Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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One of the key functions of blood vessels is to transport nutrients and oxygen to distant tissues and organs in the body. When blood supply is insufficient, new vessels form to meet the metabolic tissue demands and to re-establish cellular homeostasis. Expansion of the vascular network through sprouting angiogenesis requires the specification of ECs into leading (sprouting) tip and following (non-sprouting) stalk cells. Attracted by guidance cues tip cells dynamically extend and retract filopodia to navigate the nascent vessel sprout, whereas trailing stalk cells proliferate to form the extending vascular tube. All of these processes are under the control of environmental signals (e.g. hypoxia, metabolism) and numerous cytokines and peptide growth factors. The Dll4/Notch pathway coordinates several critical steps of angiogenic blood vessel growth. Even subtle alterations in Notch activity can profoundly influence endothelial cell behavior and blood vessel formation, yet little is known about the intrinsic regulation and dynamics of Notch signaling in endothelial cells. In addition, it remains an open question, how different growth factor signals impinging on sprouting ECs are coordinated with local environmental cues originating from nutrient-deprived, hypoxic tissue to achieve a balanced endothelial cell response. Acetylation of lysines is a critical posttranslational modification of histones, which acts as an important regulatory mechanism to control chromatin structure and gene transcription. In addition to histones, several non-histone proteins are targeted for acetylation reversible acetylation is emerging as a fundamental regulatory mechanism to control protein function, interaction and stability. Previous studies from our group identified the NAD+-dependent deacetylase SIRT1 as a key regulator of blood vessel growth controlling endothelial angiogenic responses. These studies revealed that SIRT1 is highly expressed in the vascular endothelium during blood vessel development, where it controls the angiogenic activity of endothelial cells. Moreover, in this work SIRT1 has been shown to control the activity of key regulators of cardiovascular homeostasis such as eNOS, Foxo1 and p53. The present study describes that SIRT1 antagonizes Notch signaling by deacetylating the Notch intracellular domain (NICD). We showed that loss of SIRT1 enhances DLL4-induced endothelial Notch responses as assessed by different luciferase responsive elements as well as transcriptional analysis of Notch endogenous target genes activation. Conversely, SIRT1 gain of function by overexpression of pharmacological activation decreases induction of Notch targets in response to DLL4 stimulation. We also showed that the NICD can be directly acetylated by PC AF and p300 and that SIRT1 promotes deacetylation of NICD. We have identified 14 lysines that are targeted for acetylation and their mutation abolishes the effects of SIRT1 of Notch responses. Furthermore, over-expression or activation of SIRT1 significantly reduces the levels of NICD protein. Moreover, SIRT1-mediated NICD degradation can be reversed by blockade of the proteasome suggesting a mechanism resulting from ubiquitin-mediated proteolysis. Indeed, we have shown that SIRT1 knockdown or pharmacological inhibition decreased NICD ubiquitination. We propose a novel molecular mechanism of modulation of the amplitude and duration of Notch responses in which acetylation increases NICD stability and therefore permanence at the promoters, while SIRT1, by inducing NICD degradation through its deacetylation, shortens Notch responses. In order to evaluate the physiological relevance of our findings we used different models in which the Notch functions during blood vessel formation have been extensively characterized. First, retinal angiogenesis in mice lacking SIRT1 activity shows decreased branching and reduced endothelial proliferation, similar to what happens after Notch gain of function mutations. ECs from these mice exhibit increased expression of Notch target genes. Second, these results were reproducible during intersomitic vessel growth in sirt1-deficient zebrafish. In both models, the defects could be partially rescued by inhibition of Notch activation. Third, we used an in vitro model of vessel sprouting from differentiating embryonic bodies in response to VEGF in a collagen matrix. Our results showed that Sirt1-deficient cells shows impaired sprouting which correlated with increased NICD levels. In addition, when in competition with wild-type cells in this assay, Sirt1-deficient cells are more prone to occupy the stalk cell position. Taken together, our study identifies reversible acetylation of NICD as a novel molecular mechanism to adapt the dynamics of Notch signaling and suggest that SIRT1 acts as a rheostat to fine-tune endothelial Notch responses. The NAD+-dependent feature of SIRT1 activity possibly links endothelial Notch responses to environmental cues and metabolic changes during nutrient deprivation in ischemic environments or upon other cellular stresses.
Succinate:quinone oxidoreductases (SQORs) are integral membrane protein complexes, which couple the two-electron oxidation of succinate to fumarate (succinate → fumarate + 2H+ + 2e-) to the two-electron reduction of quinone to quinol (quinone + 2H+ + 2e- → quinol) as well as catalyzing the opposite reaction, the reduction of fumarate by quinol. In mitochondria and some aerobic bacteria, succinate:ubiquinone reductase, also known as complex II of the aerobic respiratory chain or as succinate dehydrogenase from the tricarboxylic acid (TCA or Krebs) cycle, catalyzes the oxidation of succinate by ubiquinone, which is mildly exergonic under standart conditions and not directly associated with energy storage in the form of a transmembrane electrochemical proton potential (Δp). Gram-positive bacteria do not contain ubiquinone but rather menaquinone, a quinone with significantly lower oxidation-reduction (“redox”) midpoint potential. In these cases, the catalyzed oxidation of succinate by quinone is endergonic under standard conditions. Consequently, these bacteria face a thermodynamic problem in supporting the catalysis of this reaction in vivo. Based on experimental evidence obtained on whole cells and purified membranes, it had previously been proposed that the SQR from Gram-positive bacteria supports this reaction at the expense of the protonmotive force, Δp. Nonetheless, it has been argued that the observed Δp dependence is not associated specifically with the activity of SQR because the occurrence of artifacts in experiments with bacterial membranes and whole cells can not be fully excluded. Clearly, definitive insight into the mechanism of catalysis of this intriguing reaction required a corresponding functional characterization of an isolated, membranebound SQR from a Gram-positive bacterium. The first aim of the present work addresses the question if the general feasibility of the energetically uphill electron transfer from succinate to menaquinone is associated specifically to a single enzyme complex, the SQR. The prerequisite to achieve this goal was stable preparation of this enzyme.
