At present we have no open positions, next openings in 2010.

Diploma/master thesis projects

Our research group studies the structure and function of VEGF family growth factors and their receptors. For details see the research section on our homepage, VEGF structure and VEGF signaling.

We investigate the structure and function of VEGF family growth factors, Vascular Endothelial Growth Factors, and their receptors, VEGFR-1 -3. VEGF family growth factors are vasculogenic and angiogenic polypeptides that stimulate endothelial cell growth, migration and differentiation in blood and lymphatic vessels during embryo development. VEGFs also regulate the repair of damaged vessels in disease and aberrant angiogenesis is the basis of many disease. We study signal output from VEGF receptors both in vitro and in live cells using biochemical and cell biological methods. In particular, we use live cell fluorescence videomicroscopy to investigate the interaction between the various ligands and their receptors and the subsequent activation of downstream signaling molecules.

The role of distinct receptor domains in endothelial cell signaling by VEGF receptors

Project A: In this project we focus on the cloning and production of variants of VEGF receptor 2 that will subsequently be used for crystallographic, electron microscopic and small angle solution scattering analysis (SAXS). Specific extracellular domains of the receptor will be produced in insect and mammalian cells and in the yeast Pichia pastoris. Expression of intracellular and secreted proteins will be optimized in the various expression systems and the proteins will be purified by affinity and size exclusion chromatography. The proteins are subsequently analyzed biophysically, biochemically and in biological assays. Finally, the material will be submitted to structural analysis using the techniques mentioned above at the Swiss Light Source at PSI.

Project B: VEGF receptors are dimerized upon ligand stimulation followed by receptor kinase activation. Active dimers signal to downstream targets such as MAP kinases, phospholipases and phosphatidyl-inositol 3-kinase and are routed via specific intracellular vesicles to degradation-promoting cellular compartments such as lysosomes or proteasomes. VEGF receptors continue to signal to downstream targets while being transported through endosomal vesicles. We have evidence suggesting that specific coreceptors activated by distinct VEGF family proteins determine vesicle targeting and thereby modulate receptor signaling. VEGF receptor traffic is studied by fluorescence microscopy and live cell videomicroscopy using GFP tagged marker proteins and fluorescent antibody labelling. Variants of green fluorescent protein that allow FRET (fluorescence resonance energy transfer) analysis are used to determine protein-protein interaction in live cells. Receptor signaling to downstream targets will be analyzed using fluorescent sensor molecules specifically recognizing phosphorylated proteins via SH2 or PTB domains.

Project in biotechnology; production and characterization of recombinant proteins

Our group uses pro- and eukaryotic protein expression systems for large scale production of complex proteins. For mammalian proteins we currently use yeast (Pichia pastoris), mammalian cells (HEK293 cells) and insect cell expression systems (transfected Sf9 or 21 cells or baculovirus infected Sf9 or 21 or Hy-5 cells). The project offers the possibility to become familiar with one or several of these expression systems. In each project the corresponding expression vectors will be constructed using state of the art PCR technology, followed by transfection of the host cells and selection of appropriate well expressing clones. Large scale protein production and purification using chromatographic methods and biochemical as well as biophysical charaterization of the produced proteins give the candidate insigth into the practice of recombinant protein production. For details of the expression systems used see our publication list and the link 'research projects'.

Ph. D. thesis project

Developing new highly specific DARPIN-based isoform-specific VEGF antagonists as anti-angiogenic tumor therapeutics

Introduction

The role of VEGF in angiogenesis: VEGF family polypeptide growth factors are among the most important biomolecules regulating blood and lymphatic vessel formation during embryogenesis and under pathological conditions such as in wound healing, arthritis, retinopathy or in tumor vascularization [1]. VEGFs interact with target cells through three receptor tyrosine kinases (RTKs) and bind additional surface molecules such as neuropilins and heparansulfate glycosaminoglycans acting as co-receptors. VEGFR-1 to -3 (Flt-1, Flk1/KDR, Flt-4) have been characterized in considerable detail so far [2]. VEGFs vary in their ability to bind to and activate these receptors. VEGF-A for instance has mitogenic activity mediated by VEGFR-2 and regulates vessel morphogenesis through VEGFR-1. The importance of VEGF-A and its receptors in vascular development has been best illustrated in knockout mice. Both VEGF-A [3,4] and VEGFR-2 [5,6] knockouts were lethal due to a deficiency in blood vessel formation. A VEGFR-1 knockout was also lethal because of apparent overgrowth of immature vessels suggesting that this receptor modulates vessel maturation [7]. A VEGFR-1 splice variant lacking the intracellular tyrosine kinase and the transmembrane domain (TMD), sFlt or sVEGFR-1, was shown to be deficient in signaling and is apparently expressed in many tissues during normal embryonic development and in a disease called preeclampsia [8]. This molecule might behave as a decoy for VEGF thereby attenuating VEGF signaling. The proposal that VEGFR-1 kinase activity is dispensable for vascular development in some circumstances is further supported by the finding that a kinase-inactive VEGFR-1 mutant rescues VEGFR-1 null mutant mice [9]. Full-length kinase active VEGFR-1 may also play a role in pathological angiogenesis upon binding of PlGF as suggested recently [10,11]. For the formation of functional vessels, VEGF activity must be complemented by additional factors, such as basic fibroblast growth factors (bFGF), platelet derived growth factors (PDGF) and angiopoietins. During the normal life cycle of an adult mammal, endothelial cells in blood and lymphatic vessels assume a resting state and express only low levels of VEGF receptors. Only few tissues, such as the endometrium or the corpus luteum undergo cycles of de novo vascularization which depend on VEGF receptor expression. Under pathological conditions, such as for instance after myocardial infarction, in tumor tissue, after endothelial injury, in the regenerating liver, or in retinopathy, expression of receptors is upregulated. Blocking VEGF signaling to downstream targets might therefore be a means to destroy aberrantly developing vessels without harming the mature vasculature (reviewed in [12]).

