RESUMEN
I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), making plausible the following implications. The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. Indeed, as suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages, as analyzed here. Furthermore, this late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. Indeed, this polyphyletic origin would imply that the genetic code was still evolving because at least two ARSs, i.e. proteins that make the genetic code possible today, were still evolving. This would imply that the evolutionary stages involved were characterized not by cells but by protocells, that is, by progenotes because this is precisely the definition of a progenote. This conclusion would be strengthened by the observation that both GlyRS and LysRS originating in the phyletic lineages leading to bacteria and archaea, would demonstrate that, more generally, proteins were most likely still in rapid and progressive evolution. Namely, a polyphyletic origin of proteins which would qualify at least the initial phase of the evolutionary stage of the ancestor of bacteria and that of archaea as stages belonging to the progenote.
Asunto(s)
Evolución Molecular , Código Genético , Glicina-ARNt Ligasa , Lisina-ARNt Ligasa , Filogenia , Código Genético/genética , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Archaea/genética , Archaea/enzimología , Bacterias/genética , Bacterias/enzimologíaRESUMEN
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
Glycyl-tRNA synthetases (GlyRSs) have different oligomeric structures depending on the organisms. While a dimeric α2 GlyRS species is present in archaea, eukaryotes and some eubacteria, a heterotetrameric α2ß2 GlyRS species is found in most eubacteria. Here, we present the crystal structure of heterotetrameric α2ß2 GlyRS, consisting of the full-length α and ß subunits, from Lactobacillus plantarum (LpGlyRS), gram-positive lactic bacteria. The α2ß2LpGlyRS adopts the same X-shaped structure as the recently reported Escherichia coli α2ß2 GlyRS. A tRNA docking model onto LpGlyRS suggests that the α and ß subunits of LpGlyRS together recognize the L-shaped tRNA structure. The α and ß subunits of LpGlyRS together interact with the 3'-end and the acceptor region of tRNAGly, and the C-terminal domain of the ß subunit interacts with the anticodon region of tRNAGly. The biochemical analysis using tRNA variants showed that in addition to the previously defined determinants G1C72 and C2G71 base pairs, C35, C36 and U73 in eubacterial tRNAGly, the identification of bases at positions 4 and 69 in tRNAGly is required for efficient glycylation by LpGlyRS. In this case, the combination of a purine base at Position 4 and a pyrimidine base at Position 69 in tRNAGly is preferred.
Asunto(s)
Glicina-ARNt Ligasa , Lactobacillus plantarum , ARN de Transferencia , Lactobacillus plantarum/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/metabolismo , Cristalografía por Rayos XRESUMEN
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.
Asunto(s)
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
Macrophages play important roles in cancer microenvironment. Human cytosolic glycyl-tRNA synthetase (GARS1) was previously shown to be secreted via extracellular vesicles (EVs) from macrophages to trigger cancer cell death. However, the effects of GARS1-containing EVs (GARS1-EVs) on macrophages as well as on cancer cells and the working mechanisms of GARS1 in cancer microenvironment are not yet understood. Here we show that GARS1-EVs induce M1 polarization and facilitate phagocytosis of macrophages. GARS1-EVs triggers M1 polarization of macrophage via the specific interaction of the extracellular cadherin subdomains 1-4 of the cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2) with the N-terminal WHEP domain containing peptide region of GARS1, and activates the RAF-MEK-ERK pathway for M1 type cytokine production and phagocytosis. Besides, GARS1 interacted with cadherin 6 (CDH6) of cancer cells via its C-terminal tRNA-binding domain to induce cancer cell death. In vivo model, GARS1-EVs showed potent suppressive activity against tumor initiation via M1 type macrophages. GARS1 displayed on macrophage-secreted extracellular vesicles suppressed tumor growth in dual mode, namely through pro-apoptotic effect on cancer cells and M1 polarization effect on macrophages. Collectively, these results elucidate the unique tumor suppressive activity and mechanism of GARS1-EVs by activating M1 macrophage via CELSR2 as well as by direct killing of cancer cells via CDH6.
