Identification and verification of protein-protein interactions using automated two hybrid screens
The identification and analysis of protein-protein interaction is a proteomics strategy to gain insight into protein function and to connect proteins to cellular pathways important for disease development. Within NGFN1 (platform 4-T03) a human protein interaction network was systematically generated, screening more than 5,500 human proteins for pair wise interactions by an automated Y2H procedure. 3,186 interactions among 1,705 human proteins were identified, resulting in a large, highly connected interaction map. The network revealed mostly novel interactions and provided functional links for 258 uncharacterized gene products and 218 human disease proteins. Within NGNF2 the network will serve as a basis for the more detailed analysis and characterization of regulatory protein networks and cellular complexes, relevant to human diseases. Bioinformatic analysis will include a quality assessment of the high throughput data and identify important functional subnetworks (“modules”). Protein interactions within network modules, will be verified applying in vitro binding, coimmunoprecipitation and colocalisation assays in mammalian cell culture systems. In cooperation with subproject 3.2 (B. Lange/J. Gobom), large protein complexes will be isolated from mammalian cells and analyzed for their protein composition. A novel Y2H variant will be developed to characterize more complex relationships between proteins, e.g. phosphorylation-dependent interactions or interactions that involve a third protein counterpart. Data from the SMP e.g. complex data and other research projects will be integrated in a database. It is further planned to analyze selected protein complexes using in vivo models (collaboration with SMP model). In this project we aim at connecting results generated by high throughput proteomics techniques to physiological/pathological cellular pathways, thus contributing to a better understanding of disease processes, the development of novel disease biomarkers, and accelerated drug development.
