Identification of Genes Involved in Breast Tumor Invasion Utilizing a Ubiquitin-Mediated Proteolysis in Nitro Screen
Abstract
In this proposal, we explore the potential use of ubiquitin-dependent proteolysis as a "reverse genetics" tool in functional genomics studies. We will develop a retroviral-based system that artificially targets random cellular proteins to the proteolytic machinery for degradation. To achieve this, a randomized peptide library will be linked to a segment of the F-box motif of beta- TrCP, the F-box protein that mediates the ubiquitination of l(kappa)B(alpha) and (beta)-catenin via the multimeric SOF ubiquitin ligase. The resultant chimeric proteins are expected to direct any interacting proteins that are otherwise stable, to the SCF ligase for ubiquitination. As proof of principle, we will use 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 will be 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 chimeras 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, 2005
- Accession Number
- ADA446291
Entities
People
- Charles H. Spruck