RESUMEN
BACKGROUND: Inhibition of HDAC6 has been proposed as a broadly applicable therapeutic strategy for Charcot-Marie-Tooth disease (CMT). Inhibition of HDAC6 increases the acetylation of proteins important in axonal trafficking, such as α-tubulin and Miro, and has been shown to be efficacious in several preclinical studies using mouse models of CMT. AIMS: Here, we sought to expand on previous preclinical studies by testing the effect of genetic deletion of Hdac6 on mice carrying a humanized knockin allele of Gars1, a model of CMT-type 2D. METHODS: Gars1ΔETAQ mice were bred to an Hdac6 knockout strain, and the resulting offspring were evaluated for clinically relevant outcomes. RESULTS: The genetic deletion of Hdac6 increased α-tubulin acetylation in the sciatic nerves of both wild-type and Gars1ΔETAQ mice. However, when tested at 5 weeks of age, the Gars1ΔETAQ mice lacking Hdac6 showed no changes in body weight, muscle atrophy, grip strength or endurance, sciatic motor nerve conduction velocity, compound muscle action potential amplitude, or peripheral nerve histopathology compared to Gars1ΔETAQ mice with intact Hdac6. INTERPRETATION: Our results differ from those of two previous studies that demonstrated the benefit of the HDAC6 inhibitor tubastatin A in mouse models of CMT2D. While we cannot fully explain the different outcomes, our results offer a counterexample to the benefit of inhibiting HDAC6 in CMT2D, suggesting additional research is necessary.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Modelos Animales de Enfermedad , Histona Desacetilasa 6 , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Histona Desacetilasa 6/genética , Ratones , Humanos , Nervio Ciático , Ratones Noqueados , Eliminación de Gen , Masculino , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Glicina-ARNt Ligasa/genética , Conducción Nerviosa/fisiología , Conducción Nerviosa/efectos de los fármacosRESUMEN
Psoriasis is characterized by excessive keratinocyte proliferation and immunocyte infiltration, but the underlying pathogenesis remains unclear. Aminoacyl-tRNA synthetases are universally expressed enzymes that catalyze the first step of protein synthesis. Glycyl-tRNA synthetase (GARS) is a member of the aminoacyl-tRNA synthetase family. In addition to its canonical function, we found that GARS was overexpressed in the serum and skin lesions of patients with psoriasis. Moreover, GARS was highly expressed in human skin keratinocytes, and GARS knockdown in keratinocytes suppressed cell proliferation and promoted apoptosis through NF-κB/MAPK signaling pathway. Moreover, intradermal injection of recombinant GARS protein caused skin thickening, angiogenesis, and IFN/TNF-driven skin inflammation. Intriguingly, the reported functional receptor for GARS, cadherin 6 (CDH6), was specifically expressed in vascular endothelial cells, and we found that keratinocyte-derived GARS promotes inflammation and angiogenesis of vascular endothelial cells through CDH6. In addition, intradermal injection of GARS aggravated the phenotype and angiogenesis in imiquimod-induced psoriasiform dermatitis models, whereas the psoriatic phenotype and angiogenesis were relieved after knockdown of GARS by adeno-associated virus. Taken together, the results of this study identify the critical role of GARS in the pathogenesis of psoriasis and suggest that blocking GARS may be a therapeutic approach for alleviating psoriasis.
Asunto(s)
Dermatitis , Glicina-ARNt Ligasa , Psoriasis , Humanos , Angiogénesis , Dermatitis/patología , Células Endoteliales/patología , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Inflamación/patología , Queratinocitos/metabolismo , Psoriasis/patología , Piel/patologíaRESUMEN
Two types of glycyl-tRNA synthetase (GlyRS) are known, the α2 and the α2ß2 GlyRSs. Both types of synthetase employ a class II catalytic domain to aminoacylate tRNAGly. In plastids and some bacteria, the α and ß subunits are fused and are designated as (αß)2 GlyRSs. While the tRNA recognition and aminoacylation mechanisms are well understood for α2 GlyRSs, little is known about the mechanisms for α2ß2/(αß)2 GlyRSs. Here we describe structures of the (αß)2 GlyRS from Oryza sativa chloroplast by itself and in complex with cognate tRNAGly. The set of structures reveals that the U-shaped ß half of the synthetase selects the tRNA in a two-step manner. In the first step, the synthetase engages the elbow and the anticodon base C35 of the tRNA. In the second step, the tRNA has rotated â¼9° toward the catalytic centre. The synthetase probes the tRNA for the presence of anticodon base C36 and discriminator base C73. This intricate mechanism enables the tRNA to access the active site of the synthetase from a direction opposite to that of most other class II synthetases.
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Glicina-ARNt Ligasa , Glicina-ARNt Ligasa/genética , Anticodón , ARN de Transferencia de Glicerina/química , ARN de Transferencia , PlastidiosRESUMEN
Glycyl-tRNA synthetase (GARS) is a potential oncogene associated with poor overall survival in various cancers. However, its role in prostate cancer (PCa) has not been investigated. Protein expression of GARS was investigated in benign, incidental, advanced, and castrate-resistant PCa (CRPC) patient samples. We also investigated the role of GARS in vitro and validated GARS clinical outcomes and its underlying mechanism, utilizing The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD) database. Our data revealed a significant association between GARS protein expression and Gleason groups. Knockdown of GARS in PC3 cell lines attenuated cell migration and invasion and resulted in early apoptosis signs and cellular arrest in S phase. Bioinformatically, higher GARS expression was observed in TCGA PRAD cohort, and there was significant association with higher Gleason groups, pathological stage, and lymph nodes metastasis. High GARS expression was also significantly correlated with high-risk genomic aberrations such as PTEN, TP53, FXA1, IDH1, SPOP mutations, and ERG, ETV1, and ETV4 gene fusions. Gene Set Enrichment Analysis (GSEA) of GARS through the TCGA PRAD database provided evidence for upregulation of biological processes such as cellular proliferation. Our findings support the oncogenic role of GARS involved in cellular proliferation and poor clinical outcome and provide further evidence for its use as a potential biomarker in PCa.
Asunto(s)
Glicina-ARNt Ligasa , Neoplasias de la Próstata , Humanos , Masculino , Glicina-ARNt Ligasa/genética , Mutación , Proteínas Nucleares/genética , Próstata/patología , Neoplasias de la Próstata/metabolismo , Proteínas Represoras/genéticaRESUMEN
Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.
Asunto(s)
Actinas , Tirosina-ARNt Ligasa , Animales , Humanos , Actinas/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Drosophila/genética , Glicina-ARNt Ligasa/genética , Mutación , ARN de Transferencia , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismo , Línea Celular TumoralRESUMEN
As a class of essential enzymes in protein translation, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are organized into two classes of 10 enzymes each, based on two conserved active site architectures. The (αß)2 glycyl-tRNA synthetase (GlyRS) in many bacteria is an orphan aaRS whose sequence and unprecedented X-shaped structure are distinct from those of all other aaRSs, including many other bacterial and all eukaryotic GlyRSs. Here, we report a cocrystal structure to elucidate how the orphan GlyRS kingdom specifically recognizes its substrate tRNA. This structure is sharply different from those of other aaRS-tRNA complexes but conforms to the clash-free, cross-class aaRS-tRNA docking found with conventional structures and reinforces the class-reconstruction paradigm. In addition, noteworthy, the X shape of orphan GlyRS is condensed with the largest known spatial rearrangement needed by aaRSs to capture tRNAs, which suggests potential nonactive site targets for aaRS-directed antibiotics, instead of less differentiated hard-to-drug active site locations.
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Aminoacil-ARNt Sintetasas , Glicina-ARNt Ligasa , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Ligasas/metabolismo , ARN de Transferencia , Dominio CatalíticoRESUMEN
Heterozygosity for missense variants and small in-frame deletions in GARS1 has been reported in patients with a range of genetic neuropathies including Charcot-Marie-Tooth disease type 2D (CMT2D), distal hereditary motor neuropathy type V (dHMN-V), and infantile spinal muscular atrophy (iSMA). We identified two unrelated patients who are each heterozygous for a previously unreported missense variant modifying amino-acid position 336 in the catalytic domain of GARS1. One patient was a 20-year-old woman with iSMA, and the second was a 41-year-old man with CMT2D. Functional studies using yeast complementation assays support a loss-of-function effect for both variants; however, this did not reveal variable effects that might explain the phenotypic differences. These cases expand the mutational spectrum of GARS1-related disorders and demonstrate phenotypic variability based on the specific substitution at a single residue.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Glicina-ARNt Ligasa , Humanos , Enfermedad de Charcot-Marie-Tooth/genética , Codón , Glicina-ARNt Ligasa/genética , Mutación , FenotipoRESUMEN
OBJECTIVE: Hepatocellular carcinoma (HCC) is a malignant cancer with poor survival rates. Glycyl-tRNA synthetase (GARS) is a tRNA-charging enzyme that can serve as a biomarker for multiple tumors. Nevertheless, the role of GARS in HCC remains unclear. METHODS: The expression, clinical significance, prognostic value, genetic alterations, immune infiltration and histone modification of GARS in HCC were assessed using multiple databases. The role of GARS in HCC cells was also verified by CCK-8, cell cycle analysis and apoptosis assays in vitro and by a xenograft model in vivo. RESULTS: GARS levels were upregulated in HCC tissues and cells. GARS was confirmed to be a prognostic factor in HCC patients and was significantly correlated with immune infiltration. Enhanced GARS expression in HCC was induced by histone modification of the GARS promotor. Functional network analysis showed that GARS and its coexpressed genes regulate the cell cycle, lysosome and spliceosome. Furthermore, we found that GARS depletion inhibited HCC cell proliferation and cell cycle progression and promoted apoptosis in vitro. GARS overexpression promoted growth, reduced xenograft apoptosis and enhanced CD206+ tumor-associated macrophage infiltration in vivo. CONCLUSION: Our study indicates that GARS is a promising prognostic and therapeutic marker in HCC and might provide new directions and strategies for HCC treatment.