Asunto(s)
Vesículas Extracelulares , Glicina-ARNt Ligasa , Macrófagos , Neoplasias , Cadherinas/metabolismo , Polaridad Celular , Vesículas Extracelulares/enzimología , Vesículas Extracelulares/metabolismo , Glicina-ARNt Ligasa/análisis , Glicina-ARNt Ligasa/metabolismo , Glicina-ARNt Ligasa/farmacología , Humanos , Macrófagos/enzimología , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias/enzimología , Neoplasias/metabolismo , Fagocitosis , Microambiente TumoralRESUMEN
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
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
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
Glycyl-tRNA synthetase (GlyRS) has non-canonical roles beyond aminoacylation, but the molecular mechanism is largely unknown. We have previously found that GlyRS is phosphorylated in the cytoplasm of bovine mammary epithelial cells (bMECs) in response to amino acid stimulation, and the phosphorylated GlyRS enters nucleus to stimulate gene expression for milk synthesis. In this study, we aim to uncover the upstream kinase of GlyRS and reveal the signaling pathways that methionine (Met) stimulates GlyRS phosphorylation. We show that mitogen-activated protein kinase 10 (MAP3K10) interacts with GlyRS in bMECs by Co-IP, mass spectrometry, and Western blotting analysis. We further identify that MAP3K10 is an upstream kinase of GlyRS by in vitro kinase assay and MAP3K10 stimulates NFκB1 phosphorylation via activating GlyRS. We also uncover that Met stimulates GlyRS phosphorylation via the GPR87-CDC42/Rac1-MAP3K10 signaling pathway. Our findings help to understand the molecular mechanism of GlyRS in cellular signaling transduction.
Asunto(s)
Glicina-ARNt Ligasa/metabolismo , Metionina/farmacología , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Bovinos , Activación Enzimática/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.
Asunto(s)
Codón Iniciador , Factor 1 Eucariótico de Iniciación/metabolismo , Sistemas de Lectura Abierta , Iniciación de la Cadena Peptídica Traduccional , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
OBJECTIVES: Glyphosate (N-phosphonomethyl glycine) and its commercial herbicide formulations have been shown to exert toxicity via various mechanisms. It has been asserted that glyphosate substitutes for glycine in polypeptide chains leading to protein misfolding and toxicity. However, as no direct evidence exists for glycine to glyphosate substitution in proteins, including in mammalian organisms, we tested this claim by conducting a proteomics analysis of MDA-MB-231 human breast cancer cells grown in the presence of 100 mg/L glyphosate for 6 days. Protein extracts from three treated and three untreated cell cultures were analysed as one TMT-6plex labelled sample, to highlight a specific pattern (+/+/+/-/-/-) of reporter intensities for peptides bearing true glyphosate treatment induced-post translational modifications as well as allowing an investigation of the total proteome. RESULTS: Comparative statistical analysis of global proteome changes between glyphosate treated and non-treated samples did not show significant differences. Crucially, filtering of data to focus analysis on peptides potentially bearing glycine for glyphosate replacement revealed that the TMT reporter intensity pattern of all candidates showed conclusively that they are all false discoveries, with none displaying the expected TMT pattern for such a substitution. Thus, the assertion that glyphosate substitutes for glycine in protein polypeptide chains is incorrect.
Asunto(s)
Glicina/análogos & derivados , Glicina/metabolismo , Herbicidas/química , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Línea Celular Tumoral , Expresión Génica , Glicina/química , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Herbicidas/metabolismo , Humanos , Modelos Moleculares , Proteínas de Neoplasias/genética , Proteoma/genética , GlifosatoRESUMEN
The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.
Asunto(s)
Glicina-ARNt Ligasa/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , Ribosomas/metabolismo , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Glicina-ARNt Ligasa/genética , Oryza/genética , Proteínas de Plantas/genética , Plantones/genética , Plantones/metabolismoRESUMEN
This study reports the X-ray crystallographic structure of the glycyl-tRNA synthetase (GlyRS) of Nanoarchaeum equitans - a hyperthermophilic archaeal species. This is the first archaeal GlyRS crystal structure elucidated. The GlyRS comprises an N-terminal catalytic domain and a C-terminal anticodon-binding domain with a long ß-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNAGly was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNAGly with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNAGly elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNAGly G71 substitution mutants and a GlyRS mutant where Arg223 is mutated to alanine strengthen the possibility that the carbonyl oxygen at position 6 of G71 would hydrogen-bond with the guanidine nitrogen of Arg223 in N. equitans GlyRS.
