RESUMEN
Phosphatidylserine (PS) synthase 1 (PSS1) of mammalian cells is a multiple membrane-spanning protein of the endoplasmic reticulum (ER) and regulated by inhibition with the product PS. Alanine-scanning mutagenesis of PSS1 has revealed eight amino acid residues as those crucial for its activity and six as those important for its regulation. Furthermore, three missense mutations in the human PSS1 gene, which lead to regulatory dysfunctions of PSS1 and are causative of Lenz-Majewski syndrome, have been identified. In this study, we investigated the membrane topology of PSS1 by means of epitope insertion and immunofluorescence. According to a 10-transmembrane segment model supported by topology analysis of PSS1, all the 8 amino acid residues crucial for the enzyme activity were localized to the luminal side of the lipid bilayer or the lumen of the ER, whereas all the 9 amino acid residues involved in the enzyme regulation were localized to the cytosol or the cytoplasmic side of the lipid bilayer of the ER. This localization of the functional amino acid residues suggests that PSS1 is regulated by inhibition with PS in the cytoplasmic leaflet of the ER membrane and synthesizes PS at the luminal leaflet.
Asunto(s)
Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , Membrana Dobles de Lípidos/metabolismo , Transferasas de Grupos Nitrogenados/metabolismo , Retículo Endoplásmico/genética , Células HeLa , Humanos , Transferasas de Grupos Nitrogenados/genéticaRESUMEN
Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mutación , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Transferasas de Grupos Nitrogenados/genética , Biosíntesis de Proteínas/genética , Humanos , ARN de Transferencia de Glutamina/metabolismo , Aminoacilación de ARN de Transferencia , Tuberculosis/microbiologíaRESUMEN
The function of the glutaminyl-tRNA synthetase and Glu-tRNAGln amidotransferase might be related to the origin of the genetic code because, for example, glutaminyl-tRNA synthetase catalyses the fundamental reaction that makes the genetic code. If the evolutionary stage of the origin of these two enzymes could be unambiguously identified, then the genetic code should still have been originating at that particular evolutionary stage because the fundamental reaction that makes the code itself was still evidently evolving. This would result in that particular evolutionary moment being attributed to the evolutionary stage of the progenote because it would have a relationship between the genotype and the phenotype not yet fully realized because the genetic code was precisely still originating. I then analyzed the distribution of the glutaminyl-tRNA synthetase and Glu-tRNAGln aminodotrasferase in the main phyletic lineages. Since in some cases the origin of these two enzymes can be related to the evolutionary stages of ancestors of archaea and eukaryotes, this would indicate these ancestors as progenotes because at that evolutionary moment the genetic code was evidently still evolving, thus realizing the definition of progenote. The conclusion that the ancestor of archaea and that of eukaryotes were progenotes would imply that even the last universal common ancestor (LUCA) was a progenote because it appeared, on the tree of life, temporally before these ancestors.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Archaea/genética , Eucariontes/genética , Evolución Molecular , Transferasas de Grupos Nitrogenados/genética , Filogenia , Aminoacil-ARNt Sintetasas/metabolismo , Archaea/enzimología , Eucariontes/enzimología , Transferasas de Grupos Nitrogenados/metabolismoRESUMEN
BACKGROUND: Researchers have identified about 40 genes with mutations that result in the most common cause of CKD in children, congenital anomalies of the kidney and urinary tract (CAKUT), but approximately 85% of patients with CAKUT lack mutations in these genes. The anomalies that comprise CAKUT are clinically heterogenous, and thought to be caused by disturbances at different points in kidney development. However, identification of novel CAKUT-causing genes remains difficult because of their variable expressivity, incomplete penetrance, and heterogeneity. METHODS: We investigated two generations of a family that included two siblings with CAKUT. Although the parents and another child were healthy, the two affected siblings presented the same manifestations, unilateral renal agenesis and contralateral renal hypoplasia. To search for a novel causative gene of CAKUT, we performed whole-exome and whole-genome sequencing of DNA from the family members. We also generated two lines of genetically modified mice with a gene deletion present only in the affected siblings, and performed immunohistochemical and phenotypic analyses of these mice. RESULTS: We found that the affected siblings, but not healthy family members, had a homozygous deletion in the Cobalamin Synthetase W Domain-Containing Protein 1 (CBWD1) gene. Whole-genome sequencing uncovered genomic breakpoints, which involved exon 1 of CBWD1, harboring the initiating codon. Immunohistochemical analysis revealed high expression of Cbwd1 in the nuclei of the ureteric bud cells in the developing kidneys. Cbwd1-deficient mice showed CAKUT phenotypes, including hydronephrosis, hydroureters, and duplicated ureters. CONCLUSIONS: The identification of a deletion in CBWD1 gene in two siblings with CAKUT implies a role for CBWD1 in the etiology of some cases of CAKUT.
