RESUMEN
Glutamine synthetase (GS) is the enzyme responsible for the biosynthesis of glutamine, providing the only source of endogenous glutamine necessary for several critical metabolic and developmental pathways. GS deficiency, caused by pathogenic variants in the glutamate-ammonia ligase (GLUL) gene, is a rare autosomal recessive inborn error of metabolism characterized by systemic glutamine deficiency, persistent moderate hyperammonemia, and clinically devastating seizures and multi-organ failure shortly after birth. The four cases reported thus far were caused by homozygous GLUL missense variants. We report a case of GS deficiency caused by homozygous GLUL gene deletion, diagnosed prenatally and likely representing the most severe end of the spectrum. We expand the known phenotype of this rare condition with novel dysmorphic, radiographic and neuropathologic features identified on post-mortem examination. The biallelic deletion identified in this case also included the RNASEL gene and was associated with immune dysfunction in the fetus. This case demonstrates that total absence of the GLUL gene in humans is viable beyond the embryonic period, despite the early embryonic lethality found in GLUL animal models.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/genética , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/patología , Femenino , Feto , Glutamina/genética , Homocigoto , Humanos , Recién Nacido , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/patologíaRESUMEN
Streptomyces coelicolor is a Gram-positive soil bacterium with a high metabolic and adaptive potential that is able to utilize a variety of nitrogen sources. However, little is known about the utilization of the alternative nitrogen source ethanolamine. Our study revealed that S. coelicolor can utilize ethanolamine as a sole nitrogen or carbon (N/C) source, although it grows poorly on this nitrogen source due to the absence of a specific ethanolamine permease. Heterologous expression of a putative ethanolamine permease (SPRI_5940) from Streptomycespristinaespiralis positively influenced the biomass accumulation of the overexpression strain grown in defined medium with ethanolamine. In this study, we demonstrated that a glutamine synthetase-like protein, GlnA4 (SCO1613), is involved in the initial metabolic step of a novel ethanolamine utilization pathway in S. coelicolor M145. GlnA4 acts as a gamma-glutamylethanolamide synthetase. Transcriptional analysis revealed that expression of glnA4 was induced by ethanolamine and repressed in the presence of ammonium. Regulation of glnA4 is governed by the transcriptional repressor EpuRI (SCO1614). The ΔglnA4 mutant strain was unable to grow on defined liquid Evans medium supplemented with ethanolamine. High-performance liquid chromatography (HPLC) analysis demonstrated that strain ΔglnA4 is unable to utilize ethanolamine. GlnA4-catalyzed glutamylation of ethanolamine was confirmed in an enzymatic in vitro assay, and the GlnA4 reaction product, gamma-glutamylethanolamide, was detected by HPLC/electrospray ionization-mass spectrometry (HPLC/ESI-MS). In this work, the first step of ethanolamine utilization in S. coelicolor M145 was elucidated, and a putative ethanolamine utilization pathway was deduced based on the sequence similarity and genomic localization of homologous genes.IMPORTANCE Until now, knowledge of the utilization of ethanolamine in Streptomyces was limited. Our work represents the first attempt to reveal a novel ethanolamine utilization pathway in the actinobacterial model organism S. coelicolor through the characterization of the key enzyme gamma-glutamylethanolamide synthetase GlnA4, which is absolutely required for growth in the presence of ethanolamine. The novel ethanolamine utilization pathway is dissimilar to the currently known ethanolamine utilization pathway, which occurs in metabolome. The novel ethanolamine utilization pathway does not result in the production of toxic by-products (such as acetaldehyde); thus, it is not encapsulated. We believe that this contribution is a milestone in understanding the ecology of Streptomyces and the utilization of alternative nitrogen sources. Our report provides new insight into bacterial primary metabolism, which remains complex and partially unexplored.
