Validation of interactions between aminoacyl- tRNA synthetases and cancer-associated factors using FRET-based screening system

Abstract

Aminoacyl-tRNA synthetases(ARSs) are essential proteins that are involved in cellular protein synthesis and viability. They catalyse the specific amino acids to their cognate tRNAs with a high fidelity. However, previous study shows that these enzymes are multi-functional proteins that are involved in diverse cellular processes. According to the recent study, a systematic analysis of the expression of ARSs indicates that these proteins are associated with cancer. Because of the need to identify the correlation, we developed a cell-based high throughput screen method using förster resonance energy transfer (FRET) for anticancer activity in the living cells. Fluorescence resonance energy transfer (FRET) is unique in generating fluorescence signals that are sensitive to molecular conformation, association, and separation in the 1-10nm range. At first, we constructed vectors that expressed cyan fluorescent protein (CFP) tagged ARSs and yellow fluorescent protein (YFP) tagged CAGs. Then we used a confocal microscope and calculated the FRET efficiency. As a result, we found that AIMP3 and RPSA protein pair which has higher FRET efficiency than the control. To confirm this protein-protein interaction, we conducted co-immunoprecipitation and in vitro pull down assay. From the previous experiments, we identified that the AIMP3 and RPSA has a direct interaction. Accordingly, our results indicated that FRET-based validating system could be used to detect protein-protein interactions. Moreover, with our assay, it could help with the development of new anti-cancer drugs in the future.