Development of a Universal FRET Technology for Determining Biochemical and Pharmaceutical Parameters and Application in Deciphering the Interplay Between Influenza Viruses and SUMOylation Pathway
- Author(s): Xiong, Zhehao
- Advisor(s): Liao, Jiayu
- et al.
SUMOylation is one of the most important post-translational modifications, which plays pivotal roles in many physiological processes. SUMOylation is a multi-step enzymatic cascade, which involves multiple protein-protein interactions and regulates protein activity in many aspects. Misregulation of the SUMO pathway has been associated with many types of diseases, including viral infection, tumorigenesis, and neurodegenerative diseases. Thus, SUMOylation has great potential to be a target for the development of novel antiviral and anticancer agents. Förster Resonance Energy Transfer (FRET) is an energy transfer process that occurs between two interacting fluorophores with overlapping spectra, and is widely used to study protein-protein interactions. Previously, our group has developed quantitative FRET assay for determining protein interaction affinity and enzymatic kinetics. Here, my research goal is to further develop quantitative FRET (qFRET) technology into a robust and reliable method for determining biochemical parameters, and to investigate the interplay between SUMOylation and influenza replication. Specifically, I have proven our high-sensitive FRET technology can be used to determine protein interaction affinity even without protein purification, and utilizing this strategy I have for the first time measured the Kd between SUMO E3-PIAS1 with SUMO E2 or SUMO substrate-NS1 which elucidates the mechanism of how SUMO E3 regulates SUMOylation of different substrates. Then, I have developed a systematic method for determining mechanism of enzyme inhibition through our qFRET technology, and characterized the inhibition type and inhibition constant of our newly found SUMOylation inhibitor- STE. We have also identified Lys131 of influenza NS1 protein as the SUMOylated lysine residue which is important for virus replication through an in vitro FRET-based SUMOylation assay. And lastly, I have shown our SUMOylation inhibitor-STE exhibits great inhibition on influenza growth and can serve as a potential new anti-influenza drug. In summary, these findings prove our qFRET technology provides a powerful tool for determining biochemical parameters and dissects the role of SUMOylation in influenza viral life cycle.