Abstract
Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNAGlu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.
Highlights
Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis
Each of the two mutations was carried by one parent, who are asymptomatic, and both are present in the patients but not in their unaffected healthy brother
P14 is located in the N-terminal GST-l domain, whereas E205 is in the E205G in the glutamyl-tRNA synthetase (ERS) catalytic domain, proximal to the site of ATP binding (Fig. 1, A, C, and D). We hypothesize that these residues are likely to play a critical function in the canonical role of encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) in tRNA aminoacylation, and in this study we investigated the effect of these mutations on canonical ERS enzymatic activities
Summary
Patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics. Two compound heterozygous EPRS variants were identified, resulting in P14R and E205G substitutions in the N-terminal GST-l and ERS catalytic domains, respectively To establish whether these mutations affect the canonical function of EPRS in protein synthesis, we purified WT and mutant recombinant ERS proteins and assessed their impact on enzyme structure and function in vitro, as well as on MSC association in cells. Our results provide mechanistic insights into patient-derived EPRS mutants and their linkage with stress sensitivity and disease
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