Structure-based identification of salicylic acid derivatives as malarial threonyl tRNA-synthetase inhibitors.

Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Threonyl t-RNA synthetase (ThrRS) is one of the enzymes involved in this pathway, and it has been validated as an anti-malarial drug target. Here, we present 9 structurally diverse low micromolar Plasmodium falciparum ThrRS inhibitors that were identified using high-throughput virtual screening (HTVS) and were verified in a FRET enzymatic assay. Salicylic acid-based compound (LE = 0.34) was selected as a most perspective hit and was subjected to hit-to-lead optimisation. A total of 146 hit analogues were synthesised or obtained from commercial vendors and were tested. Structure-activity relationship study was supported by the crystal structure of the complex of a salicylic acid analogue with a close homologue of the plasmodium target, E. coli ThrRS (EcThrRS). Despite the availability of structural information, the hit identified via virtual screening remained one of the most potent PfThrRS inhibitors within this series. However, the compounds presented herein provide novel scaffolds for ThrRS inhibitors, which could serve as starting points for further medicinal chemistry projects targeting ThrRSs or structurally similar enzymes.

Beyond protein synthesis: non-translational functions of threonyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARSs) play an indispensable role in the translation of mRNAs into proteins. It has become amply clear that AARSs also have non-canonical or non-translational, yet essential, functions in a myriad of cellular and developmental processes. In this mini-review we discuss the current understanding of the roles of threonyl-tRNA synthetase (TARS) beyond protein synthesis and the underlying mechanisms. The two proteins in eukaryotes - cytoplasmic TARS1 and mitochondrial TARS2 - exert their non-canonical functions in the regulation of gene expression, cell signaling, angiogenesis, inflammatory responses, and tumorigenesis. The TARS proteins utilize a range of biochemical mechanisms, including assembly of a translation initiation complex, unexpected protein-protein interactions that lead to activation or inhibition of intracellular signaling pathways, and cytokine-like signaling through cell surface receptors in inflammation and angiogenesis. It is likely that new functions and novel mechanisms will continue to emerge for these multi-talented proteins.

Coordination between aminoacylation and editing to protect against proteotoxicity.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

Secreted Akkermansia muciniphila threonyl-tRNA synthetase functions to monitor and modulate immune homeostasis.

Commensal bacteria are critically involved in the establishment of tolerance against inflammatory challenges, the molecular mechanisms of which are just being uncovered. All kingdoms of life produce aminoacyl-tRNA synthetases (ARSs). Thus far, the non-translational roles of ARSs have largely been reported in eukaryotes. Here, we report that the threonyl-tRNA synthetase (AmTARS) of the gut-associated bacterium Akkermansia muciniphila is secreted and functions to monitor and modulate immune homeostasis. Secreted AmTARS triggers M2 macrophage polarization and orchestrates the production of anti-inflammatory IL-10 via its unique, evolutionary-acquired regions, which mediates specific interactions with TLR2. This interaction activates the MAPK and PI3K/AKT signaling pathways, which converge on CREB, leading to an efficient production of IL-10 and suppression of the central inflammatory mediator NF-κB. AmTARS restores IL-10-positive macrophages, increases IL-10 levels in the serum, and attenuates the pathological effects in colitis mice. Thus, commensal tRNA synthetases can act as intrinsic mediators that maintain homeostasis.

Synthesis and evaluation of an agrocin 84 toxic moiety (TM84) analogue as a malarial threonyl tRNA synthetase inhibitor.

An analogue of a toxic moiety (TM84) of natural product agrocin 84 containing threonine amide instead of 2,3-dihydroxy-4-methylpentanamide was prepared and evaluated as a putative Plasmodium falciparum threonyl t-RNA synthetase (PfThrRS) inhibitor. This TM84 analogue features submicromolar inhibitory potency (IC50 = 440 nM) comparable to that of borrelidin (IC50 = 43 nM) and therefore complements chemotypes known to inhibit malarial PfThrRS, which are currently limited to borrelidin and its analogues. The crystal structure of the inhibitor in complex with the E. coli homologue enzyme (EcThrRS) was obtained, revealing crucial ligand-protein interactions that will pave the way for the design of novel ThrRS inhibitors.

Loss of threonyl-tRNA synthetase-like protein Tarsl2 has little impact on protein synthesis but affects mouse development.

Aminoacyl-tRNA synthetases (aaRSs) are essential components for mRNA translation. Two sets of aaRSs are required for cytoplasmic and mitochondrial translation in vertebrates. Interestingly, TARSL2 is a recently evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase) and represents the only duplicated aaRS gene in vertebrates. Although TARSL2 retains the canonical aminoacylation and editing activities in vitro, whether it is a true tRNA synthetase for mRNA translation in vivo is unclear. In this study, we showed that Tars1 is an essential gene since homozygous Tars1 KO mice were lethal. In contrast, when Tarsl2 was deleted in mice and zebrafish, neither the abundance nor the charging levels of tRNAThrs were changed, indicating that cells relied on Tars1 but not on Tarsl2 for mRNA translation. Furthermore, Tarsl2 deletion did not influence the integrity of the multiple tRNA synthetase complex, suggesting that Tarsl2 is a peripheral member of the multiple tRNA synthetase complex. Finally, we observed that Tarsl2-deleted mice exhibited severe developmental retardation, elevated metabolic capacity, and abnormal bone and muscle development after 3 weeks. Collectively, these data suggest that, despite its intrinsic activity, loss of Tarsl2 has little influence on protein synthesis but does affect mouse development.

ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels.

Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNAThr). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNAThr levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS.

Tyrosine-targeted covalent inhibition of a tRNA synthetase aided by zinc ion.

Aminoacyl-tRNA synthetases (AARSs), a family of essential protein synthesis enzymes, are attractive targets for drug development. Although several different types of AARS inhibitors have been identified, AARS covalent inhibitors have not been reported. Here we present five unusual crystal structures showing that threonyl-tRNA synthetase (ThrRS) is covalently inhibited by a natural product, obafluorin (OB). The residue forming a covalent bond with OB is a tyrosine in ThrRS active center, which is not commonly modified by covalent inhibitors. The two hydroxyl groups on the o-diphenol moiety of OB form two coordination bonds with the conserved zinc ion in the active center of ThrRS. Therefore, the ß-lactone structure of OB can undergo ester exchange reaction with the phenolic group of the adjacent tyrosine to form a covalent bond between the compound and the enzyme, and allow its nitrobenzene structure to occupy the binding site of tRNA. In addition, when this tyrosine was replaced by a lysine or even a weakly nucleophilic arginine, similar bonds could also be formed. Our report of the mechanism of a class of AARS covalent inhibitor targeting multiple amino acid residues could facilitate approaches to drug discovery for cancer and infectious diseases.

Threonyl-tRNA synthetase gene, a potential target for RNAi-based control of three rice planthoppers.

BACKGROUND: RNA interference (RNAi) has potential as a new strategy for pest control. However, the current overemphasis on the control of a single pest increased control costs. The aim of this study was to find a green method of controlling several pests without affecting the natural enemies with a single target gene. One possible RNAi target is the threonyl-tRNA synthetase (ThrRS), which is conserved and plays a significant role in protein biosynthesis. RESULTS: In this study, one threonyl-tRNA synthetase gene (NlthrS) was identified from the brown planthopper (Nilaparvata lugens). Spatio-temporal expression pattern analysis showed that NlthrS was highly expressed in the ovary, late embryogenesis, nymphs and female adults. In addition, RNAi-mediated knockdown of NlthrS caused 85.6% nymph mortality, 100% female infertility, molting disorder, extended nymph duration and shortened adult longevity. Target-specific results were obtained when dsNlthrS was used to interfere with the whiteback planthopper (Sogatella furcifera), small brown planthopper (Laodelphax striatellus), zig-zag winged leafhopper (Inazuma dorsalis) and their natural enemy (green mirid bug, Cyrtorhinus lividipennis). In addition, dsNlthrS could cause high mortalities of three species of planthoppers (85.6-100%), while only dsNlthrS-1 led to the death (97.3%) of I. dorsalis that was not affected by dsNlthrS-2. Furthermore, neither dsNlthrS-1 nor dsNlthrS-2 could influence the survival of C. lividipennis. CONCLUSION: The results reveal the biological functions of ThrRS in N. lugens in addtion to its protein synthesis, deepening our understanding of tRNA synthase in insects and providing a new method for the control of several rice pests via one dsRNA design. © 2022 Society of Chemical Industry.

Comparative QM/MM study on the inhibition mechanism of ß-Hydroxynorvaline to Threonyl-tRNA synthetase.

ß-Hydroxynorvaline (ßHNV) is unnatural amino acid structurally identical to the threonine amino acid with branched ethyl group instead of threonine's methyl. It is a known competitive inhibitor that readily bind to Threonyl-tRNA synthetase's (ThrRS) catalytic site and blocks its function. In this work, we utilized a combination of Molecular Dynamics simulation (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) methodologies to provide mechanistic insights into its inhibition reaction for ThrRS. Due to the presence of Zn(II) with its Lewis acidity character, only the ionized form of ßHNV gives an enzymatically feasible energy barrier. Furthermore, in consistence with the homochirality behavior of this active site, we observed only one conformation of ßHNV that could be acylated in the active site of ThrRS. Considering these new findings together with the recent search for new antibacterial agents, our findings should guide pharmaceutical scientists with further knowledge regarding the chemical nature of this drug. Moreover, benchmarking analysis of the utilized DFT functional has also been performed to identify the impact of various DFT functionals on representing the geometry and kinetics of our system. Notably, our Zn(II) containing chemical models are found to be responsive to the %HF contribution included together with the dispersion correction. Importantly, the BP86(0%HF)-D3 functional is found to display the greatest impact on the rate-limiting step kinetically. The crucial role played by Zn(II) is further enriched when its mutation with the chemically similar Cd(II) led to dramatic difference via obtaining less feasible reaction mechanism from thermodynamic and kinetic perspectives.

Knockdown of mitochondrial threonyl-tRNA synthetase 2 inhibits lung adenocarcinoma cell proliferation and induces apoptosis.

Lung cancer is a significant global burden. Aminoacyl-tRNA synthetases (aaRSs) can be reliably identified by the occurrence and improvement of tumors. Threonyl-tRNA synthetase (TARS) and mitochondrial threonyl-tRNA synthetase 2 (TARS2) are both aaRSs. Many studies have shown that TARS are involved in tumor angiogenesis and metastasis. However, TARS2 has not yet been reported in tumors. This study explored the role of TARS2 in the proliferation and apoptosis of lung adenocarcinoma (LUAD). TARS2 expression in lung adenocarcinoma and non-cancerous lung tissues was detected via immunohistochemistry. Cell proliferation was detected using MTS, clone formation, and EdU staining assays. Flow cytometry was used to detect cell cycle, mitochondria reactive oxygen species (mROS) production, and apoptosis. Mitochondrial membrane potential (MMP ΔΨm) was detected using JC-1 fluorescent probes. Cell cycle, apoptosis-related pathway, and mitochondrial DNA (mtDNA) -encoded protein expression was detected via Western blotting. Finally, the effect of TARS2 on tumor growth was examined using a xenotransplanted tumor model in nude mice. We found that TARS2 was highly expressed in lung adenocarcinoma tissues and associated with poor overall survival (OS). Mechanistic analysis showed that knockdown of TARS2 inhibited proliferation through the retinoblastoma protein (RB) pathway and promoted mROS-induced apoptosis. Knockdown of TARS2 inhibits tumor growth in a xenotransplanted tumor model. TARS2 plays an important role in LUAD cell proliferation and apoptosis and may be a new therapeutic target.

Elucidating the molecular mechanisms associated with TARS2-related mitochondrial disease.

TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. In addition, we document seven novel TARS2 variants-one nonsense variant and six missense variants-that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modeling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.