Asunto(s)
Proteínas Arqueales/química , Glicina-ARNt Ligasa/química , Nanoarchaeota/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Glicina-ARNt Ligasa/metabolismo , Modelos Moleculares , Nanoarchaeota/química , Nanoarchaeota/metabolismo , Conformación Proteica , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Alineación de SecuenciaRESUMEN
Amino acids are required for the activation of mammalian target of rapamycin (mTOR) to increase cell growth, protein and lipid synthesis, and inhibit autophagy. However, the mechanism through which amino acids activate the mTOR signaling is still largely unknown. In our previous study, we discovered that glycyl-tRNA synthetase (GlyRS) is a key mediator of amino-acid-induced mTOR expression and activation in bovine mammary epithelial cells (BMECs). Here we show that amino acids stimulate GlyRS nuclear localization for mTOR expression in BMECs. Met stimulates GlyRS nuclear localization, and the nuclear GlyRS is cleaved into a C-terminus-containing truncated form. We prove that GlyRS has a bipartite nuclear leading sequences, and GlyRS is phosphorylated at Thr544 and Ser704 in the cytoplasm under the stimulation of amino acids (Met, Leu, and Lys). The nuclear GlyRS physically binds to nuclear factor kappa B1, triggers its phosphorylation, thereby enhancing mRNA expression of its target genes including mTOR, S6K1, and 4EBP1. We further demonstrate that GlyRS is required for the inhibition of autophagy by Met. Thus our work elucidates that amino acids trigger GlyRS phosphorylation and nuclear localization to enhance the mRNA expression of mTOR.
Asunto(s)
Aminoácidos/metabolismo , Células Epiteliales/metabolismo , Glicina-ARNt Ligasa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/fisiología , Bovinos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Fosforilación/fisiología , Transducción de Señal/fisiologíaRESUMEN
The origin and evolution of the genetic code is a fundamental challenge in modern biology. At the center of this problem is the correct interaction between amino acids and tRNAs. Aminoacyl-tRNA synthetase is the enzyme responsible for the correct binding between amino acids and tRNAs. Among the 20 canonical amino acid, glycine was the most abundant in prebiotic condition and it must have been one of the first to be incorporated into the genetic code. In this work, we derive the ancestral sequence of Glycyl-tRNA synthetase (GlyRS) and predict its 3D-structure. We show, via molecular docking experiments, the capacity of ancestral GlyRS to bind the tRNA anticodon stem loop, cofactors and substrates. These bindings exhibit high affinity and specificity. We propose that the primordial function of these interactions was to stabilize both compounds to make possible the catalysis. In this context, the anticodon stem loop did contribute to the encoding system and just with the emergence of the mRNA it was co-opted for codification. Thus, we present a model for the origin of the genetic code in which the operational and the anticodon codes did not evolve independently.
Asunto(s)
Glicina-ARNt Ligasa/metabolismo , Simulación del Acoplamiento Molecular/métodos , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Anticodón/metabolismo , Sitios de Unión , Bases de Datos Genéticas , Evolución Molecular , Código Genético , Pliegue de Proteína , Estructura Secundaria de ProteínaRESUMEN
During tissue repair, the injury site releases various bioactive molecules as damage signals to actively recruit stem cells to the damaged region. Despite convincing evidence that mesenchymal stem cells (MSCs) can sense damage signals and promote repair processes, the identity of these signals and how these signals regulate stem cell-mediated tissue repair remain unknown. Glycyl tRNA synthetase (GRS) is a ubiquitously expressed enzyme that catalyzes the first step of protein synthesis in all organisms. In addition to this canonical function, we identified for the first time that GRS is released by damaged tissues or cells in response to various injury signals and may function as a damage signal that activates the proliferative, differentiation, and migratory potential of MSCs, possibly through its identified receptor, cadherin-6 (CDH-6). Binding between GRS and CDH-6 activates survival signals, such as those of the PI3K/Akt and/or FAK/ERK1/2 pathways. More importantly, we also found that MSCs stimulated with GRS show significantly improved homing and differentiation potential and subsequent in vivo therapeutic effects, in a liver fibrosis animal model. Collectively, our findings provide compelling evidence for a novel function of GRS in enhancing the multiple beneficial functions of stem cells via a non-canonical mechanism as a damage signal.
