In situ proteomics for brain using genetically encoded probes.

摘要 这一探索性的提议将发展一种原位遗传学的交叉方法。 基于生物素连接酶裂解片段的哺乳动物脑蛋白质组学研究 小鼠的二聚化。目前的蛋白质组数据库从高通量提取 在非生理性环境中的实验，通常涉及在 高度转化的细胞系或体外系统。开发一种更具生理学意义的方法 对于基因复杂疾病的关键蛋白质网络，我们将改造一种分裂的生物素 小鼠脑内诱导邻近原位标记的连接酶方法。我们将标记一个 利用生殖系分离的生物素连接酶的一段内源目的蛋白 在小鼠身上进行编辑，并使用遗传杂交将该融合基因与 从一个不同的基因座表达的互补分裂片段。二聚化反应形成 活性酶受重叠表达和雷帕霉素给药的控制。 这将使我们能够识别感兴趣的蛋白质在生理上的物理网络 背景。作为初始演示，我们将标记内源性Zfp804a，其人类 同源基因由一个复制良好的精神疾病风险基因编码。同时，我们 将开发基于细胞的模型来改进方法，将结果集中在选定的 亚细胞隔区，并将该方法扩展到其他神经发育风险 基因座和Zfp804a配对蛋白。

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.