RESUMEN
The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Enfermedades Pulmonares/genética , Mutación/genética , Adolescente , Alelos , Enfermedad de Charcot-Marie-Tooth/genética , Preescolar , Femenino , Genes Recesivos/genética , Heterocigoto , Humanos , Lactante , Masculino , Biosíntesis de Proteínas/genéticaRESUMEN
Many human tRNA synthetases evolved alternative functions outside of protein synthesis. These functions are associated with over 200 splice variants (SVs), most of which are catalytic nulls that engender new biology. While known to regulate non-translational activities, little is known about structures resulting from natural internal ablations of any protein. Here, we report analysis of two closely related, internally deleted, SVs of homodimeric human tyrosyl-tRNA synthetase (TyrRS). In spite of both variants ablating a portion of the catalytic core and dimer-interface contacts of native TyrRS, each folded into a distinct stable structure. Biochemical and nuclear magnetic resonance (NMR) analysis showed that the internal deletion of TyrRSΔE2-4 SV gave an alternative, neomorphic dimer interface 'orthogonal' to that of native TyrRS. In contrast, the internal C-terminal splice site of TyrRSΔE2-3 prevented either dimerization interface from forming, and yielded a predominantly monomeric protein. Unlike ubiquitous TyrRS, the neomorphs showed clear tissue preferences, which were distinct from each other. The results demonstrate a sophisticated structural plasticity of a human tRNA synthetase for architectural reorganizations that are preferentially elicited in specific tissues.
Asunto(s)
Empalme Alternativo , Multimerización de Proteína , Estructura Secundaria de Proteína , Tirosina-ARNt Ligasa/química , Tirosina-ARNt Ligasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Dominio Catalítico/genética , Línea Celular , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Tirosina-ARNt Ligasa/metabolismoRESUMEN
Misfolded proteins are an emerging hallmark of cardiac diseases. Although some misfolded proteins, such as desmin, are associated with mutations in the genes encoding these disease-associated proteins, little is known regarding more general mechanisms that contribute to the generation of misfolded proteins in the heart. Reduced translational fidelity, caused by a hypomorphic mutation in the editing domain of alanyl-tRNA synthetase (AlaRS), resulted in accumulation of misfolded proteins in specific mouse neurons. By further genetic modulation of the editing activity of AlaRS, we generated mouse models with broader phenotypes, the severity of which was directly related to the degree of compromised editing. Severe disruption of the editing activity of AlaRS caused embryonic lethality, whereas an intermediate reduction in AlaRS editing efficacy resulted in ubiquitinated protein aggregates and mitochondrial defects in cardiomyocytes that were accompanied by progressive cardiac fibrosis and dysfunction. In addition, autophagic vacuoles accumulated in mutant cardiomyocytes, suggesting that autophagy is insufficient to eliminate misfolded proteins. These findings demonstrate that the pathological consequences of diminished tRNA synthetase editing activity, and thus translational infidelity, are dependent on the cell type and the extent of editing disruption, and provide a previously unidentified mechanism underlying cardiac proteinopathy.
Asunto(s)
Alanina-ARNt Ligasa/deficiencia , Alanina-ARNt Ligasa/genética , Cardiopatías/genética , Deficiencias en la Proteostasis/genética , Edición de ARN , Alelos , Animales , Proteínas Bacterianas/genética , Ecocardiografía , Homeostasis , Humanos , Hidrólisis , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Moleculares , Mutación , Miocitos Cardíacos/ultraestructura , Parafina/química , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain. Despite the large deletions, the two SVs cross-react with a substantial population of anti-Jo-l antibodies from myositis patients. Moreover, expression of at least one of the SVs is up-regulated in dermatomyositis patients, and cell-based experiments show that both SVs and HisRS can be secreted. We suggest that, in patients with inflammatory myositis, anti-Jo-1 antibodies may have extracellular activity.
