Development and Application of Endogenous Epitope Tagging Technology in Human Cel

在未来十年，通过下一代人类癌症基因组测序，几乎所有的致癌基因和肿瘤抑制基因都可能被鉴定出来。随着这些正在进行的癌症基因组计划的完成，人们的注意力将不可避免地从癌症基因的鉴定转向确定它们的功能和它们控制的途径。为了开发鉴定癌症基因/途径功能的新技术，我们最近开发了一种新技术，可以快速轻松地鉴定内源性人类蛋白质(i)在人类细胞中的相互作用伙伴，（ii）不需要对感兴趣的单个蛋白质的高质量抗体，（iii）不需要表位标记转基因的异位表达。这种方法，我们称之为“内源性表位标记”，利用了人类基因组修饰的最新进展，使得通过修饰其基因的内源性等位基因，相对快速和容易地将表位标签添加到蛋白质的氨基或羧基端成为可能。在培养等基因亲本细胞和表位标记衍生物后，制备蛋白裂解物并进行免疫沉淀/质谱分析。通过这种方法，可以以无偏倚的方式鉴定来自表位标记细胞的免疫沉淀蛋白，而不是来自缺乏表位标记的等基因亲本细胞，从而鉴定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低级真核生物酿酒酵母（Saccharomyces cerevisiae）的完整相互作用组，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发展这项技术，使更有效的蛋白质生产和纯化（Specific Aim #1），并将该技术应用于癌症途径相互作用组的初步鉴定（Specific Aim #2）。这些研究的长期目标是为最终扩大这些努力在完整信号转导途径的规模，并最终整个人类蛋白质组提供基础。

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.