Development of Quantitative FRET Technology for SENP Enzyme Kinetics Determinations and High-Sensitive High-Throughput Screening Assay for Protease Inhibitor Discovery
- Author(s): Liu, Yan
- Advisor(s): Liao, Jiayu
- et al.
SUMOylation is an important post-translational protein modification mechanism which plays important roles in a variety of biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. SENP, SUMO specific protease, can either mature pre-SUMO or deconjugate SUMO from its target proteins. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. FRET is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores in close proximity. The efficiency of FRET is highly dependent on the distance between the donor and acceptor fluorophores, which makes FRET very powerful in the detection of molecular interactions and biomolecular conformational changes.
A novel highly sensitive FRET-based protease assay was designed and developed to study the activities and specificities of SENP in SUMOylation signaling pathway and to identify bioactive chemical compounds which can specifically inhibit SENPs' activities. The endopeptidase and isopeptidase activities of SENP1/2/5/6/7c toward SUMO1/2/3 were studied and verified by western-blot assay. A novel quantitative FRET analysis was performed to study the protease kinetics to compare the specificities to substrate. The direct emissions of donor and acceptor were considered in FRET signal analysis. The mathematical algorithm dependent on pre-established standard curves or later optimized internal calibration method related the hydrolyzed substrate concentration to detected fluorescent signals during the dynamic processes, and thus derived the kinetics constants. Then, the developed protease assay and protease kinetics analysis methods were applied to study the pre-SUMO4's maturation and the product inhibition in pre-SUMO's maturation, which indicated the potential application in biomechanism study and chemical compounds' inhibition characterization. Finally, the FRET-based assay was developed to HTS assay to screen small chemical SENP inhibitors. The screening conditions were optimized to achieve satisfactory Z' factor value and high signal-to-noise ratio. 55,000 compounds were screened by the developed HTS assay. The general principles of the quantitative FRET analysis in protease kinetics, including the characterization of inhibition, can be applied to other substrate-protease with inhibitors. The FRET-based protease assay as well as the HTS assay provided a powerful tool for large-scale and high-throughput applications.