Asunto(s)
Empalme Alternativo , Autoanticuerpos/metabolismo , Epítopos/metabolismo , Histidina-ARNt Ligasa/metabolismo , Miositis/enzimología , Autoanticuerpos/inmunología , Línea Celular Tumoral , Epítopos/genética , Epítopos/inmunología , Histidina-ARNt Ligasa/genética , Histidina-ARNt Ligasa/inmunología , Humanos , Inflamación/enzimología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Miositis/genética , Miositis/inmunología , Miositis/patología , Estructura Terciaria de ProteínaRESUMEN
Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Exosomas/genética , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , Empalme Alternativo , Aminoacil-ARNt Sintetasas/metabolismo , Western Blotting , Neoplasias de la Mama , Línea Celular Tumoral , Citoplasma/genética , Citoplasma/metabolismo , Exosomas/metabolismo , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Células MCF-7 , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immunity has evolved to balance the destructive nature of inflammation with wound healing to overcome trauma, infection, environmental insults, and rogue malignant cells. The inflammatory response is marked by overlapping phases of initiation, resolution, and post-resolution remodeling. However, the disruption of these events can lead to prolonged tissue damage and organ dysfunction, resulting long-term disease states. Macrophages are the archetypic phagocytes present within all tissues and are important contributors to these processes. Pleiotropic and highly plastic in their responses, macrophages support tissue homeostasis, repair, and regeneration, all while balancing immunologic self-tolerance with the clearance of noxious stimuli, pathogens, and malignant threats. Neuropilin-2 (Nrp2), a promiscuous co-receptor for growth factors, semaphorins, and integrins, has increasingly been recognized for its unique role in tissue homeostasis and immune regulation. Notably, recent studies have begun to elucidate the role of Nrp2 in both non-hematopoietic cells and macrophages with cardiothoracic disease. Herein, we describe the unique role of Nrp2 in diseases of the heart and lung, with an emphasis on Nrp2 in macrophages, and explore the potential to target Nrp2 as a therapeutic intervention.
RESUMEN
Neuropilin-2 (NRP2) is a cell surface receptor that plays key roles in lymphangiogenesis, but also in pathophysiological conditions such as cancer and inflammation. NRP2 targeting by efzofitimod, a novel immunomodulatory molecule, is currently being tested for the treatment of pulmonary sarcoidosis. To date, no anti-NRP2 antibodies are available for companion diagnostics. Here we describe the development and characterization of a novel NRP2 antibody. Using a variety of research techniques, that is, enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry, we demonstrate that our antibody detects all major NRP2 isoforms and does not cross-react with NRP1. Using this antibody, we show high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies. Our novel anti-NRP2 antibody could prove to be a useful clinical tool for sarcoidosis and other indications where NRP2 has been implicated. Clinical Trial Registration: clinicaltrials.gov NCT05415137.
Asunto(s)
Neoplasias , Sarcoidosis , Humanos , Neuropilina-2/metabolismo , Anticuerpos Monoclonales , Neoplasias/diagnóstico , Inmunohistoquímica , Sarcoidosis/diagnósticoRESUMEN
Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.
Asunto(s)
Neuropilina-2 , Neoplasias de la Mama Triple Negativas , Humanos , Neuropilina-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Unión Proteica , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Neuropilina-1/metabolismoRESUMEN
Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.
Asunto(s)
Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Enfermedades Neurodegenerativas/enzimología , Pliegue de Proteína , Acetilación , Alanina/genética , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Animales , Catálisis , Escherichia coli/genética , Fibroblastos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Enfermedades Neurodegenerativas/genética , Fenotipo , Estructura Terciaria de Proteína , Células de Purkinje/metabolismo , Células de Purkinje/patología , ARN de Transferencia de Alanina/genética , Serina/genética , Serina/metabolismoRESUMEN
His-tRNA synthetase (HARS) is targeted by autoantibodies in chronic and acute inflammatory anti-Jo-1-positive antisynthetase syndrome. The extensive activation and migration of immune cells into lung and muscle are associated with interstitial lung disease, myositis, and morbidity. It is unknown whether the sequestration of HARS is an epiphenomenon or plays a causal role in the disease. Here, we show that HARS circulates in healthy individuals, but it is largely undetectable in the serum of anti-Jo-1-positive antisynthetase syndrome patients. In cultured primary human skeletal muscle myoblasts (HSkMC), HARS is released in increasing amounts during their differentiation into myotubes. We further show that HARS regulates immune cell engagement and inhibits CD4+ and CD8+ T-cell activation. In mouse and rodent models of acute inflammatory diseases, HARS administration downregulates immune activation. In contrast, neutralization of extracellular HARS by high-titer antibody responses during tissue injury increases susceptibility to immune attack, similar to what is seen in humans with anti-Jo-1-positive disease. Collectively, these data suggest that extracellular HARS is homeostatic in normal subjects, and its sequestration contributes to the morbidity of the anti-Jo-1-positive antisynthetase syndrome.
