RESUMO
Aminoacyl-tRNA synthetases (aaRSs), essential components of the protein synthesizing machinery, have been often chosen for devising therapeutics against parasitic diseases. Due to their relevance in drug development, the current study was designed to explore functional and structural aspects of Leishmania donovani glutamyl-tRNA synthetase (LdGluRS). Hence, LdGluRS was cloned into an expression vector and purified to homogeneity using chromatographic techniques. Purified protein showed maximum enzymatic activity at physiological pH, with more binding capacity towards its cofactor (Adenosine triphosphate, 0.06 ± 0.01 mM) than the cognate substrate (L-glutamate, 9.5 ± 0.5 mM). Remarkably, salicylate inhibited LdGluRS competitively with respect to L-glutamate and exhibited druglikeness with negligible effect on human macrophages. The protein possessed more α-helices (43 %) than ß-sheets (12 %), whereas reductions in thermal stability and cofactor-binding affinity, along with variation in mode of inhibition after mutation signified the role of histidine (H60) as a catalytic residue. LdGluRS could also generate a pro-inflammatory milieu in human macrophages by upregulating cytokines. The docking study demonstrated the placement of salicylate into LdGluRS substrate-binding site, and the complex was found to be stable during molecular dynamics (MD) simulation. Altogether, our study highlights the understanding of molecular inhibition and structural features of glutamyl-tRNA synthetase from kinetoplastid parasites.
Assuntos
Aminoacil-tRNA Sintetases , Leishmania donovani , Humanos , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Ácido Glutâmico , Aminoacil-tRNA Sintetases/química , Trifosfato de Adenosina , Leishmania donovani/metabolismo , SalicilatosRESUMO
Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (â¼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be â¼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.
Assuntos
Proteínas de Escherichia coli , Fosforilação , Proteínas de Escherichia coli/química , Escherichia coli/genética , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismoRESUMO
Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation machinery that provide the charged tRNAs needed for protein synthesis. Over the past decades, aaRSs have been studied as anti-parasitic, anti-bacterial, and anti-fungal drug targets. This study focused on the cytoplasmic glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum, which belongs to class Ib in aaRSs. GluRS unlike most other aaRSs requires tRNA to activate its cognate amino acid substrate L-Glutamate (L-Glu), and fails to form an intermediate adenylate complex in the absence of tRNA. The crystal structures of the Apo, ATP, and ADP-bound forms of Plasmodium falciparum glutamyl-tRNA synthetase (PfGluRS) were solved at 2.1 Å, 2.2 Å, and 2.8 Å respectively. The structural comparison of the Apo- and ATP-bound holo-forms of PfGluRS showed considerable conformational changes in the loop regions around the ATP-binding pocket of the enzyme. Biophysical characterization of the PfGluRS showed binding of the enzyme substrates L-Gluand ATP.. The sequence and structural conservation were evident across GluRS compared to other species. The structural dissection of the PfGluRS gives insight into the critical residues involved in the binding of ATP substrate, which can be harvested to develop new antimalarial drugs.
Assuntos
Aminoacil-tRNA Sintetases , Glutamato-tRNA Ligase , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , RNA de Transferência/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.
Assuntos
Aminoacil-tRNA Sintetases , Isoleucina-tRNA Ligase , Isoleucina-tRNA Ligase/química , Aminoacil-tRNA Sintetases/metabolismo , Glutamato-tRNA Ligase/química , RNA de Transferência/metabolismoRESUMO
The existence of chlorine-resistant bacteria (CRB) in drinking water supply systems (DWSSs) results in significant challenges to the biological security of drinking water. However, little is known about the intrinsic chlorine-resistant molecular metabolic mechanism of bacteria in DWSSs. This research explored the microbial interactions and the key metabolic pathways that modulate the chlorine resistance of bacteria in full-scale chloraminated DWSSs. The dominant CRB, including Bdellovibrio, Bradyrhizobium, Peredibacter, Sphingomonas, and Hydrogenophaga, strongly interacted with each other to maintain basic metabolism. A total of 4.21% of the bacterial metabolic pathways were key and specific to chlorine-resistant bacteria. Glutaminyl-tRNA biosynthesis was the dominant metabolic pathway of CRB in the target DWSSs. After chloramine disinfection, the relative abundance of glutamate-tRNA ligase (GlnRS) and the related orthologous genes increased by 10.11% and 14.58%, respectively. The inactivation rate of the GlnRS overexpression strain (81.40%) was lower than that of the wild-type strain (90.11%) after exposure to chloramine. Meanwhile, the growth rate of the GlnRS overexpression strain was higher than that of the wild-type strain. Glutaminyl-tRNA biosynthesis can enhance chlorine resistance in DWSSs.