Asunto(s)
Eliminación de Gen , Transferasas de Grupos Nitrogenados/genética , Anomalías Urogenitales/genética , Reflujo Vesicoureteral/genética , Adulto , Animales , Femenino , Humanos , Masculino , Ratones , LinajeRESUMEN
While hundreds of consistently altered metabolic genes had been identified in hepatocellular carcinoma (HCC), the prognostic role of them remains to be further elucidated. Messenger RNA expression profiles and clinicopathological data were downloaded from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma and GSE14520 data set from the Gene Expression Omnibus database. Univariate Cox regression analysis and lasso Cox regression model established a novel four-gene metabolic signature (including acetyl-CoA acetyltransferase 1, glutamic-oxaloacetic transaminase 2, phosphatidylserine synthase 2, and uridine-cytidine kinase 2) for HCC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was significantly correlated with other negative prognostic factors such as higher α-fetoprotein. The signature was found to be an independent prognostic factor for HCC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. Furthermore, gene set enrichment analyses revealed several significantly enriched pathways, which might help explain the underlying mechanisms. Our study identified a novel robust four-gene metabolic signature for HCC prognosis prediction. The signature might reflect the dysregulated metabolic microenvironment and provided potential biomarkers for metabolic therapy and treatment response prediction in HCC.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Transcriptoma/genética , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Adulto , Anciano , Aspartato Aminotransferasas/genética , Aspartato Aminotransferasas/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo , Nomogramas , Pronóstico , Uridina Quinasa/genética , Uridina Quinasa/metabolismoRESUMEN
The B vitamins provide essential co-factors for central metabolism in all organisms. In plants, B vitamins have surprising emerging roles in development, stress tolerance and pathogen resistance. Hence, there is a paramount interest in understanding the regulation of vitamin biosynthesis as well as the consequences of vitamin deficiency in crop species. To facilitate genetic analysis of B vitamin biosynthesis and functions in maize, we have mined the UniformMu transposon resource to identify insertional mutations in vitamin pathway genes. A screen of 190 insertion lines for seed and seedling phenotypes identified mutations in biotin, pyridoxine and niacin biosynthetic pathways. Importantly, isolation of independent insertion alleles enabled genetic confirmation of genotype-to-phenotype associations. Because B vitamins are essential for survival, null mutations often have embryo lethal phenotypes that prevent elucidation of subtle, but physiologically important, metabolic consequences of sub-optimal (functional) vitamin status. To circumvent this barrier, we demonstrate a strategy for refined genetic manipulation of vitamin status based on construction of heterozygotes that combine strong and hypomorphic mutant alleles. Dosage analysis of pdx2 alleles in endosperm revealed that endosperm supplies pyridoxine to the developing embryo. Similarly, a hypomorphic bio1 allele enabled analysis of transcriptome and metabolome responses to incipient biotin deficiency in seedling leaves. We show that systemic pipecolic acid accumulation is an early metabolic response to sub-optimal biotin status highlighting an intriguing connection between biotin, lysine metabolism and systemic disease resistance signaling. Seed-stocks carrying insertions for vitamin pathway genes are available for free, public distribution via the Maize Genetics Cooperation Stock Center.
Asunto(s)
Complejo Vitamínico B/genética , Complejo Vitamínico B/metabolismo , Zea mays/genética , Zea mays/metabolismo , Alelos , Vías Biosintéticas/genética , Vías Biosintéticas/fisiología , Elementos Transponibles de ADN/genética , Endospermo/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Transferasas de Grupos Nitrogenados/genética , Fenotipo , Hojas de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Piridoxina/metabolismo , Semillas/genética , TranscriptomaRESUMEN
Lenz-Majewski syndrome (LMS) is an extremely rare type of cutis laxa caused by dominant mutations in PTDSS1 gene. We report an Egyptian patient who presented with cutis laxa, brachydactyly, and progeroid features. LMS syndrome was suspected and a previously reported de novo heterozygous missense mutation (c.284G > T, p.R95L) in PTDSS1 was identified. To the best of our knowledge, nine molecularly proven patients with LMS from different ethnicities have been reported. Our patient is the first report from the Middle East and the tenth molecularly proven patient reported to date. His clinical features were in accordance with LMS syndrome. In addition, his hands X-ray images showed hypoplastic or absent middle and proximal phalanges but sparing the thumbs. This hand patterning was similarly observed before. Further, he had relatively large and convex fingernails. Our report highlights this unique hand patterning and suggests these signs should be considered among the diagnostic criteria of LMS. Further reports of patients with PTDSS1 mutations are necessary to further elucidate the detailed clinical features of LMS syndrome.
