Systematic Exploration of the Human Interactome

 描述(申请人提供)：蛋白质组可以被视为相互作用的蛋白质模块的星座，这些模块被组织成细胞器、分子机器和信号转导网络。到目前为止，只有一小部分表达的蛋白质的相互作用伙伴是已知的，但蛋白质-蛋白质关联是理解蛋白质功能和活性的主要驱动因素。在过去的两年里，我们在NHGRI和Biogen-IDEC的联合支持下，实施了一条高通量的蛋白质相互作用管道，用于分析293T细胞中C端标记的大约10,000个人类蛋白质的蛋白质相互作用。我们把这称为“第一次通过”的交互作用组分析。到目前为止，这已经导致了对~5900种蛋白质的分析(9月20号，2014年)，其中2594个已经存入BioGRID数据库，因为它们被收录在描述管道的第一篇论文中。其他互动每季度发布一次，所有内容都是免费向公众开放的。在相互作用蛋白质组学领域，执行这种规模的项目所需的基础设施的发展是前所未有的。事实上，我们已经开发了许多新的信息和操作元素到一个管道中，允许一个小型科学家团队每月通过AP-MS对500-600个诱饵进行分析。已经制定了许多质量控制措施，并将第一批2594个诱饵蛋白的相互作用伙伴与现有的 蛋白质相互作用数据库揭示了与最高质量的CORUM数据库的广泛重叠，同时也为20,000多个以前没有报道的新的相互作用提供了实验证据。该网络识别了250多个蛋白质簇，包括已知和新的复合体。在这份提案中，我们寻求继续生产这条管道，有三个主要目标，这三个目标是根据与NHGRI的讨论而被选为优先目标的。在具体目标1中，我们将完成对其余可用的ORF克隆(约4000个克隆)的分析。我们还将重新访问在第一次通过分析中未能产生稳定细胞系的1,000个ORF，并将使用一种不同的洗涤剂来检测1,000种膜蛋白，这种洗涤剂比“第一次通过”分析中使用的洗涤剂更容易溶解某些类别的膜蛋白。在特定的目标2中，我们将测试细胞类型 第二细胞系(HCT116)中前2600个诱饵的相互作用的特异性，从而为相互作用的子集提供验证并揭示候选细胞类型特定的相互作用。在具体目标3中，我们将使用串联质量标签方法来比较~50个重要疾病基因和每个基因的几个突变等位基因的相互作用，从而实现一个高度可比性的平台，以了解疾病突变如何改变信号网络、分子机器和途径。所有数据将通过公共数据库传播，并将接受广泛的生物信息学和网络分析。

 DESCRIPTION (provided by applicant): The proteome can be viewed as constellations of interacting protein modules, which are organized into organelles, molecular machines, and signal transduction networks. To date, the interaction partners for only a small fraction of expressed proteins are known, yet protein-protein associations are a major driving factor in understanding protein function and activity. During the last 2 years, we have implemented a high-throughput protein interaction pipeline for analysis of protein interactions for ~10,000 human proteins in 293T cells tagged at their C-terminus with an HA tag with joint support by NHGRI and Biogen-Idec. We refer to this as a "first- pass" interactome analysis. This has led so far to the analysis of ~5900 proteins (Sept. 20th, 2014) by AP-MS, 2594 of which have already been deposited in the BioGRID database based on their inclusion in a first paper describing the pipeline. Additional interactions are being released quarterly and all are free to the public. The development of the infrastructure required to perform a project of this scale is unprecedented in the field of interaction proteomics. Indeed, we have developed numerous new informatic and operational elements into a pipeline that allows a small team of scientists to perform an analysis of 500-600 baits by AP-MS per month. Numerous quality control measures have been developed, and comparison of the interaction partners of the first 2594 bait proteins with existing protein interaction databases reveals extensive overlap with the highest quality CORUM database, while also providing experimental evidence for over 20,000 novel interactions that have not been previously reported. The network identifies more than 250 clusters of proteins, and includes both known and novel complexes. In this proposal, we seek to continue the production of this pipeline with 3 major goals, which were chosen as priority objectives based on discussions with NHGRI. In Specific Aim 1, we will complete the analysis of the remaining ORF clones that are available (~4000 clones). We will also re- visit 1,000 ORFs that failed to generate stable cell lines in the first pass analysis, and will examine 1,000 membrane proteins using a different detergent that more readily solubilizes certain classes of membrane proteins than the detergent used in the "first-pass" analysis. In Specific Aim 2, we will test the cell-type specificity of interactions for the first 2600 baits in a second cell line (HCT116), thereby providing validation for a subset of interactions and revealing candidate cell-type-specific interactions. In Specific Aim 3, we will employ a Tandem Mass Tagging approach to compare interactions for ~50 important disease genes and several mutant alleles for each, thereby implementing a highly comparative platform for understanding how disease mutations alter signaling networks, molecular machines, and pathways. All data will be disseminated through public databases and will be subject to extensive bioinformatics and network analysis.