Targeted protein degradation via ubiquitination-proteasome pathway plays an important role in many areas of biology including cancer biology. Protein ubiquitination is carried out by a set of three enzymes (an E1 ubiquitin-activating enzyme E1, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin protein ligase), which transfers the ubiquitin to the target protein to signal for destruction by the 26S proteasome. A number of tumor suppressor genes and oncogenes encode either E3 ligases (including pVHL, FBW7, MDM2, BRCA1, Park2 and E6) or ubiquitination targets (including p53, E2F-1, the Cyclins, c-Myc and c-Jun). Moreover, the potential substrates of all cancer-related E3 ligases have never systematically investigated in a high throughput manner. Establishing a complete list of potential substrates of all E3 ligases encoded by human genome will not only advance our understanding of biology but also help develop more effective therapeutics for treating human diseases caused by aberrant targeted protein degradation. It is well established that unstable ubiquitination targets, when fused with a bioluminescence reporter (such as firefly luciferase), can promote the degradation of their fusion partners. Therefore, measuring the reporter activity of a Protein-X firefly luciferase fusion protein could serve as an effective means to monitor the change of protein stability of endogenous Protein-X in response to a given signal. To this end, I have developed a high throughput, dual- luciferase reporter assay for measuring protein stability. The objective of this proposal is to extend the application of this approach to a genome-wide scale by generating a library of human open reading frame (ORF) firefly luciferase fusion clones with built-in reniall luciferase control and performing proof-of-principle experiments to establish the utility of this library for screening substrates and/or biomarkers of E3 ligases involved in cancer or other human diseases.
Ubiquitin-dependent protein degradation is involved in many aspects of cancer research since many tumor suppressor genes and oncogenes encode ubiquitination enzymes and substrates. The goal of this research proposal is to establish a high throughput screening approach for measuring protein stability genome-widely and to test the utility of this approach to identify potential substrates of cancer-related ubiquitination enzymes. Therefore, it holds great promise to facilitate the development of effective therapeutic cures for treating cancer and other human diseases caused by aberrant ubiquitin-dependent protein degradation.