RESUMEN
We report the case of a 78-year-old patient with late diagnosis of hyperoxaluria type III (PH3). He developed renal failure after nephrectomy for clear cell papillary renal carcinoma and complained of recurrent urolithiasis for some 30 years, whose aetiology was never identified. Biochemical laboratory investigations of urine and urolithiasis composition revealed marked hyperoxaluria but normal concentrations of urinary glyceric and glycolic acid as well as stones of idiopathic calcium-oxalate appearance. Furthermore, the dietary survey showed excessive consumption of food supplements containing massive amounts of oxalate precursors. However, the persistence of excessive hyperoxaluria after his eating habits was changed leading us to perform molecular genetic testing. We found heterozygous mutations of the recently PH3-associated HOGA1 gene when sequencing PH genes. This is the first description of late diagnosis primary PH3 in a patient with several additional pro-lithogenic factors. This case illustrates the importance of undertaking a complete biological work-up to determine the aetiology of hyperoxaluria. This may reveal underdiagnosed primary hyperoxaluria, even in older patients.
Asunto(s)
Diagnóstico Tardío , Hiperoxaluria Primaria/diagnóstico , Mutación , Oxo-Ácido-Liasas/genética , Urolitiasis/diagnóstico , Anciano , Carcinoma de Células Renales/diagnóstico , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/cirugía , Expresión Génica , Ácidos Glicéricos/orina , Glicolatos/orina , Humanos , Hiperoxaluria Primaria/complicaciones , Hiperoxaluria Primaria/genética , Hiperoxaluria Primaria/orina , Riñón/metabolismo , Riñón/patología , Riñón/cirugía , Neoplasias Renales/diagnóstico , Neoplasias Renales/patología , Neoplasias Renales/cirugía , Masculino , Nefrectomía , Oxo-Ácido-Liasas/metabolismo , Urolitiasis/complicaciones , Urolitiasis/genética , Urolitiasis/orinaRESUMEN
Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.
Asunto(s)
Proteínas de Homeodominio/genética , Intrones/genética , Oxigenasas de Función Mixta/genética , Obesidad/genética , Oxo-Ácido-Liasas/genética , Proteínas/genética , Factores de Transcripción/genética , Tejido Adiposo/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Metabolismo Basal/genética , Índice de Masa Corporal , Peso Corporal/genética , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta , Genes Dominantes/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipotálamo/metabolismo , Masculino , Ratones , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Delgadez/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
BACKGROUND: The Fat mass and obesity gene (FTO) has been identified through genome wide association studies as an important genetic factor contributing to a higher body mass index (BMI). However, the molecular context in which this effect is mediated has yet to be determined. We investigated the potential molecular network for FTO by analyzing co-expression and protein-protein interaction databases, Coxpresdb and IntAct, as well as the functional coupling predicting multi-source database, FunCoup. Hypothalamic expression of FTO-linked genes defined with this bioinformatics approach was subsequently studied using quantitative real time-PCR in mouse feeding models known to affect FTO expression. RESULTS: We identified several candidate genes for functional coupling to FTO through database studies and selected nine for further study in animal models. We observed hypothalamic expression of Profilin 2 (Pfn2), cAMP-dependent protein kinase catalytic subunit beta (Prkacb), Brain derived neurotrophic factor (Bdnf), neurotrophic tyrosine kinase, receptor, type 2 (Ntrk2), Signal transducer and activator of transcription 3 (Stat3), and Btbd12 to be co-regulated in concert with Fto. Pfn2 and Prkacb have previously not been linked to feeding regulation. CONCLUSIONS: Gene expression studies validate several candidates generated through database studies of possible FTO-interactors. We speculate about a wider functional role for FTO in the context of current and recent findings, such as in extracellular ligand-induced neuronal plasticity via NTRK2/BDNF, possibly via interaction with the transcription factor CCAAT/enhancer binding protein ß (C/EBPß).
