO pathway and should examine the molecular mechanisms behind Ssi-mediated 5-LO inhibiton. As COX-2 is upregulated during carcinogenesis and is inhibited by Ssi, further investigations should show regulatory effects of Ssi on 5-LO gene expression in MM6-cells and whether Sp1 as a common transcriptional factor is involved in such a regulation. As the use of NO-NSAIDs seem to be a promising strategy concerning their chemopreventive and gastroprotective effects compared to the parent NSAIDs, a possible interaction with the 5-LO pathway as a second, potent target should additionally be elucidated. In the first section it was demonstrated that the pharmacologically active metabolite of sulindac, Ssi, targets 5-LO. Ssi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human PMNL (IC50 ≈ 8 -10 μM). Importantly, Ssi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC50 = 18.7 μM). Ssi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone, failed to inhibit 5-LO. Mechanistic analysis demonstrated that Ssi directly suppresses 5-LO with an IC50 of 20 μM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy. In the second part of the work dealing with the analysis of Ssi’s inhibitory mechanism on 5-LO it was presented that Ssi shows a lack of potency in cellular systems where membrane constituents are existent. The addition of microsomal fractions of PMNLto crude 5-LO enzyme were able to recover enzyme activity to ~ 100 %. Selectively 5-LO activity stimulating lipids like PC, participating in 5-LO membrane interactions within the regulatory C2-like domain of 5-LO, counteracted the Ssimediated inhibition on 5-LO-wt in a concentration-dependent manner. Lastly, a protein mutant lacking three trp resudies essential for linking the enzyme to nuclear membranes and deploying catalytic activity was not influenced by Ssi and shows enzyme activity in a cell-free assay. Ssi displays the first 5-LO inhibitor on the market interacting with the C2-like domain of the enzyme and therfore can stand for a novel lead structure of 5-LO inhibitors. An influence on 5-LO gene expression by Ssi could be detected in differentiated MM6-cells, described in the results chapter 3 (4.3). Ssi downregulated the 5-LO mRNA level after 72 hrs of incubation in differentiated MM6-cells to ~ 20 % of output control at concentrations of 10 μM. Concomitantly, mRNA levels of Sp1 were suppressed. Reporter gene studies revealed Sp1 most probably as a regulating agent involved in the Ssi-mediated 5-LO mRNA downregulation as co-transfection of increasing amounts of Sp1 could abrogate the effect. A ChIP assay could identify Sp1 as a critical transcriptional factor as Sp1 binding to the 5-LO promoter decreased in presence of Ssi. Lastly, three NO-NSADIs (NO-sulindac, NOnaproxen, NO-aspirin) were tested for the ability of 5-LO product inhibition. In intact PMNL, all compounds showed effective inhibition of 5-LO activity and NO-sulindac was most potent with an IC50 value of ~ 3 μM. NO-ASA inhibited 5-LO with IC50 values of ~ 30 μM and showed a non-competitive mode of action in cell-based assays. On human recombinant 5-LO all compounds again showed inhibitory potency whereas NO-sulindac again suppressed LT biosynthesis with an IC50 vaue comparable to intact cellular systems. Unfortunately, all inhibitors showed a loss of potency when tested for inhibition of 5-LO product synthesis in human whole blood as higher concentrations up to 100 μM were needed to reach at least 55 % enzyme inhibition. However, this strategy of 5-LO inhibition seems promising and needs further experimental approaches to gain more insight into the mechanism of 5-LO inhibition by NONSAIDs.
Macroautophagy, herein referred to as autophagy, is an evolutionarily conserved homeostatic process that normally occurs inside eukaryotic cells which involves degradation of cytoplasmic substances via lysosomes. It can be induced by various conditions such as starvation and drug exposure, as well as be inhibited by numerous compounds. Under normal conditions, the doublemembrane autophagosomes engulf the cytosolic substrates and deliver them to lysosomes for digestion. These substrates include unnecessary or dysfunctional cell components, such as faulty macromolecules, organelles and even invading pathogens. Autophagosomes are formed through the co-operative work of various autophagy-related (ATG) proteins organized into complexes. Upon closure of the autophagosomes, they fuse with the acidic lysosomes, resulting in formation of autolysosomes and the delivery of lysosomal hydrolases to degrade the engulfed contents. The fusion of the autophagosome with lysosome is carried out by specific SNARE proteins, small GTPases and their effectors including tethers, adaptors and motor proteins. Autophagy is impaired in many human diseases including cancer, neurodegenerative diseases, aging and inflammation. Therefore, manipulation of autophagy pathway holds a great promise for new therapeutic applications ...
