Proximity- and complementation-based analysis of signaling complexes

摘要 绘制细胞内蛋白质相互作用网络图是系统水平的最终目标 蛋白质组学。最近，使用混杂生物素连接酶Bira*的基于接近的方法 已经开发出直接在活细胞中确定蛋白质相互作用的方法，克服了 其他蛋白质-蛋白质相互作用分析的一些概念和技术限制。 该应用程序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检测新的蛋白质 与已知的信号复合体相互作用。为了做到这一点，成对的分裂的Bira*片段 只有当接近时才能恢复酶活性，这将被定义并 在基于细胞的生物素连接酶检测中得到验证。候选人积极分裂的Bira*片段将 然后在体内用来鉴定与信号受体和平面相互作用的新蛋白 非洲爪哇胚胎神经中空间分离的细胞极性(PCP)复合体 板材，一种新型的PCP。非洲爪哇的胚胎非常适合这些研究， 通过以下组合实现对蛋白质定位和功能的快速分析 生物化学、胚胎学和细胞生物学方法。这些研究将提供一个 多功能通用平台，用于绘制不同实验环境中的蛋白质相互作用图 促进我们对PCP信号机制的理解，并有助于建立新的 脊椎动物PCP模型。由于PCP蛋白与神经管缺陷有关， 纤毛疾病、多囊肾病和转移性癌症，拟议的研究是 与人类健康相关，并将促进预防五氯苯酚的必要知识- 相关疾病和先天畸形。 第1条

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