Asunto(s)
Histidina-ARNt Ligasa/sangre , Inmunidad , Especificidad de Órganos , Animales , Autoanticuerpos/sangre , Estudios de Casos y Controles , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Histidina-ARNt Ligasa/inmunología , Humanos , Inmunidad/efectos de los fármacos , Inmunomodulación/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Pulmón/efectos de los fármacos , Pulmón/patología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Células Musculares/efectos de los fármacos , Células Musculares/enzimología , Músculos/efectos de los fármacos , Músculos/patología , Miositis/sangre , Miositis/diagnóstico por imagen , Miositis/inmunología , Especificidad de Órganos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tomografía Computarizada por Rayos XRESUMEN
Of the many inherited Charcot-Marie-Tooth peripheral neuropathies, type 2D (CMT2D) is caused by dominant point mutations in the gene GARS, encoding glycyl tRNA synthetase (GlyRS). Here we report a dominant mutation in Gars that causes neuropathy in the mouse. Importantly, both sensory and motor axons are affected, and the dominant phenotype is not caused by a loss of the GlyRS aminoacylation function. Mutant mice have abnormal neuromuscular junction morphology and impaired transmission, reduced nerve conduction velocities, and a loss of large-diameter peripheral axons, without defects in myelination. The mutant GlyRS enzyme retains aminoacylation activity, and a loss-of-function allele, generated by a gene-trap insertion, shows no dominant phenotype in mice. These results indicate that the CMT2D phenotype is caused not by reduction of the canonical GlyRS activity and insufficiencies in protein synthesis, but instead by novel pathogenic roles for the mutant GlyRS that specifically affect peripheral neurons.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Modelos Animales de Enfermedad , Glicina-ARNt Ligasa/genética , Mutación/genética , Enfermedades del Sistema Nervioso Periférico/genética , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Axones/patología , Axones/ultraestructura , Secuencia de Bases , Enfermedad de Charcot-Marie-Tooth/clasificación , Enfermedad de Charcot-Marie-Tooth/enzimología , Mapeo Cromosómico , Femenino , Genes Dominantes/genética , Glicina-ARNt Ligasa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Enfermedades del Sistema Nervioso Periférico/enzimología , Enfermedades del Sistema Nervioso Periférico/patología , Nervio Ciático/metabolismo , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Homología de Secuencia de AminoácidoRESUMEN
Aminoacyl-tRNA synthetases prevent mistranslation, or genetic code ambiguity, through specialized editing reactions. Mutations that disrupt editing in bacteria adversely affect cell growth and viability, and recent work in the mouse supports the idea that translational errors caused by an editing defect lead to a neurological disease-like phenotype. To further investigate the connection of mistranslation to cell pathology, we introduced an inducible transgene expressing an editing-deficient valyl-tRNA synthetase into mammalian cells. Introducing mistranslation precipitated a disruption of cell morphology and membrane blebbing, accompanied by activation of caspase-3, consistent with an apoptotic response. Addition of a noncanonical amino acid that is misactivated, but not cleared, by the editing-defective enzyme exacerbated these effects. A special ambiguity-detecting sensor provided direct readout of mistranslation in vivo, supporting the possibility that decreased translational fidelity could be associated with disease.
