Poster abstracts
Poster number 18 submitted by Danni Jin
Disease-associated point mutations in a bifunctional aminoacyl-tRNA synthetase elicit the integrated stress response
Danni Jin (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210), Nathan Kudlapur (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210), Ronald Wek (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, 46202), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus OH 43210)
Abstract:
Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that catalyze the charging of specific amino acids onto cognate tRNAs. Human glutamyl-prolyl-tRNA synthetase (EPRS) is a bifunctional AARS with two catalytic domains (ERS and PRS), an N-terminal GST domain and a linker region connecting the catalytic cores. Previous work revealed a role for EPRS in the cellular integrated stress response (ISR), wherein the translation of eprs and a few other genes are selectively upregulated under stress conditions1. In this work, we investigate the effects of two EPRS point mutations recently linked to human diseases: P14R in the GST domain and E205G in the ERS catalytic domain. Patients possessing P14R/E205G compound heterozygous mutations suffer from diabetes and bone disease. WT ERS constructs were first purified and shown to display robust tRNA binding and catalytic activity in vitro. When tested individually, both point mutants displayed similar tRNA binding affinity as WT but were defective in amino acid activation. The E205G mutation also resulted in a significant loss of aminoacylation activity. The P14R mutant displayed near-WT levels of overall aminoacylation efficiency, but a 10-fold reduced kcat and similarly reduced KM, suggesting an altered catalytic mechanism. When expressed in HEK293T cells, EPRS P14R displayed significant proteolysis in the linker region, suggesting a long-range conformational effect of the point mutation. This conformational effect was confirmed by limited-protease digestion analyses in vitro. Preliminary studies also showed increased expression of ISR factors ATF4 and Chop in patient fibroblasts. We propose that the aminoacylation defects and conformational changes in EPRS mutants sensitize patient cells to stress, triggering an increased ISR that diminishes cell viability. This work has important implications for understanding AARS-associated human disease mechanisms and development of new therapeutics.
References:
1. Young, S. K. & Wek, R. C. Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response. J. Biol. Chem. 291, 16927–16935 (2016).
Keywords: Aminoacyl-tRNA synthetase, EPRS , Integrated stress response