Identification, characterization and inhibition of protein interactions in immune responses (172.42)

Abstract

Abstract Protein interaction mapping provides molecular basis to protein functions and signaling pathways. We have developed a high-throughput yeast two-hybrid technology that allows the exhaustive screening of highly complex, domain-based cDNA libraries relevant to the immune system (e.g. leukocytes and activated mononuclear cells, thymocytes CD4+/CD8+ and T-cells). These libraries are screened to saturation thanks to an optimized mating procedure, allowing to test on average 100 million interactions per screen. This corresponds to a 10-fold coverage of the libraries, and ensures exhaustive and reproducible results. Moreover, multiple independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score. A database containing over five thousand cDNA screens realized with this technique permits a rigorous calculation of relative prey connectivity and understanding of true/false positives and sticky proteins. We have developed additional methods to probe and characterize protein interactions in vitro with a medium to high throughput, using Surface Plasmon Resonance and a miniaturized time-resolved FRET assay which has been used for drug discovery projects targeting protein-protein interactions. Here, we will present results that illustrate the strength and benefits of our protein interaction screening and validation technologies to decipher the molecular components of immune responses.