Myxobacteria are on order of Gram-negative, soil dwelling bacteria that feature an impressive number of properties: they can glide on solid surfaces by using two different motility motors, subsist by preying on other microorganisms, are often producers of multiple natural products, and upon adverse environmental conditions, they are able to form multicellular structures called “fruiting bodies”. The process, in which these macroscopically visible structures arise from independent single cells, has been the predominant subject of myxobacterial research for many decades. More precisely, researchers have strived for the discovery of genes, proteins and small molecules that act as signals, receivers or modulators of this complex process. In this regard, the species Myxococcus xanthus has evolved into the model organism due to its relatively simple and reliable handling in a laboratory environment. The research underlying this thesis focused on the identification and biosynthesis of lipids that may act as intercellular signaling molecules during the course of fruiting body formation of the myxobacterium Myxococcus xanthus as part of the “E-signal” system. In general, lipids containing branched-chain fatty acids with an uneven number of carbon atoms were found to be important players in this particular process. Nevertheless, their exact roles remain largely unknown as of this day. The first publication that is part of this thesis deals with an aspect that even strengthened the importance of role of iso-branched compounds in myxobacteria: myxobacterial metabolism is able to transform precursors of iso-lipids to isoprenoids. It addresses the question whether isoprenoids in general are important for fruiting body formation. Phenotypic analysis of mutants impaired in the biosynthesis of the central isoprenoid precursor 3-hydroxymethylglutaryl-Coenzyme A (3-HMG-CoA) from acetate and/or branched chain keto acids and their genetic and metabolic complementation clearly showed that isoprenoids are essential for fruiting body formation and confirmed that leucine derived isovalerate is an important source for isoprenoid precursors in myxobacteria. The second, and by far and away most tedious and sophisticated study, addressed the question as to how myxobacteria form fatty acid derived iso-branched ether lipids and to what extent they are important for fruiting body formation and sporulation. In a previous study, those unusual lipids were identified as specific biomarkers for myxobacterial development. No biochemical pathways to ether lipids specific for prokaryotes were known by then. In this study, a putative candidate gene that may be in involved in ether lipid biosynthesis was investigated. A combination of gene disruption and complementation experiments, phenotypic analysis and monitoring of ether lipid formation by means of GC-MS demonstrated its involvement in myxobacterial ether lipid biosynthesis and the importance of these lipids for the developmental process. Heterologous expression and biochemical testing of this gene together with in-silico sequence analysis and docking experiments confirmed the functions of its predicted domains. The discussion section provides an additional suggestion on how the ether bond formation is performed. Furthermore and most importantly, iso-branched ether lipids were found to be essential for sporulation but not for fruiting body formation. In summary, one or several molecules derived from an iso-branched alkylglycerol seem to play a role during sporulation in M. xanthus and a multidomain enzyme unique for myxobacteria is involved in their biosynthesis. The last manuscript addresses the complexity of lipid metabolism in myxobacteria. Prior to this work, there was limited knowledge about the exact composition of the myxobacterial lipidome and no method was available to monitor putative changes in the myxobacterial lipidome down to the single molecular species for studying lipid biosynthesis or regulation. An ultra-performance liquid chromatography coupled with mass spectrometry based method with electrospray ionization (UPLC-ESI-MS) utilizing standard equipment and a water/acetonitrile/isopropanol based eluent system proved to be geared for the construction of lipid profiles for wild type and mutant cells of M. xanthus and to show their differences. Fragmentation spectra based structure elucidation of lipid molecular species resulted in the identification of 99 molecular species comprising glycerophosphoethanolamines, glycerophosphoglycerols, glycerolipids, ceramides and ceramide phosphoinositols. The latter have never been described for any prokaryotes before. Three dimensional plots were created from the relative intensity differences of the single molecular ion species between the different samples to provide an efficient and versatile visualization of the data and enable the researcher to quickly detect differences.
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.