Rice TSV2 encoding threonyl-tRNA synthetase is needed for early chloroplast development and seedling growth under cold stress.

Threonyl-tRNA synthetase (ThrRS), one of the aminoacyl-tRNA synthetases (AARSs), plays a crucial role in protein synthesis. However, the AARS functions on rice chloroplast development and growth were not fully appraised. In this study, a thermo-sensitive virescent mutant tsv2, which showed albino phenotype and lethal after the 4-leaf stage at 20°C but recovered to normal when the temperatures rose, was identified and characterized. Map-based cloning and complementation tests showed that TSV2 encoded a chloroplast-located ThrRS protein in rice. The Lys-to-Arg mutation in the anticodon-binding domain hampered chloroplast development under cold stress, while the loss of function of the ThrRS core domain in TSV2 fatally led to seedling death regardless of growing temperatures. In addition, TSV2 had a specific expression in early leaves. Its disruption obviously resulted in the downregulation of certain genes associated with chlorophyll biosynthesis, photosynthesis, and chloroplast development at cold conditions. Our observations revealed that rice nuclear-encoded TSV2 plays an important role in chloroplast development at the early leaf stage under cold stress.

A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation.

Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.

Contribution of upregulated aminoacyl-tRNA biosynthesis to metabolic dysregulation in gastric cancer.

BACKGROUND AND AIM: Metabolic reprogramming is characterized by dysregulated levels of metabolites and metabolic enzymes. Integrated metabolomic and transcriptomic data analysis can help to elucidate changes in the levels of metabolites and metabolic enzymes, screen the core metabolic pathways, and develop novel therapeutic strategies for cancer. METHODS: Here, the metabolome of gastric cancer tissues was determined using liquid chromatography-mass spectrometry. The transcriptome data from The Cancer Genome Atlas dataset were integrated with the liquid chromatography-mass spectrometry data to identify the common dysregulated gastric cancer-specific metabolic pathways. Additionally, the protein expression and clinical significance of key metabolic enzymes were examined using a gastric cancer tissue array. RESULTS: Metabolomic analysis of 16 gastric cancer tissues revealed that among the 15 dysregulated metabolomic pathways, the aminoacyl-tRNA biosynthesis pathway in the gastric tissues was markedly upregulated relative to that in the adjacent noncancerous tissues, which was consistent with the results of transcriptome analysis. Bioinformatic analysis revealed that among the key regulators in the aminoacyl-tRNA biosynthesis pathway, the expression levels of threonyl-tRNA synthetase (TARS) and phenylalanyl-tRNA synthetase (FARSB) were correlated with tumor grade and poor survival, respectively. Additionally, gastric tissue array data analysis indicated that TARS and FARSB were upregulated in gastric cancer tissues and were correlated with poor prognosis and tumor metastasis. CONCLUSIONS: This study demonstrated that the aminoacyl-tRNA biosynthesis pathway is upregulated in gastric cancer and both TARS and FARSB play key roles in the progression of gastric cancer. Additionally, a novel therapeutic strategy for gastric cancer was proposed that involves targeting the aminoacyl-tRNA biosynthesis pathway.

miR-720 is a key regulator of glioma migration and invasion by controlling TARSL2 expression.

Glioblastoma (GBM) is the most lethal type of primary brain tumor and is characterized by diffuse infiltrative growth. However, the mechanisms that control this phenotype remain largely unknown. Emerging evidence has demonstrated that the abnormal expression of microRNAs and their target genes are involved in the migration and invasion of glioma cells. In this study, we demonstrated that microRNA-720 (miR-720) was significantly upregulated in glioma tissues and cells. Functional experiments showed that overexpression of miR-720 promotes glioma migration and invasion, while downregulation of miR-720 inhibits glioma migration and invasion. Meanwhile, we found that threonyl-tRNA synthetase like-2 (TARSL2) was a direct and functional target of miR-720 in glioma. Reintroduction of TARSL2 into glioma cells repressed the invasion promoting function of miR-720, whereas downregulation of TARSL2 reversed the anti-invasion function of anti-miR-720. Furthermore, quantitative real-time polymerase chain reaction results showed that miR-720 was inversely correlated with TARSL2 expression in 40 GBM tissues. Finally, in vivo experiments showed that miR-720 promotes glioma growth and upregulates invasion-related genes in nude mice. Overall, our findings suggest increasing miR-720 enhances glioma migration and invasion through downregulation of TARSL2, which may provide novel insight into the treatment of glioma.

