Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

Elucidating the structure-function attributes of a trypanosomal arginyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2.

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

Preparation of tRNAArg for Arginylation Assay by In Vitro Transcription.

This chapter describes the preparation of tRNAArg by in vitro transcription. tRNA produced by this method can be efficiently utilized for in vitro arginylation assays, following aminoacylation with Arg-tRNA synthetase, either directly during the arginylation reaction or separately to produce the purified preparation of Arg-tRNAArg. tRNA charging is described in other chapters of this book.

Enzymatic Aminoacylation of tRNAArg Using Recombinant Arg-tRNA Synthetase.

This chapter describes the preparation of pre-charged Arg-tRNA that can be used in arginylation reaction. While in a typical arginylation reaction arginyl-tRNA synthetase (RARS) is normally included as a component of the reaction and continually charges tRNA during arginylation, it is sometimes necessary to separate the charging and the arginylation step, in order to perform each reaction under controlled conditions, e.g., for measuring the kinetics or determining the effect of different compounds and chemicals on the reaction. In such cases, tRNAArg can be pre-charged with Arg and purified away from the RARS enzyme prior to arginylation.

Synthesis of Stably Charged Arg-tRNAArg for Structural Analysis.

Posttranslational protein arginylation catalyzed by arginyl transferases is a mechanism to regulate multiple physiological processes. This protein arginylation reaction uses a charged Arg-tRNAArg as the donor of arginine (Arg). The inherent instability of the ester linkage of the arginyl group to the tRNA, which is sensitive to hydrolysis at the physiological pH, makes it difficult to obtain structural information on how the arginyl transfer reaction is catalyzed. Here, we describe a methodology to synthesize stably charged Arg-tRNAArg that would facilitate structural analysis. In the stably charged Arg-tRNAArg, the ester linkage is replaced with an amide linkage, which is resistant to hydrolysis even at alkaline pH.

Functional Interplay between Arginyl-tRNA Synthetases and Arginyltransferase.

Protein arginylation, mediated by arginyltransferase ATE1, is a post-translational modification of emerging biological importance that consists of transfer of the amino acid Arg to protein and peptide substrates. ATE1 utilizes charged tRNAArg as the donor of the arginyl group, which depends on the activity of Arg-tRNA synthetases (RARS) and is also utilized in translation. The mechanisms that regulate the functional balance among ATE1, RARS and translation are unknown. Here, we addressed the question of how these two enzymes can partition Arg-tRNAArg to functionally distinct pathways using an intracellular arginylation sensor in cell lines with overexpression or deletion of ATE1 and RARS isoforms. We found that arginylation levels depend on the physiological state of the cells but are not directly affected by translation activity or the availability of RARS isoforms. However, displacement of RARS from the multi-synthetase complex leads to an increase in intracellular arginylation independently of RARS enzymatic activity. This effect is accompanied by ATE1's redistribution into the cytosol. Our results provide the first comprehensive analysis of the interdependence among translation, arginyl-tRNA synthesis and arginylation.

The Evolutionary Fate of Mitochondrial Aminoacyl-tRNA Synthetases in Amitochondrial Organisms.

During the endosymbiotic evolution of mitochondria, the genes for aminoacyl-tRNA synthetases were transferred to the ancestral nucleus. A further reduction of mitochondrial function resulted in mitochondrion-related organisms (MRO) with a loss of the organelle genome. The fate of the now redundant ancestral mitochondrial aminoacyl-tRNA synthetase genes is uncertain. The derived protein sequence for arginyl-tRNA synthetase from thirty mitosomal organisms have been classified as originating from the ancestral nuclear or mitochondrial gene and compared to the identity element at position 20 of the cognate tRNA that distinguishes the two enzyme forms. The evolutionary choice between loss and retention of the ancestral mitochondrial gene for arginyl-tRNA synthetase reflects the coevolution of arginyl-tRNA synthetase and tRNA identity elements.

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease.

Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5A>G, p.D2G; c.1367C>T, p.S456L; c.1535G>A, p.R512Q and c.1846_1847del, p. Y616Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-tRNA synthetase (hArgRS), are linked to Pelizaeus-Merzbacher-like disease (PMLD) with unclear pathogenesis. Among these mutations, c.5A>G is the most extensively reported mutation, leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS. Here, we showed the detrimental effects of R512Q substitution and ΔC mutations on the structure and function of hArgRS, while the most frequent mutation c.5A>G, p.D2G acted in a different manner without impairing hArgRS activity. The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.

Interactions of Destruxin A with Silkworms' Arginine tRNA Synthetase and Lamin-C Proteins.

Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus Metarhizium anisopliae, has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms' arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with KD value 5.53 × 10-5 and 8.64 × 10-5 M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect.

A patient with pontocerebellar hypoplasia type 6: Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome.

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.

Newly acquired N-terminal extension targets threonyl-tRNA synthetase-like protein into the multiple tRNA synthetase complex.

A typical feature of eukaryotic aminoacyl-tRNA synthetases (aaRSs) is the evolutionary gain of domains at either the N- or C-terminus, which frequently mediating protein-protein interaction. TARSL2 (mouse Tarsl2), encoding a threonyl-tRNA synthetase-like protein (ThrRS-L), is a recently identified aaRS-duplicated gene in higher eukaryotes, with canonical functions in vitro, which exhibits a different N-terminal extension (N-extension) from TARS (encoding ThrRS). We found the first half of the N-extension of human ThrRS-L (hThrRS-L) is homologous to that of human arginyl-tRNA synthetase. Using the N-extension as a probe in a yeast two-hybrid screening, AIMP1/p43 was identified as an interactor with hThrRS-L. We showed that ThrRS-L is a novel component of the mammalian multiple tRNA synthetase complex (MSC), and is reliant on two leucine zippers in the N-extension for MSC-incorporation in humans, and mouse cell lines and muscle tissue. The N-extension was sufficient to target a foreign protein into the MSC. The results from a Tarsl2-deleted cell line showed that it does not mediate MSC integrity. The effect of phosphorylation at various sites of hThrRS-L on its MSC-targeting is also explored. In summary, we revealed that ThrRS-L is a bona fide component of the MSC, which is mediated by a newly evolved N-extension domain.

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNAArg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNAArg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNAArg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNAArg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.

The RARS-MAD1L1 Fusion Gene Induces Cancer Stem Cell-like Properties and Therapeutic Resistance in Nasopharyngeal Carcinoma.

Purpose: Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in Southeast Asia. Because local recurrence and distant metastasis are still the main causes of NPC treatment failure, it is urgent to identify new tumor markers and therapeutic targets for advanced NPC.Experimental Design: RNA sequencing (RNA-seq) was applied to look for interchromosome translocation in NPC. PCR, FISH, and immunoprecipitation were used to examine the fusion gene expression at RNA, DNA, and protein levels in NPC biopsies. MTT assay, colony formation assay, sphere formation assay, co-immunoprecipitation, chromatin immunoprecipitation assay, and in vivo chemoresistance assay were applied to explore the function of RARS-MAD1L1 in NPC.Results: We demonstrated that RARS-MAD1L1 was present in 10.03% (35/349) primary NPC biopsies and 10.7% (9/84) in head and neck cancer (HNC) samples. RARS-MAD1L1 overexpression increased cell proliferation, colony formation, and tumorigenicity in vitro, and the silencing of endogenous RARS-MAD1L1 reduced cancer cell growth and colony formation in vitro In addition, RARS-MAD1L1 increased the side population (SP) ratio and induced chemo- and radioresistance. Furthermore RARS-MAD1L1 interacted with AIMP2, which resulted in activation of FUBP1/c-Myc pathway. The silencing of FUBP1 or the administration of a c-Myc inhibitor abrogated the cancer stem cell (CSC)-like characteristics induced by RARS-MAD1L1. The expression of c-Myc and ABCG2 was higher in RARS-MAD1L1-positive HNC samples than in negative samples.Conclusions: Our findings indicate that RARS-MAD1L1 might contribute to tumorigenesis, CSC-like properties, and therapeutic resistance, at least in part, through the FUBP1/c-Myc axis, implying that RARS-MAD1L1 might serve as an attractive target for therapeutic intervention for NPC. Clin Cancer Res; 24(3); 659-73. ©2017 AACR.