Asunto(s)
Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/genética , Discapacidad Intelectual/genética , Egipto , Exones/genética , Humanos , Lactante , Intrones/genética , Masculino , Transferasas de Grupos Nitrogenados/genética , SíndromeRESUMEN
Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.
Asunto(s)
CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa , Transferasas de Grupos Nitrogenados/genética , Síndrome de Costilla Pequeña y Polidactilia , Pez Cebra , Animales , Animales Modificados Genéticamente , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/genética , Linaje de la Célula , Células HeLa , Humanos , TransgenesRESUMEN
Vitamin B6 (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B6 content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B6 in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B6 biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B6 levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B6 biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B6 in leaves (up to 28.3-fold) and roots (up to 12-fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B6 did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B6 content could also be achieved in rice seeds (up to 3.1-fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B6 -enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.
Asunto(s)
Arabidopsis/genética , Oryza/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Vitamina B 6/biosíntesis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Liasas de Carbono-Nitrógeno/genética , Liasas de Carbono-Nitrógeno/metabolismo , Endospermo/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo , Oryza/genética , Oryza/crecimiento & desarrollo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Estrés Salino/fisiología , Semillas/metabolismo , Transgenes , Vitamina B 6/metabolismo , Xanthomonas/patogenicidadRESUMEN
Non-typhoidal Salmonella can colonize the gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 ( yfeJ) encodes one of these proteins, and it is currently annotated as a type I glutamine amidotransferase. STM2437 was purified to homogeneity, and its catalytic properties with a wide range of γ-glutamyl derivatives were determined. The catalytic efficiency toward the hydrolysis of l-glutamine was extremely weak with a kcat/ Km value of 20 M-1 s-1. γ-l-Glutamyl hydroxamate was identified as the best substrate for STM2437, with a kcat/ Km value of 9.6 × 104 M-1 s-1. A homology model of STM2437 was constructed on the basis of the known crystal structure of a protein of unknown function (Protein Data Bank entry 3L7N ), and γ-l-glutamyl hydroxamate was docked into the active site based on the binding of l-glutamine in the active site of carbamoyl phosphate synthetase. Acivicin was shown to inactivate the enzyme by reaction with the active site cysteine residue and the subsequent loss of HCl. Mutation of Cys91 to serine completely abolished catalytic activity. Inactivation of STM2437 did not affect the ability of this strain to colonize mice, but it inhibited the growth of S. enterica Typhimurium in bacteriologic media containing γ-l-glutamyl hydroxamate.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/metabolismo , Salmonelosis Animal/microbiología , Animales , Proteínas Bacterianas/genética , Bovinos , Enfermedades de los Bovinos/microbiología , Colitis/microbiología , Colitis/veterinaria , Activación Enzimática , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Glutamatos/metabolismo , Glutamatos/farmacología , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/farmacología , Hidroxilamina/farmacología , Isoxazoles/farmacología , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Transferasas de Grupos Nitrogenados/genética , Conformación Proteica , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Especificidad por SustratoRESUMEN
Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.
Asunto(s)
Cardiomiopatías/enzimología , Cardiomiopatías/genética , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Mutación/genética , Transferasas de Grupos Nitrogenados/genética , Subunidades de Proteína/genética , Secuencia de Aminoácidos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Lactante , Recién Nacido , Lentivirus/metabolismo , Masculino , Modelos Moleculares , Miocardio/patología , Miocardio/ultraestructura , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/metabolismo , Fosforilación Oxidativa , Linaje , Biosíntesis de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise in developed countries due to overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-diabetes, we evaluated the impact of a diet high in saturated fat (HFD) on O-GlcNAcylation of retinal proteins, as dysregulated O-GlcNAcylation contributes to diabetic complications and evidence supports a role in DR. Protein O-GlcNAcylation was increased in the retina of mice fed a HFD as compared to littermates receiving control chow. Similarly, O-GlcNAcylation was elevated in retinal Müller cells in culture exposed to the saturated fatty acid palmitate or the ceramide analog Cer6. One potential mechanism responsible for elevated O-GlcNAcylation is increased flux through the hexosamine biosynthetic pathway (HBP). Indeed, inhibition of the pathway's rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) prevented Cer6-induced O-GlcNAcylation. Importantly, expression of the mRNA encoding GFAT2, but not GFAT1 was elevated in both the retina of mice fed a HFD and in retinal cells in culture exposed to palmitate or Cer6. Notably, expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) was increased in the retina of mice fed a HFD and NR4A1 expression was sufficient to promote GFAT2 mRNA expression and O-GlcNAcylation in retinal cells in culture. Whereas palmitate or Cer6 addition to culture medium enhanced NR4A1 and GFAT2 expression, chemical inhibition of NR4A1 transactivation repressed Cer6-induced GFAT2 mRNA expression. Overall, the results support a model wherein HFD increases retinal protein O-GlcNAcylation by promoting NR4A1-dependent GFAT2 expression.
