UC Riverside

SUMOylation is an important post-translational protein modification mechanism which plays important roles in a variety of biological processes. Conjugation and deconjugation of SUMO is a cascade event requiring multiple protein-protein interactions between SUMO, SUMO E1-E3 enzymes and SUMO substrates. The nature of the SUMOylation network indicates a great potential for small chemical inhibitors to be used in the investigation and manipulation of this important pathway. 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. The goal of my research is to develop FRET-based techniques to study protein-protein interactions in the SUMOylation network and to identify bioactive chemical compounds which can inhibit the activity of this pathway. Using engineered fluorescent proteins to modify protein components involved in the SUMOylation pathway, I set up a FRET-based assay and developed mathematical algorithms to determine the binding affinity of non-covalent protein-protein interactions. I then developed this FRET-based assay into high-throughput screening platforms to look for small chemical inhibitors which can disrupt the interaction of SUMO1 and its E2 enzyme, Ubc9. I further used FRET-based strategy to set up a whole-pathway screening assay to identify SUMOylation/deSUMOylation inhibitors. Over 200,000 compounds were screened and the positive hits were characterized for their activities in vitro and in cells. Finally, I was able to use FRET to detect the formation of intermediate protein complex during SUMOylation reactions, and used the assay to map the targets of the SUMOylation inhibitors identified from the high-throughput screening. These FRET-based applications provide powerful tools for the study of the SUMOylation pathway and can be used in the investigation of protein-protein interactions in other biological pathways.