Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Adenosine-Dependent Activation Mechanism of Prodrugs Targeting an Aminoacyl-tRNA Synthetase.

Prodrugs have little or no pharmacological activity and are converted to active drugs in the body by enzymes, metabolic reactions, or through human-controlled actions. However, prodrugs promoting their chemical bioconversion without any of these processes have not been reported before. Here, we present an enzyme-independent prodrug activation mechanism by boron-based compounds (benzoxaboroles) targeting leucyl-tRNA synthetase (LeuRS), including an antibiotic that recently has completed phase II clinical trials to cure tuberculosis. We combine nuclear magnetic resonance spectroscopy and X-ray crystallography with isothermal titration calorimetry to show that these benzoxaboroles do not bind directly to their drug target LeuRS, instead they are prodrugs that activate their bioconversion by forming a highly specific and reversible LeuRS inhibition adduct with ATP, AMP, or the terminal adenosine of the tRNALeu. We demonstrate how the oxaborole group of the prodrugs cyclizes with the adenosine ribose at physiological concentrations to form the active molecule. This bioconversion mechanism explains the remarkably good druglike properties of benzoxaboroles showing efficacy against radically different human pathogens and fully explains the mechanism of action of these compounds. Thus, this adenosine-dependent activation mechanism represents a novel concept in prodrug chemistry that can be applied to improve the solubility, permeability and metabolic stability of challenging drugs.

Identification of LARS as an essential gene for osteosarcoma proliferation through large-Scale CRISPR-Cas9 screening database and experimental verification.

BACKGROUND: Osteosarcoma is one of the most malignant tumors, and it occurs mostly in children and adolescents. Currently, surgery and chemotherapy are the main treatments. The recurrence rate is high and the prognosis is often poor. Finding an effective target gene therapy for osteosarcoma may effectively improve its prognosis. METHOD: In this study, genes essential for the survival of osteosarcoma cells were identified by genome-wide screening of CRISPR-Cas9 based on the DepMap database. The expression of these essential genes in osteosarcoma patients' tissues and normal tissues was identified in the GSE19276 database. Functional pathway enrichment analysis, protein interaction network construction, and LASSO were performed to construct a prognostic risk model based on these essential genes. CCK8 assay was used to detect the effect of essential gene-LARS (Leucyl-TRNA Synthetase 1) on the proliferation of osteosarcoma. RESULTS: In this study, 785 genes critical for osteosarcoma cell proliferation were identified from the DepMap. Among these 785 essential genes, 59 DEGs were identified in osteosarcoma tissues. In the functional enrichment analysis, these 59 essential genes were mainly enriched in cell cycle-related signaling pathways. Furthermore, we established a risk score module, including LARS and DNAJC17, screened from these 59 genes, and this module could divide osteosarcoma patients into the low-risk and high-risk groups. In addition, knockdown of LARS expression inhibited the proliferative ability of osteosarcoma cells. A significant correlation was found between LARS expression and Monocytic lineage, T cells, and Fibroblasts. CONCLUSION: In conclusion, LARS was identified as an essential gene for survival in osteosarcoma based on the DepMap database. Knockdown of LARS expression significantly inhibited the proliferation of osteosarcoma cells, suggesting that it is involved in the formation and development of osteosarcoma. The results are useful as a foundation for further studies to elucidate a potential osteosarcoma diagnostic index and therapeutic targets.

A Novel Leucyl-tRNA Synthetase Inhibitor, MRX-6038, Expresses Anti-Mycobacterium abscessus Activity In Vitro and In Vivo.

Therapeutic options for Mycobacterium abscessus infections are extremely limited, and new drugs are needed. The anti-M. abscessus activity of MRX-6038, a new leucyl-tRNA synthetase inhibitor, was evaluated in vitro and in vivo. Antimicrobial susceptibility testing was performed on 12 nontuberculosis mycobacteria (NTM) reference strains and 227 clinical NTM isolates. A minimum bactericidal concentration assay was conducted to distinguish the bactericidal versus bacteriostatic activity of MRX-6038. The synergy between MRX-6038 and 12 clinically important antibiotics was determined using a checkerboard assay. The activity of MRX-6038 against M. abscessus residing inside macrophages was also evaluated. Finally, the potency of MRX-6038 in vivo was determined in a neutropenic mouse model that mimicked a pulmonary M. abscessus infection. MRX-6038 exhibited high anti-M. abscessus activity against extracellular M. abscessus in culture, with a MIC50 of 0.063 mg/L and a MIC90 of 0.125 mg/L. Fifty percent of the activity was bactericidal, and fifty percent was bacteriostatic. A synergy between MRX-6038 and clarithromycin or azithromycin was found in 25% of strains. No antagonism was evident between MRX-6038 and 12 antibiotics commonly used to treat NTM infections. MRX-6038 also exhibited activity against intracellular NTM, which caused a significant reduction in the bacterial load in the lungs of M. abscessus-infected neutropenic mice. In conclusion, MRX-6038 was active against M. abscessus in vitro and in vivo, and it represents a potential candidate for incorporation into strategies by which M. abscessus infections are treated.

