Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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Clathrin-mediated endocytosis (CME) involves spatially and temporally restricted molecular dynamics.
Although protein kinases and the actin cytoskeleton contribute to the process, whether and how
functions of kinases and actin are integrated remains unknown. Here, we demonstrate that neural
Wiskott-Aldrich syndrome protein (N-WASP) and protein kinase CK2 form a complex and localize on
clathrin-coated vesicles (CCVs). N-WASP binds to and is phosphorylated by CK2, thereby reducing the
kinase activity of CK2. By contrast, N-WASP-promoted actin polymerization is decreased upon both
phosphorylation and binding of CK2. Knockdown of N-WASP and CK2, alone or in combination, results
in impaired endocytosis of epidermal growth factor (EGF) and increased cell-surface levels of EGF
receptor (EGFR). In order to rescue the phenotype of N-WASP-CK2 knockdown cells, both N-WASP and
CK2 activities and abilities to assemble in a complex are required. In summary, this study shows that the
N-WASP-CK2 complex integrates in a single circuit different activities contributing to CME of EGFR and
that the interplay between the two proteins optimizes this process.
5-lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. In a two step reaction the enzyme oxygenates arachidonic acid (AA) to form the highly unstable epoxide leukotriene A4 (LTA4) in dehydrating a hydroperoxide intermediate (20). LTA4 can then be further metabolized by two terminal synthases yielding either the potent chemoattractant leukotriene B4 (LTB4) or the cysteinyl leukotrienes (CysLTs). 5-LO enzyme expression is primarily found in mature leukocytes (22) where it can either reside in the cytoplasm or in the nucleus associated with euchromatin (29). Its enzymatic activity is embedded in a complicated network in intact cells regulating LT synthesis by various factors dependent on the cell type and nature of stimulus. Factors such as the amount of free AA released by phospholipase A2 enzymes, levels of enzymes involved, catalytic activity per enzyme molecule and availability of different small molecules influence 5-LO activity (36).
The 5-LO derived LTs are lipid mediators which were shown to primarily mediate inflammatory and allergic reactions and their role in the pathogenesis of asthma is well defined. CysLTs are among the most potent bronchoconstrictors yet studied in man and play an important role in airway remodeling. LTB4 has no bronchoconstrictory effects in healthy and asthmatic humans but displays potent chemoattractant properties on neutrophils and increases leukocyte adhesion to the vessel wall endothelium (22). Therefore, LTB4 enhances the capacity of macrophages and neutrophils to ingest and kill microbes. In concert with LTB4, histamine and prostaglandin E2 (PGE2) CysLTs are thought to maintain the tone of the human airways (82).
Besides their well studied role in asthma, 5-LO derived LTs have also been implicated to play a role in cardiovascular diseases and cancer. In contrast to healthy tissues, LT pathway enzymes and receptors were found to be abundantly expressed in cancer tissues, atherosclerotic lesions in the aorta, heart and carotid artery (86). Pharmacological inhibition of 5-LO potently suppressed tumour cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway (92, 93). In several studies LTs were found to exhibit cardiovascular actions by promotion of plasma leakage in postcapillary venules, coronary artery vasoconstriction and impaired ventricular contraction leading to reduced coronary blood flow and cardiac output (24). Unfortunately, the precise molecular mechanisms through which LTs influence carcinogenesis and cardiovascular diseases are still incompletely understood.
In contrast, an increasing number of studies questions the correlation between 5-LO and cancer (95-97) since extreme LT concentrations were applied to induce proliferative effects in the majority of the publications. A few studies exist which show susceptibility towards 5-LO products in physiological concentrations or achieve anti-proliferation by applying low concentrations of 5-LO inhibitors (98) ...
Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.