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Hipotálamo/metabolismo , Glicoproteínas de Membrana/genética , Oxigenasas de Función Mixta/genética , Obesidad/genética , Oxo-Ácido-Liasas/genética , Proteínas Tirosina Quinasas/genética , Transducción de Señal/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Biología Computacional/métodos , Estudio de Asociación del Genoma Completo/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismoRESUMEN
Optimizing the productivity of bioengineered strains requires balancing ATP generation and carbon atom conservation through fine-tuning cell respiration and metabolism. Traditional approaches manipulate cell respiration by altering air feeding, which are technically difficult especially in large bioreactors. An approach based on genetic regulation may better serve this purpose. With excess oxygen supply to the culture, we efficiently manipulated Escherichia coli cell respiration by adding different amount of coenzyme Q1 to strains lacking the ubiCA genes, which encode two critical enzymes for ubiquinone synthesis. As a proof-of-concept, the metabolic effect of the ubiCA gene knockout and coenzyme Q1 supplementation were characterized, and the metabolic profiles of the experimental strains showed clear correlations with coenzyme Q1 concentrations. Further proof-of-principle experiments were performed to illustrate that the approach can be used to optimize cell respiration for the production of chemicals of interest such as ethanol. This study showed that controlled respiration through genetic manipulation can be exploited to allow much larger operating windows for reduced product formation even under fully aerobic conditions.
Asunto(s)
Escherichia coli/metabolismo , Oxígeno/metabolismo , Reactores Biológicos , Etanol/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Metaboloma , Oxidación-Reducción , Oxo-Ácido-Liasas/genética , Ubiquinona/administración & dosificaciónRESUMEN
The first intron of FTO contains common single nucleotide polymorphisms associated with body weight and adiposity in humans. In an effort to identify the molecular basis for this association, we discovered that FTO and RPGRIP1L (a ciliary gene located in close proximity to the transcriptional start site of FTO) are regulated by isoforms P200 and P110 of the transcription factor, CUX1. This regulation occurs via a single AATAAATA regulatory site (conserved in the mouse) within the FTO intronic region associated with adiposity in humans. Single nucleotide polymorphism rs8050136 (located in this regulatory site) affects binding affinities of P200 and P110. Promoter-probe analysis revealed that binding of P200 to this site represses FTO, whereas binding of P110 increases transcriptional activity from the FTO as well as RPGRIP1L minimal promoters. Reduced expression of Fto or Rpgrip1l affects leptin receptor isoform b trafficking and leptin signaling in N41 mouse hypothalamic or N2a neuroblastoma cells in vitro. Leptin receptor clusters in the vicinity of the cilium of arcuate hypothalamic neurons in C57BL/6J mice treated with leptin, but not in fasted mice, suggesting a potentially important role of the cilium in leptin signaling that is, in part, regulated by FTO and RPGRIP1L. Decreased Fto/Rpgrip1l expression in the arcuate hypothalamus coincides with decreased nuclear enzymatic activity of a protease (cathepsin L) that has been shown to cleave full-length CUX1 (P200) to P110. P200 disrupts (whereas P110 promotes) leptin receptor isoform b clustering in the vicinity of the cilium in vitro. Clustering of the receptor coincides with increased leptin signaling as reflected in protein levels of phosphorylated Stat3 (p-Stat3). Association of the FTO locus with adiposity in humans may reflect functional consequences of A/C alleles at rs8050136. The obesity-risk (A) allele shows reduced affinity for the FTO and RPGRIP1L transcriptional activator P110, leading to the following: 1) decreased FTO and RPGRIP1L mRNA levels; 2) reduced LEPR trafficking to the cilium; and, as a consequence, 3) a diminished cellular response to leptin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Hipotálamo/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Leptina/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Tejido Adiposo/metabolismo , Adiposidad/genética , Alelos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Catepsina L/genética , Catepsina L/metabolismo , Línea Celular Tumoral , Proteínas de Homeodominio/genética , Humanos , Intrones/genética , Ratones , Ratones Mutantes , Oxigenasas de Función Mixta , Proteínas Nucleares/genética , Obesidad/genética , Obesidad/metabolismo , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Fosforilación/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Leptina/genética , Proteínas Represoras/genética , Elementos de Respuesta , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Sequence variants in the first intron of FTO are strongly associated with human obesity and human carriers of the risk alleles show evidence for increased appetite and food intake. Mice globally lacking Fto display a complex phenotype characterised by both increased energy expenditure and increased food intake. The site of action of FTO on energy balance is unclear. Fasting reduces levels of Fto mRNA in the arcuate nucleus (ARC) of the hypothalamus, a site where Fto expression is particularly high. In this study, we have extended this nutritional link by demonstrating that consumption of a high fat diet (45%) results in a 2.5 fold increase in Arc Fto expression. We have further explored the role of hypothalamic Fto in the control of food intake by using stereotactic injections coupled with AAV technology to bi-directionally modulate Fto expression. An over expression of Fto protein by 2.5-fold in the ARC results in a 14% decrease in average daily food intake in the first week. In contrast, knocking down Arc Fto expression by 40% increases food intake by 16%. mRNA levels of Agrp, Pomc and Npy, ARC-expressed genes classically associated with the control of food intake, were not affected by the manipulation of Fto expression. However, over expression of Fto resulted in a 4-fold increase in the mRNA levels of Stat3, a signalling molecule critical for leptin receptor signalling, suggesting a possible candidate for the mediation of Fto's actions. These data provide further support for the notion that FTO itself can influence key components of energy balance, and is therefore a strong candidate for the mediation of the robust association between FTO intronic variants and adiposity. Importantly, this provide the first indication that selective alteration of FTO levels in the hypothalamus can influence food intake, a finding consistent with the reported effects of FTO alleles on appetite and food intake in man.