LmrA is a member of the ATP Binding Cassette (ABC) transporter family of membrane proteins and a structural and functional homologue of P-glycoprotein1, 2. ABC-transporters share a common architecture of two transmembrane domains and two nucleotide binding domains. The NBDs are highly conserved in this transporter family whereas the TMDs are highly diverse3. The TMDs recognize the substrate and the NBDs bind and hydrolyze ATP and thus contribute the energy for substrate translocation. ABC transporters as a protein family transport a high number of substrates including peptides, nutrients, ions, bile acids, lipids and other lipophilic compounds. LmrA is a multidrug transporter that recognizes a number of hydrophobic substrates including fluorescent dyes and antibiotics1, 4-6. LmrA is a native protein of the gram-positive bacterium Lactococcus lactis. In this thesis, L. lactis was used as a homologous expression host for the preparation of LmrA for a variety of experiments. Wildtype LmrA as well as a number of cysteine mutants were successfully expressed in L. lactis, purified and subsequently characterized by a variety of biochemical assays (Chapter 4). LmrA can be expressed to very high amounts in L. lactis. The purification and reconstitution were optimized for the requirements of solid-state NMR experiments in this thesis. For the first time, an ABC transporter has been reconstituted in synthetic lipids to a ratio of up to 1:150 (mol/mol). LmrA was shown to be active under magic angle spinning conditions with these reconstitution ratios. By taking advantage of the slower ATP hydrolysis by LmrA ΔK388 (lysine deletion in the Walker A motif), a real-time 31P solid-state NMR ATPase assay was established (Chapter 5). This assay allowed, for the first time, the investigation of all phosphor nuclei during the ATP hydrolysis cycle of a membrane protein simultaneously and in real time7. This assay has been successfully adapted to investigate both ATP hydrolysis and substrate phosphorylation of diacylglycerol kinase (together with S. Wollschlag) and ATP hydrolysis at high temperatures of the thermophilic ABC transporter ABC1 from Thermos thermophilus (together with A. Zutz). In the course of this thesis, the gene for LmrA has been cloned into expression vectors suitable for Escherichia coli and the heterologous expression of LmrA was established (Chapter 4). The functionality of the heterologously expressed protein has been investigated and compared to L. lactis LmrA. In these experiments, LmrA was shown to yield a distinct multidrug resistance phenotype in its E. coli host and to show secondary active multidrug transport in the absence of ATP and presence of a proton gradient [Hellmich et al, in prep] (Chapter 4). Previously, it had been shown that LmrA acts as a seconadary active transporter when the NBDs are truncated8. The overexpression in minimal and defined medium and the purification of LmrA from E. coli have been optimized. Isotope labeling for ssNMR has been established and the first multinuclear ssNMR experiments have been carried out on a functional ABC transporter (Chapter 8). ABC transporters couple two cycles: upon ATP binding, the NBDs dimerize, hydrolyze the ATP, subsequently release Pi and ADP and finally dissociate. During this cycle, conformational changes are relayed to the TMDs which utilize the energy from ATP binding and/or hydrolysis to translocate the respective substrate. The prehydrolysis state can be trapped by beryllium fluoride, whereas the post-hydrolysis state of this cycle can be trapped by vanadate9-12. Trapping protocols for these reagents were successfully established for LmrA in this thesis (Chapter 4). This allowed for the investigation of different catalytic states by both ssNMR and EPR. A general 19F labeling protocol for membrane proteins has been established in the course of this thesis and successfully applied to proteorhodopsin (together with N. Pfleger)13 and LmrA (chapter 6). Single cysteine mutants of LmrA that line out the dimer interface have been labeled with a fluorine label for ssNMR. In the apo state, the 19F labeling indicates highly flexible transmembrane domains, a finding that is supported by 13C ssNMR and EPR measurements. The addition of drugs has a different effect on different positions within the LmrA dimer, therefore indicating that different drugs are recognized at a different position within the protein. For P-glycoprotein and LmrA it has been previously shown by biochemical methods that different drug binding sites co-exist. For a 19F label attached at position 314 (LmrA E314C), the spectra showed two distinct peaks with similar populations. This could hint towards a structural asymmetry within the LmrA dimer that might also be reflected in the alternating ATP hydrolysis at the NBDs. E314 has been specifically implicated with drug transport. Thus, structural asymmetry at this position might be functionally relevant for guiding a substrate through the transporter. Structural asymmetry within a homodimeric ABC transporter has also been shown for BtuCD, the E. coli vitamin B12 importer14. In addition, the conserved glutamates in EmrE, a small multidrug resistance protein, were shown to be asymmetric in the drug bound state15. Both, uniformly 13C/15N labeled as well as selectively amino acid type labeled LmrA has been investigated in different conformational states. Interestingly, significant dynamic changes in the b-sheet regions of LmrA (confined to the NBDs) were observed in the pre-hydrolysis (beryllium fluoride) and transition state (vanadate trapped) state. These were interpreted as the transition from a domain in fast conformational exchange in the apo state to one of intermediate exchange in the nucleotide bound state. A significant change in NBD mobility upon nucleotide binding was previously also shown with 2H ssNMR on LmrA16. By EPR it was shown that LmrA in both the vanadate and BeFx trapped states displays a significantly higher rigidity and therefore defined distances, whereas the apo state resembled a “floppy” protein with no preferred distance distribution. This concurs with data obtained from 19F ssNMR with fluorine labeled single-cysteine mutants. Here, in agreement with the EPR data, a higher label (and possibly) protein mobility was observed in the apo state displaying rather broad line widths. Upon trapping with vanadate, the line widths of the majority of fluorine-labeled mutants decreased due to an enhanced protein rigidity and a more homogenous environment of the fluorine labels. A similar observation was made when increasing the temperature that can be explained due to higher protein flexibility at increased temperatures. Solution NMR was employed to investigate the isolated soluble NBD of LmrA (Chapter 9). First 2D and 3D spectra were successfully obtained and could be utilized for a preliminary assignment of a significant fraction of residues. Additionally, binding of ATP and ADP in absence and presence of magnesium was investigated. Finally, the effects of peptides emulating the coupling helices of the full-length transporter on the soluble NBD were investigated. Strikingly, binding of one of these peptides only occurred in the presence of nucleotides (whereas the other showed no binding at all) hinting towards a tightly coupled regulation of the NBD and TMD during the substrate translocation/ATP hydrolysis cycle based on nucleotide binding.
Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV
infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer”
tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an
HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10
HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral
combination therapy. T cell infusions were tolerated well with no severe side effects. A
significant increase of CD4 counts was observed post infusion. At the end of the one-year
follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified
cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear
follow-up, whereby marking levels correlated with the cell dose. No significant changes of
viral load were observed during the first four months. Four of the seven patients that changed
their antiviral drug regimen thereafter responded with a significant decline in plasma viral load.
In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene
marking and may improve immune competence in HIV-infected patients with advanced disease
and multidrug resistant virus. However, the low level of gene marking and the lack of substantial
long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate
the requirement for new vectors with new strategy.
In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements
were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from
this work provide the basis for the selection of a suitable candidate vector for extensive
preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors
incorporate a number of additional features that are of potential value for gene therapeutic
applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors
and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration
profile. The use of internal promoters to drive expression of the therapeutic transgene maC46
should further improve the safety profile of these new‐generation vectors, while an additional
artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene
expression. The rationale for this is that hematopoietic stem and progenitor cells will be
Summary
98
protected from enhancer‐mediated transactivation effects and also from potential side effects due
to the aberrant expression of maC46 while at the same time the full clinical benefit for the
patients is maintained.
In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors
harboring different regulatory elements were selected based on results from pilot experiments.
The internal promoters used to drive expression of codon optimized maC46 were the PGK
promoter and MPSV promoter. This work focuses on the transgene expression levels in
lymphoid cells and antiviral activity. The issues of long term expression, propensity to
methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first
step the performance of different vectors was evaluated in the human T cell lines. Based on
promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the
MPSV promoter along with intron were selected for in vivo transplantation experiments.