Asunto(s)
Etanolamina/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glutamato-Amoníaco Ligasa/metabolismo , Redes y Vías Metabólicas , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Glutamato-Amoníaco Ligasa/deficiencia , Nitrógeno/metabolismo , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
Mutations in the human GLUL gene, which encodes the enzyme glutamine synthetase (GS), may cause congenital glutamine synthetase deficiency. The disease was first described in 2005 and only three patients have been reported to date. We report a fourth patient suffering from congenital GS deficiency who was found to have some distinctive clinical findings. The patient was a 30-month-old girl who was referred to us due to developmental delay and seizures which began at 5 months of age. She was seizure free for 5 months with valproic acid and vigabatrin. At presentation, she was found to have microcephaly and hypotonia. Her plasma glutamine concentration was near normal and she had mild hyperammonemia. Cranial magnetic resonance imaging (MRI) showed mild changes. Whole exome sequencing (WES) revealed a homozygous c.121C > T (p.R41C) (p.Arg41Cys) pathogenic variant of the GLUL gene. The diagnosis of this patient underlines the importance of careful evaluation of patients with borderline low glutamine levels. Treatment was begun with L-glutamine and nicotinamide and biochemical improvements have been observed at 6 months of follow-up. The outcome of this patient may provide important data about the effectiveness of glutamine and nicotinamide treatment in patients with congenital GS deficiency.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/complicaciones , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico por imagen , Glutamato-Amoníaco Ligasa/deficiencia , Hipotonía Muscular/diagnóstico por imagen , Hipotonía Muscular/etiología , Convulsiones/diagnóstico por imagen , Convulsiones/etiología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Preescolar , Femenino , Glutamato-Amoníaco Ligasa/genética , Humanos , Hipotonía Muscular/genética , Convulsiones/genéticaRESUMEN
The cellular, molecular, and metabolic mechanisms that underlie the development of mesial temporal lobe epilepsy are incompletely understood. Here we review the role of astrocytes in epilepsy development (a.k.a. epileptogenesis), particularly astrocyte pathologies related to: aquaporin 4, the inwardly rectifying potassium channel Kir4.1, monocarboxylate transporters MCT1 and MCT2, excitatory amino acid transporters EAAT1 and EAAT2, and glutamine synthetase. We propose that inhibition, dysfunction or loss of astrocytic glutamine synthetase is an important causative factor for some epilepsies, particularly mesial temporal lobe epilepsy and glioblastoma-associated epilepsy. We postulate that the regulatory mechanisms of glutamine synthetase as well as the downstream effects of glutamine synthetase dysfunction, represent attractive, new targets for antiepileptogenic interventions. Currently, no antiepileptogenic therapies are available for human use. The discovery of such interventions is important as it will fundamentally change the way we approach epilepsy by preventing the disease from ever becoming manifest after an epileptogenic insult to the brain.
Asunto(s)
Astrocitos/fisiología , Encéfalo/enzimología , Encéfalo/fisiopatología , Epilepsia del Lóbulo Temporal/enzimología , Glutamato-Amoníaco Ligasa/metabolismo , Animales , Astrocitos/enzimología , Epilepsia del Lóbulo Temporal/fisiopatología , Glutamato-Amoníaco Ligasa/deficiencia , HumanosRESUMEN
Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO2-provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Epilepsia/enzimología , Glutamato-Amoníaco Ligasa/deficiencia , Ácido Glutámico/metabolismo , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Modelos Animales de Enfermedad , Epilepsia/genética , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Neuroglía/metabolismo , Receptores de Glutamato/metabolismoRESUMEN
Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
Asunto(s)
Células Endoteliales/enzimología , Células Endoteliales/patología , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/biosíntesis , Neovascularización Patológica , Actinas/metabolismo , Animales , Movimiento Celular , Células Endoteliales/metabolismo , Femenino , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lipoilación , Ratones , Ácido Palmítico/metabolismo , Procesamiento Proteico-Postraduccional , Fibras de Estrés/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
In cells derived from several types of cancer, a transcriptional program drives high consumption of glutamine (Gln), which is used for anaplerosis, leading to a metabolic addiction for the amino acid. Low or absent expression of Glutamine Synthetase (GS), the only enzyme that catalyzes de novo Gln synthesis, has been considered a marker of Gln-addicted cancers. In this study, two human cell lines derived from brain tumors with oligodendroglioma features, HOG and Hs683, have been shown to be GS-negative. Viability of both lines depends from extracellular Gln with EC50 of 0.175 ± 0.056 mM (Hs683) and 0.086 ± 0.043 mM (HOG), thus suggesting that small amounts of extracellular Gln are sufficient for OD cell growth. Gln starvation does not significantly affect the cell content of anaplerotic substrates, which, consistently, are not able to rescue cell growth, but causes hindrance of the Wnt/ß-catenin pathway and protein synthesis attenuation, which is mitigated by transient GS expression. Gln transport inhibitors cause partial depletion of intracellular Gln and cell growth inhibition, but do not lower cell viability. Therefore, GS-negative human oligodendroglioma cells are Gln-auxotrophic but do not use the amino acid for anaplerosis and, hence, are not Gln addicted, exhibiting only limited Gln requirements for survival and growth.
