Identification of Genes Involved in Breast Tumor Invasion Utilizing a Ubiquitin-Mediated Proteolysis in Vitro Screen
Abstract
In this proposal, we explored the potential use of ubiquitin-dependent proteolysis as a reverse genetics tool in functional genomics studies. We developed a retroviral-based system that artificially targets random cellular proteins to the proteolytic machinery for degradation. To achieve this, a randomized peptide library was linked to a segment of the F-box motif of beta- TrCP, the F-box protein that mediates the ubiquitination of I(kappa)B(alpha) and (beta)-catenin via the multimeric SCF ubiquitin ligase. The resultant chimeric proteins were expected to direct any interacting proteins that are otherwise stable, to the SCF ligase for ubiquitination. As proof of principle, we used this system in a loss-of-function in vitro assay to identify putative genes involved in breast tumor invasion. MDA-MB-231 breast tumor-derived cells were tranduced with the retroviral chimera library and peptides that confer the ability to invade through an artificial extracellular matrix will be isolated using a modified Boyden chamber assay. Following multiple rounds of selection, the fusion proteins that provide invasion properties will be confirmed using tumorgenicity assays in nude mice. If successful, ubiquitination-based functional assays will undoubtedly contribute to the identification of potential protein targets for therapeutic intervention in breast cancer.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2006
- Accession Number
- ADA463461
Entities
People
- Charles H. Spruck