In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells,
along with maintained expression of introduced genes. It was observed that the expression of
these constructs depends strongly on the activation and differentiation status of the targeted T
cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46
can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed
at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no
sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified
lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with
the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed
engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. .
In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of
T cell activation status as related to the desired clinical effect must be addressed. These results
might open the way for a gene therapy targeting mainly or exclusively activated T cells and
could be exploited for immunostimulatory as well as suppressive approaches.
Wastewater treatment plants (WWTPs) do not eliminate micropollutants completely and are thus important point sources for these substances. In particular, concerns about en-docrine disrupting compounds in WWTP effluents give rise to the implementation of advanced treatment steps for the elimination of trace organic contaminants. The present study investigated ozonation (O3) and activated carbon treatment (AC) at two WWTPs. For an ecotoxicological assessment at WWTP Regensdorf, conventionally treated wastewater, wastewater after ozonation, and ozonated wastewater after sand filtration were evaluated in parallel via the fish early life stage toxicity test (FELST) using rainbow trout (Oncorhynchus mykiss). Additionally, a comparative toxicity evalu-ation of ozonated and activated carbon treated effluents was performed at the pilot scale treatment plant in Neuss (WWTP Neuss). For this purpose, four invertebrate tests and one higher plant toxicity test were selected to assess potential biological effects on or-ganisms [Lemna minor growth inhibition test, chironomid toxicity test with Chironomus riparius, Lumbriculus variegatus toxicity test, comet assay with haemolymph of the zebra mussel (Dreissena polymorpha), reproduction test with Potamopyrgus antipo-darum]. All in vivo assays were performed on site at the treatment plants in flow-through test systems. Furthermore, the present study investigated the effects of ozona-tion and activated carbon treatment on endocrine activities [estrogenicity, anti-estrogenicity, androgenicity, anti-androgenicity, aryl-hydrocarbon receptor (AhR) agonistic activity] with yeast based bioassays using solid phase extracted water samples. To evaluate the removal of in vitro non-specific toxicity, a cytotoxicity assay using a rat cell line was applied. The FELST at WWTP Regensdorf revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW, and to the ozonated water after sand filtration. Hence sand filtration obvi-ously prevents from adverse ecotoxicological effects of ozonation. An additional test – starting with yolk-sac larvae – resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treat-ed wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. At WWTP Neuss, the reproduction test with the mudsnail P. antipodarum exhibited a decreased reproductive output after advanced treatment compared to conventional treatment. This indicates an effective estrogenicity removal by ozonation and activated carbon treatment and is confirmed by results of the yeast estrogen screen with a reduc-tion of in vitro estrogenic activity by > 75%. The L. variegatus test revealed a signifi-cantly enhanced toxicity after ozonation compared to conventional treatment, whereas this effect was reduced following subsequent sand filtration. When ozonation was applied, a significantly increased genotoxicity was observed, detected with the comet assay using haemolymph of the zebra mussel. Again, this effect was removed by subsequent sand filtration to the level of conventional treatment. Activated carbon treatment even resulted in a significant reduction of genotoxicity. At both treatment plants, adverse effects after ozonation may have been a result of the formation of toxic oxidation by-products. However, sand filtration reduced toxication effects, indicating that these oxidation by-products are readily degradable or adsorbable. The results point out that, in any case, ozonation should not be applied without subsequent biologically active post treatment appropriate for oxidation by-products removal (e.g. sand filtration). However, only activated carbon achieved a toxicity reduction compared to the conventional treated wastewater. Thus, it cannot be excluded that po-tential beneficial effects due to ozonation might be masked by residual toxic oxidation by-products passing the sand filter or ozonation is not as effective in toxicity removal as PAC treatment. The yeast based assays with solid phase extracted samples revealed an effective endo-crine activity removal during ozonation and activated carbon filtration (estrogenicity: 77 – 99%, anti-androgenicity: 63 – 96%, AhR agonistic activity: 79 – 82%). The cyto-toxicity assay exhibited a 32% removal of non-specific toxicity after ozonation com-pared to conventional treatment. Ozonation in combination with sand filtration reduced cytotoxic effects by 49%, indicating that sand filtration contributes to the removal of toxicants. Activated carbon treatment was the most effective technology for cytotoxici-ty removal (61%). Sample evaporation reduced cytotoxic effects by 52% (after activated carbon treatment) to 73% (after ozonation), demonstrating that volatile substances contribute considerably to toxic effects, particularly after ozone treatment. These results confirm an effective removal or transformation of toxicants with receptor mediated mode of action and non-specific toxicants during both investigated treatment steps. However, due to the limited extractability, polar ozonation by-products were neglected for toxicity analysis, and hence non-specific toxicity after O3 is underestimated. In the long run, only on-site comparisons at WW receiving water bodies (e.g. communi-ty analysis of fish, macroinvertebrates, plants, microorganisms) – before and after up-grading WWTPs – allow drawing environmentally relevant conclusions regarding bene-fits and risks of advanced WW treatment methods. Conclusively, the benefits and possible negative impacts have to be carefully evaluated to prove that not more environmental impact will be induced than removed by advanced treatment technologies as each additional treatment requires considerable amounts of energy, resources, and infrastructure facilities. Accordingly, comprehensive sustainable approaches for pollution prevention and wastewater treatment (e.g. source control and source separation) are preferable compared to end-of-pipe treatment systems.
EGFL7 regulates adult neural stem cell maintenance and differentiation by inhibition of Notch1
(2010)
In neurobiology the preexisting dogma on the unchangeability of the adult mammalian brain and its inability to give rise to new neurons has been challenged since the early nineties. Generally, it is now accepted that neurogenesis persists in adults. Progress in developmental and stem cell biology in recent years led to an increasing interest in regeneration-based treatment strategies for damaged tissue of the central nervous system. Thus, the enhancement of the endogenous potential of the brain to repair itself is potentially a feasible therapeutic strategy to treat various types of brain damage. Therefore, it is of great interest to understand the molecular mechanism that regulate adult neurogenesis. One of the prominent pathways suggested to be involved here is the Notch signaling cascade. Previously, it has been shown that various components of Notch signaling are expressed in the stem cell niche of the adult subventricular zone (SVZ) in vivo. Interestingly, a recent study demonstrated that the self-renewal potential of adult neural stem cells (NSCs) isolated from the SVZ depend on Notch signaling in vitro.
Recently, we identified a novel non-canonical Notch ligand termed epidermal growth factor-like domain 7 (EGFL7), which was originally described as a protein secreted by endothelial cells and functionally implicated in cellular responses of the vascular system such as cell migration and blood vessel formation. We were able to show that secreted EGFL7 binds to a region in Notch that is involved in ligand-mediated receptor activation, thus acting as an antagonist of Notch signaling.