Asunto(s)
Glutamato-Amoníaco Ligasa/deficiencia , Glutamina/metabolismo , Oligodendroglioma/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Técnicas de Inactivación de Genes , Glutamato-Amoníaco Ligasa/deficiencia , Proteínas Recombinantes/metabolismo , Western Blotting , Glutamato-Amoníaco Ligasa/análisis , Células HEK293 , HumanosRESUMEN
Glutamine-synthetase (GS), the glutamine-synthesizing enzyme from glutamate, controls important events, including the release of inflammatory mediators, mammalian target of rapamycin (mTOR) activation, and autophagy. However, its role in macrophages remains elusive. We report that pharmacologic inhibition of GS skews M2-polarized macrophages toward the M1-like phenotype, characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis, which could be partly related to HIF1α activation. As a result of these metabolic changes and HIF1α accumulation, GS-inhibited macrophages display an increased capacity to induce T cell recruitment, reduced T cell suppressive potential, and an impaired ability to foster endothelial cell branching or cancer cell motility. Genetic deletion of macrophagic GS in tumor-bearing mice promotes tumor vessel pruning, vascular normalization, accumulation of cytotoxic T cells, and metastasis inhibition. These data identify GS activity as mediator of the proangiogenic, immunosuppressive, and pro-metastatic function of M2-like macrophages and highlight the possibility of targeting this enzyme in the treatment of cancer metastasis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glutamato-Amoníaco Ligasa/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Metionina Sulfoximina/farmacología , Neovascularización Patológica/prevención & control , Animales , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/patología , Glucosa/metabolismo , Glutamato-Amoníaco Ligasa/deficiencia , Glutamina/metabolismo , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Inyecciones Subcutáneas , Interleucina-10/genética , Interleucina-10/inmunología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/patología , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Cultivo Primario de Células , Ácido Succínico/metabolismo , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patologíaRESUMEN
Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion changes in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of [Formula: see text] and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The purpose of the current study was to determine the role of proximal tubule GS in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low-protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule GS expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.
Asunto(s)
Dieta con Restricción de Proteínas , Proteínas en la Dieta/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Túbulos Renales Proximales/enzimología , Adaptación Fisiológica , Amoníaco/orina , Animales , Biomarcadores/orina , Proteínas de Transporte de Catión/metabolismo , Genotipo , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/genética , Glicoproteínas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Eliminación Renal , Factores de Tiempo , Urea/orinaRESUMEN
Genetic defects in ammonia metabolism can produce irreversible damage of the developing CNS causing an impairment of cognitive and motor functions. We investigated alterations in behavior, synaptic plasticity and gene expression in the hippocampus and dorsal striatum of transgenic mice with systemic hyperammonemia resulting from conditional knockout of hepatic glutamine synthetase (LGS-ko). These mice showed reduced exploratory activity and delayed habituation to a novel environment. Field potential recordings from LGS-ko brain slices revealed significantly reduced magnitude of electrically-induced long-term potentiation (LTP) in both CA3-CA1 hippocampal and corticostriatal synaptic transmission. Corticostriatal but not hippocampal slices from LGS-ko brains demonstrated also significant alterations in long-lasting effects evoked by pharmacological activation of glutamate receptors. Real-time RT-PCR revealed distinct patterns of dysregulated gene expression in the hippocampus and striatum of LGS-ko mice: LGS-ko hippocampus showed significantly modified expression of mRNAs for mGluR1, GluN2B subunit of NMDAR, and A1 adenosine receptors while altered expression of mRNAs for D1 dopamine receptors, the M1 cholinoreceptor and the acetylcholine-synthetizing enzyme choline-acetyltransferase was observed in LGS-ko striatum. Thus, inborn systemic hyperammonemia resulted in significant deficits in novelty acquisition and disturbed synaptic plasticity in corticostriatal and hippocampal pathways involved in learning and goal-directed behavior.
