Proximity- and complementation-based analysis of signaling complexes

总结 绘制细胞内蛋白质相互作用网络是系统级研究的最终目标 蛋白质组学最近，使用混杂的生物素连接酶BirA* 的基于邻近的方法 已经被开发用于直接在活细胞中确定蛋白质相互作用，克服了 其他蛋白质-蛋白质相互作用测定的一些概念和技术限制。 本申请旨在开发一种新的通用技术，该技术结合了 基于邻近的生物素化和分裂蛋白互补来检测新的蛋白质， 与已知的信号复合物相互作用。为了实现这一点，成对的分裂BirA* 片段 将定义仅当紧密接近时才恢复酶活性， 在基于细胞的生物素连接酶测定中得到验证。候选阳性分裂BirA* 片段将 然后在体内用于鉴定与信号受体相互作用的新蛋白质， 细胞极性（PCP）复合物在空间上分离的非洲爪蟾胚胎神经 板，一种新型的PCP。非洲爪蟾的胚胎非常适合这些研究， 允许通过以下组合快速分析蛋白质定位和功能： 生物化学、胚胎学和细胞生物学方法。这些研究将提供一个 通用平台，以映射蛋白质相互作用在不同的实验设置，将 推进我们对PCP信号机制的理解，并帮助建立一个新的 脊椎动物PCP模型。由于PCP蛋白与神经管缺陷有关， 纤毛病，多囊肾病和转移性癌症，拟议的研究是 与人类健康相关，并将增进预防五氯苯酚的必要知识， 相关疾病和先天性畸形。 1

Summary Mapping protein interaction networks of the cell is the ultimate goal of systems-level proteomics. Recently, proximity-based approaches using the promiscuous biotin ligase BirA* have been developed to determine protein interactions directly in live cells, overcoming some of the conceptual and technical limitations of other protein-protein interaction assays. This application aims to develop a new general technique that combines the advantages of proximity-based biotinylation and split protein complementation to detect novel proteins that interact with known signaling complexes. To accomplish this, pairs of split BirA* fragments that restore enzymatic activity only when brought into close proximity will be defined and validated in cell-based biotin ligase assays. Candidate positive split BirA* fragments will then be used in vivo to identify novel proteins interacting with signaling receptors and planar cell polarity (PCP) complexes that are spatially segregated in the Xenopus embryonic neural plate, a new model of PCP. Xenopus embryos are uniquely suited for these studies, allowing rapid analysis of protein localization and function through a combination of biochemical, embryological and cell biological approaches. These studies will provide a versatile general platform to map protein interactions in different experimental settings, will advance our understanding of PCP signaling mechanisms and help establish a new vertebrate PCP model. Since PCP proteins have been implicated in neural tube defects, ciliopathies, polycystic kidney disease and metastatic cancers, the proposed research is relevant to human health and will advance the knowledge necessary for preventing PCP- associated diseases and congenital abnormalities.   1