Assuntos
Água Potável , Sphingomonas , Cloraminas , Cloro/farmacologia , Desinfecção , Água Potável/microbiologia , Glutamato-tRNA Ligase/genética , RNA de Transferência , Abastecimento de ÁguaRESUMO
Elizabethkingia bacteria are globally emerging pathogens that cause opportunistic and nosocomial infections, with up to 40% mortality among the immunocompromised. Elizabethkingia species are in the pipeline of organisms for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include the structure-function analysis of potential therapeutic targets. Glutamyl-tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined high-resolution structures of GluRS from E. meningosepticum (EmGluRS) and E. anopheles (EaGluRS). EmGluRS was co-crystallized with glutamate, while EaGluRS is an apo structure. EmGluRS shares â¼97% sequence identity with EaGluRS but less than 39% sequence identity with any other structure in the Protein Data Bank. EmGluRS and EaGluRS have the prototypical bacterial GluRS topology. EmGluRS and EaGluRS have similar binding sites and tertiary structures to other bacterial GluRSs that are promising drug targets. These structural similarities can be exploited for drug discovery.
Assuntos
Anopheles , Infecções por Flavobacteriaceae , Sequência de Aminoácidos , Animais , Anopheles/metabolismo , Cristalografia por Raios X , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismoRESUMO
Aminoacylation reaction is the first step of protein biosynthesis. Transfer RNA (tRNA) is charged with an amino acid in this reaction and the reaction is catalyzed by aminoacyl tRNA synthetase enzyme (aaRS). In the present work, we use classical molecular dynamics simulation to show that the tRNA bound Mg2+ ions significantly influence the charging step of class I TtGluRS: Glu-AMP: tRNAGlu and class II dimeric TtSerRS: Ser-AMP: tRNASer. The CCA end of the acceptor terminal is disordered in the absence of coordinated Mg2+ ions and the CCA end can freely explore beyond the specific conformational space of the tRNA in its precharging state. A balance between the conformational disorder of the tRNA and the restriction imposed on the CCA terminal via coordination with the Mg2+ ions is needed for the placement of the CCA terminal in a precharging state organization. This result provides a molecular-level explanation of the experimental observation that the presence of Mg2+ ions is a necessary condition for a successful aminoacylation reaction.Communicated by Ramaswamy H. Sarma.
Assuntos
Aminoacil-tRNA Sintetases , Serina-tRNA Ligase , Monofosfato de Adenosina/metabolismo , Aminoácidos/química , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Íons , Ligases/metabolismo , Magnésio , RNA de Transferência/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , RNA de Transferência de Serina/metabolismo , Serina-tRNA Ligase/químicaRESUMO
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.
Assuntos
Doenças Ósseas , Diabetes Mellitus , Doenças Genéticas Inatas , Glutamato-tRNA Ligase , Mutação de Sentido Incorreto , Estresse Fisiológico/genética , Substituição de Aminoácidos , Doenças Ósseas/enzimologia , Doenças Ósseas/genética , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Células HEK293 , Humanos , MasculinoRESUMO
Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome-and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion's capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.
Assuntos
Capsídeo/metabolismo , Citomegalovirus/ultraestrutura , Fosfoproteínas/metabolismo , RNA de Transferência/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/ultraestrutura , Anticódon/metabolismo , Sequência de Bases , Linhagem Celular , Microscopia Crioeletrônica , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/metabolismo , Humanos , Modelos Moleculares , Fosfoproteínas/ultraestrutura , RNA Viral/ultraestrutura , Proteínas da Matriz Viral/ultraestruturaRESUMO
BACKGROUND: Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is a hereditary disorder caused by biallelic variants in the EARS2 gene. Patients exhibit developmental delay, hypotonia, and hyperreflexia. Brain magnetic resonance imaging (MRI) reveals T2-hyperintensities in the deep white matter, thalamus, and brainstem, which generally stabilize over time. Herein, we report a case of LTBL, showing remitting and exacerbating white matter lesions. CASE DESCRIPTION: A non-consanguineous Japanese boy exhibited unsteady head control with prominent hypotonia, with no family history of neurological diseases. Brain MRI at one year of age revealed extensive T2-hyperintensities on the cerebral white matter, cerebellum, thalamus, basal ganglia, pons, and medulla oblongata. Magnetic resonance spectroscopy of the lesions showed lactate and myoinositol peaks. Whole-exome sequencing yielded novel compound heterozygous EARS2 variants of c.164G>T, p.Arg55Leu and c.484C>T, p.Arg162Trp. Interestingly, the lesions were reduced at three years of age, and new lesions emerged at eight years of age. At 10 years of age, the lesions were changed in the corpus callosum, deep cerebral white matter, and cerebellum, without physical exacerbation. The lesions improved one year later. CONCLUSION: We present the first case with remitting and exacerbating brain lesions in LTBL. EARS2 could relate to selective and specific brain regions and age dependency. Although the exact role of EARS2 remains unknown, the remitting and exacerbating imaging changes may be a clue in elucidating a novel EARS2 function in LTBL.
