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- Production of the human adenosine A2A receptor in Pichia pastoris, its solubilization and purification, and the selection of a specific single-chain Fv fragment by phage display (2006)
- G protein-coupled receptors (GPCRs) comprise the largest membrane protein family and play an essential role in signal transduction through the cell membrane. They are currently the targets of approximately 50 % of the pharmaceuticals on the market (Klabunde and Hessler, 2002). However, only one high-resolution GPCR structure has been determined up to now, that of bovine rhodopsin (Palczewski et al., 2000). The GPCR activation and regulation mechanisms are still unknown and other GPCR structures are thus required. MePNet (Membrane Protein Network) was a European consortium dedicated to structural studies of GPCRs. The approach was to produce 100 GPCRs in three expression systems (Escherichia coli, Pichia pastoris and Semliki Forest Virus infected mammalian cells) in order to select at each step of the process (production, solubilization, purification) the constructs that fulfilled quantity and quality (functionality) requirements for crystallization trials. In our team, we screened 38 of the 100 targets in P. pastoris. For each receptor, the clone with the highest production level was identified by dot-blot. The size and homogeneity of each receptor were then analyzed by Western-blot. The human adenosine A2A receptor showed a well-defined and pronounced single band and was thus selected for further characterization. The adenosine A2A receptor is a GPCR mainly localized in the central nervous system and, as it antagonizes dopaminergic activity, it has great potential as a drug target for the treatment of Parkinson’s disease. Functional characterization by binding assays with the specific antagonist [3H]-ZM241385 demonstrated a Bmax of 56 +/- 3 pmol/mg i.e. pmol of binder per milligram of total membrane protein, and a KD of 0.40 +/- 0.02 nM. Receptor production was then improved by lowering the induction temperature, decreasing the induction time and adding DMSO to the medium. For large-scale production, fermention reached around 300 g cells (wet weight)/L culture, which provided 43 mg of functional receptor in membranes per liter of culture. Functional solubilization was achieved with dodecyl-β-D-maltoside and the soluble yield was increased to 70-80 % of the membrane content by addition of cholesteryl hemisuccinate and increasing the ionic strength. The receptor was successfully purified via Ni-NTA and monomeric avidin chromatography in the presence of the antagonist ZM241385. This strategy produced a pure, homogeneous and stable receptor preparation with functionality demonstrated by radioligand binding assays. The total receptor yield after purification was routinely around 20 % of the membrane functional receptor content and 2 g of membranes provided 4 mg of pure receptor for crystallization trials. GPCRs are very difficult targets for crystallization, and co-crystallization with antibody fragments has been shown to be a successful method for crystallization of membrane proteins. In order to develop such a tool for the adenosine A2A receptor, a single-chain Fv (scFv) fragment specific to the purified receptor was selected by phage display. The receptor was functionally immobilized on the surface of streptavidin beads and after two rounds of selection, 6 different phages were identified several times. After production in E. coli and purification via Ni-NTA affinity chromatography, 4 out of the 6 scFv fragments were sufficiently enriched to be tested by ELISA. For the ELISA, the receptor was functionally immobilized via the biotinylation domain of the construct in a 96-well streptavidin-coated plate. The antibody fragments binding to the receptor were identified based on interaction with HRP-conjugated protein L. One scFv fragment gave a positive ELISA signal 10 fold above background and titration of the scFv fragment binding to the receptor was specific and saturable. However no complex of scFv fragment and receptor was observed on gel filtration. In order to have a more sensitive detection method, the scFv fragment was labeled with fluorescein: a complex was then observed up on gel filtration but the binding appeared to be non-specific. A pull-down assay with immobilized non-labeled scFv fragment finally confirmed the specificity of the binding, but also the low affinity of the interaction. Affinity maturation of this specific scFv fragment by a random mutagenesis and selection process should improve this parameter in order to obtain an adapted tool for co-crystallization.