Asunto(s)
Carcinoma Hepatocelular , Glicina-ARNt Ligasa , Neoplasias Hepáticas , Apoptosis/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Humanos , Neoplasias Hepáticas/patologíaRESUMEN
BACKGROUND AND AIMS: Both Charcot-Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V) are GARS1 disease phenotypes involving axonal peripheral neuropathy. Patients often develop clinical symptoms in their teens. Herein, we reported a Chinese family with infantile-onset CMT2D/dSMA-V. METHODS: Clinical evaluation and laboratory examination were performed in our proband, the older sister from this family, and trio exome sequencing (ES) was conducted on the proband and her parents, followed by Sanger sequencing. RESULTS: A novel GARS1 mutation (c.997G>C, p.E333Q; NM_002047) was identified in this patient and her younger sister but not in her parents; thus, it is presumed that this mutation is inherited from a germline mosaic parent. The younger sister began to exhibit weakness of her hands and feet at the age of 1 year old. CONCLUSION: This is the first report of infantile CMT2D/dSMA-V in China. Our study increases the number of infantile-onset cases, as well as reported pathogenic variants in the GARS1 gene, and highlights the important role of exome sequencing in the clinical diagnosis of disease and enabling subsequent prenatal diagnosis. Our study reminds us to consider the possibility of parent germline mosaicism in the subsequent prenatal genetic diagnosis when identifying a de novo variant.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Glicina-ARNt Ligasa , Atrofia Muscular Espinal , Atrofias Musculares Espinales de la Infancia , Adolescente , Enfermedad de Charcot-Marie-Tooth/genética , Femenino , Células Germinativas , Glicina-ARNt Ligasa/genética , Humanos , Lactante , Mosaicismo , Atrofia Muscular Espinal/genética , MutaciónRESUMEN
Charcot-Marie-Tooth disease type 2D (CMT2D), is a hereditary peripheral neuropathy caused by mutations in the gene encoding glycyl-tRNA synthetase (GARS1). Here, human induced pluripotent stem cell (hiPSC)-based models of CMT2D bearing mutations in GARS1 and their use for the identification of predictive biomarkers amenable to therapeutic efficacy screening is described. Cultures containing spinal cord motor neurons generated from this line exhibit network activity marked by significant deficiencies in spontaneous action potential firing and burst fire behavior. This result matches clinical data collected from a patient bearing a GARS1P724H mutation and is coupled with significant decreases in acetylated α-tubulin levels and mitochondrial movement within axons. Treatment with histone deacetylase 6 inhibitors, tubastatin A and CKD504, improves mitochondrial movement and α-tubulin acetylation in these cells. Furthermore, CKD504 treatment enhances population-level electrophysiological activity, highlighting its potential as an effective treatment for CMT2D.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Glicina-ARNt Ligasa , Células Madre Pluripotentes Inducidas , Transporte Axonal , Enfermedad de Charcot-Marie-Tooth/tratamiento farmacológico , Glicina-ARNt Ligasa/genética , Histona Desacetilasa 6/genética , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Tubulina (Proteína)/genéticaRESUMEN
Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell typespecific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Glicina-ARNt Ligasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico , Tirosina-ARNt Ligasa/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Genes Dominantes , Glicina-ARNt Ligasa/genética , Masculino , Ratones , Ratones Mutantes , Neuronas Motoras/fisiología , Biosíntesis de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Médula Espinal/fisiopatología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Transcriptoma , Tirosina-ARNt Ligasa/genéticaRESUMEN
Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. We found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, we found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. Our findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Glicina-ARNt Ligasa/metabolismo , ARN de Transferencia de Glicerina/metabolismo , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Modelos Animales de Enfermedad , Drosophila melanogaster , Femenino , Glicina-ARNt Ligasa/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/fisiología , ARN de Transferencia de Glicerina/genéticaRESUMEN