Since combinatorial chemistry and high throughput screening have become a common technique in the drug discovery phase the number of compounds being considered has increased frequently. These structures are often characterized by high molecular weight, high lipophilicity and low solubility in aqueous and physiological media. Due to the generally poor bioavailability, new in vitro techniques were needed for screening of pharmacokinetic properties. An important parameter for these screening methods is the implementation at an early state of drug discovery phase, to find potential lead structures, before investment costs become significant. The established in vitro methods for the prediction of membrane interaction are not reliable especially for poorly soluble compounds. A new method that is fast and easy to use, requires only small amounts of NCE and which can provide more reliable predictions is needed. In this study, a new screening technique based on surface activity profiling for the prediction of oral drug absorption was evaluated with special emphasis on the predictability of biological membrane interaction of poorly soluble drug compounds. It was demonstrated that drug absorption through a bilayer membrane can be modeled by the orientation of compounds at the air/water interface. Thus amphilicity of a drug is generally related to both oral absorption and blood brain barrier penetration. In turn, amphiphilicity is influenced by the lipophilicity, size and charge distribution of a drug. Surface activity profiling was determined by analysis of surface pressure profiles using the Gibbs adsorption isotherm. The surface activity measurements were carried out using a multichannel tensiometer Delta 8, which was developed by Kibron to be utilized in conjugation high throughput screening in early drug discovery processes. For this study two test sets were analyzed, one for the prediction of gastrointestinal wall interaction and the second for the prediction of the penetration behavior at the blood brain barrier. Both test sets consist of drug compounds with a wide range of absorption properties and consist mainly of compounds with poor water solubility. Since the drugs characteristics varied, they were classified according to water solubility and surface activity and a sample preparation method for each group was established. For the prediction of oral drug absorption, three different methods were established to model the interaction of compound and gastrointestinal wall. For drug compounds with solubility above 1mmol/L the traditional shake-flask method enabled the determination of the amphiphilic properties of drug compounds in pure aqueous media. Compounds with solubility below 1mmol/L tend to not to exhibit any increase in surface activity. Thus surface tension measurements of compounds, which exhibited a limited surface activity due to poor aqueous solubility, were conducted from stock solutions prepared with various organic solvents. Mainly polar organic solvents were used. A mixture of DMSO and DMF resulted in the best combination of properties: the intensive solubility enhancing effect of DMF and the lower intrinsic surface activity of DMSO. The polar solvent ruptured the water clusters, so that highly lipophilic structures had a higher affinity to the solvent and higher concentrations could be obtained. For these compounds higher maximum surface pressure were generated than was possible in pure aqueous media. The surface pressure data were correlated with the fraction absorbed values in vivo. However it was found that poor water solubility is not the only limiting step to exhibiting any surface activity. Some compounds were showed no surface activity in either solvent system. Therefore a micelle vehicle method was established using short chain phospholipids to mimic the gastrointestinal wall. It could be concluded from the results, that non surface active drugs can interact with the phospholipids micelle vehicle in a way analogous to their interaction with the membrane bilayer. The relative critical micelle concentration was correlated with the fraction absorbed of this test set. A sample preparation schema based on the three types of drugs was established. This schema enabled us to predict the absorbance of slightly soluble and poorly soluble drugs with acceptable reliability for early compound screening. For the prediction of blood brain barrier penetration using surface activity profiling as analyzing method, a test set with very poorly soluble characteristics was chosen. The sample preparation method was based on a strictly aqueous approach using the ‘shake flask’ method. The surface tension measurements enabled correlation of the amphiphilic properties of the very poorly soluble drug compounds with BBB uptake. From the aqueous surface pressure profiles and the determination of physicochemical parameters, it was found that blood brain barrier is more likely when a drug provides a small cross-sectional area, As, at the interface. The cross-sectional area is the only parameter which is independent from the maximal concentration in aqueous media and it is particularly suitable for lower solubility compounds. In summary, it was shown that amphilicity is related to biological membrane interaction in the human body and that surface activity profiling with appropriate sample preparation can be used as a reliable screening tool for the prediction of oral drug absorption of poorly soluble drugs. Furthermore an in vitro screening method of blood-brain-barrier penetration was established.
NK cells are part of the innate immune system, and are important players in the body’s first defence line against virus-infected and malignantly transformed cells. While T cells recognize neoplastic cells in an MHC-restricted fashion, NK cells do not require prior sensitization and education about the target. In leukemia and lymphoma patients undergoing allogeneic hematopoietic stem cell transplantation not only T cells but also NK cells have been found to mediate potent graft-versus-tumor effects. Hence, autologous or donor-derived NK cells hold great promise for cancer immunotherapy. Since the generation of highly purified NK cell products for clinical applications is labor-intensive and time consuming, established human NK cell lines such as NK-92 are also being considered for clinical protocols. NK-92 cells display phenotypic and functional characteristics similar to activated primary NK cells. While NK-92 cells are highly cytotoxic towards malignant cells of hematologic origin, they do not affect healthy human tissues. NK-92 cells can be expanded under GMP-compliant conditions, and can therefore be provided in sufficient numbers with defined phenotypic characteristics for clinical applications. Safety of NK-92 cells for adoptive immunotherapy was already shown in two phase I/II clinical trials...