Asunto(s)
Encéfalo/fisiopatología , Conducta Exploratoria , Glutamato-Amoníaco Ligasa/deficiencia , Hiperamonemia/genética , Hiperamonemia/psicología , Plasticidad Neuronal , Animales , Encéfalo/metabolismo , Corteza Cerebral/fisiopatología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Glutamato-Amoníaco Ligasa/genética , Habituación Psicofisiológica , Hipocampo/metabolismo , Hipocampo/fisiopatología , Hiperamonemia/congénito , Hígado/metabolismo , Masculino , Ratones Noqueados , Receptores de Dopamina D2/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión SinápticaRESUMEN
Astrocytes play crucial roles in maintaining brain homeostasis and in orchestrating neural development, all through tightly coordinated steps that cooperate to maintain the balance needed for normal development. Here, we review the alterations in astrocyte functions that contribute to a variety of developmental neurometabolic disorders and provide additional data on the predominant role of astrocyte dysfunction in the neurometabolic neurodegenerative disease glutaric acidemia type I. Finally, we describe some of the therapeutical approaches directed to neurometabolic diseases and discuss if astrocytes can be possible therapeutic targets for treating these disorders.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Astrocitos/patología , Encefalopatías Metabólicas/diagnóstico , Encefalopatías Metabólicas/terapia , Encéfalo/patología , Glutaril-CoA Deshidrogenasa/deficiencia , Enfermedad de Alexander/diagnóstico , Enfermedad de Alexander/metabolismo , Enfermedad de Alexander/patología , Enfermedad de Alexander/terapia , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Antioxidantes/uso terapéutico , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/patología , Ceruloplasmina/deficiencia , Ceruloplasmina/metabolismo , Dieta/métodos , Manejo de la Enfermedad , Glucosa/uso terapéutico , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/metabolismo , Glutaril-CoA Deshidrogenasa/metabolismo , Encefalopatía Hepática/diagnóstico , Encefalopatía Hepática/metabolismo , Encefalopatía Hepática/patología , Encefalopatía Hepática/terapia , Homeostasis , Humanos , Trastornos del Metabolismo del Hierro/diagnóstico , Trastornos del Metabolismo del Hierro/metabolismo , Trastornos del Metabolismo del Hierro/patología , Trastornos del Metabolismo del Hierro/terapia , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/terapia , Neurogénesis/efectos de los fármacos , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Enfermedad de Niemann-Pick Tipo C/terapia , Enfermedad por Deficiencia de Piruvato Carboxilasa/diagnóstico , Enfermedad por Deficiencia de Piruvato Carboxilasa/metabolismo , Enfermedad por Deficiencia de Piruvato Carboxilasa/patología , Enfermedad por Deficiencia de Piruvato Carboxilasa/terapia , Desintoxicación por SorciónRESUMEN
Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Glutamato-Amoníaco Ligasa/química , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/genética , Mutación/genética , Adenosina Trifosfato/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Unión Proteica/genéticaRESUMEN
The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p<0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.
Asunto(s)
Amígdala del Cerebelo/enzimología , Anhedonia/fisiología , Encéfalo/enzimología , Núcleo Amigdalino Central/enzimología , Epilepsia del Lóbulo Temporal/enzimología , Glutamato-Amoníaco Ligasa/deficiencia , Análisis de Varianza , Anhedonia/efectos de los fármacos , Animales , Encéfalo/fisiopatología , Comorbilidad , Trastorno Depresivo/enzimología , Modelos Animales de Enfermedad , Electroencefalografía , Inhibidores Enzimáticos/farmacología , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/fisiopatología , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Hipocampo/fisiología , Masculino , Metionina Sulfoximina/farmacología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Convulsiones/enzimologíaRESUMEN
It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+) accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+) when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4(+). Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+) when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.