AaRSs (aminoacyl-tRNA synthetases) group into two ten-member classes throughout evolution, with unique active site architectures defining each class. Most are monomers or homodimers but, for no apparent reason, many bacterial GlyRSs are heterotetramers consisting of two catalytic α-subunits and two tRNA-binding ß-subunits. The heterotetrameric GlyRS from Escherichia coli (EcGlyRS) was historically tested whether its α- and ß-polypeptides, which are encoded by a single mRNA with a gap of three in-frame codons, are replaceable by a single chain. Here, an unprecedented X-shaped structure of EcGlyRS shows wide separation of the abutting chain termini seen in the coding sequences, suggesting strong pressure to avoid a single polypeptide format. The structure of the five-domain ß-subunit is unique across all aaRSs in current databases, and structural analyses suggest these domains play different functions on α-subunit binding, ATP coordination and tRNA recognition. Moreover, the X-shaped architecture of EcGlyRS largely fits with a model for how two classes of tRNA synthetases arose, according to whether enzymes from opposite classes can simultaneously co-dock onto separate faces of the same tRNA acceptor stem. While heterotetrameric GlyRS remains the last structurally uncharacterized member of aaRSs, our study contributes to a better understanding of this ancient and essential enzyme family.
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Dominio Catalítico/genética , Escherichia coli/genética , Glicina-ARNt Ligasa/genética , ARN de Transferencia de Glicerina/química , Adenosina Trifosfato/metabolismo , Cristalografía por Rayos X , Glicina/química , Modelos Moleculares , ARN de Transferencia de Glicerina/genéticaRESUMEN
Toxic gain-of-function mutations in aminoacyl-tRNA synthetases cause a degeneration of peripheral motor and sensory axons, known as Charcot-Marie-Tooth (CMT) disease. While these mutations do not disrupt overall aminoacylation activity, they interfere with translation via an unknown mechanism. Here, we dissect the mechanism of function of CMT mutant glycyl-tRNA synthetase (CMT-GARS), using high-resolution ribosome profiling and reporter assays. We find that CMT-GARS mutants deplete the pool of glycyl-tRNAGly available for translation and inhibit the first stage of elongation, the accommodation of glycyl-tRNA into the ribosomal A-site, which causes ribosomes to pause at glycine codons. Moreover, ribosome pausing activates a secondary repression mechanism at the level of translation initiation, by inducing the phosphorylation of the alpha subunit of eIF2 and the integrated stress response. Thus, CMT-GARS mutant triggers translational repression via two interconnected mechanisms, affecting both elongation and initiation of translation.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/genética , Extensión de la Cadena Peptídica de Translación/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Ribosomas/metabolismo , Línea Celular , Factor 2 Eucariótico de Iniciación/metabolismo , Mutación con Ganancia de Función/genética , Expresión Génica/genética , Glicina/genética , Células HEK293 , Humanos , Fosforilación , Biosíntesis de Proteínas/genética , ARN de Transferencia de Glicerina/genéticaRESUMEN
Charcot-Marie-Tooth disease is the most common inherited peripheral neuropathy. Dominant mutations in the glycyl-tRNA synthetase (GARS) gene cause peripheral nerve degeneration and lead to CMT disease type 2D. The underlying mechanisms of mutations in GARS (GARSCMT2D ) in disease pathogenesis are not fully understood. In this study, we report that wild-type GARS binds the NAD+ -dependent deacetylase SIRT2 and inhibits its deacetylation activity, resulting in the acetylated α-tubulin, the major substrate of SIRT2. The catalytic domain of GARS tightly interacts with SIRT2, which is the most CMT2D mutation localization. However, CMT2D mutations in GARS cannot inhibit SIRT2 deacetylation, which leads to a decrease of acetylated α-tubulin. Genetic reduction of SIRT2 in the Drosophila model rescues the GARS-induced axonal CMT neuropathy and extends the life span. Our findings demonstrate the pathogenic role of SIRT2-dependent α-tubulin deacetylation in mutant GARS-induced neuropathies and provide new perspectives for targeting SIRT2 as a potential therapy against hereditary axonopathies.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Sirtuina 2/metabolismo , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Drosophila , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Células HEK293 , Humanos , Sirtuina 2/genéticaRESUMEN