A 64-Year-Old Man With Multifocal Infiltrates.

CASE PRESENTATION: A 64-year-old previously healthy man presented with 8 weeks of progressive dyspnea on exertion and cough. Prior to presentation, the patient was able to bicycle > 60 miles per week and work full-time in a home improvement store. He was up-to-date with age-appropriate cancer screening and immunizations, and home medications included famotidine for reflux and nonsteroidal antiinflammatories for osteoarthritis, both as-needed. He had no significant respiratory exposure, aside from previous work as an electrician. His symptoms began in mid-February 2020 amid the coronavirus disease 2019 pandemic, although he had no known exposure to the virus.

Clinical features of anti-synthetase syndrome associated interstitial lung disease: a retrospective cohort in China.

BACKGROUND: Anti-synthetase syndrome (ASSD) is a chronic autoimmune condition characterized by antibodies directed against an aminoacycl transfer RNA synthetase (ARS) along with a group of clinical features including the classical clinical triad: inflammatory myopathy, arthritis, and interstitial lung disease (ILD). ASSD is highly heterogenous due to different organ involvement, and ILD is the main cause of mortality and function loss, which presents as different patterns when diagnosed. We designed this retrospective cohort to describe the clinical features and disease behaviour of ASSD associated ILD. METHODS: Data of 108 cases of ASSD associated ILD were retrospectively collected in Beijing Chaoyang Hospital from December 2017 to March 2019. Data were obtained from the Electronic Medical Record system. Patients were divided into 5 groups according to distinct aminoacyl tRNA synthetase (ARS) antibodies. RESULTS: Overall, 108 consecutive patients were recruited. 33 were JO-1 positive, 30 were PL-7 positive, 23 were EJ positive, 13 were PL-12 positive and 9 were OJ positive. The JO-1 (+) group had a significant higher rate of mechanic's hand (57.6%) than other 4 groups. Polymyositis/dermatomyositis (PM/DM) was diagnosed in 25 (23.1%) patients and no difference was observed among the 5 groups. The PL-7 (+) group had a higher frequency of UIP pattern (13.3%) than the other 4 groups but the difference was not significant, and the EJ (+) group had the most frequent OP pattern (78.2%), which was significantly higher than the PL-7 (+) (P < 0.001) and PL-12 (+) groups (P = 0.025). The median follow-up time was 10.7 months, during which no patients died. All received prednisone treatment, with or without immunosuppressants. At the 6-month follow-up, 96.3% of all patients (104/108) had a positive response to therapy, the JO-1 (+) and EJ (+) groups had a significantly higher improvement of forced vital capacity than the other 3 groups (P < 0.05), and the PL-7 group had the lowest FVC improvement (P < 0.05). The JO-1 (+) group and EJ (+) group had significantly higher anti-Ro-52 positive occurrence than the other 3 groups (P < 0.05). CONCLUSION: Anti PL-7 antibody had the same frequency as anti-JO-1 in ASSD-ILD, in which the ILD pattern was different with distinct anti-ARS antibodies. Most ASSD-ILD had a positive response to steroid therapies, with or without immunosuppressants. The PL-7 (+) group had the highest occurrence of UIP pattern, and a significantly lower response to therapy.

Hypoxia: Uncharged tRNA to the Rescue!

A new study has identified genes that protect Caenorhabditis elegans from hypoxic stress. Genomic approaches and whole-organism proteomics reveal a regulatory interaction between a threonyl-tRNA synthetase and ribosome biogenesis that modulates global translation and hypoxic sensitivity.