Asunto(s)
Acetilglucosamina/metabolismo , Dieta Alta en Grasa/efectos adversos , Proteínas del Ojo/metabolismo , Transferasas de Grupos Nitrogenados/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Retina/metabolismo , Regulación hacia Arriba , Acilación , Animales , Línea Celular , Ceramidas/metabolismo , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Retinopatía Diabética/etiología , Retinopatía Diabética/metabolismo , Proteínas del Ojo/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Transferasas de Grupos Nitrogenados/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Ácido Palmítico/metabolismo , RatasRESUMEN
Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterized by fatigable muscle weakness owing to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) owing to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1-/- knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggest a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity and highlight that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit α and muscle-specific kinase, which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.
Asunto(s)
Debilidad Muscular/genética , Enfermedades Musculares/genética , Síndromes Miasténicos Congénitos/genética , Transferasas de Grupos Nitrogenados/genética , Animales , Modelos Animales de Enfermedad , Expresión Génica/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Glicosilación , Humanos , Ratones , Ratones Noqueados , Debilidad Muscular/fisiopatología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Enfermedades Musculares/fisiopatología , Mutación , Síndromes Miasténicos Congénitos/fisiopatología , Unión Neuromuscular/genética , Unión Neuromuscular/fisiopatología , Transmisión Sináptica/genéticaRESUMEN
This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.
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Cardiomiopatías/genética , ADN Mitocondrial/genética , Discapacidades del Desarrollo/genética , Enfermedades Mitocondriales/genética , Mutación , Alanina-ARNt Ligasa/genética , Cardiomiopatías/diagnóstico , Proteínas Portadoras/genética , Discapacidades del Desarrollo/diagnóstico , Femenino , Feto , Humanos , Lactante , Masculino , Enfermedades Mitocondriales/diagnóstico , Transferasas de Grupos Nitrogenados/genética , Fosforilación Oxidativa , Linaje , Proteínas de Unión al ARN , SíndromeRESUMEN
The cutis laxa syndromes are multisystem disorders that share loose redundant inelastic and wrinkled skin as a common hallmark clinical feature. The underlying molecular defects are heterogeneous and 13 different genes have been involved until now, all of them being implicated in elastic fiber assembly. We provide here molecular and clinical characterization of three unrelated patients with a very rare phenotype associating cutis laxa, facial dysmorphism, severe growth retardation, hyperostotic skeletal dysplasia, and intellectual disability. This disorder called Lenz-Majewski syndrome (LMS) is associated with gain of function mutations in PTDSS1, encoding an enzyme involved in phospholipid biosynthesis. This report illustrates that LMS is an unequivocal cutis laxa syndrome and expands the clinical and molecular spectrum of this group of disorders. In the neonatal period, brachydactyly and facial dysmorphism are two early distinctive signs, later followed by intellectual disability and hyperostotic skeletal dysplasia with severe dwarfism allowing differentiation of this condition from other cutis laxa phenotypes. Further studies are needed to understand the link between PTDSS1 and extra cellular matrix assembly.
Asunto(s)
Cutis Laxo/diagnóstico , Cutis Laxo/genética , Hiperostosis/diagnóstico , Hiperostosis/genética , Mutación , Transferasas de Grupos Nitrogenados/genética , Fenotipo , Adulto , Alelos , Niño , Preescolar , Exones , Facies , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , RadiografíaRESUMEN
AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.