Asunto(s)
Ingestión de Energía , Conducta Alimentaria , Hipotálamo/metabolismo , Oxo-Ácido-Liasas/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Metabolismo Energético , Homeostasis , Oxigenasas de Función Mixta , RatasRESUMEN
3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder due to a deficiency of the 3-hydroxy-3-methylglutarylCoA lyase (HMG-CoA lyase), a mitochondrial enzyme involved in ketogenesis and in the final step of l-leucine catabolism. HMG-CoA lyase deficiency can lead, in particular circumstances, such as fever, prolonged fasting or digestive disorders, to brutal and severe hypoglycemia with metabolic acidosis and sometimes fatal coma. We report on a new case of 3-hydroxy-3-methylglutaric aciduria particular by its late onset in a 3-year-old patient. Molecular investigation identified two new sequence modifications in the HMGCL gene: c.494G>A (p.Arg165Gln) and c.820G>A (p.Gly274Arg). We remind about this case report that the therapeutical is mainly preventive and allows a very good prognosis for this disease. Long-term treatment consists in limited fasting time, continuous low protein diet and l-carnitine supplementation. Preventive measures are essential: prevention of fasting and emergency treatment during intercurrent infections.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Aberraciones Cromosómicas , Genes Recesivos/genética , Hipoglucemia/genética , Meglutol/orina , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/genética , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Carnitina/administración & dosificación , Preescolar , Terapia Combinada , Análisis Mutacional de ADN , Dieta con Restricción de Proteínas , Exones/genética , Humanos , Hipoglucemia/orina , Leucina/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Enfermedades Raras/terapia , Análisis de Secuencia de ADNRESUMEN
Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.
Asunto(s)
Genes Bacterianos , Genoma de Planta/genética , Lotus/genética , Lotus/metabolismo , Fijación del Nitrógeno/genética , Rhizobium/metabolismo , Simbiosis/genética , Azotobacter vinelandii , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Prueba de Complementación Genética , Ácidos Cetoglutáricos/metabolismo , Lotus/enzimología , Datos de Secuencia Molecular , Mutación/genética , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rhizobium/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Ácidos Tricarboxílicos/metabolismoRESUMEN
BACKGROUND: Polymorphism in the FTO gene is strongly associated with obesity, but little is known about the molecular bases of this relationship. We investigated whether hypothalamic FTO is involved in energy-dependent overconsumption of food. We determined FTO mRNA levels in rodent models of short- and long-term intake of palatable fat or sugar, deprivation, diet-induced increase in body weight, baseline preference for fat versus sugar as well as in same-weight animals differing in the inherent propensity to eat calories especially upon availability of diverse diets, using quantitative PCR. FTO gene expression was also studied in organotypic hypothalamic cultures treated with anorexigenic amino acid, leucine. In situ hybridization (ISH) was utilized to study FTO signal in reward- and hunger-related sites, colocalization with anorexigenic oxytocin, and c-Fos immunoreactivity in FTO cells at initiation and termination of a meal. RESULTS: Deprivation upregulated FTO mRNA, while leucine downregulated it. Consumption of palatable diets or macronutrient preference did not affect FTO expression. However, the propensity to ingest more energy without an effect on body weight was associated with lower FTO mRNA levels. We found that 4-fold higher number of FTO cells displayed c-Fos at meal termination as compared to initiation in the paraventricular and arcuate nuclei of re-fed mice. Moreover, ISH showed that FTO is present mainly in hunger-related sites and it shows a high degree of colocalization with anorexigenic oxytocin. CONCLUSION: We conclude that FTO mRNA is present mainly in sites related to hunger/satiation control; changes in hypothalamic FTO expression are associated with cues related to energy intake rather than feeding reward. In line with that, neurons involved in feeding termination express FTO. Interestingly, baseline FTO expression appears linked not only with energy intake but also energy metabolism.