The present study identifies neurons of the human and murine brain as a novel source of EGFL7, which suggests functions of EGFL7 in the neural system. Expression analyses by quantitative RT-PCR (qRT-PCR) revealed EGFL7 is down regulated in the adult SVZ, which suggests that endogenous EGFL7 may act as a Notch modulator of NSCs. We assessed the expression of Notch pathway components in adult NSCs isolated from the SVZ of adult mice and demonstrated that inhibition of Notch activity by the γ-secretase inhibitor DAPT reduced the self-renewal potential of NSCs. Accordingly, adenoviral-mediated expression of EGFL7 in vitro decreased Notch-specific signaling and reduced proliferation and self-renewal of NSCs. Conversely, activation of Notch by a constitutive active form of Notch (NICD) rescued the EGFL7-mediated reduction of NSC self-renewal verifying that this effect was directly linked to Notch signaling. Congruent to the reduced proliferation rate measured in vitro, induced expression of EGFL7 in vivo significantly reduced the number of Ki-67 positive cells within the SVZ upon cerebroventricular injection of EGFL7 adenovirus.
Expression analyses in the developing brain showed single EGFL7-positive cells within the marginal zone of the neocortex as measured by in situ hybridization. These cells might be Cajal-Retzius cells, specialized neurons, which specifically express Reelin, which is a protein of the extracellular matrix known to control neuronal migration and differentiation. Interstingly, we could show that Reelin and EGFL7 are expressed in a subtype of neurons of the adult mouse cortex. This implied an interaction of both proteins and was verified by co-immunoprecipitation assays, suggesting an additional role for EGFL7 in neuronal maintenance. QRT-PCR based expression analyses in vitro comparing differentiated and non-differentiated NSCs displayed an increase in EGFL7 expression during the differentiation process, which was paralled by reduced Notch signaling. NSCs differentiated on coverslips coated with EGFL7 differentiated into all three cell types - neurons, oligodendrocytes and astrocytes. EGFL7 favored the formation of neurons as compared to control comparable to the effect of the Notch-inhibitor DAPT. Furthermore, additional oligodendrocytes were formed. These cells displayed a mature morphology with distinct sprouts and branches in contrast to the small and round oligodendrocytes that formed on control coverslips, which resembled us of precursor cells. Neurons and oligodendrocytes were formed at the expense of astrocytes. Congruently to the effect observed in vitro, adenoviral-based expression of EGFL7 in the SVZ yielded a slight induction of neuronal differentiation in vivo. Taken together, these results show for the first time a previously unrecognized role for EGFL7 in the brain by modulation of the Notch pathway in adult NSCs, which changes the proliferation and differentiation potential of adult NSCs in vitro and in vivo.
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.
ABCB9 is a peptide transporter belonging to the ATP-binding cassette (ABC) transporter subfamily B. Due to its high sequence identity to the transporter associated with antigen processing (TAP) the protein was named TAP-like (TAPL). The primary aim of this PhD thesis was the functional characterization of the TAPL transport complex. Despite the lack of TAPL function in the classical MHC class I pathway an involvement of TAPL in antigen presentation was still suggested. Apart from the crucial role of TAP for peptide delivery into the ER, TAP-independent translocation pathways in professional antigen presenting cells (pAPC) have been proposed, but not identified so far. Remarkably, TAPL mRNA and protein expression is strongly induced during differentiation of monocytes to immature and mature dendritic cells. This result was confirmed in the promonocytic cell line THP-1, which was used as a model system for monocyte to macrophage differentiation. By using quantitative immunofluorescence microscopy and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome associated membrane protein 2 (LAMP-2) thus excluding the ER-localization formerly reported. Furthermore, by in vitro assays, a TAPL-specific and ATPdependent translocation of peptides into isolated lysosomes was demonstrated. Hence, TAPL is a candidate mediating peptide transport in alternative antigen presentation pathways in pAPCs. The presence of an extra N-terminal transmembrane domain (TMD0) lacking sequence homology to any known protein distinguishes TAPL from most other ABC transporters of its subfamily. By dissecting the TAPL translocation complex into its four putative transmembrane helices containing TMD0 and the core complex, distinct functions to the core complex and TMD0 were assigned. The core-TAPL complex composed of six predicted transmembrane helices and the nucleotide-binding domain (NBD) was expressed transiently in HeLa or stably in Raji cells. Crude membranes containing core-TAPL showed the same peptide transport activity as wt-TAPL demonstrating that the six core helices and the NBD are sufficient for peptide transport. This result also shows that the core transport complex is correctly targeted to and assembled in the membrane. Strikingly, in contrast to the wt transporter, the core complex localizes only partially to lysosomes and is mistargeted to the plasma membrane as observed by immunofluorescence microscopy and confirmed biochemically by cell surface biotinylation. Thus, a crucial role for TMD0 in proper subcellular targeting can be postulated. The vast majority of biological processes are mediated by protein complexes, hence characterization of such protein-protein-interactions is essential for understanding protein function on the cellular level. To identify interaction partners of TAPL, the transporter was isolated by tandem affinity purification. By tandem mass spectrometry the membrane proteins LAMP-1 and LAMP-2 were deciphered as specific proteins interacting with wt-TAPL. Notably, core-TAPL lacks these interactions indicating a role for TMD0 in recruiting other proteins. These results were verified for endogenous TAPL by co-immunoprecipitation. Using cells deficient in LAMP-1 and/or in LAMP-2 an escort function for the LAMP proteins was excluded. Very importantly, the physiological function of the LAMP-1and LAMP-2 interaction with TAPL is an increase in stability, since in their absence half-life of TAPL is drastically reduced.
The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080 cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.
Project I: The progression of rod and cone degeneration in retinally degenerate (rd) mice ultimately results in a complete loss of photoreceptors and blindness. The inner retinal neurons survive and several recent studies using genetically targeted, light activated channels have made these neurons intrinsically light sensitive. We crossbred a transgenic mouse line expressing channelrhodopsin2 (ChR2) under the control of the Thy1 promoter with the Pde6b(rd1) mouse, a model for retinal degeneration (rd1/rd1). Approximately 30-40% of the ganglion cells of the offspring expressed ChR2. Extracellular recordings from ChR2-expressing ganglion cells in degenerated retinas revealed their intrinsic light sensitivity which was approximately 7 log U less sensitive than the scotopic threshold and approximately 2 log U less sensitive than photopic responses of normal mice. All ChR2-expressing ganglion cells were excited at light ON. The visual performance of rd1/rd1 mice and ChR2 rd1/rd1 mice was compared. Behavioral tests showed that both mouse strains had a pupil light reflex and they were able to discriminate light fields from dark fields in the visual water task. Cortical activity maps were recorded with optical imaging. The ChR2rd1/rd1 mice did not show a better visual performance than rd1/rd1 mice. In both strains the residual vision was correlated with the density of cones surviving in the peripheral retina. The expression of ChR2 under the control of the Thy1 promoter in retinal ganglion cells does not rescue vision. Project II: Lentiviral vectors are becoming the vector of choice for transgene delivery into cells due to their ability to infect non- dividing cells and stably integrate the gene into the genome of the host. Two different viral vector systems, namely HIV-1 and SIV and three different viral vectors PLECYT, PHRCMVChR2 of HIV-1 family and PBjChR2 of SIV were used in this study. The efficiency of the vectors was analyzed by applying them onto the retinal explants in culture and checking the transgene expression. The transgene in the PLECYT lentiviral vector was driven by the EF1A promoter. Upon administration of 5.2 X 106 infectious units of PLECYT viral vector suspension onto the retinal explant resulted in the transduction of retinal ganglion cells. Very few other retinal neurons were found transduced. In the case of PHRCMVChR2, approximately 5 X 105 TU/ml of the vector was used and resulted in the transduction of different neuronal subtypes. Many amacrine cells, ganglion cells and Müller cells were found expressing the transgene. For PBjChR2, 5.6 X104 TU/ml was used which resulted in Müller cell- specific transduction. Very few or no other retinal neurons were found transduced. This study demonstrates the transduction efficiency of different viral vectors on the retinal neurons in vitro. An interesting observation on these viral vectors is their altered tropism. The glycoprotein of the virus is critical for determining their tropism and in this study, all the viral vectors generated were pseudotyped with VSVG, which confers a broad non-specific spectrum of infection. However, analyzing the transgene expression, the viral vectors differ from one another and show remarkable difference in their transduction pattern. To list a few factors that might possibly responsible for the drastic transduction difference exerted by the viral vectors include; 1. Promoters used to drive the transgene expression. 2. HIV or SIV component of the vector in combination with the promoter 3. Titre of the vector used and 4. Other factors like pH and serum used in the study. Therefore optimizing the viral vectors and generating high titers would increase the efficiency and cell-type specific expression of the transgene.