Partitioning of the initial catalytic steps of leucyl-tRNA synthetase is driven by an active site peptide-plane flip.

To correctly aminoacylate tRNALeu, leucyl-tRNA synthetase (LeuRS) catalyzes three reactions: activation of leucine by ATP to form leucyl-adenylate (Leu-AMP), transfer of this amino acid to tRNALeu and post-transfer editing of any mischarged product. Although LeuRS has been well characterized biochemically, detailed structural information is currently only available for the latter two stages of catalysis. We have solved crystal structures for all enzymatic states of Neisseria gonorrhoeae LeuRS during Leu-AMP formation. These show a cycle of dramatic conformational changes, involving multiple domains, and correlate with an energetically unfavorable peptide-plane flip observed in the active site of the pre-transition state structure. Biochemical analyses, combined with mutant structural studies, reveal that this backbone distortion acts as a trigger, temporally compartmentalizing the first two catalytic steps. These results unveil the remarkable effect of this small structural alteration on the global dynamics and activity of the enzyme.

O-GlcNAc modification of leucyl-tRNA synthetase 1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5'AMP.

Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing ~120-130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNALeu aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5'AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5'AMP unusually slowly, relative to the two phosphorylation reactions.

Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.

Tumourigenesis and cancer progression require enhanced global protein translation1-3. Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase-along with its downstream cognate tRNAs-elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

Tissue-specific alternative splicing separates the catalytic and cell signaling functions of human leucyl-tRNA synthetase.

The aminoacyl-tRNA synthetases are an ancient and ubiquitous component of all life. Many eukaryotic synthetases balance their essential function, preparing aminoacyl-tRNA for use in mRNA translation, with diverse roles in cell signaling. Herein, we use long-read sequencing to discover a leukocyte-specific exon skipping event in human leucyl-tRNA synthetase (LARS). We show that this highly expressed splice variant, LSV3, is regulated by serine-arginine-rich splicing factor 1 (SRSF1) in a cell-type-specific manner. LSV3 has a 71 amino acid deletion in the catalytic domain and lacks any tRNA leucylation activity in vitro. However, we demonstrate that this LARS splice variant retains its role as a leucine sensor and signal transducer for the proliferation-promoting mTOR kinase. This is despite the exon deletion in LSV3 including a portion of the previously mapped Vps34-binding domain used for one of two distinct pathways from LARS to mTOR. In conclusion, alternative splicing of LARS has separated the ancient catalytic activity of this housekeeping enzyme from its more recent evolutionary role in cell signaling, providing an opportunity for functional specificity in human immune cells.

Improvement of substrate recognition in branched-chain aminoacyl-tRNA synthetases from Escherichia coli under conditions of pyrophosphate amplification.

Isoleucyl-tRNA synthetase (IleRS), leucyl-tRNA synthetase (LeuRS), and valyl-tRNA synthetase (ValRS) are enzymes that have potential for the determination of l-isoleucine, l-leucine, and l-valine in food products and plasma. However, the disadvantages of these enzymes are their specificity and sensitivity. Here, we examined the substrate specificity of IleRS, LeuRS, and ValRS under various conditions of pyrophosphate amplification to improve their specificity and sensitivity. The amount of pyrophosphate produced in IleRS, LeuRS, and ValRS reactions was amplified after the addition of excess adenosine-5'-triphosphate and magnesium ions, and was approximately 9-, 8-, and 7-fold higher, respectively, for each of the initial l-amino acid substrates (50 µM). However, in addition to their target amino acids, IleRS, LeuRS, and ValRS also reacted with l-valine, l-lysine, and l-threonine, respectively. This substrate misrecognition was overcome by making the reaction pH more acidic and by increasing the magnesium ion concentration. The pyrophosphate amplification in IleRS, LeuRS, and ValRS reactions resulted in the production of p1, p4-di (adenosine) 5'-tetraphosphate. We also observed a strong positive correlation (R = 0.99) between the amount of pyrophosphate produced and the initial concentration of l-amino acid with 5 and 50 µM l-isoleucine, l-leucine, and l-valine. Our results suggest that amino acid assays using IleRS, LeuRS, and ValRS are promising methods to accurately measure l-valine, l-isoleucine, and l-leucine in food products and plasma.

