Application of Homogeneous Time-resolved Fluorescence (HTRFTM) to Monitor Poly-ubiquitination of Wild-type P53

Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome. We have established a high throughput assay system to monitor poly-ubiquitination of wild-type p53 using a new fluorescence homogeneous technology known as Homogeneous Time-Resolved Fluorescence (HTRFTM). The Europium Cryptate [Eu(K)]-labeled ubiquitins are incorporated into poly-ubiquitin chains conjugated with the biotinylated p53. In the HTRF assay, Europium cryptate-labeled ubiquitin and streptavidin-labeled allophycocyanin (XL665) are used as the fluorescence donor and acceptor, respectively. The biotinylated p53 is ubiquitinated by ubiquitination enzymes, then by the addition of streptavidin-labeled XL665, the donor and acceptor molecules are brought in close proximity, thereby generating fluorescent signals. This time-resolved fluorescence assay system shows a sufficient signal for its application in synthetic compound screening and having almost the same level of sensitivity as that monitored by the scintillation proximity assay (SPA) using 125I-labeled ubiquitin. The detection of poly-ubiquitination of wild-type p53 by using the HTRFTM or SPA systems described here is much easier and quicker than by using conventional methods. Therefore, these new systems would be appropriate for high throughput screening of compounds for the discovery of new inhibitors of poly-ubiquitination of wild-type p53.