In this thesis, the structure of the C-terminal domain of presenilin-1, the catalytic component of the y-secretase complex, is investigated by NMR spectroscopy. The ysecretase complex has a definitive role in the pathogenic development of Alzheimer's disease, in that it mediates the cleavage of aprecursor to create the amyloid ß peptide. Aggregates of amyloid ß which form amyloid plaques are the most overt clinieal feature observed in the post-mortem brains of Alzheimer's patient. In addition, many of the mutations found in the aggressive early onset familial Alzheimer's disease have been linked to presenilin-1, highlighting its importance in disease progression and deeming it an important target for investigation. One of the greatest challenges for the structural investigation of the y-secretase components is their low expression yields in cell-based systems. We therefore applied continuous-exchange cell-free expression to obtain sufficient amounts of protein for our structural studies. An added benefit of the cell-free expression system is the freedom to incorporate any desired combination of stable-isotope labels directly into sampies. We were therefore able to develop a labeling scheme which targets the amino acid composition of transmembrane a-helices, allowing us to simplify an assignment procedure whieh tends to be cumbersome and diffieult for most a-helical transmembrane proteins. The y-secretase complex is a member of the intramembrane cleaving proteases which, as their name implies, cleave their transmembrane substrates within the bilayer. Single particle analysis of the y-secretase (1) as weil as crystal structures of rhomboid (2) and S2P (3) have revealed the presence of hydrophilie po res within the membrane where catalysis occurs. In light of evidence that certain elements of CTF reside in close proximity or even contribute to the formation of the hydrophilic pore, we chose to study the structure of CTF in mieelles, whieh may be better suited to accommodate CTF in isolation as compared with solid membranes in the absence of the other y-secretase components. The structure of CTF was solved to 1.7 A (backbone r.m.s.d) and revealed the presence of unusual features, including a partially membrane-spanning helix which situates the catalytic asparte at its N-terminus in what would be the center of the membrane where catalysis is proposed to occur, as weil as a severely kinked helix which is partially embedded beneath the surface of the membrane (P6). Interestingly, similar features have been observed in the crystal structure of the GlpG rhomboid. In addition, a soluble helix was found in the long N-terminal loop of CTF which until now has been described as unstructured. The first part of the thesis is designed to provide an introduction to Alzheimer's disease, the role of y-secretase and its presenilin-l catalytic component in disease progression, as weil as cell-free expression and liquid-state NMR techniques involved in the structural investigation of membrane proteins. In chapter 2, the reader is familiarized with the history, the clinical manifestation, and biochemical features of Alzheimer's disease. The chapter goes further to describe the role of the y-secretase complex and its individual components in disease progression and substrate processing. Chapter 3 focuses more specifically on presenilin-l in the context of the newly emerging class of intramembrane proteases. In chapter 4, attention is shifted to the cell-free expression system with special focus on the expression of membrane proteins, and chapter 5 explores the various liquid-state NMR techniques that were required for the characterization of CTF. The second part of the thesis is cumulative and contains original research, method, and review articles that were produced during the course of study. Chapter 6 explores the various techniques and innovations used to study membrane proteins using continuous exchange cell-free expression coupled with NMR spectroscopy. In chapter 7, a new technique, transmembrane segment targeted labeling, is described as a tool that facilitates the backbone assignment of transmembrane proteins which display severe overlap in NMR spectra. Chapter 8 presents the novel NMR structure of the C-terminal fragment of presenilin-l solved in SOS micelles.
Paleoecology is the study of organismal interactions with the environment in the geological past. Organisms are influenced in their distribution and abundance by abiotic factors such as temperature and precipitation. A change in these factors, for example by major climatic shifts, would then affect the communities of organisms. Studying this hypothesized causal link between climatic and faunal change is especially interesting for the Plio-Pleistocene of East Africa due to the fact that our own ancestors also inhabited these regions. Both the Turkana basin in Kenya and the Lake Albert region in Uganda offer unique opportunities to investigate these paleoecological issues. Their late Miocene through Pleistocene deposits provide a very good record of climatic, vegetation and faunal change in East Africa (Pickford et al. 1993, Leakey et al. 1995, 1998, McDougall & Feibel 2003, Wynn 2004). This study focuses on the mammal family Bovidae as they are good indicator of vegetation and environment (e.g. Vrba 1980, 1995, Shipman & Harris 1988, Bobe & Eck 2001, Bobe & Behrensmeyer 2004, Bobe et al. 2007). Bovidae are quite species-rich and inhabit a wide range of habitats from tropical rain forests to deserts which predicates their array of morphological adaptations (ecovariables) to these environments. Diet is the ecovariable that is most to climate and thus habitat change. Therefore, the fossil Bovidae are especially suitable for reconstructing past environments. The objective of this thesis is to test the hypothesis that, from the late Miocene through the Holocene, Africa has experienced an overall increase in aridity and concomitant pulses of habitat change. The hypothesis predicts that increasing aridity causes a likewise growth in the abundance of taxa adapted to open arid environments. In particular, an increase in bovid grazers should be observed in combination with a decrease of bovid browsers. To test this hypothesis, I examine the fossil bovid communities from each stratigraphic member of Lake Turkana (Lothagam, Kanapoi, West Turkana and Koobi Fora) and Lake Albert (Nkondo-Kaiso region) and through a taxonomic and a functional perspective reconstruct the paleoenvironments and -climates from approximately 8 to 0.6 Ma. This study is the first to use taxonomic and ecomorphological data together to reconstruct the paleoenvironments of the Turkana basin and the Nkondo-Kaiso region of Lake Albert. In a first analysis, mesowear, as introduced by Fortelius & Solounias (2000), is used to gather information about the diet of bovids. As a result of my preliminary investigations on upper vs. lower molars of recent species, the sample of fossil bovid specimens from the Turkana basin and Lake Albert were found to be unsuitable to reveal a meaningful diet reconstruction. Therefore, the bovids are assigned to diet categories based on literature. For each member of the time period from 8.0 to 0.6 Ma, I provide a detailed characterization of the bovid fauna in terms of α- and β- diversity both on tribe and diet level based on presence-absence as well as for the Turkana basin on abundance data. Statistical comparisons between the fossil bovid communities and those in modern protected areas with known vegetation and climatic conditions have yielded modern analogues for each stratigraphic member. Following