Assuntos
Tronco Encefálico , Progressão da Doença , Glutamato-tRNA Ligase/genética , Ácido Láctico/metabolismo , Leucoencefalopatias , Exacerbação dos Sintomas , Tálamo , Adolescente , Fatores Etários , Tronco Encefálico/diagnóstico por imagem , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Leucoencefalopatias/patologia , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Remissão Espontânea , Tálamo/diagnóstico por imagem , Tálamo/metabolismo , Tálamo/patologiaRESUMO
Glutamyl-tRNA synthetase 2 (encoded by EARS2) is a mitochondrial aminoacyl-tRNA synthetase required to translate the 13 subunits of the electron transport chain encoded by the mitochondrial DNA. Pathogenic EARS2 variants cause combined oxidative phosphorylation deficiency, subtype 12 (COXPD12), an autosomal recessive disorder involving lactic acidosis, intellectual disability, and other features of mitochondrial compromise. Patients with EARS2 deficiency present with variable phenotypes ranging from neonatal lethality to a mitigated disease with clinical improvement in early childhood. Here, we report a neonate homozygous for a rare pathogenic variant in EARS2 (c.949G>T; p.G317C). Metabolomics in primary fibroblasts from this patient revealed expected abnormalities in TCA cycle metabolites, as well as numerous changes in purine, pyrimidine, and fatty acid metabolism. To examine genotype-phenotype correlations in COXPD12, we compared the metabolic impact of reconstituting these fibroblasts with wild-type EARS2 versus four additional EARS2 variants from COXPD12 patients with varying clinical severity. Metabolomics identified a group of signature metabolites, mostly from the TCA cycle and amino acid metabolism, that discriminate between EARS2 variants causing relatively mild and severe COXPD12. Taken together, these findings indicate that metabolomics in patient-derived fibroblasts may help establish genotype-phenotype correlations in EARS2 deficiency and likely other mitochondrial disorders.
Assuntos
Variação Genética/genética , Glutamato-tRNA Ligase/genética , Leucoencefalopatias/genética , Erros Inatos do Metabolismo/genética , Acidose Láctica/etiologia , Aminoacil-tRNA Sintetases/genética , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Glutamato-tRNA Ligase/metabolismo , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/etiologia , Leucoencefalopatias/metabolismo , Masculino , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , MutaçãoRESUMO
The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient's clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease.
Assuntos
Variação Genética/genética , Glutamato-tRNA Ligase/genética , Ácido Láctico/metabolismo , Leucoencefalopatias/genética , Adulto , Aminoacil-tRNA Sintetases/genética , Tronco Encefálico/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Leucoencefalopatias/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/genética , Fenótipo , RNA de Transferência/genética , Tálamo/metabolismo , Adulto JovemRESUMO
Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.
Assuntos
Caulobacter crescentus/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Sistemas Toxina-Antitoxina/genética , Aminoacil-tRNA Sintetases/genética , Antibacterianos/farmacologia , Caulobacter crescentus/enzimologia , Escherichia coli/genética , Genoma Bacteriano/genética , Glutamato-tRNA Ligase/genética , Óperon/genética , Proteínas Quinases/genéticaRESUMO
The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.