Asunto(s)
Muridae/genética , Edición de ARN , Transcripción Genética , Valina-ARNt Ligasa/genética , Animales , Apoptosis/genética , Técnicas Biosensibles , Caspasa 3/metabolismo , Células Cultivadas , Ratones , Modelos Moleculares , Células 3T3 NIH , Conformación Proteica , Transgenes , Valina-ARNt Ligasa/metabolismoRESUMEN
Genetic efficiency in higher organisms depends on mechanisms to create multiple functions from single genes. To investigate this question for an enzyme family, we chose aminoacyl tRNA synthetases (AARSs). They are exceptional in their progressive and accretive proliferation of noncatalytic domains as the Tree of Life is ascended. Here we report discovery of a large number of natural catalytic nulls (CNs) for each human AARS. Splicing events retain noncatalytic domains while ablating the catalytic domain to create CNs with diverse functions. Each synthetase is converted into several new signaling proteins with biological activities "orthogonal" to that of the catalytic parent. We suggest that splice variants with nonenzymatic functions may be more general, as evidenced by recent findings of other catalytically inactive splice-variant enzymes.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Dominio Catalítico , Empalme Alternativo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Catálisis , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Especificidad de Órganos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of tRNAs in the cytoplasm. Surprisingly, AARSs also have critical extracellular and nuclear functions. Evolutionary pressure for new functions might be manifested by splice variants that skip only an internal catalytic domain (CD) and link noncatalytic N- and C-terminal polypeptides. Using disease-associated histidyl-tRNA synthetase (HisRS) as an example, we found an expressed 171-amino acid protein (HisRSΔCD) that deleted the entire CD, and joined an N-terminal WHEP to the C-terminal anticodon-binding domain (ABD). X-ray crystallography and three-dimensional NMR revealed the structures of human HisRS and HisRSΔCD. In contrast to homodimeric HisRS, HisRSΔCD is monomeric, where rupture of the ABD's packing with CD resulted in a dumbbell-like structure of flexibly linked WHEP and ABD domains. In addition, the ABD of HisRSΔCD presents a distinct local conformation. This natural internally deleted HisRS suggests evolutionary pressure to reshape AARS tertiary and quaternary structures for repurposing.
Asunto(s)
Evolución Molecular , Histidina-ARNt Ligasa/química , Eliminación de Secuencia , Anticuerpos/sangre , Anticuerpos/inmunología , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , ADN Complementario/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Histidina-ARNt Ligasa/genética , Histidina-ARNt Ligasa/inmunología , Humanos , Enfermedades Pulmonares Intersticiales/sangre , Enfermedades Pulmonares Intersticiales/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Miositis/sangre , Miositis/inmunología , Isoformas de Proteínas , Estructura Secundaria de Proteína , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Dominio Catalítico , Modelos Moleculares , Triptófano/metabolismo , Aminoacil-ARNt Sintetasas/genética , Western Blotting , Humanos , Inmunoprecipitación , Mutagénesis , Relación Estructura-ActividadRESUMEN
Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot-Marie-Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structures are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located approximately 30 A away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/genética , Mutación , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Charcot-Marie-Tooth (CMT) diseases are the most common heritable peripheral neuropathy. At least 10 different mutant alleles of GARS (the gene for glycyl-tRNA synthetase) have been reported to cause a dominant axonal form of CMT (type 2D). A unifying connection between these mutations and CMT has been unclear. Here, mapping mutations onto the recently determined crystal structure of human GlyRS showed them within a band encompassing both sides of the dimer interface, with two CMT-causing mutations being at sites that are complementary partners of a "kissing" contact across the dimer interface. The CMT phenotype is shown here to not correlate with aminoacylation activity. However, most mutations affect dimer formation (to enhance or weaken). Seven CMT-causing variants and the wild-type protein were expressed in transfected neuroblastoma cells that sprout primitive neurites. Wild-type GlyRS distributed into the nascent neurites and was associated with normal neurite sprouting. In contrast, all mutant proteins were distribution-defective. Thus, CMT-causing mutations of GlyRS share a common defect in localization. This defect may be connected in some way to a change in the surfaces at the dimer interface.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/genética , Mutación , Neuritas/enzimología , Neuritas/patología , Sustitución de Aminoácidos/genética , Animales , Axones/enzimología , Axones/patología , Línea Celular Tumoral , Enfermedad de Charcot-Marie-Tooth/patología , Dimerización , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/metabolismo , Humanos , Ratones , Neuritas/metabolismo , Propiedades de Superficie , Transfección , Tirosina-ARNt Ligasa/genéticaRESUMEN
The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids. Ambiguity in the code is prevented by editing activities whereby incorrect aminoacylations are cleared by specialized hydrolytic reactions of aminoacyl tRNA synthetases. Whereas editing reactions have long been known, their significance for cell viability is still poorly understood. Here we investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS). The four mutant enzymes were shown to differ quantitatively in the severity of the defect in their ability to clear mischarged tRNA in vitro. Strikingly, in the presence of excess concentrations of alpha-aminobutyrate, one of the amino acids that is misactivated by ValRS, growth of bacterial strains bearing these mutant alleles is arrested. The concentration of misactivated amino acid required for growth arrest correlates inversely in a rank order with the degree of the editing defect seen in vitro. Thus, cell viability depends directly on the suppression of genetic code ambiguity by these specific editing reactions and is finely tuned to any perturbation of these reactions.