5-lipoxygenase (5-LO) is the key enzyme in the formation of inflammatory leukotrienes, which are mediators of inflammation and allergy. The 5-LO catalyses the oxidation of arachidonic acid to 5-HPETE and subsequently to LTA4. The leukotrienes are involved in the development and maintenance of inflammatory diseases, like asthma and allergic rhinitis. Additionally, 5-LO is overexpressed in some cancer types, although its relevance is still not fully understood. 5-LO expressing cells are B- lymphocytes and cells of myeloid origin like monocytes, macrophages and granulocytes. The 5-LO promoter lacks a TATA or CCAT box and covers two CpG islands. These are characteristics of a housekeeping gene, but as the 5-LO is not expressed ubiquitiously, the expression of the 5-LO is tightly regulated. Epigenetic mechanisms were known to be involved in the control of the 5-LO expression. The HDAC inhibitor TsA significantly induced the transcriptional activity of the 5-LO promoter in reporter gene assays as well as on 5-LO mRNA transcript level in MM6 cells. The GC-boxes GC4 and GC5 in the proximal 5-LO promoter were identified to be essential for the TsA effect, as deletion of these element led to an attenuated TsA effect in reporter gene assay. Recruitment of the transcription factors Sp1 and Sp3 and the RNA polymerase II to the 5-LO promoter was detectable after TsA treatment in MM6 cells by chromatin immunoprecipitation assays (ChIP), while the acetylation status of histone H4 remained unchanged. Likewise it is known that DNA methylation leads to silencing of 5-LO expression in-vitro and in-vivo. The 5-LO promoter is densely methylated in the cell line U937, but unmethylated in HL-60 cells and - elucidated in this study - also in MM6 cells. Reporter gene assays with in-vitro methylated 5-LO promoter containing plasmids revealed that the frequency of methylated CpGs is directly proportional to reduction of 5-LO promoter activity. Incubation of U937 cells with 5-AdC, an inhibitor of DNA methyltransferases, was able to reactivate 5-LO transcription and to demethylate CpG dinucleotides. In the first part of this study the mechanism of TsA induced promoter activation was further investigated. I elucidated the mechanism of Sp1 and Sp3 recruitment to the 5-LO promoter after TsA treatment. Immnoprecipitation assay was used to detect a transcription factor complex containing Sp1 or Sp3 interacting with HDAC proteins, which might change its composition after TsA treatment. Besides the posttranslational modifications of the transcription factors Sp1 and Sp3 after TsA treatment were investigated, potentially causing an increased interaction of the proteins with the 5-LO promoter. Both aspects and their response in HDAC inhibition have been described. TsA did not affect the composition of the Sp1/HDAC1/HDAC2 complex. Sp3 was not located in a complex with the HDAC enzymes. Acetylation of Sp1 and Sp3 was detectable, but no change occurred after TsA treatment. Since neither release of the transcription factors off a complex, nor alterations in posttranslational modifications of Sp1 and Sp3 are the reason for the increased Sp1 and Sp3 binding to the 5-LO promoter, I elucidated alterations in the chromatin structure. The acetylation status of the histone proteins H3 and H4, as well as the chromatin marks H3K4me3, representing active chromatin, and H3K9me, representative for repressive state, were investigated. Additionally, the time course of the TsA effect was determined on 5-LO mRNA level using real-time PCR. The acetylation status of the histone proteins on the 5-LO core promoter correlated with the basal 5-LO mRNA transcript expression in MM6, HL-60 and U937 cells. The highest 5-LO mRNA level was detectable in MM6 cells, followed by HL-60 cells. The lowest 5-LO mRNA level was detected in 5-LO promoter methylated U937 cells. The order of the basal 5-LO mRNA expression of the three cell lines correlates with the basal acetylation status of histone proteins H3 and H4. In MM6 cells the highest basal levels in acH3 and acH4 were detected, followed by HL-60 and U937 cells. Moreover, the data obtained in U937 cells revealed that the correlation between DNA methylation and histone hypoacetylation is alike on the 5-LO promoter. TsA treatment induced the 5-LO mRNA level in the three cell lines with different intensity: 5-LO mRNA level in MM6 cells was induced 11-fold, in HL-60 cells 6- fold and in U937 cells 4- fold. The histone acetylation and methylation levels on the 5-LO promoter after TsA incubation were investigated. No increase in acH3 and acH4, but in H3K4me3 was detectable in MM6 cells by ChIP assay. HL-60 cells showed an increase in acH3 and acH4 as well as in H3K4me3. H3K9me was only detectable in untreated U937 cells, but disappeared after TsA treatment, while acH3, acH4 and H3K4me3 increased constantly after TsA treatme nt. A strong correlation between the histone modifications and the time course of the mRNA expression was detectable in all three cell lines. The combination of the posttranslational modifications acH3, acH4 and H3K4me3 led to a fast effect in transcriptional activation and the maxima of acH3 and acH4 were