Isolation of Regulators of Immune Cell Behavior

Johns Hopkins University, Baltimore MD

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Abstract

? DESCRIPTION (provided by applicant): The molecular understanding of how signal transduction pathways govern immune cell function depends upon elucidating the physical interactions between pathway components and defining how these interactions transmit signals and provide regulation. Several approaches toward discovering relevant protein-protein interactions in signaling pathways have become standard tools in the last few decades, including yeast-two-hybrid screening with a bait protein of interest, affinity purification of a bat followed by mass spectrometry of associated proteins, the probing of protein chips with a recombinant bait protein, and others. Each technique has yielded important findings in a variety of fields and each has its particular advantages and disadvantages depending on the application. To enable our studies of antigen receptor signaling and how its dysregulation contributes to immunodeficiency, autoimmunity and lymphoma, we have sought novel screening approaches for identifying protein-protein interactions. Specifically, we desired a technique that would allow us to screen for protein-protein interactions a) in live mammalian cells, b) with a quantitative measure of the interaction, c) in a cost-efficient and d) high-throughput manner, that e) would be highly modular and therefore easily adaptable for many applications. In this application we propose exploratory and developmental studies to establish such a screening approach based upon Bioluminescence Resonance Energy Transfer (BRET), which occurs between a Renilla luciferase derivative, Rluc8, and YPet, a YFP variant, provided the distance between the proteins is equal to or less than ~100 Å. Our pilot studies suggest that a library of YPet-cDNA fusions can be screened easily in a high-throughput manner in live mammalian cells for proteins that associate and permit BRET with an individual Bait-Rluc8 fusion protein. We propose to demonstrate the advantages of this technique, develop it into a standardized approach, and generate reagents for screening that will be of general use to the field and freely distributed.