E6-AP: p53 (hect) Protein Degradation Redistribution Assay

• Designed to assay compounds for their ability to modulate activation of E6-AP ubiquitin E3 ligase
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Conjugation of ubiquitin to a protein substrate during protein degradation by the proteasome follows a three-step mechanism. First, the ubiquitin activating enzyme E1 activates ubiquitin to generate a high-energy thiol-ester intermediate in a reaction dependent on ATP. The activated ubiquitin is then transferred to an E2 ubiquitin conjugating enzyme, that finally transfers the ubiquitin to a substrate protein that is specifically bound by an E3 ligase.

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins. The ubiquitin E3 ligase E6-AP is utilized by the E6 oncoprotein to target p53 for degradation. Downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines.

Figure 1. Inhibition of proteasomal degradation of p53(44-Ct)-EGFP. Cells were transfected with siRNAs or treated with the proteasome inhibitor MG-132. Knockdown of E6-AP ligase or treatment with MG-132 induces stabilization of p53(44-Ct)-EGFP, while knockdown of other E3 ligases results in no accumulation (Skp2 and βTRCP).

Figure 2. MG-132 concentration response in the E6-AP:p53 Protein Degradation assay: The EC₅₀ is ~3 μM. Concentration response was measured in 9 point half log dilution series (n=8). Cells were treated with test compound for 24 hr. Cells were then fixed and increase in fluorescence intensity was measured using the Cellomics ArrayScan V^TI Reader and the MolecularTranslocationV2 BioApplication. % activity was calculated relative to the positive (5 μM MG-132) and negative control (0.25% DMSO).