usually associated with the maximum in 5-LO mRNA transcript level. An increase in H3K4me3 alone, as detected in MM6 cells, led to continuous increase in the 5-LO mRNA expression with a late maximum. Additionally, we detected a slight overall decrease in 5-LO promoter methylation in U937 cells after TsA treatment. This fact taken together with the observed histone modifications could explain the 4- fold response in 5-LO mRNA level to TsA treatment of the methylated cell line U937. Another aim of the present study was to identify the specific HDAC enzymes involved in the 5-LO promoter regulation. Reporter gene assays and real-time PCR with selective HDAC inhibitors revealed that HDACs of class I are involved in 5-LO promoter regulation, namely HDAC 1, 2 and 3. The influence of each of the enzymes seemed to depend on the cell type, as inhibition of HDACs 2, 3 strongly induced 5-LO promoter activity in reporter gene assay in HeLa cells, whereas in MM6 cells HDACs 1 and 2, 3 seemed to be responsible for the 5-LO promoter regulation, measured as 5-LO mRNA level. The HDACs of class IIa and class III are not involved in the regulation of 5-LO mRNA expression. The second part of this study investigated the influence of MBD proteins on the methylated 5-LO promoter and the 5-LO mRNA expression. ChIP assays revealed MBD1, 2 and MeCP2 protein binding to the proximal 5-LO promoter in U937 cells. MBD1 was detectable on the 5-LO promoter in unmethylated HL-60 cells, while no MBD protein was located on the 5-LO promoter in MM6 cells. To elucidate the functional role of the MBD proteins, stable knocked down of MBD proteins was established in U937 cells. 5-LO mRNA transcript level was determined in the knock down clones by real-time PCR. The 5-LO transcript level was increased in all knock down samples. MBD2 knock down clones showed the highest effect in activating 5-LO with a 3- and 4.4-fold increase in the 5-LO mRNA level, followed by MBD1 (3.5- fold) and MeCP2 (2.5-fold) knock down clones. A combined participation of these three enzymes in the corepression of the methylated 5-LO promoter is indicated. Taken together, the data reveal that epigenetic mechanisms are strongly involved in the regulation of 5-LO transcription and might function as a crucial control mechanism of 5-LO expression.
This work investigated the influence of the CRISPR/Cas9 mediated knockout of 5-lipoxygenase (5-LO) on different adherent tumour cell lines derived from solid tumours. For this, the 5-LO expressing tumour cell lines HCT-116, HT-29, and U-2 OS were transiently transfected using a plasmid carrying the CRISPR/Cas9 complex sequence to the ALOX5 gene. Subsequently, cells were selected using Puromycin and analysed via Western blotting and DNA Sanger sequencing. Cells that were transfected with a control plasmid missing the guide RNA sequence, were used as a control for all experiments.
Differential gene expression analysis, performed after next-generation RNA sequencing, revealed that the expression of various genes was altered after the knockout of 5-LO. In HCT-116 cells, 28 genes were expressed differentially in all 5-LO knockout single-cell clones, while in HT-29 cells the expression of 18 genes and in U-2 OS cells of 234 genes was influenced by the knockout of 5-LO. These findings were validated by real-time qPCR. A lot of the genes that were influenced by the 5-LO knockout are known to be connected to epithelial-mesenchymal-transition (EMT), a process necessary for tumour metastasis. The results from RNA sequencing were the starting point for further investigations. In the following, different aspects of the tumour cell lines were examined. In HT-29, as
well as in U-2 OS cells, it was shown that knockout of the 5-LO resulted in impaired cell proliferation. Also, the formation of three-dimensional tumour spheroids was altered. In HT-29 cells, the knockout of 5-LO increased the number of cells in spheroids. In contrast, in U-2 OS cells, the number of cells per spheroid was decreased, even though the diameter of the spheroids was increased, due to more loosely packed spheroids. The difference between 5-LO positive and negative U-2 OS cells became even more obvious after embedding the spheroids in an artificial extracellular matrix. In that scenario, cells lacking the 5-LO formed smaller spheroids that did not have the same ability to grow into the extracellular matrix as 5-LO positive cells did. Also, directed cell migration was strongly influenced by the knockout of 5-LO. In both, HCT-116 and U-2 OS cells, directed cell migration towards a serum gradient was increased in 5-LO knockout single-cell clones. Pharmacological inhibition of the enzyme was used to investigate, whether canonical or non-canonical functions were responsible for the previously mentioned effects.
Therefore, vector control cells were treated with the 5-LO inhibitors Zileuton and CJ-13610 in different concentrations. Interestingly, only some of the effects mediated by the complete knockout of 5-LO could be reproduced by inhibiting the enzyme, leading to the suggestion, that canonical, as well as non-canonical functions of 5-LO, play a role in these tumour cells.
