Effective Mammalian Two Hybrid Screening Approach

描述(申请人提供)：本申请是对RFA-CA-07-001的响应，是一种有效的哺乳动物双杂交筛选方法。识别新的蛋白质-蛋白质相互作用是了解蛋白质功能和信号网络的基本步骤，从而有效地实施靶向癌症治疗。目前，大多数蛋白质-蛋白质相互作用是通过酵母双杂交(Y2H)、免疫共沉淀和质谱学或蛋白质文库来鉴定的。这些方法中的每一种都有其自己的一组主要限制，未能模拟自然的生理条件(Y2H和蛋白质文库)或有效地识别细胞骨架或膜上的蛋白质相互作用，要么是由于相互作用的位置(Y2H)，要么是由于细胞骨架或膜蛋白的免疫共沉淀困难。此外，传统的Y2H方法产生假阳性信号，转录因子排除了筛选。因此，一种新的筛选方法，有效地识别与生物相关的蛋白质相互作用，绕过现有筛选方法的局限性，将具有广泛的适用性。我们建议开发和验证一种易于应用的、依赖于上下文、亚细胞定位、cDNA文库和细胞类型独立的基于逆转录病毒的哺乳动物双杂交(ReMTH)筛选方法，用于鉴定新的蛋白质-蛋白质相互作用，包括细胞骨架和膜蛋白，在哺乳动物细胞中允许天然蛋白质折叠和翻译后修饰。得到的细胞将成为研究新的蛋白质-蛋白质相互作用复合体的定位和功能的试剂，以及高含量药物或siRNA筛选的资源。这项全面开发的技术将比现有方法更有效地识别功能性蛋白质-蛋白质相互作用，并识别目前方法无法发现的相互作用，特别是在上下文相关的哺乳动物屏幕上。此外，拟议的ReMTH筛查具有稳定或捕获瞬时/弱相互作用(如酶/底物相互作用)的独特潜力，允许识别以前无法检测到的癌症中信号通路和网络的组件。我们已经在HeLa细胞中完成了初步的概念验证筛选，以确定癌基因AKT1的相互作用伙伴。我们鉴定了一系列已知的AKT1相互作用伙伴和底物，以及新的相互作用伙伴，包括细胞骨架和膜蛋白。我们已经证实了一个新的相互作用伙伴，ACTN4，与AKT1在物理和功能上相互作用。因此，这项技术将揭示其他方法无法检测到的功能性蛋白质-蛋白质相互作用，并促进我们对癌症中蛋白质功能和信号网络的理解。

DESCRIPTION (provided by applicant): This application, Effective Mammalian Two Hybrid Screening Approach, is in response to RFA-CA-07-001. Identification of novel protein-protein interactions is a fundamental step to understanding protein function and signaling networks allowing efficient implementation of targeted cancer therapy. The majority of protein-protein interactions are currently identified using yeast two hybrid (Y2H), co-immunoprecipitation and mass spectrometry or protein libraries. Each of these approaches has its own set of major limitations, failing to mimic native physiological conditions (Y2H and protein libraries) or to efficiently identify protein interactions on the cytoskeleton or membrane, either due to the location of the interaction (Y2H) or due to difficulties in co-immunoprecipitation of cytoskeletal or membrane proteins. Furthermore, conventional Y2H approaches yield false positive signals with transcription factors precluding screening. Therefore, a novel screening method that efficiently identifies biologically-relevant protein interactions, bypassing the limitations of present screening methods, would have wide applicability. We propose to develop and validate a readily applicable, context-dependent, subcellular localization-, cDNA library- and cell type-independent retrovirus-based mammalian two hybrid (ReMTH) screen method for identification of novel protein-protein interactions, including cytoskeletal and membrane proteins, in allowing native protein folding and post-translational modifications in mammalian cells. The resultant cells will be reagents for the study of the localization and function of the novel protein-protein interaction complex as well as resources for high content drug or siRNA screening. The fully developed technology will identify functional protein-protein interactions more efficiently than present methods and identify interactions not discoverable by present methods, particularly in context-dependent mammalian screens. Furthermore, the proposed ReMTH screen has the unique potential to stabilize or trap transient/weak interactions such as enzyme/substrate interactions, allowing identification of components of signaling pathways and networks in cancers previously undetectable. We have completed an initial proof-of-concept screen in HeLa cells for identification of interaction partners of the oncogene AKT1. We identified a series of previously known AKT1 interaction partners and substrates, as well as novel interaction partners, including cytoskeleton and membrane proteins. We have confirmed that one novel interaction partner, ACTN4, interacts physically and functionally with AKT1. Thus the technology will uncover functional protein-protein interactions not detectable by other approaches and advance our understanding of protein functions and signaling networks in cancer.