Proximity- and complementation-based analysis of signaling complexes

基于邻近和互补的信号复合物分析

• 批准号: 9227603
• 负责人: Sergei Sokol
• 金额: $ 20.13万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-05 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9227603
• 关键词: Address; Affinity; Animal Model; Binding; Biochemical; Biological; Biological Assay; Biological Preservation; Biotin; Biotinylation; Birth; Cell Polarity; Cells; Complement; Complex; Congenital Abnormality; Data; Development; Dimerization; Disease; Disseminated Malignant Neoplasm; Embryo; Enzymes; Goals; Health; Human; In Vitro; Individual; Knowledge; Label; Length; Ligands; Ligase; Light; Link; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Metastatic to; Modeling; Molecular; Neural Tube Defects; Neuroepithelial Cells; Operating System; Pathologic Processes; Polycystic Kidney Diseases; Prevention; Protein Analysis; Protein Fragment; Proteins; Proteomics; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Research; Signal Transduction; Sirolimus; Syndrome; System; Tacrolimus Binding Proteins; Techniques; Technology; Testing; Tissue Model; Vitronectin; Whole Organism; Xenopus; base; cell behavior; ciliopathy; dimer; embryo tissue; enzyme activity; experimental study; human disease; in vivo; loss of function; neural plate; neuroepithelium; novel; planar cell polarity; prevent; protein complex; protein protein interaction; reconstitution

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

总结