Systematic Exploration of the Human Interactome IV

总结 蛋白质组的复杂性是巨大的和动态的，反映了不同的，特定的背景 单个细胞中的基因表达和许多蛋白质的不同同种型。此外，个别蛋白质 可能参与几个不同的蛋白质组装在他们的生活和经历动态信号- 依赖性重组，以赋予独特的功能和细胞属性。此外，许多 蛋白质组装体自我组合和划分，以产生细胞器和信号模块， 细胞，它们是内在动态的。在过去的9年里，我们设计、验证并应用了 使用亲和纯化-质谱法大规模分析蛋白质相互作用伴侣的平台 （AP-MS）称为生物质谱，它使我们能够为超过10，000个非冗余的相互作用伙伴进行分析。 人诱饵蛋白在两种对比的人细胞系HEK 293T和HCT 116中的表达。此外，我们还重申， 2000这些IP的在三个额外的细胞系-U2OS，RPE 1，和HeLa-扩大我们的探索细胞- 特定的相互作用组。总的来说，在五种不同的细胞系中进行的25，000次AP-MS实验使我们能够 定义了一个包含15,500种人类蛋白质中近280,000种蛋白质相互作用的图谱。大多数 没有通过独立的努力报告。当以下列标准为基准时， 其他研究和跨细胞系比较时，平行或超过可用资源，使我们能够 广泛定义人类蛋白质群落，预测功能和定位的基础上未研究的蛋白质 互动伙伴，并定义了大量的领域领域丰富，开始赋予结构 架构在网络上。最后，通过在多个单元中对数千个诱饵重复AP-MS实验， 线，我们已经创建了第一个也是最大的蛋白质组规模，人类相互作用组的上下文特定模型， 揭示了广泛的重塑，反映了每个细胞系的独特生物学。然而，这些珍贵的 生物网络一直是生物学的发现，它们仍然是人类的不完整模型 相互作用体近四分之一的蛋白质根本没有出现在生物医学网络中， 在这些网络中只作为猎物出现，只提供了它们相互作用的部分视图。最后，我们只有 刚刚开始在其他互补基因组学资源的背景下探索生物信息学。在这次更新中，我们 我们会从以下三个主要方面加强和扩展我们的工作：第一，我们会检控多一名 HEK293T细胞中的7，500个基因。通过这种方式，生物燃料网络将从利用所有人类的50% 基因作为诱饵，约90%用作诱饵，揭示了许多新的相互作用以前不可能。 其次，将在HCT 116细胞中检查相同的7，500个克隆，以提供验证和探索细胞系 的特异性第三，我们将整合BioFold与包括AlphaFold在内的互补基因组规模资源 和人类蛋白质图谱，以从分子相互作用到 细胞结构

SUMMARY The complexities of the proteome are both immense and dynamic, reflecting diverse, context-specific expression of genes in individual cells and distinct isoforms for many proteins. In addition, individual proteins may participate in several distinct protein assemblies during their lifetimes and undergo dynamic signal- dependent re-organization in order to impart unique 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 9 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 over 10,000 nonredundant human bait proteins in two contrasting human cell lines: HEK 293T and HCT116. In addition, we repeated 2000 of these IP’s in three additional cell lines – U2OS, RPE1, and HeLa – to broaden our exploration of cell- specific interactomes. In aggregate, these 25,000 AP-MS experiments in five distinct cell lines have enabled us to define an atlas of nearly 280,000 protein-protein interactions among 15,500 human proteins. The majority have not been reported through independent efforts. The robustness of BioPlex, when benchmarked against other studies and when compared across cell lines, 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. Finally, by repeating AP-MS experiments for thousands of baits in multiple cell lines, we have created the first and largest proteome-scale, context-specific models of the human interactome, revealing extensive remodeling that reflects the unique biology of each cell line. Yet, as valuable as these BioPlex networks have been for biological discovery, they remain incomplete models of the human interactome. Nearly 1 in 4 proteins do not appear in the BioPlex network at all, and a similar number of proteins appear in these networks as preys only, providing just a partial view of their interactions. Finally, we have only just begun to explore BioPlex in the context of other complementary genomics resources. In this renewal, we seek to greatly enhance and extend our efforts in three major ways: First, we will prosecute an additional 7,500 genes in HEK293T cells. In this way, the BioPlex network will transition from utilizing 50% of all human genes as baits to approximately 90% used as baits, revealing many novel interactions not possible previously. Second, the same 7,500 clones will be examined in HCT116 cells to provide validation and explore cell line specificity. Third, we will integrate BioPlex with complementary genome-scale resources including AlphaFold and the Human Protein Atlas to model protein interactions at scales ranging from molecular interactions to cellular structures.