Development and Application of Endogenous Epitope Tagging Technology in Human Cel

人细胞内源表位标记技术的开发及应用

• 批准号: 8044714
• 负责人: TODD A WALDMAN
• 金额: $ 19.43万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8044714
• 关键词: Alleles; Antibodies; Antineoplastic Agents; Area; Attention; Cancer Etiology; Cell Line; Cells; Complex; Computer software; Data; Data Set; Development; Ectopic Expression; Effectiveness; Epitopes; Eukaryota; Foundations; Funding; Gene Proteins; Gene Targeting; Gene-Modified; Generations; Genes; Genomics; Goals; Human; Human Cell Line; Immunoprecipitation; Individual; Link; MAP Kinase Gene; MDM2 gene; Malignant Neoplasms; Mass Spectrum Analysis; Modification; Molecular; Oncogenes; PTEN gene; Pathogenesis; Pathway interactions; Play; Production; Protein p53; Proteins; Proteome; Proteomics; Proteomics Shared Resource; Publishing; Research Personnel; Role; Saccharomyces cerevisiae; Signal Pathway; Signal Transduction Pathway; Somatic Cell; Specificity; Staging; Suspension substance; Suspensions; Technology; Time; Transgenes; Tumor Suppressor Genes; cancer genome; cancer type; design; homologous recombination; improved; insight; interest; new technology; next generation; novel; p53-binding protein; protein complex; protein function; public health relevance; success; technology development

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.

描述（由申请人提供）：项目概要几乎所有的癌基因和肿瘤抑制基因都将在未来十年内通过人类癌症基因组的下一代测序来鉴定。随着这些正在进行的癌症基因组计划的完成，注意力将不可避免地从癌症基因的识别转移到确定它们的功能和它们控制的途径。为了开发用于鉴定癌症基因/途径功能的新技术，我们最近开发了一种新技术，该技术使得可以快速且容易地鉴定（i）人细胞中内源性人蛋白质的相互作用伴侣，（ii）不需要针对感兴趣的单个蛋白质的高质量抗体，并且（iii）不需要表位标记的转基因的异位表达。我们称之为“内源性表位标记”的这种方法利用了人类基因组修饰的最新进展，使得可以通过修饰蛋白质基因的内源性等位基因相对快速且容易地将表位标记添加到蛋白质的氨基或羧基末端。在生长亲本细胞和表位标记的衍生物的等基因组后，制备蛋白质裂解物并进行免疫沉淀/质谱分析。以这种方式，有可能以无偏的方式鉴定从表位标记的细胞免疫沉淀的蛋白质，但不鉴定从缺乏表位标记的其他等基因亲本细胞免疫沉淀的蛋白质，从而鉴定可能与标记的蛋白质相互作用的候选蛋白质。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整相互作用组，但由于同源重组技术的局限性，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该技术，实现更有效的蛋白质生产和纯化（具体目标#1），并将该技术应用于癌症途径相互作用组的初步鉴定（具体目标#2）。这些研究的长期目标是为最终在完整的信号转导通路的规模上扩展这些努力提供基础，并最终扩展到整个人类蛋白质组。 公共卫生相关性：在本申请中，我们提出进一步开发和应用一种新技术，该技术使得有可能确定与癌症发病机制密切相关的蛋白质的功能。这些见解将有望：i）提供有关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，iii）帮助发现新的致癌基因和蛋白质。除了实现这些目标，该项目的资金将使我们能够证明启动更大规模的方法来持续识别动态和不断发展的癌症相互作用组所需的可行性。