Asunto(s)
Ingestión de Energía/fisiología , Conducta Alimentaria/fisiología , Hipotálamo/metabolismo , Oxo-Ácido-Liasas/metabolismo , Recompensa , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Análisis de Varianza , Animales , Peso Corporal , Dieta , Ingestión de Alimentos/fisiología , Emulsiones Grasas Intravenosas/administración & dosificación , Hipotálamo/efectos de los fármacos , Hibridación in Situ , Leucina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Oxigenasas de Función Mixta , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Oxo-Ácido-Liasas/genética , Oxitocina/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sacarosa/administración & dosificaciónRESUMEN
Layer and broiler chickens demonstrate striking differences in body weight and body composition. However, the mechanism underlying such difference is elusive. Hypothalamus-pituitary-adrenal (HPA) axis regulates energy homeostasis and body size in mammals, but information in birds is scarce. Here we test the hypothesis that such breed difference is more associated with hypothalamic expression of genes related to HPA axis, rather than orexigenic neuropeptides. Broiler chicks exhibit significantly higher body weight and food intake at day (D) 7 posthatching, but the food intake relative to body weight gain was actually lower. No breed differences were observed for hypothalamic expression of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), orexin (ORX), leptin receptor (LEPR), acetyl-CoA carboxylase (ACC) or fatty acid synthase (FAS). However, broiler chicks expressed significantly higher glucocorticoid receptor (GR) mRNA (P<0.05) and protein (P<0.01) in hypothalamus compared to layer chicks, which is associated with lower corticotropin-releasing hormone (CRH) mRNA (P<0.05) yet higher accumulation of CRH peptide in hypothalamus, suggesting an augmented GR-mediated negative feedback regulation of CRH transcription and release in broiler chicks. Furthermore, fat mass and obesity associated (FTO) gene was also more highly expressed in hypothalamus of broiler chicks (P<0.05). These results suggest that the genes related to energy homeostasis and obesity, such as GR, CRH and FTO, rather than orexigenic neuropeptides, are impacted by the genetic selection practices and play a role in breed-specific body weight setpoint regulation in the chicken.
Asunto(s)
Regulación del Apetito/genética , Pollos/genética , Pollos/metabolismo , Metabolismo Energético/genética , Hipotálamo/metabolismo , Neuropéptidos/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Peso Corporal/genética , Pollos/anatomía & histología , Hormona Liberadora de Corticotropina/genética , Hormona Liberadora de Corticotropina/metabolismo , Retroalimentación/fisiología , Regulación de la Expresión Génica/genética , Homeostasis/genética , Hipotálamo/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Oxigenasas de Función Mixta , Neuropéptidos/metabolismo , Obesidad/genética , Obesidad/metabolismo , Obesidad/fisiopatología , Orexinas , Oxo-Ácido-Liasas/genética , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Especificidad de la EspecieRESUMEN
Polyunsaturated fatty acids (PUFA), at high doses, have been demonstrated to possess anticonvulsant properties in animal seizure models. Little is known, however, about the possible metabolic or adverse effects of PUFA at these high, anticonvulsant doses. The goal of the present study was to assess the metabolic and potential adverse effects of high-dose PUFA administration to rats. Adult male rats received a fatty acid mixture containing alpha-linolenic and linoleic acid in a 1 to 4 ratio, intraperitoneally, for 3 wk. After sacrifice, livers were isolated and analyzed for fatty acid composition and for mRNA expression of HMG-CoA lyase, catalase, and glutathione S-transferases A1 and A4, markers for ketosis, antioxidant defense, and phase II xenobiotic metabolism, respectively. Chronic administration of the PUFA mixture decreased hepatic levels of total lipids--and several fatty acids within total lipids--without altering mRNA expression of HMG-CoA lyase, a metabolic marker of ketosis. The PUFA mixture did not affect mRNA expression of catalase or glutathione S-transferases A1 and A4, which are involved in antioxidant defense and phase II xenobiotic metabolism. These findings suggest that PUFA, given for 3 wk at anticonvulsant doses, result in significant changes in liver lipid metabolism, but do not alter measured genetic markers of liver toxicity.
