Development and Application of Endogenous Epitope Tagging Technology in Human Cel

描述(由申请人提供)：项目概述几乎所有的癌基因和肿瘤抑制基因都可能在未来十年通过人类癌症基因组的下一代测序来识别。随着这些正在进行的癌症基因组计划接近完成，人们的注意力将不可避免地从识别癌症基因转移到确定它们的功能和它们控制的途径上。为了开发新的癌症基因/途径功能鉴定技术，我们最近开发了一种新技术，它使得快速和容易地鉴定内源性人类蛋白的相互作用伙伴成为可能：(I)在人类细胞中，(Ii)不需要针对感兴趣的单个蛋白的高质量抗体，(Iii)不需要异位表达表位标记的转基因。这种方法，我们称之为“内源性表位标记”，利用人类基因组修饰的最新进展，使得通过修改其基因的内源性等位基因，相对快速和容易地将表位标记添加到蛋白质的氨基或羧基末端成为可能。在培养同基因组的亲本细胞和标记表位的衍生物后，制备蛋白质裂解产物，并进行免疫沉淀/质谱分析。通过这种方式，可以以无偏见的方式鉴定来自表位标记的细胞而不是来自缺乏表位标记的同源亲本细胞的免疫沉淀蛋白，从而识别可能与标记蛋白相互作用的候选蛋白。值得注意的是，这种方法已经成功地应用于产生低等真核生物酿酒酵母的完全相互作用，但由于同源重组技术的局限性，直到最近才应用于人类细胞。在这里，我们建议在人类细胞中进一步开发这项技术，使蛋白质能够更有效地生产和纯化(特定目标1)，并将该技术应用于癌症途径相互作用组的初步鉴定(特定目标2)。这些研究的长期目标是为最终将这些努力扩展到完整的信号转导途径，并最终扩展到整个人类蛋白质组奠定基础。 公共卫生相关性：在这项应用中，我们建议进一步开发和应用一项新技术，使确定与癌症发病密切相关的蛋白质的功能(S)成为可能。这种洞察力有望：i)提供关于新抗癌药物潜在有效性的重要线索，ii)提供新的癌症途径联系，以及iii)帮助发现新的致癌基因和蛋白质。除了实现这些目标，这个项目的资金将使我们能够证明启动一个更大规模的方法来持续识别动态和不断演变的癌症相互作用组所需的可行性。

DESCRIPTION (provided by applicant): Project Summary Virtually all oncogenes and tumor suppressor genes will likely be identified over the next decade via next- generation sequencing of human cancer genomes. As these ongoing cancer genome projects move to completion, attention will invariably shift away from the identification of cancer genes and towards determining their functions and the pathways they control. In an effort to develop new technologies for the identification of cancer gene/pathway function, we have recently developed a new technology that makes it possible to quickly and easily identify the interaction partners of endogenous human proteins (i) in human cells, (ii) without requiring high quality antibodies to the individual proteins of interest, and (iii) without the need for ectopic expression of epitope-tagged transgenes. This approach, which we refer to as "endogenous epitope tagging," exploits recent advances in human genomic modification, making it possible to relatively quickly and easily add an epitope tag to the amino or carboxyl terminus of a protein via modification of the endogenous allele of its gene. After growing isogenic sets of parental cells and epitope-tagged derivatives, protein lysates are prepared and immunoprecipitation/mass spectrometry performed. In this way it is possible to identify, in an unbiased way, proteins immunoprecipitated from epitope-tagged cells but not from the otherwise isogenic parental cells that lack the epitope tag, thereby identifying candidate proteins that may interact with the tagged protein. Of note, such an approach has been successfully applied to generate complete interactomes of the lower eukaryote Saccharomyces cerevisiae, but because of limitations in homologous recombination technology has only very recently been applied to human cells. Here we propose to further develop the technology in human cells, enabling more efficient protein production and purification (Specific Aim #1), and to apply the technology to the initial identification of a cancer-pathway interactome (Specific Aim #2). The long-term goal of these studies is to provide a foundation for the eventual expansion of these efforts at the scale of complete signal transduction pathways, and eventually the entire human proteome. PUBLIC HEALTH RELEVANCE: In this application we propose to further develop and apply a new technology that makes it possible to determine the function(s) of proteins that are intimately linked to the pathogenesis of cancer. Such insights would be expected to: i) provide important clues about the potential effectiveness of new anticancer drugs, ii) provide new linkages cancer pathways, and iii) aid in the discovery new cancer-causing genes and proteins. In addition to accomplishing these goals, funding for this project will enable us to demonstrate the feasibility needed to initiate a larger scale approach to the ongoing identification of a dynamic and evolving cancer interactome.