DEVELOPMENT AND APPLICATION OF ENDOGENOUS EPITOPE TAGGING TECHNOLOGY IN HUMAN CEL


Year of Award:
2010
Award Type:
R21
Project Number:
CA143282
RFA Number:
RFA-CA-09-006
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
WALDMAN, TODD A
Other PI or Project Leader:
N/A
Institution:
GEORGETOWN UNIVERSITY
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.