Deep phenotyping of MARS1 (interstitial lung and liver disease) and LARS1 (infantile liver failure syndrome 1) recessive multisystemic disease using Human Phenotype Ontology annotation: Overlap and differences. Case report and review of literature.

INTRODUCTION: Aminoacyl transfer RNA (tRNA) synthetases are associated with diseases when mutations occur in their encoding genes. Pulmonary alveolar proteinosis can be caused by mutation in the methionyl-tRNA synthetase (MARS) gene while mutations in the leucine-tRNA synthetase (LARS) gene lead to infantile liver failure syndrome type 1. We report the case of a patient with LARS1 pathogenics variants and two patients with MARS1 pathogenics variants. The aim of this study was to analyze the phenotypes of our three patients in detail and classify cases in the literature using Human Phenotype Ontology (HPO) terms. RESULTS: The first patient has two previously undescribed heterozygous variants in LARS1 (c.1818dup and c.463A>G). The other two patients' MARS1 variants (c.1177G>A and c.1700C>T) have already been described in the literature. All three patients had anemia, hepatomegaly, feeding difficulties, failure to thrive and hypoalbuminemia. Including ours, 65 patients are described in total, for whom 117 phenotypic abnormalities have been described at least once, 41.9% of which both in patients with LARS1 and MARS1 mutations. CONCLUSION: Patients with LARS1 and MARS1 mutations seem to share a common phenotype but further deep phenotyping studies are required to clarify the details of these complex pathologies.

Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants.

Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNALeu for protein synthesis and plays an important role in mTORC1 activation by sensing intracellular leucine concentrations. Here, we describe a protocol for the purification, reductive methylation, binding affinity measurement by microscale thermophoresis, T i value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed us to build the RagD binding domain, which was shown to be a dynamic region of LARS1 refractory to crystallization. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).

Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells.

Uncontrolled cell proliferation associated with cancer depends on the functional abrogation of at least one of tumor suppressor. In response to nutrient cue, tuberous sclerosis complex (TSC) works as a tumor suppressor which inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). However, the regulation mechanism of nutrient-dependent cell proliferation in TSC-null cells remains unclear. Here, we demonstrate that leucine is required for cell proliferation through the activation of leucyl-tRNA synthetase (LARS1)-mTORC1 pathway in TSC-null cells. Cell proliferation and survival were attenuated by LARS1 knock-down or inhibitors in TSC-null cells. In addition, either rapamycin or LARS1 inhibitors significantly decreased colony formation ability while their combined treatment drastically attenuated it. Taken together, we suggest that LARS1 inhibitors might considered as novel tools for the regression of tumor growth and proliferation in TSC-null tumor cells which regrow upon discontinuation of the mTORC1 inhibition.

Structure-based modification of pyrazolone derivatives to inhibit mTORC1 by targeting the leucyl-tRNA synthetase-RagD interaction.

The enzyme leucyl-tRNA synthetase (LRS) and the amino acid leucine regulate the mechanistic target of rapamycin (mTOR) signaling pathway. Leucine-dependent mTORC1 activation depends on GTPase activating protein events mediated by LRS. In a prior study, compound BC-LI-0186 was discovered and shown to interfere with the mTORC1 signaling pathway by inhibiting the LRS-RagD interaction. However, BC-LI-0186 exhibited poor solubility and was metabolized by human liver microsomes. In this study, in silico physicochemical properties and metabolite analysis of BC-LI-0186 are used to investigate the addition of functional groups to improve solubility and microsomal stability. In vitro experiments demonstrated that 7b and 8a had improved chemical properties while still maintaining inhibitory activity against mTORC1. The results suggest a new strategy for the discovery of novel drug candidates and the treatment of diverse mTORC1-related diseases.

tRNA-Dependent Import of a Transit Sequence-Less Aminoacyl-tRNA Synthetase (LeuRS2) into the Mitochondria of Arabidopsis.

