Evaluation of ULK-1 as a Target for non-canonical TARS Regulation of the Autophagy Pathway in Ovarian Cancer
Bellotte, Jacob Ward
Bellotte, Jacob Ward
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Abstract
Previous experiments have suggested that Threonyl tRNA Synthetase (TARS) has a regulatory role within the autophagy cell-signaling pathway, a crucial mechanism of survival that enables cancer cells to overcome both nutrient deprivation and pharmaceutical intervention. It is speculated that TARS performs a noncanonical enzymatic reaction, whereupon it is able to aminoacylate autophagy activating kinase (ULK-1), effectively inhibiting its subsequent activation and downstream signal transduction. To assess the effects of TARS on autophagy, we measured autophagy in the absence of TARS using siRNA with quantitative western blotting, and we examined a direct modification of the autophagy protein ULK-1 by TARS. Western blotting results indicated that TARS knockdown was associated with an increase in phosphorylated/activated ULK-1, although another autophagy marker p62 was unaffected. Purified proteins were used to determine if TARS modifies ULK-1. The kinase domain of human ULK-1 was expressed in E. Coli and purified via FPLC. Once purified, ULK-1 was subjected to an in vitro aminoacylation reaction with purified TARS, and the sample products were separated using SDS-PAGE. The extracted bands are in the process of being analyzed via mass spectrometry in order to determine possible “threonylation” of ULK-1. The results of these experiments have the potential to expand our knowledge of the noncanonical regulatory functions of tRNA synthetases in biochemical signaling pathways, as well as providing a potential avenue for drug intervention in the search for effective cancer therapies. Future studies should aim to elucidate an in vivo model for this reaction.
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9:00 AM
1:00pm-3:00pm
Undergraduate
1:00pm-3:00pm
Undergraduate
Date
2019-01-01