Asunto(s)
Compuestos de Amonio/metabolismo , Glutamato-Amoníaco Ligasa/genética , Lotus/enzimología , Aspartatoamoníaco Ligasa/metabolismo , Glutamato Deshidrogenasa/metabolismo , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/metabolismo , Lotus/genética , Lotus/metabolismo , Mutación , Fotosíntesis , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Plastidios/enzimología , Plastidios/genética , Plastidios/metabolismo , ARN de Planta/aislamiento & purificación , ARN de Planta/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Glutamato-Amoníaco Ligasa/deficiencia , Glutamina/sangre , Hiperamonemia/genética , NAD/sangre , NAD/deficiencia , Linfocitos B/citología , Técnicas de Cultivo de Célula , Suplementos Dietéticos , Fibroblastos/citología , Glutamato-Amoníaco Ligasa/genética , Humanos , Mutación PuntualRESUMEN
Epilepsy is a family of brain disorders with a largely unknown etiology and high percentage of pharmacoresistance. The clinical manifestations of epilepsy are seizures, which originate from aberrant neuronal synchronization and hyperexcitability. Reactive astrocytosis, a hallmark of the epileptic tissue, develops into loss-of-function of glutamine synthetase, impairment of glutamate-glutamine cycle and increase in extracellular and astrocytic glutamate concentration. Here, we argue that chronically elevated intracellular glutamate level in astrocytes is instrumental to alterations in the metabolism of glycogen and leads to the synthesis of polyglucosans. Unaccessibility of glycogen-degrading enzymes to these insoluble molecules compromises the glycogenolysis-dependent reuptake of extracellular K(+) by astrocytes, thereby leading to increased extracellular K(+) and associated membrane depolarization. Based on current knowledge, we propose that the deterioration in structural homogeneity of glycogen particles is relevant to disruption of brain K(+) homeostasis and increased susceptibility to seizures in epilepsy.
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Epilepsia/metabolismo , Glucógeno/química , Potasio/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Convulsivantes/farmacología , Susceptibilidad a Enfermedades , Gliosis/metabolismo , Glucanos/metabolismo , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/deficiencia , Glutamatos/metabolismo , Glutamina/metabolismo , Glucógeno/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Homeostasis , Humanos , Potenciales de la Membrana , Metionina Sulfoximina/farmacología , Estructura Molecular , Neuronas/metabolismo , Convulsiones/inducido químicamente , Convulsiones/etiología , Convulsiones/metabolismo , Sueño/fisiología , Privación de Sueño/fisiopatología , Relación Estructura-ActividadRESUMEN
In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume-rhizobia symbiosis by modifying nodule development and metabolic processes.
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Glutamato-Amoníaco Ligasa/genética , Lotus/metabolismo , Lotus/microbiología , Mesorhizobium/fisiología , Fijación del Nitrógeno , Simbiosis , Glutamato-Amoníaco Ligasa/deficiencia , Mesorhizobium/genética , Mesorhizobium/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Glutamine synthetase (GS) is ubiquitously expressed in mammalian organisms and is a key enzyme in nitrogen metabolism. It is the only known enzyme capable of synthesising glutamine, an amino acid with many critical roles in the human organism. A defect in GLUL, encoding for GS, leads to congenital systemic glutamine deficiency and has been described in three patients with epileptic encephalopathy. There is no established treatment for this condition.Here, we describe a therapeutic trial consisting of enteral and parenteral glutamine supplementation in a four year old patient with GS deficiency. The patient received increasing doses of glutamine up to 1020 mg/kg/day. The effect of this glutamine supplementation was monitored clinically, biochemically, and by studies of the electroencephalogram (EEG) as well as by brain magnetic resonance imaging and spectroscopy.Treatment was well tolerated and clinical monitoring showed improved alertness. Concentrations of plasma glutamine normalized while levels in cerebrospinal fluid increased but remained below the lower reference range. The EEG showed clear improvement and spectroscopy revealed increasing concentrations of glutamine and glutamate in brain tissue. Concomitantly, there was no worsening of pre-existing chronic hyperammonemia.In conclusion, supplementation of glutamine is a safe therapeutic option for inherited GS deficiency since it corrects the peripheral biochemical phenotype and partially also improves the central biochemical phenotype. There was some clinical improvement but the patient had a long standing severe encephalopathy. Earlier supplementation with glutamine might have prevented some of the neuronal damage.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/terapia , Aminoácidos/metabolismo , Glutamato-Amoníaco Ligasa/deficiencia , Glutamina/administración & dosificación , Encéfalo/patología , Niño , Humanos , Imagen por Resonancia MagnéticaRESUMEN
Glutamine deficiency with hyperammonemia due to an inherited defect of glutamine synthetase (GS) was found in a 2 year old patient. He presented neonatal seizures and developed chronic encephalopathy. Thus, GS deficiency leads to severe neurological disease but is not always early lethal.