In situ proteomics for brain using genetically encoded probes.

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Summary This exploratory proposal will develop an intersectional genetics approach to in situ proteomics in a mammalian brain based on a split biotin ligase fragments with induced dimerization in mice. Current proteomic databases draw from high-throughput experiments in non-physiological settings that typically involve high expression in a highly transformed cell line or in vitro system. To develop a more physiological approach to key protein networks for genetically complex disorders, we will adapt a split biotin ligase approach for inducible proximity labeling in situ in mouse brain. We will tag an endogenous protein of interest with one fragment of the split biotin ligase by germline editing in mice and use genetic crosses to pair this fusion gene with one for the complementary split fragment expressed from a distinct locus. Dimerization to form the active enzyme is controlled by overlapping expression and rapamycin administration. This will allow us to identify the protein of interest’s physical network in physiological context. As an initial demonstration we will tag endogenous Zfp804a, whose human cognate is encoded by a well-replicated risk locus for psychiatric illness. In parallel, we will develop cell-based models to refine the approach, focus results on selected subcellular compartments, and extend the approach to other neurodevelopmental risk loci and Zfp804a partner proteins.