VEGF isoforms in angiogenic signaling: Our lab focuses on VEGF signaling and on the structures of VEGFs and VEGF receptors. A significant part of our efforts has been dedicated to establish efficient and economical protein expression systems using insect and mammalian cells, and, whenever possible, the yeast Pichia pastoris. The application of biofermenters allowed us to optimize the production cycle for recombinant VEGFs and to produce large amounts of properly folded and active protein, which is a precondition for our structural studies. We recently clarified the mechanism by which a new set of VEGF splice variants that carry sequences encoded by exon 8b instead of exon 8a at their carboxyterminus, interact with VEGFR-2 and neuropilin-1. These VEGF isoforms, originally discovered by Bates and collaborators [13-15], are now considered to behave as partial receptor agonists that only transiently activate receptor signaling and therefore lead to blunted activation of endothelial cells [15-20]. Exon 8b VEGFs show only weak angiogenic activity and are apparently required to maintain vessel homeostasis under normal conditions in healthy organisms. The 8a isoforms are highly angiogenic and are produced by cells in tissues undergoing angiogensis [21-23].

VEGF receptor activation: The role of the various extracellular Ig-like domains in dimerization and activation of VEGFR-2 was investigated by electron microscopy (EM) with purified recombinant proteins. We could show that the entire extracellular domain (ECD) encompassing Ig-like domains 1-7 assumes a random structure in the absence of ligand. Upon ligand binding receptor molecules became highly compacted assuming a well defined three dimensional structure [24]. Most interestingly, our data show that ligand binding promotes additional receptor interactions in the ECD in subdomains 4 and 7 that were not known before and that are essential for regulating receptor activity. Taken together our data suggest that domains 4 and 7 are required for ligand-induced receptor activation [24].

VEGF structure: In collaboration with Structural Biology and SLS at PSI we determined the 3D structure of VEGF-E at 2.3 Å resolution [25,26]. The data allowed us to design receptor specific VEGF variants which gave insight into the mechanism by which receptor specificity of ligands is determined. A NMR structure is known for the heparin binding domain encoded by exon 7 [27,28], but so far no crystallographic data are available for the exon 7 and 8 encoded part of the longer VEGF-As.

Single chain antibodies for targeted tumor therapy: Single chain antibodies (scFv) have been used exentsively to develop specific high affinity target molecule binders for therapeutic application in a variety of disease such as in tumor therapy [29,30]. Our lab has developed a series of scFvs specific for tumor endothelail markers or molecules involved in promoting angiogenesis such as VEGF [31-34]. One of the most prominent antibody-based drugs in the clinic these days used in anti-angiogenic tumor therapy and in macular degeneration is Avastin, a recombinant antibody developed by Genentech/Roche [35].

Research plan

‘Designed ankyrin repeat protein’ libraries used for the selection of high affinity VEGF binders:
We currently develop a new generation of VEGF antagonists based on DARPIN technology [36]. DARPINs are selected from complex libraries. They are used as protein binding molecules similar to scFvs or receptor-based ‘VEGF trap’ reagents. DARPINs interact with their target with affinities as low as 20 pmol. Our major goal is to develop isoform-specific VEGF antagonists that specifically block angiogenesis without the dramatic side effects observed with the current reagents used in the clinic after longterm treatment [37]. With the discovery of the new functionally different exon 8 VEGF splice variants this is now possible. VEGF-A variants such as VEGF-A165 and VEGF-A165b  differing in exon 8 will be produced in Pichia pastoris, purified by affinity chromatography, deglycosylated and used for DARPIN selection.

In vitro and  in vivo evaluation of DARPIn activity: The selected DARPINs will be characterized initially in vitro using a variety of assays such as the chicken chorioallantois membrane angiogenesis assay [38], vessel formation in matrigel by endothelial cells in vitro and in vivo [39-41], spheroid cell cultures and 2D and 3D embryoid body sprouting angiogenesis assays [41-45]. DARPINs with the expected inhibitory activity, i.e. molecules that bind the ligand with high affinity and block VEGF-A165 but not VEGF-A165b signaling, will then be applied systemically in mice to inhibit tumor growth and vessel formation. In vivo inhibition of normal angiogenesis will be tested in matrigel implant angiogenesis assays. Imaging of angiogenesis in live animals will be performed by PET and SPECT imaging with radioisotope-tagged DARPINs in tumor bearing mice [46].

Structural analysis of VEGF/DARPIN complexes: DARPIN/VEGF complexes generated in vitro will be analyzed structurally using X-ray crystallography together with other group members in our laboratory. This analysis will give us a deeper insight into the molecular basis of inhibition of VEGF signaling and will allow us to further optimize inhibitory DARPINs in subsequent library pannings.

References

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