- Regulation of IL-18 binding protein by IFN-gamma (2003)
- In this study we investigated the regulation of IL-18BPa by IFN-y in the context of colon cancer and human autoimmune diseases. IL-18BPa is a naturally occuring inhibitor that counteracts IL-18 bioactivity. By enhancing IFN-y production IL-18 has been introduced as pivotal mediator of TH1 immune responses. Indeed, many IL-18 effects are mediated by IFN-y. IL-18 bioactivity is connected with the pathogenesis of different inflammatory diseases, for instance, septic shock, colitis, Crohn's disease, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, atherosclerosis, and organ transplant rejection. In addition, IL-18 has tumor-suppressive properties. IFN-y induced IL-18BPa expression was shown on protein and mRNA level in different colon carcinoma cell lines, organ cultures of colonic intestinal biopsy specimens, HaCaT keratinocytes as well as rheumatoid arthritis fibroblastlike synoviocytes (RA-FLS). The IFN-y-mediated induction of IL-18BPa appears to be a more general phenomenom. The capability of IFN-y to induce IL-18BPa also has been confirmed on the promoter level by performing luciferase reporter gene studies with two IL- 18BP promoter fragments. A GAS-site proximal to the transcription start site has been identified to be relevant for IFN-y-mediated induction of these two IL18BP promoter fragments. The induction of IL-18BPa is most likely mediated by STAT-1 in DLD-1 colon carcinoma cells. Sodium butyrate inhibited IFN-y-induced IL-18BPa expression in these cells. On the basis of our observations, we postulate a negative feedback mechanism, by which IFN-y-dependent and -independent IL-18 action might be counterregulated. In this model sodium butyrate is an additional player, that may interrupt the postulated negative feedback loop. A coculture system was performed to simulate an inflammatory TH1 response. This model which is more close to the in vivo situation, confirmed upregulation of IL-18BPa by endogenously produced IFN-y. The role of IL-18BPa is manifold and depends on IL-18 function in each particular case. In autoimmune diseases, for instance, which are often characterized by a TH1 polarized immune response, IL-18BPa might counterregulate IL-18 and/or IL-18-induced IFN-y bioactivity. Important examples are Crohn's disease and rheumatoid arthritis. In CD therapeutic use of IL-18BPa may therefore restore a hypothetically disturbed IL-18/IL-18BP balance. Concerning RA, IL-18BPa expression might contribute to protective functions of IFN-y, observed in different murine models for arthritis and in rheumatoid arthritis patients. Moreover, IL-18BPa might inhibit IL-18-mediated induction of subsequent cardinal inflammatory cytokines responsible for the pathogenesis of these diseases. Indeed, the pharmaceutical industry successfully used IL-18BP as therapeutic agent in a murine model of RA and in phase I clinical trials. On the contrary, in the context of carcinogenesis IFN-y- mediated IL-18BPa expression might be disadvantageous. By counterregulating the IL-18 arm of immune defenses against tumors, IL-18BP may have the potential to promote carcinogenesis. Our hypothesis is underlined by the observation that sodium butyrate, known to be protective in colon cancer, inhibited IFN-y-induced IL-18BPa expression. In parallel, IL-18-induced IFN-y is also responsible for iNOS induction. iNOS-derived NO provides a second possible way for inhibition of IFN-y-dependent and -independent tumor suppressive effects of IL-18. Finally, IFN-y-induced IL-18BPa expression was confirmed on the promoter level. This induction on the promoter level was associated with STAT-1 binding to the GAS element proximal to the start of transcription. It is tempting to speculate that blockage of the cytokine cascade upstream of IL-1 and TNF- a on the level of IL-18 may be of therapeutic benefit. Our data reflect the relationship between inflammation and cancer, in that inflammatory cells and cytokines found in tumors are likely to contribute to tumor growth, progression, and immunosuppression than they are to mount an effective host antitumour response.