Assuntos
Aspartato-tRNA Ligase/genética , Glutamato-tRNA Ligase/genética , RNA de Transferência de Asparagina/genética , RNA de Transferência de Ácido Aspártico/genética , Sequência de Aminoácidos/genética , Asparagina/química , Asparagina/genética , Aspartato-tRNA Ligase/química , Código Genético/genética , Glutamato-tRNA Ligase/química , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Conformação Proteica , Proteoma/química , Proteoma/genética , Aminoacil-RNA de Transferência/genética , RNA de Transferência de Asparagina/química , RNA de Transferência de Ácido Aspártico/química , Homologia de Sequência de AminoácidosRESUMO
About 1 billion years ago, in a single-celled holozoan ancestor of all animals, a gene fusion of two tRNA synthetases formed the bifunctional enzyme, glutamyl-prolyl-tRNA synthetase (EPRS). We propose here that a confluence of metabolic, biochemical, and environmental factors contributed to the specific fusion of glutamyl- (ERS) and prolyl- (PRS) tRNA synthetases. To test this idea, we developed a mathematical model that centers on the precursor-product relationship of glutamic acid and proline, as well as metabolic constraints on free glutamic acid availability near the time of the fusion event. Our findings indicate that proline content increased in the proteome during the emergence of animals, thereby increasing demand for free proline. Together, these constraints contributed to a marked cellular depletion of glutamic acid and its products, with potentially catastrophic consequences. In response, an ancient organism invented an elegant solution in which genes encoding ERS and PRS fused to form EPRS, forcing coexpression of the two enzymes and preventing lethal dysregulation. The substantial evolutionary advantage of this coregulatory mechanism is evidenced by the persistence of EPRS in nearly all extant animals.
Assuntos
Aminoacil-tRNA Sintetases/química , Proteínas de Bactérias/química , Evolução Molecular , Modelos Químicos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Fusão Gênica , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Prolina/química , Prolina/metabolismo , Biossíntese de Proteínas/genéticaRESUMO
The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high-copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence.
Assuntos
Tolerância a Medicamentos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glutamato-tRNA Ligase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Glutamato-tRNA Ligase/genética , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais/genética , Especificidade por SubstratoRESUMO
Aminoacyl-tRNA synthetases (aaRSs) have housekeeping roles in protein synthesis, but little is known about how these aaRSs are involved in organ development. Here, we report that a rice (Oryza sativa) glutamyl-tRNA synthetase (OsERS1) maintains proper somatic cell organization and limits the overproliferation of male germ cells during early anther development. The expression of OsERS1 is specifically detectable in meristematic layer 2-derived cells of the early anther, and osers1 anthers exhibit overproliferation and disorganization of layer 2-derived cells, producing fused lobes and extra germ cells in early anthers. The conserved biochemical function of OsERS1 in ligating glutamate to tRNAGlu is enhanced by its cofactor aaRS OsARC. Furthermore, metabolomics profiling revealed that OsERS1 is an important node for multiple metabolic pathways, indicated by the accumulation of amino acids and tricarboxylic acid cycle components in osers1 anthers. Notably, the anther defects of the osers1 mutant are causally associated with the abnormal accumulation of hydrogen peroxide, which can reconstitute the osers1 phenotype when applied to wild-type anthers. Collectively, these findings demonstrate how aaRSs affect male organ development in plants, likely through protein synthesis, metabolic homeostasis, and redox status.
Assuntos
Flores/citologia , Glutamato-tRNA Ligase/metabolismo , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Divisão Celular , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Glutamato-tRNA Ligase/genética , Ácido Glutâmico/metabolismo , Meristema/citologia , Meristema/genética , Mutação , Oryza/citologia , Proteínas de Plantas/genética , Plantas Geneticamente ModificadasRESUMO
Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH-S-transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Caspases/metabolismo , Aminoacil-tRNA Sintetases/química , Domínio Catalítico , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , ProteóliseRESUMO
Glutaminyl-tRNA synthetase (GlnRS) catalyzes the aminoacylation of glutamine to the corresponding tRNAGln. However, most bacteria and all archaea lack GlnRS and thus an indirect noncanonical aminoacylation is required. With the assistance of a non-discriminating version of Glutamyl-tRNA synthetases (ND-GluRS) the tRNAGln is misaminoacylated by glutamate. In this study, we have computationally investigated the aminoacylation mechanism in GlnRS and ND-GluRS employing Molecular Dynamics (MD) simulations, Quantum Mechanics (QM) cluster and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations. Our investigations demonstrated the feasibility of a water-mediated, substrate-assisted catalysis pathway with rate limiting steps occurring at energy barriers of 25.0 and 25.4 kcal mol-1 for GlnRS and ND-GluRS, respectively. A conserved lysine residue participates in a second proton transfer to facilitate the departure of the adenosine monophosphate (AMP) group. Thermodynamically stable (-29.9 and -9.3 kcal mol-1 for GlnRS and ND-GluRS) product complexes are obtained only when the AMP group is neutral.