that I provide paleoclimatic conditions such as assumed mean annual temperature for each member. Based on abundance of diet categories in the bovid communities, the paleoclimate of the Turkana basin was in general cooler and considerably more humid during the late Miocene to the Pleistocene than today. The mean annual temperature at Lothagam is assumed as 22.2 °C, the annual precipitation as 685 mm for 8.0 – 6.54 Ma and 4.9 – 3.4 Ma. The intervening time period is characterized by a slightly lower mean annual temperature and precipitation (20.3 °C, 583 mm). From 4.17 to 4.07 Ma Kanapoi faced 21.3 °C and 592 mm rainfall. In the eastern part of the basin the climate was warmer and more humid (3.4 – 2.68 Ma: 26.2, 961 mm; 2.68 – 1.3 Ma: 27.1 °C, 935 mm) from 3.4 to 1.3 Ma than in the preceeding eras. In the western part, the climate became warmer and more humid ~500,000 years later and was more variable than that in the eastern basin. From 2.94 to 2.52 Ma the mean annual temperature was 26.2 °C and the annual precipitation 961 mm. Between 2.34 and 1.6 Ma the climate again cooled and became drier as before 2.94 Ma. A second shift to higher temperature and precipitation occurred after 1.6 Ma (27.1 °C, 935 mm) lasted until 1.34 Ma. The results of the bovid community analyses do not support the hypothesis of increasing aridity in Eastern Africa during the late Mio- to Pleistocene. Instead, the results show that the bovid communities differed much over time and on a relatively small spatial scale. Regional paleovegetation and paleoclimate exhibit fluctuations through the studied time period at western Turkana and differences between the western and eastern part of the Turkana basin. This is indicative of a patchy habitat distribution both on temporal and spatial levels. Increased climate variability predicts an increase in landscape complexity as proposed by the ‘variability selection hypothesis’ (Potts 1998a+b). Therefore, this thesis research supports the hypothesis of increased landscape complexity on the spatial level. This study has important implications for future research. First, an analysis based on ecovariable characteristics such as diet may be preferred to a taxonomic analysis. Second, abundance data should be used for an ecovariable analysis because the results then provide more precise information on the paleovegetation and –climate than just the presence of these adaptations in the faunal community. Lastly, as this study is based on one mammal family, further studies on other mammal groups should be conducted to increase the database of exploited resource by the entire faunal community. Most significantly this study provides a basis for new interpretations of faunal community distributions. It also raises the question whether small scale spatial community variability is also to be expected at other fossil sites. If so then this methodology has important implications for reconstructions of paleovegetation and paleoclimate.
Employing NMR spectroscopy, it is not only possible to calculate the three dimensional structures of single proteins, but also to study dynamics and conformational changes of protein-complexes. In fact that is an important aspect, since the protein function depends on dynamics and interactions with other molecules. Therefore the study of protein-protein interactions is of highest importance for a better understanding of biological processes. Based on NMR methods, in this thesis we were able to determine protein-protein interactions within the enterobacterial Rcs signalling complex which is regulated via a phosphorelay. Originally identified as regulator of capsule synthesis, the Rcs phosphorelay is now considered to be implicated in stress response caused by disturbances in the peptidoglycan layer. Beyond that the Rcs system is involved in multiplex transcriptional networks including cell division, motility, biofilm formation and virulence. Because of such global nature and its extraordinary structural organisation involving membrane integrated sensor proteins (RcsC, RcsD), coactivators (RcsF, RcsA) and a transcription factor (RcsB), the Rcs system is one of the most remarkable phosphorelays in the family of enterobacteriacaea. During the complex phosphotransfer the histidine phosphotransferase (HPt) domain of the intermediary RcsD protein mediates the phosphotransfer between RcsC and RcsB, and probably modulates the phosphorylation state of the response regulator RcsB. Therefore the present work has been focused on the interface between RcsD and RcsB in more detail. In the first part of the thesis a new domain within the RcsD protein has been identified and structurally analysed by liquid NMR spectroscopy. RcsD is an inner membrane bound hybrid sensor like-kinase composed of a periplasmic sensor domain and a cytoplasmic portion. The cytoplasmic part contains the histidine like-kinase (HK) domain and the histidine phosphotransferase (HPt) domain. By analysis of the secondary structure in more detail, it was shown here that the two domains are intermitted by an additional 13.3 kDa domain. Corresponding to the position of the ABL (α−β−loop) domain of RcsC, located C-terminal to the RcsC-HK domain, the new identified domain was named RcsD-ABL. The central structural element of RcsD-ABL is a β-sheet composed of six strands with a β1−β2−β3−β4−β6−β5 topology and surrounded by two α-helices α1 and α2. In the second part of the thesis, RcsD-ABL is identified as a binding domain for the response regulator RcsB by NMR titration experiments. Such a binding domain for a response regulator has so far only been described for the histidine kinase CheA. In reportergene assays with β-galactosidase and ONPG as substrate it was shown that overexpression of RcsD-ABL in high amounts inhibited binding of RcsB to its target promoter. The β-galactosidase activity was reduced by 80 % with respect to cells carrying no plasmid encoding RcsD-ABL. The mapping of the binding interface was successfully achieved by chemical shift perturbations, a fast mapping protocol and selective labelling. It was shown that the interaction between RcsD-ABL and RcsB takes place via a binding interface comprising mainly the two α-helices of RcsD-ABL and the α-helices α7, α8 and α10 in the effector domain of RcsB. In the third part of the thesis, the interaction of RcsB with RcsD-ABL was related to that with RcsD-HPt. Using NMR titration experiments and ITC measurements, a comparison of the binding constants (Kd) of RcsB interacting either with the isolated RcsD-ABL (2 PM) or the isolated RcsDHPt domain (40 PM) revealed a higher affinity of RcsD-ABL to RcsB. A conjugate of RcsD-ABL-HPt interacting with RcsB decreased the Kd in the one-site fitting mode to 10 PM. However, the two-site fitting mode applied for RcsD-ABL-HPt/RcsB interaction resulted in a Kd (RcsD-ABL) of 2 PM and a Kd (RcsD-HPt) of 8 PM, indicating that RcsD-ABL enhances the binding of RcsD-HPt to RcsB. In the last part of the thesis, it was partly possible together with the data obtained from NMR titration experiments, PRE measurements and a HADDOCK protocol to develop a geometrical model for the interaction of RcsD with RcsB. In this model the receiver domain of RcsB interacts with the RcsD-HPt domain and the RcsB effector domain interacts with the RcsD-ABL domain. These results lead to surprising insights on the regulation of phosphorelays, since normally the effector domain binds to DNA. Here the effector domain is recognized by the newly identified RcsD-ABL domain. Prospectively, further investigations of phosphorylation affects and mutational studies will be of great interest.
The power to dissociate : molecular function of the twin-ATPase ABCE1 in archaeal ribosome recycling
(2010)
Fuer die schlechte Prognose von Glioblastompatienten mit einer ueberlebenszeit von 9-15 Monaten (Norden and Wen, 2006) ist vor allem die hohe Invasivitaet dieser Tumore verantwortlich. Nach operativer Entfernung des Haupttumors entstehen aus den verbleibenden invadierten Zellen sekundaere Tumore, die sich mitunter ueber weite Bereiche des Hirns verteilen. Des Weitern sind die hochinvasiven Tumorzellen oft resistent gegen Chemo- und Strahlentherapie (Drappatz et al., 2009; Lefranc et al., 2005). In Maustumormodellen und Pateinten konnte zudem gezeigt werden, dass die neuartige antiangiogenetische Therapie zwar das Tumorwachstum verringert, jedoch die Invasivitaet stark erhoeht. (Norden et al., 2008; Ebos et al., 2009; Paez-Ribes et al., 2009). Ueber die Mechanismen die diese hohen Invasivitaet induzieren, ist bislang nur sehr wenig bekannt. Die durch Reduktion von Blutgefaessen steigende Hypoxie des Tumors foerdert die Expression von Matrix-Metalloproteinasen (MMPs). Dies fuehrt zum Abbau der extrazelluaeren Matrix des umgebenden gesunden Gewebes und beguenstigt dadurch die Tumorzellinvasion (Indelicato et al., 2010; Miyazaki et al., 2008; Shyu et al., 2007). Die Umformung des Aktinzytoskeletts und damit die Mobilitaet von Zellen wird vorwiegend durch ein akkurates Zusammenspeil der Rho GTPasen Rac, Rho und Cdc42, kontrolliert (Ridley et al., 2003). Fuer die Organisation von Axonen im Nervensystem und fuer die Blut- und Lymphgefaessbildung wurde gezeigt, dass die Interaktion der Eph-Rezeptortyrosinkinasen und Ihrer Ephrin-Liganden Signalwege induziert, die in die Regulation dieses Zusammenspiels involviert sind (Egea and Klein, 2007; Makinen et al., 2005; Palmer et al., 2002; Sawamiphak et al., 2010). Des Weiteren zeigt die Analyse der Genloci von Eph-Rezeptoren und Ephrinen in verschieden Hirntumoren eine gehaeufte Deletionen des Ephrin-B2-Gens. Die Quantifizierung von Ephrin-B2 mRNA in diesen Tumoren hat ausserdem ergeben, dass mit zunehmender Malignitaet die Expression von Ephrin-B2 sinkt. Aus diesen Gruenden wurden die Untersuchungen in dieser Arbeit auf die Rolle von Ephrin-B2 anhaengigen Signalwegen in der Glioblastomzellinvasion konzentriert. In einem modifiziertem Boyden-Chamber-Assay konnte gezeigt werden, dass das Ephrin-B2 induzierte EphB4 forward signaling und EphB4 induzierte Ephrin-B2 reverse signaling die Invasivitaet der human Glioblastomzelllinien LN-229, G55 und SNB-19 reduziert. In einem Maustumormodel konnte weiterhin gezeigt werden, dass Ephrin-B2 Knock-Out (KO) Astrozytomzellen, im Vergleich zu Wild-Typ (WT) Zellen, Tumore mit einem groesseren Volumen und einer erhoehten Invasivitaet bilden. Da die Expressionslevel fuer die Ephrin-B2 bindenden Rezeptoren EphA4, EphB1 EphB3 und EphB6 auch im adulten Hirn hoch sind (Hafner et al., 2004), weisen diese in vitro und in vivo Ergebnisse auf eine Tumorsupressorfunktion von Ephrin-B2 hin, die durch repulsive Effekte des Ephrin-B2 reverse signaling vermittelte werden koennten. Dies geht mit Erkenntnissen ueber kolorektale Tumore einher (Batlle et al., 2005). Die in einem Sphaeroid-Invasionsassay mit einer EphB-Rezeptoren freien Umgebung beobachtete verminderte Invasion von Ephrin-B2 WT deutet auf eine zusaetzliche invasionsblockierende Rolle der Ephrin-B2-Eph-Rezeptor Interaktion zwischen benachbarten Tumorzellen hin, wie sie auch in Brusttumoren gefunden wurde (Noren et al., 2006). Es scheint als sei Tumorprogression und Invasion erst moeglich, nachdem die Expression von Ephrin-B2 vermindert wurde. Es konnte weiterhin gezeigt werden, dass in hypoxischen Glioblastomzellen die Ephrin-B2 Expression durch die direkte Bindung des den Transkriptionsfaktors ZEB2 an den Ephrin-B2 Promoter reprimiert wird. In einem Weiteren Maustumormodel konnte gezeigt werden, dass die Blockierung der ZEB2 Expression mittels shRNA und die damit einhergehenden Inhibition der hypoxie induzierten Ephrin-B2 Repression das Wachstum und die Invasivitaet von Glioblastomen verringert. Zusaetzlich wurde gezeigt, dass der Verlust von ZEB2 ausreicht, die durch antiangiogenetische Therapie induzierte stark erhoehte Invasivitaet zu vermeiden. Die in dieser Arbeit gewonnen Erkenntnisse fuehren zu folgendem Modelmechanismus. In kleinen normoxischen Tumoren koennen repulsive Effekte des Ephrin-B2 reverse signalings und EphB forward signalings zwischen Tumorzellen und Zellen des umgebenden Gewebes die Ausbreitung und Invasion des Tumors unterdruecken. Zusaetzlich koennte das Ephrin-B2 induzierte EphB forward signaling zwischen benachbarten Tumorzellen die Mobilitaet der Tumorzellen wie in Brusttumoren inhibieren. Beim Erreichen einer bestimmten Tumorgroesse tritt Hypoxie auf, wodurch HIF-1alpha stabilisiert wird. Dies fuehrt dann zur ZEB2 Expression und leitet die Repression von Ephrin-B2 ein, was wiederum zur erhoehten Tumorzellemobilitaet und im Zusammenspiel mit MMPs zu Invasion fuehren kann. Gleichzeitig werden durch den HIF-induzierten VEGF-Gradienten neue Blutgefaesse rekrutiert. Damit wird der hypoxie-induzierten Invasivitaet entgegengewirkt. Wird mittels antiangiogenetischer Behandlung versucht Tumorprogression entgegenzuwirken, resultiert daraus eine erneut gesteigerte Hypoxie, die dann durch die ZEB2 vermittelte Repression von Ephrin-B2 wieder eine erhoehte Invasivitaet induzieren kann. Das Blockieren der ZEB2 Expression kann dieser durch antiangiogenetischen Behandlung induzierten Invasivitaet entgegenwirken.