Aminoacyl-tRNA synthetases play a critical role in protein synthesis by catalyzing the covalent attachment of amino acids to their cognate tRNAs. However, the role of aminoacyl-tRNA synthetases in the transition from vegetative to reproductive growth in plants remains poorly understood. In this study, a rice (Oryza sativa) glycyl-tRNA synthetase 3, OsGlyRS3, was found to impact heading date in rice. Flowering in osglyrs3, a mutant line containing a T-DNA insertion in OsGlyRS3, was advanced by approximately 2 weeks compared to wild type. Expression analysis of flowering regulator genes showed that transcript levels of Heading date 1 (Hd1), Heading date 3a (Hd3a), and OsMADS51 were elevated in osglyrs3. These data indicate that the loss of OsGlyRS3 activity induces the expression of flowering-activating genes, resulting in early flowering.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Glicina-ARNt Ligasa/genética , Oryza/enzimología , Oryza/genética , Proteínas de Plantas/genética , Flores/genética , Flores/crecimiento & desarrollo , Genes de Plantas , Glicina-ARNt Ligasa/fisiología , Mutación , Oryza/crecimiento & desarrollo , Desarrollo de la Planta/genética , Proteínas de Plantas/fisiologíaRESUMEN
Aminoacyl-RNA synthetases (aaRSs) are among the key enzymes of protein biosynthesis. They are responsible for conducting the first step in the protein biosynthesis, namely attaching amino acids to the corresponding tRNA molecules both in cytoplasm and mitochondria. More and more research demonstrates that mutations in the genes encoding aaRSs lead to the development of various neurodegenerative diseases, such as incurable Charcot-Marie-Tooth disease (CMT) and distal spinal muscular atrophy. Some mutations result in the loss of tRNA aminoacylation activity, while other mutants retain their classical enzyme activity. In the latter case, disease manifestations are associated with additional neuron-specific functions of aaRSs. At present, seven aaRSs (GlyRS, TyrRS, AlaRS, HisRS, TrpRS, MetRS, and LysRS) are known to be involved in the CMT etiology with glycyl-tRNA synthetase (GlyRS) being the most studied of them.
Asunto(s)
Glicina-ARNt Ligasa/genética , Mutación , Enfermedades del Sistema Nervioso/enzimología , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Femenino , Humanos , Masculino , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/fisiopatología , Neuronas/enzimología , Neuronas/fisiologíaRESUMEN
Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.
Asunto(s)
Acetiltransferasas/genética , Escherichia coli O157/genética , Glicina-ARNt Ligasa/genética , Biosíntesis de Proteínas/genética , Antitoxinas/genética , Toxinas Bacterianas/genética , Escherichia coli O157/patogenicidad , Sistemas Toxina-Antitoxina/genéticaRESUMEN
The majority of patients with spinal muscular atrophy (SMA) identified to date harbor a biallelic exonic deletion of SMN1. However, there have been reports of SMA-like disorders that are independent of SMN1, including those due to pathogenic variants in the glycyl-tRNA synthetase gene (GARS1). We report three unrelated patients with de novo variants in GARS1 that are associated with infantile-onset SMA (iSMA). Patients were ascertained during inpatient hospital evaluations for complications of neuropathy. Evaluations were completed as indicated for clinical care and management and informed consent for publication was obtained. One newly identified, disease-associated GARS1 variant, identified in two out of three patients, was analyzed by functional studies in yeast complementation assays. Genomic analyses by exome and/or gene panel and SMN1 copy number analysis of three patients identified two previously undescribed de novo missense variants in GARS1 and excluded SMN1 as the causative gene. Functional studies in yeast revealed that one of the de novo GARS1 variants results in a loss-of-function effect, consistent with other pathogenic GARS1 alleles. In sum, the patients' clinical presentation, assessments of previously identified GARS1 variants and functional assays in yeast suggest that the GARS1 variants described here cause iSMA. GARS1 variants have been previously associated with Charcot-Marie-Tooth disease (CMT2D) and distal SMA type V (dSMAV). Our findings expand the allelic heterogeneity of GARS-associated disease and support that severe early-onset SMA can be caused by variants in this gene. Distinguishing the SMA phenotype caused by SMN1 variants from that due to pathogenic variants in other genes such as GARS1 significantly alters approaches to treatment.