Role of the Bifunctional Aminoacyl-tRNA Synthetase EPRS in Human Disease

Aminoacyl-tRNA synthetases (AARS) are a class of enzymes that catalyze the charging of tRNAs with cognate amino acids, a critical step that contributes to the fidelity of protein synthesis. Many AARSs also possess noncanonical functions such as regulation of apoptosis, mRNA translation, and RNA splicing. Some AARSs have evolved new domains with no apparent connection to their charging functions. For example, WHEP domains were originally identified in tryptophanyl-tRNA synthetase (WRS), histidyl-tRNA synthetase (HRS), and glutamyl-prolyl-tRNA synthetase (EPRS). EPRS is a unique bifunctional AARS, found only in higher eukaryotes, and consists of glutamyl-tRNA synthetase (ERS) and prolyl-tRNA synthetase (PRS) joined by a non-catalytic linker containing three WHEP domains in humans. Two compound heterozygous point mutations within human ERS (P14R and E205G) have been identified in the genomes of two patients with type 1 diabetes and bone disease. However, the mechanism by which these mutations contribute to disease is unknown. Our goal is to determine whether the point mutations affect the canonical catalytic activity of EPRS responsible for tRNA charging or noncanonical functions. Both P14 and E205 are highly conserved residues located in the GST and catalytic domain, respectively. An ERS variant appended to 2.5 WHEP domains (ERS 2.5W) has been purified and shown to display robust tRNA binding and aminoacylation activity in vitro. The P14R and E205G single mutants display the same binding affinity for tRNAGlu as WT ERS 2.5W, suggesting that the observed defect is at the catalytic step. Whereas the ERS 2.5W P14R mutant has near wild-type (WT) aminoacylation activity, the ERS 2.5W E205G variant has a severe aminoacylation defect. Both mutations, however, lead to reduced amino acid activation. Together with a collaborator, we are currently characterizing the effect of these two mutations on cell proliferation and the integrated stress response. Taken together, this work has important implications for the understanding of AARS-related human disease mechanisms and development of new therapeutics.College of Arts & SciencesOffice of Undergraduate Research & Creative InquiryNo embargoAcademic Major: Biochemistr

Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response

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

Should rapid antigen tests be first-line for COVID-19 testing? Results of a prospective urban cohort study

Abstract Background A highly accurate, rapid, and low-cost COVID-19 test is essential for guiding isolation measures. To date, the most widely used tests are either nucleic acid amplification tests or antigen tests. The objective of this study is to further assess the diagnostic performance of the Binax-CoV2 rapid antigen test in comparison to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), with additional analysis of symptomatology and cycle threshold utility. Methods This is a prospective cohort study performed between November and December 2020. Individuals who presented to COVID-19 testing events and received both RT-qPCR and a rapid antigent test were included. Testing occurred at the emergency department of an urban hospital and at a community mobile unit. No fees or appointments were required. Individuals self-reported the presence or absence of symptoms and history of positive COVID-19 test within the previous two weeks. Trained staff collected two subsequent nasopharyngeal swabs of both nares. One set of swabs underwent RT-qPCR and the other underwent Binax-CoV2 assay per manufacturer guidelines. Results A total of 390 patients were included, of which 302 were from the community site. Of these 302, 42 (14%) were RT-qPCR positive. Of the 42 RT-qPCR positive, 30 (71.4%) were also positive by Binax-CoV2. The Binax-CoV2 test had a sensitivity of 71.4% (95% CI: 55%–84%) and a specificity of 99.6% (95% CI: 98%–100%) in this population. Performance of the Binax-CoV2 test performed better in individuals with higher viral load. For symptomatic patients with cycle threshold < 20, sensitivity reached 100%. Conclusions The Binax-CoV2 assay’s specificity and sensitivity in individuals with high viral load makes it a suitable first-line test for detecting COVID-19. However, given the assay’s measured sensitivity, a negative result on the Binax-CoV2 assay may warrant additional testing with more sensitive tests, such as the RT-qPCR. This is particularly the case with high clinical suspicion for an active SARS-CoV-2 infection even after a negative Binax-CoV2 result