Mutations in RARS cause a hypomyelination disorder akin to Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a rare Mendelian disorder characterised by central nervous system hypomyelination. PMD typically manifests in infancy or early childhood and is caused by mutations in proteolipid protein-1 (PLP1). However, variants in several other genes including gap junction protein gamma 2 (GJC2) can also cause a similar phenotype and are referred to PMD-like disease (PMLD). Whole-exome sequencing in two siblings presenting with clinical symptoms of PMD revealed a homozygous variant in the arginyl-tRNA synthetase (RARS) gene: NM_002887.3: c.[5A>G] p.(Asp2Gly). Subsequent screening of a PMD cohort without a genetic diagnosis identified an unrelated individual with novel compound heterozygous variants including a missense variant c.[1367C>T] p.(Ser456Leu) and a de novo deletion c.[1846_1847delTA] p.(Tyr616Leufs*6). Protein levels of RARS and the multi-tRNA synthetase complex into which it assembles were found to be significantly reduced by 80 and 90% by western blotting and Blue native-PAGE respectively using patient fibroblast extracts. As RARS is involved in protein synthesis whereby it attaches arginine to its cognate tRNA, patient cells were studied to determine their ability to proliferate with limiting amounts of this essential amino acid. Patient fibroblasts cultured in medium with limited arginine at 30 °C and 40 °C, showed a significant decrease in fibroblast proliferation (P<0.001) compared to control cells, suggestive of inefficiency of protein synthesis in the patient cells. Our functional studies provide further evidence that RARS is a PMD-causing gene.

Acetylation of lysine ϵ-amino groups regulates aminoacyl-tRNA synthetase activity in Escherichia coli.

Previous proteomic analyses have shown that aminoacyl-tRNA synthetases in many organisms can be modified by acetylation of Lys. In this present study, leucyl-tRNA synthetase and arginyl-tRNA synthetase from Escherichia coli (EcLeuRS and EcArgRS) were overexpressed and purified and found to be acetylated on Lys residues by MS. Gln scanning mutagenesis revealed that Lys619, Lys624, and Lys809 in EcLeuRS and Lys126 and Lys408 in EcArgRS might play important roles in enzyme activity. Furthermore, we utilized a novel protein expression system to obtain enzymes harboring acetylated Lys at specific sites and investigated their catalytic activity. Acetylation of these Lys residues could affect their aminoacylation activity by influencing amino acid activation and/or the affinity for tRNA. In vitro assays showed that acetyl-phosphate nonenzymatically acetylates EcLeuRS and EcArgRS and suggested that the sirtuin class deacetylase CobB might regulate acetylation of these two enzymes. These findings imply a potential regulatory role for Lys acetylation in controlling the activity of aminoacyl-tRNA synthetases and thus protein synthesis.

Dimerization of Arginyl-tRNA Synthetase by Free Heme Drives Its Inactivation in Plasmodium falciparum.

Excess cellular heme is toxic, and malaria parasites regulate its levels during hemoglobin digestion. Aminoacyl-tRNA synthetases are ubiquitous enzymes, and of these, arginyl-tRNA synthetase (RRS) is unique as its enzymatic product of charged tRNA is required for protein synthesis and degradation. We show that Plasmodium falciparum arginyl-tRNA synthetase (PfRRS) is an active, cytosolic, and monomeric enzyme. Its high-resolution crystal structure highlights critical structural differences with the human enzyme. We further show that hemin binds to and inhibits the aminoacylation activity of PfRRS. Hemin induces a dimeric form of PfRRS that is thus rendered enzymatically dead as it is unable to recognize its cognate tRNA(arg). Excessive hemin in chloroquine-treated malaria parasites results in significantly reduced charged tRNA(arg) levels, thus suggesting deceleration of protein synthesis. These data together suggest that the inhibition of Plasmodium falciparum arginyl-tRNA synthetase can now be synergized with existing antimalarials for more potent drug cocktails against malaria parasites.

Ischemic preconditioning inhibits over-expression of arginyl-tRNA synthetase gene Rars in ischemia-injured neurons.

The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.

The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition.

Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings.