Systematic Exploration of the Human Interactome

摘要 尽管人类基因组蓝图的公布已有15年之久，但我们距离 了解人类蛋白质组的复杂性-蛋白质及其相互作用的集合 定义单个的人类细胞。蛋白质组的复杂性是巨大的和动态的，反映了 基因在单个细胞中的不同的、特定于上下文的表达，以及个别蛋白质的不同的亚型。在……里面 此外，个别蛋白质在其生命周期内可能参与几种不同的蛋白质组合。 经历动态的信号依赖的重组，以赋予不同的功能和细胞属性。 此外，许多蛋白质组合自结合和分隔产生细胞器和 细胞内的信令模块，这些模块本质上是动态的。在过去的5年里，我们设计了， 验证并应用了一个基于亲和力的大规模蛋白质相互作用伙伴分析平台 纯化-质谱学(AP-MS)被称为BioPlex，它使我们能够描述交互伙伴 在HEK293T中寻找10,000个非冗余的人诱饵蛋白。总共有近12万种蛋白质-蛋白质的图谱 相互作用是确定的。大多数都不是通过独立努力报告的。健壮性 在与其他研究以及我们对另一项类似努力的初步分析进行比较时 细胞系(HCT116)，与现有资源平行或超过现有资源，使我们能够广义地定义人类蛋白质 群落，基于相互作用伙伴预测未研究蛋白质的功能和定位，以及 定义大量的域域丰富，这些域域丰富开始赋予 网络。在这次更新中，我们寻求从三个主要方面大大加强和扩大这些努力：第一，我们 将在HCT116细胞中完成全程、10K-诱饵相互作用组，并进一步解决细胞类型多样性问题 通过对4个或更多细胞系中的2000个高优先级诱饵蛋白进行分析来进行交互作用。 第二，为了系统地解决膜蛋白相互作用的复杂性，我们将 对500种已知或预测存在于体内的蛋白质进行邻近标记(生物素化) 细胞膜，其中一半以上在UniProt中缺乏亚细胞位置。这将开始提供 对膜蛋白组装的全球理解，将有助于定义膜蛋白组装的空间结构 蛋白质组。第三，我们将开发200个高优先级互动的量化分析平台， 突变的疾病基因，从而提供了一个关于突变如何驱动关键基因重组的深入观点 网络。总而言之，这些研究将：i)确定多种细胞类型的相互作用，ii)提供空间 膜蛋白结构的观点，以及iii)开始在全球范围内定义突变等位基因如何短路的关键 促进疾病的细胞系统。 1

SUMMARY Although the publication of a human genome blueprint occurred more than 15 years ago, we remain far from understanding the complexities of the human proteome – the collections of proteins and their interactions that define individual human cells. The complexity of the proteome is both immense and dynamic, reflecting diverse, context-specific expression of genes in individual cells and distinct isoforms for individual proteins. In addition, individual proteins may participate in several distinct protein assemblies during their lifetime and undergo dynamic signal-dependent re-organization in order to impart distinct functions and cellular attributes. Moreover, numerous protein assemblies self-combine and compartmentalize to generate organelles and signaling modules within the cell, which are inherently dynamic. During the past 5 years, we have designed, validated, and applied a platform for the large scale analysis of protein interaction partners using affinity purification-mass spectrometry (AP-MS) termed BioPlex, which has allowed us to profile interaction partners for 10,000 nonredundant human bait proteins in HEK293T. In total, an atlas of nearly 120,000 protein-protein interactions was identified. The majority have not been reported through independent efforts. The robustness of BioPlex, when benchmarked against other studies as well as our initial analysis of a similar effort in another cell line (HCT116), parallels or exceeds available resources, allowing us to broadly define human protein communities, predict functions and localizations of unstudied proteins based on interaction partners, and define a large number of domain-domain enrichments that begin to impart structural architecture upon the network. In this renewal, we seek to greatly enhance and extend these efforts in three major ways: First, we will complete a full-pass, 10K-bait interactome in HCT116 cells and further address cell type diversity in interactomes by performing an analysis of 2000 high-priority bait proteins in 4 or more additional cell lines. Second, in order to systematically address the complexities of membrane protein interactomes, we will perform proximity labeling (biotinylation) across a broad range of 500 proteins known or predicted to reside in cellular membranes, more than half of which lack sub-cellular locations in UNIPROT. This will begin to provide a global understanding of membrane protein assemblies and will help to define the spatial architecture of the proteome. Third, we will develop a platform for quantitative analysis of the interactomes of 200 high-priority, mutant disease genes, thereby providing an in-depth view of how mutations drive reorganization of key networks. Together, these studies will: i) define interactomes across multiple cell types, ii) provide a spatial view of membrane protein architecture, and iii) begin to globally define how mutant alleles short-circuit key cellular systems to promote disease. 1