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Proteínas Quinasas Activadas por AMP/genética , Acetilglucosamina/metabolismo , Cardiomegalia/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Transferasas de Grupos Nitrogenados/genética , Proteínas Quinasas Activadas por AMP/deficiencia , Acetilglucosamina/farmacología , Acilación/efectos de los fármacos , Animales , Animales Recién Nacidos , Azaserina/farmacología , Compuestos Azo/farmacología , Compuestos de Bifenilo , Cardiomegalia/metabolismo , Cardiomegalia/patología , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Regulación de la Expresión Génica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Glicosilación/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Masculino , Ratones , Ratones Noqueados , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Transferasas de Grupos Nitrogenados/metabolismo , Norleucina/análogos & derivados , Norleucina/farmacología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Pironas/farmacología , Ratas , Ratas Wistar , Transducción de Señal , Tiofenos/farmacología , Troponina T/genética , Troponina T/metabolismoRESUMEN
BACKGROUND: The first global lipidomic profiles associated with urothelial cancer of the bladder (UCB) and its clinical stages associated with progression were identified. OBJECTIVE: To identify lipidomic signatures associated with survival and different clinical stages of UCB. DESIGN, SETTING, AND PARTICIPANTS: Pathologically confirmed 165 bladder-derived tissues (126 UCB, 39 benign adjacent or normal bladder tissues). UCB tissues included Ta (n=16), T1 (n=30), T2 (n=43), T3 (n=27), and T4 (n=9); lymphovascular invasion (LVI) positive (n=52) and negative (n=69); and lymph node status N0 (n=28), N1 (n=11), N2 (n=9), N3 (n=3), and Nx (n=75). RESULTS AND LIMITATIONS: UCB tissues have higher levels of phospholipids and fatty acids, and reduced levels of triglycerides compared with benign tissues. A total of 59 genes associated with altered lipids in UCB strongly correlate with patient survival in an UCB public dataset. Within UCB, there was a progressive decrease in the levels of phosphatidylserine (PS), phosphatidylethanolamines (PEs), and phosphocholines, whereas an increase in the levels of diacylglycerols (DGs) with tumor stage. Transcript and protein expression of phosphatidylserine synthase 1, which converts DGs to PSs, decreased progressively with tumor stage. Levels of DGs and lyso-PEs were significantly elevated in tumors with LVI and lymph node involvement, respectively. Lack of carcinoma in situ and treatment information is the limitation of our study. CONCLUSIONS: To date, this is the first study describing the global lipidomic profiles associated with UCB and identifies lipids associated with tumor stages, LVI, and lymph node status. Our data suggest that triglycerides serve as the primary energy source in UCB, while phospholipid alterations could affect membrane structure and/or signaling associated with tumor progression. PATIENT SUMMARY: Lipidomic alterations identified in this study set the stage for characterization of pathways associated with these altered lipids that, in turn, could inform the development of first-of-its-kind lipid-based noninvasive biomarkers and novel therapeutic targets for aggressive urothelial cancer of the bladder.
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Carcinoma de Células Transicionales/metabolismo , Ácidos Grasos/metabolismo , Fosfolípidos/metabolismo , Triglicéridos/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Estudios de Casos y Controles , Cromatografía Liquida , Biología Computacional , Diglicéridos/metabolismo , Femenino , Humanos , Metabolismo de los Lípidos/genética , Ganglios Linfáticos/patología , Lisofosfolípidos/metabolismo , Masculino , Espectrometría de Masas , Invasividad Neoplásica , Estadificación de Neoplasias , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Fosforilcolina/metabolismo , Análisis de Componente Principal , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic ß-amino acid that can be used for the synthesis of chiral materials and nonnatural peptides. The aim of this study was to accumulate DHHA by engineering Pseudomonas chlororaphis GP72, a nonpathogenic strain that produces phenazine-1-carboxylic acid and 2-hydroxyphenazine. First, the phzF deletion mutant DA1 was constructed, which produced 1.91 g/L DHHA. Moreover, rpeA and pykF were disrupted and then ppsA and tktA were co-expressed in strain DA1. The resulting strain DA4 increased DHHA concentration to 4.98 g/L, which is 2.6-fold than that of DA1. The effects of the addition of glucose, glycerol, L-tryptophan, and Fe3+on DHHA production were also investigated. Strain DA4 produced 7.48 g/L of DHHA in the culture medium in the presence of 12 g/L glucose and 3 mM Fe3+, which was 1.5-fold higher than the strain in the original fermentation conditions. These results indicate the potential of P. chlororaphis GP72 as a DHHA producer.
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Ácido 3-Hidroxiantranílico/análisis , Ácido 3-Hidroxiantranílico/metabolismo , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Ácido 3-Hidroxiantranílico/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Medios de Cultivo/química , Glucosa/farmacología , Ingeniería Metabólica/métodos , Transferasas de Grupos Nitrogenados/genética , Fenazinas/metabolismo , Pseudomonas chlororaphis/efectos de los fármacos , Eliminación de SecuenciaRESUMEN
Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.
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Hexosaminas/biosíntesis , Ácido Hialurónico/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Animales , Femenino , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Hexosaminas/genética , Ácido Hialurónico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismoRESUMEN
The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.