Aminoacyl-tRNA synthetases (AaRS) charge tRNAs with amino acids for protein translation. In plants, cytoplasmic, mitochondrial, and chloroplast AaRS exist that are all coded for by nuclear genes and must be imported from the cytosol. In addition, only a few of the mitochondrial tRNAs needed for translation are encoded in mitochondrial DNA. Despite considerable progress made over the last few years, still little is known how the bulk of cytosolic AaRS and respective tRNAs are transported into mitochondria. Here, we report the identification of a protein complex that ties AaRS and tRNA import into the mitochondria of Arabidopsis thaliana. Using leucyl-tRNA synthetase 2 (LeuRS2) as a model for a mitochondrial signal peptide (MSP)-less precursor, a ≈30 kDa protein was identified that interacts with LeuRS2 during import. The protein identified is identical with a previously characterized mitochondrial protein designated HP30-2 (encoded by At3g49560) that contains a sterile alpha motif (SAM) similar to that found in RNA binding proteins. HP30-2 is part of a larger protein complex that contains with TIM22, TIM8, TIM9 and TIM10 four previously identified components of the translocase for MSP-less precursors. Lack of HP30-2 perturbed mitochondrial biogenesis and function and caused seedling lethality during greening, suggesting an essential role of HP30-2 in planta.

Leucine-sensing mechanism of leucyl-tRNA synthetase 1 for mTORC1 activation.

Leucyl-tRNA synthetase 1 (LARS1) mediates activation of leucine-dependent mechanistic target of rapamycin complex 1 (mTORC1) as well as ligation of leucine to its cognate tRNAs, yet its mechanism of leucine sensing is poorly understood. Here we describe leucine binding-induced conformational changes of LARS1. We determine different crystal structures of LARS1 complexed with leucine, ATP, and a reaction intermediate analog, leucyl-sulfamoyl-adenylate (Leu-AMS), and find two distinct functional states of LARS1 for mTORC1 activation. Upon leucine binding to the synthetic site, H251 and R517 in the connective polypeptide and 50FPYPY54 in the catalytic domain change the hydrogen bond network, leading to conformational change in the C-terminal domain, correlating with RagD association. Leucine binding to LARS1 is increased in the presence of ATP, further augmenting leucine-dependent interaction of LARS1 and RagD. Thus, this work unveils the structural basis for leucine-dependent long-range communication between the catalytic and RagD-binding domains of LARS1 for mTORC1 activation.

Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish.

Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb-/-) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb-/- zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb-/- zebrafish. Indeed, excessive autophagy activation was observed in larsb-/- zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1.

Design and synthesis of N-(3-sulfamoylphenyl)amides as Trypanosoma brucei leucyl-tRNA synthetase inhibitors.

The protozoan parasite Trypanosoma brucei (T. brucei) causes human African trypanosomiasis (HAT), which is a fatal and neglected disease in the tropic areas, and new treatments are urgently needed. Leucyl-tRNA synthetase (LeuRS) is an attractive target for the development of antimicrobial agents. In this work, starting from the hit compound thiourea ZCL539, we designed and synthesized a series of amides as effective T. brucei LeuRS (TbLeuRS) synthetic site inhibitors. The most potent compounds 74 and 91 showed IC50 of 0.24 and 0.25 µM, which were about 700-fold more potent than the starting hit compound. The structure-activity relationship was also discussed. These compounds provided a new scaffold and lead compounds for further development of antitrypanosomal agents.

Aminoacyl-tRNA synthetases and amino acid signaling.

Aminoacyl-tRNA synthetases (ARSs) are a family of evolutionarily conserved housekeeping enzymes used for protein synthesis that have pivotal roles in the ligation of tRNA with their cognate amino acids. Recent advances in the structural and functional studies of ARSs have revealed many previously unknown biological functions beyond the classical catalytic roles. Sensing the sufficiency of intracellular nutrients such as amino acids, ATP, and fatty acids is a crucial aspect for every living organism, and it is closely connected to the regulation of diverse cellular physiologies. Notably, among ARSs, leucyl-tRNA synthetase 1 (LARS1) has been identified to perform specifically as a leucine sensor upstream of the amino acid-sensing pathway and thus participates in the coordinated control of protein synthesis and autophagy for cell growth. In addition to LARS1, other types of ARSs are also likely involved in the sensing and signaling of their cognate amino acids inside cells. Collectively, this review focuses on the mechanisms of ARSs interacting within amino acid signaling and proposes the possible role of ARSs as general intracellular amino acid sensors.