To conclude, it was shown in this study, that 5-LO affects various cellular functions when expressed in adherent tumour cell lines. These cell line-dependent effects result in altered gene expression, enhanced proliferation, and spheroid formation, as well as impaired cell motility, and can be mediated by enzymatic activity as well as other non-canonical functions.
In the first part of this study, we have identified the two steroid hormones progesterone and norgestimate as novel TRPC channel blockers. Both substances blocked TRPC-mediated Ca2+ influx with micromolar activities in fluorometric measurements. TRPC channel inhibition did not seem to be a general steroid effect since another progestin, the norgestimate metabolite levonorgestrel, was not effective. Norgestimate was 4- to 5-fold more active on the TRPC3/6/7 subfamily compared to TRPC4/5, whereas progesterone was similarly potent. This selectivity of norgestimate was confirmed by patch clamp recordings. As norgestimate blocked channels directly gated by DAG with a fast kinetic, we assume the compound acts on the channel protein itself. This view was further substantiated by the lack of effects on IP3R-mediated Ca2+ release from the endoplasmic reticulum, which is activated in parallel with TRPCs by Gq/11-coupled receptor stimulation. Norgestimate did not only block ectopically expressed TRPC channels but also native, TRPC-mediated currents in rat aortic smooth muscle cells with similar activity. The usefulness of norgestimate as a tool compound for the investigation of physiological TRPC functions was tested in isolated vessel rings. Consistent with TRPC6 being an essential component of the alpha-1-adrenoceptor-activated cation channel, we demonstrated a direct vasorelaxant, endothelium-independent effect of norgestimate on rat aortic rings precontracted with phenylephrine. Thus, our results provide further experimental support for a role of TRPC6 in alpha-1-adrenergic vessel constriction. In the second part of this study, we screened a human aorta cDNA-library for novel TRPC4-interacting proteins with a modified yeast two-hybrid (Y2H) system in which the TRPC4-C-terminus was expressed as tetrameric bait protein, thereby mimicking the native channel conformation. Of the eleven interacting proteins found SESTD1 was chosen for further analyses since it contains a phospholipid-binding Sec14p-like domain and thus could be involved in regulation of TRPC channels by phospholipids. After the biochemical validation of the found interaction, the first spectrin domain of SESTD1 was then identified to interact with the CIRB domain of TRPC4 in directed Y2H tests. SESTD1 also co-immunoprecipitated with the closely related TRPC5 protein in which the SESTD1-binding domain is highly conserved. Independent of the CIRB site, co-immunoprecipitation with TRPC6 and the distantly related TRPM8 channel was observed indicating the existence of other sites in these channel proteins that mediate interaction with SESTD1. Analysis of SESTD1 gene expression in human tissues showed that its transcripts are ubiquitously expressed and tissues with significant coexpression with TRPC4 and -5 were identified. We have generated two polyclonal antisera directed against SESTD1 that consistently detected SESTD1 protein in brain, aorta, heart, and in smooth muscle and endothelial cells. The functional consequences of the found interaction were investigated by examination of the TRPC5-mediated Ca2+ influx in a clonal HM1 cell line stably expressing the channel. Since SESTD1 overexpression had no detectable effects on TRPC5-mediated Ca2+ influx, most likely due to expression of endogenous SESTD1, we knocked-down the native protein with specific siRNA. This procedure reduced TRPC5-mediated Ca2+ influx following receptor stimulation by 50%. Parallel biotinylation experiments did not reveal any differences in cell surface expressed TRPC5-protein, suggesting that reduction of TRPC5 activity resulted from a loss of a direct SESTD1 effect on the channel. In addition, in immunofluorescence experiments we observed that reduced SESTD1 protein levels resulted in a redistribution of the multifunctional protein ß-catenin from the plasma membrane to the cytosol. This result may point to an involvement of SESTD1 in formation and maintenance of adherens junctions. SESTD1 contains a phospholipid-binding Sec14p-like domain and we were the first to demonstrate its Ca2+-dependent binding to phosphatidic acid and all physiological phosphatidylinositol mono- and bisphosphates in vitro. The physiological function of this binding activity is not known at present, but it could play a role in regulation of associated TRPC channels. TRPC4 and -5 channels are activated by phospholipid hydrolysis and also bind phospholipids directly. The identification of SESTD1 as novel TRPC-interacting protein could thus be an important step forward in the investigation and better comprehension of the complex molecular mechanisms of TRP channel regulation by lipids.