Tumor hypoxia and nutrient starvation are common phenomena in cancerous tissue. Cells that resist this hostile environment are selected for a more aggressive phenotype, usually accompanied by therapy resistance. The hypoxia inducible factors HIF-1a and HIF-2a play a key role in the adaptive homeostatic responses to these challenging conditions inducing a number of target genes that are involved in the regulation of a variety of cellular processes such as angiogenesis, proliferation, metabolism, self-renewal and cell death/cycle arrest. Thus, the HIF pathway encompasses opposing adaptive responses on tumor growthgrowth promoting abilities on the one hand and growth inhibiting on the other. A recent study in our lab uncovered that this switch between cell death and cell survival critically depends on HIF-2a protein levels. Since PHDs (HIF prolyl hydroxylases) are the main regulators of HIF protein abundance and hypoxia drives the malignant phenotype of tumors, we wanted to characterize HIF regulatory functions of PHDs under hypoxic conditions. Our intention was to reveal the importance of PHD contribution to the opposing functions of HIFs under hypoxia. Characterization of PHD1-4 mRNA and protein expression levels under normoxic and hypoxic conditions in glioblastoma cell lines led to the identification of PHD2 and PHD3 as hypoxia inducible PHD isoforms and highlighted their predominant function under hypoxia. Mechanistically, we demonstrated that HIF mediates the hypoxic induction of PHD2 and 3 within a negative feedback loop, promoting its own degradation during prolonged hypoxia. The functional impact of PHD2 and 3 abundance on cell viability under hypoxic conditions was analyzed by disrupting PHD2 and PHD3 function either through a siRNA mediated approach or by application of the PHD inhibitor DMOG. These experiments uncovered that PHD2 and 3 are protective under hypoxic conditions and that PHD inhibition expedites cell death. Combined HIF and PHD suppression under hypoxic conditions abrogated this increased susceptibility to cell death, clearly showing that PHD2 and 3 act in a negative feedback regulatory loop to limit the HIF response under prolonged hypoxia. With respect to possible future therapeutical applications we co-treated cells with a PHD inhibitor and pro-apoptotic agents staurosporine or TRAIL. Co-challenging tumor cells even potentiated the cell death response, indicating a more widespread protective function of PHD. Taken together PHD2 and 3 protect tumor cells from cell death induction, functioning in a negative feedback regulatory loop to constrain the HIF dependent cell death responses under hypoxia. Interestingly, however, when assessing the role of PHD2 and PHD3 in in vivo tumor growth using an intracranial tumor model, we identified an exclusive tumor suppressor function for PHD3. Loss of PHD3 function enhanced tumor growth whereas increased PHD3 expression diminished the tumor burden. The accelerated tumor growth following PHD3 loss could be attributed to a decrease in the induction of apoptosis and an increase in proliferation. Tumor cells are frequently exposed to temporary and spatial depletion of nutrients. Interestingly, PHD3 loss conferred a growth advantage under growth factor deprivation. The growth regulatory function of PHD3 was isoform specific, HIF independent and importantly, did not require the hydroxylase function of PHD3. Previous reports have uncovered a regulatory function of the PHD system in NF-kB signaling. However, our results demonstrated that NF- kB signaling remained unaffected by alteration in the PHD3 status of the cell. Additionally, the PHD3 tumor suppressor function proved to be independent of two putative PHD3 downstream effectors, ATF4 and KIF1Bb. Mechanistically, PHD3 suppression reduced EGFR internalization, enhancing the amount of EGFR expressed on the cell surface. We further showed that the impaired EGFR internalization during PHD3 loss resulted in receptor hyperactivation under stimulated and growth factor deprived conditions. Importantly, PHD3 physcially associated with the EGFR complex as evidenced by co-immunoprecpitation. Consequently, this extended EGFR activation in PHD3 deficient cells resulted in enhanced downstream activation of EGFR signaling and increased proliferation. Consistent with the interpretation that PHD3 loss is beneficial for tumor growth, we found PHD3 promoter methylation in glioblastoma cell lines, hinting at a epigenetic mechanism to finetune PHD3 expression on top of the hypoxic driven gene regulation. Finally, we demonstrated that PHD3 tumor suppressor function is not restricted to glioblastomas since PHD3 suppression in lung adenocarcinoma accelerated subcutaneous tumor growth. With these findings, we expand the knowledge of PHD3 action from its oxygen sensing role to a regulatory function in growth factor signaling. This clearly discriminates PHD3 from the other isoforms and supports the exclusive tumor suppressor function in glioblastoma. Taken together our results identify a complex role of PHD signaling in cancer and delineate HIF dependent and HIF independent functions of the PHD system. We think that the HIF dependent protective effect of PHD2 and 3 and the HIF independent PHD3 tumor suppressor function are not mutually exclusive, but might be activated according to the heterogeneous intra-tumoral conditions. However, PHD3 hydroxylase activity is dispensable for its HIFindependent tumor suppressor function in glioma. This uncouples PHD3 function from co-factor and co-substrate requirements and allows it to act over a broader physiological range, since its influence on cellular processes is not constrained by the availability of rate limiting factors. It might explain, why the enzymatic independent functions of PHD3 predominate in vivo. Thus, therapeutic modulation of the PHD system to inhibit tumor growth has to be based on these contrasting functions of the PHD system. However, their differential dependence on the hydroxylase activity may facilitate a therapeutic strategy to specifically inhibit or promote the protective versus suppressive functions of the PHD system.
Iron uptake is an essential process in all Gram-negative bacteria including cyanobacteria and therefore different transport systems evolved during evolution. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as cofactor in e.g. photosynthesis and nitrogen fixation. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. However, in proteobacteria TonB-dependent outer membrane transporter represent a well-explored subject whereas for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates. The heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. For Anabaena sp. PCC 7120 22 genes were identified as putative TBDTs covering almost all known TBDT subclasses. This is a high number of TBDTs compared to other cyanobacteria. The expression of the 22 putative TBDTs individually depends on the presence of iron, copper or nitrogen. The atypical dependence of TBDT gene expression on different nutrition points to a yet unknown regulatory mechanism. In addition, the hypothesis of the absence of TonB in Anabaena sp. PCC 7120 was clarified by the identification of an according sequence, all5036. Inspection of the genome of Anabaena sp. PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397 was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell. Additional two genes of Anabaena sp. PCC 7120 involved in this process were identified. SchE encoded by all4025 is a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. The mutation of schE resulted in an enhanced sensitivity to high metal concentrations and in drastically reduction of secretion of hydroxamate-type siderophores. IacT coded by all4026 is a predicted outer membrane-localized TonB-dependent iron transporter. Inactivation of iacT resulted in reduced sensitivity to elevated iron and copper levels, whereas decoupling the expression from putative regulation by exchange of the promoter resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because decrease of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in Anabaena sp. PCC 7120 where expression of all4026 is under the trc-promoter. In consequence, the results unravel a link between iron and copper homeostasis.