Asunto(s)
Glicina-ARNt Ligasa/metabolismo , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/citología , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Cadherinas/metabolismo , Tetracloruro de Carbono/toxicidad , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quinasa 1 de Adhesión Focal/metabolismo , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/farmacología , Humanos , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 2 de la Matriz/metabolismo , Células Madre Mesenquimatosas/citología , Proteína Homeótica Nanog/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
A full analysis has been conducted of the sequences and secondary structures of viral type-I or related IRESs identified in all of the elements that correspond to the previously described minimal fragment of the enterovirus C IRES, which mimics the glycine tRNA anticodon hairpin in the IRES structure and is necessary for the specific binding of glycyl-tRNA synthetase. Experiments on human glycyl-tRNA synthetase binding with the mRNA fragments of several taxonomically distant viruses showed that the binding constants of these complexes are similar. These results indicate that the regulation of translation initiation via glycyl-tRNA synthetase must be a universal mechanism for these viruses and the corresponding parts of their mRNAs must have similar spatial structures. Furthermore, at least one additional mRNA hairpin with the glycyl anticodon loop has been found in all analyzed viral type-I IRESs. It seems plausible that this extra hairpin is associated with the second RNA-binding site of the glycyl-tRNA synthetase dimer and stabilizes its complex with the viral mRNA.
Asunto(s)
Glicina-ARNt Ligasa/metabolismo , Sitios Internos de Entrada al Ribosoma , Iniciación de la Cadena Peptídica Traduccional , Humanos , ARN Mensajero/genética , ARN Viral/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Dominant mutations in glycyl-tRNA synthetase (GlyRS) cause a subtype of Charcot-Marie-Tooth neuropathy (CMT2D). Although previous studies have shown that GlyRS mutants aberrantly interact with Nrp1, giving insight into the disease's specific effects on motor neurons, these cannot explain length-dependent axonal degeneration. Here, we report that GlyRS mutants interact aberrantly with HDAC6 and stimulate its deacetylase activity on α-tubulin. A decrease in α-tubulin acetylation and deficits in axonal transport are observed in mice peripheral nerves prior to disease onset. An HDAC6 inhibitor used to restore α-tubulin acetylation rescues axonal transport deficits and improves motor functions of CMT2D mice. These results link the aberrant GlyRS-HDAC6 interaction to CMT2D pathology and suggest HDAC6 as an effective therapeutic target. Moreover, the HDAC6 interaction differs from Nrp1 interaction among GlyRS mutants and correlates with divergent clinical presentations, indicating the existence of multiple and different mechanisms in CMT2D.
Asunto(s)
Transporte Axonal/genética , Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Glicina-ARNt Ligasa/metabolismo , Histona Desacetilasa 6/metabolismo , Neuronas Motoras/metabolismo , Acetilación , Animales , Transporte Axonal/efectos de los fármacos , Enfermedad de Charcot-Marie-Tooth/genética , Modelos Animales de Enfermedad , Femenino , Glicina-ARNt Ligasa/genética , Células HEK293 , Histona Desacetilasa 6/antagonistas & inhibidores , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/metabolismo , Nervios Periféricos/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The mechanism by which dominantly inherited mutations in the housekeeping gene GARS, which encodes glycyl-tRNA synthetase (GlyRS), mediate selective peripheral nerve toxicity resulting in Charcot-Marie-Tooth disease type 2D (CMT2D) is still largely unresolved. The transmembrane receptor protein neuropilin 1 (Nrp1) was recently identified as an aberrant extracellular binding partner of mutant GlyRS. Formation of the Nrp1/mutant GlyRS complex antagonises Nrp1 interaction with one of its main natural ligands, vascular endothelial growth factor-A (VEGF-A), contributing to neurodegeneration. However, reduced extracellular binding of VEGF-A to Nrp1 is known to disrupt post-natal blood vessel development and growth. We therefore analysed the vascular system at early and late symptomatic time points in CMT2D mouse muscles, retina, and sciatic nerve, as well as in embryonic hindbrain. Mutant tissues show no difference in blood vessel diameter, density/growth, and branching from embryonic development to three months, spanning the duration over which numerous sensory and neuromuscular phenotypes manifest. Our findings indicate that mutant GlyRS-mediated disruption of Nrp1/VEGF-A signalling is permissive to maturation and maintenance of the vasculature in CMT2D mice.