Asunto(s)
Anticonvulsivantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Ácidos Grasos Insaturados/farmacología , Animales , Catalasa/genética , Catalasa/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Long-EvansRESUMEN
Two recent, large whole-genome association studies (GWAS) in European populations have associated a approximately 47-kb region that contains part of the FTO gene with high body mass index (BMI). The functions of FTO and adjacent FTM in human biology are not clear. We examined expression of these genes in organs of mice segregating for monogenic obesity mutations, exposed to underfeeding/overfeeding, and to 4 degrees C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the Ay, Lep ob, Lepr db, Cpe fat, or tub mutations, and there was a similar trend in other tissues. These effects were not due to adiposity per se. Hypothalamic Fto and Ftm expression were decreased by fasting in lean and obese animals and by cold exposure in lean mice. The fact that responses of Fto and Ftm expression to these manipulations were almost indistinguishable suggested that the genes might be coregulated. The putative overlapping regulatory region contains at least two canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 preferentially bound CUTL1[corrected] in human fibroblast DNA. 70% knockdown of CUTL1 expression in human fibroblasts decreased FTO and FTM expression by 90 and 65%, respectively. Animals and humans with various genetic interruptions of FTO or FTM have phenotypes reminiscent of aspects of the Bardet-Biedl obesity syndrome, a confirmed "ciliopathy." FTM has recently been shown to be a ciliary basal body protein.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Adiposidad/genética , Regulación de la Expresión Génica , Obesidad/genética , Oxo-Ácido-Liasas/genética , Polimorfismo de Nucleótido Simple , Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Ingestión de Alimentos , Embrión de Mamíferos/metabolismo , Metabolismo Energético/genética , Ayuno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipotálamo/metabolismo , Hipotermia Inducida , Leptina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Oxigenasas de Función Mixta , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Obesidad/metabolismo , Oxo-Ácido-Liasas/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células del Estroma/metabolismo , Factores de Transcripción , TransfecciónRESUMEN
Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.
Asunto(s)
ADN/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Núcleo Celular/enzimología , Biología Computacional , Metilación de ADN , ADN de Cadena Simple/metabolismo , Ingestión de Alimentos , Metabolismo Energético , Ayuno , Compuestos Ferrosos/metabolismo , Hipotálamo/enzimología , Hipotálamo/metabolismo , Masculino , Ratones , Oxigenasas de Función Mixta , Datos de Secuencia Molecular , Oxo-Ácido-Liasas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Ácido Succínico/metabolismo , Timina/análogos & derivados , Timina/metabolismoRESUMEN
The present study focuses on the application of immobilization technology to enzymic sugar syntheses. The paper describes an improved silica-alginate matrix established for entrapment and encapsulation. The replacement of alginate with pectate provided enhanced chemical resistance of the matrix, which allows the use of 1% (w/v) polyphosphate in reaction mixtures. Polylysine, a reagent for silica condensation, was replaced by a much cheaper alternative, namely polyethyleneimine. The proposed design was applied in the production of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid (CMP-sialic acid) by immobilized recombinant enzymes or Escherichia coli cells containing overexpressed enzymes. A comparison between these two strategies was made. On the basis of the results we conceptualized a system to synthesize sialyloligosaccharides by using a biocatalyst entrapped in calcium pectate-silica gel beads.
Asunto(s)
Células Inmovilizadas/enzimología , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Escherichia coli/enzimología , Oxo-Ácido-Liasas/metabolismo , Pectinas/química , Dióxido de Silicio/química , Técnicas de Cultivo de Célula/métodos , Materiales Biocompatibles Revestidos/química , Ácido N-Acetilneuramínico Citidina Monofosfato/síntesis química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/genética , Oxo-Ácido-Liasas/química , Oxo-Ácido-Liasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Gel de SíliceRESUMEN
Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.
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Aconitato Hidratasa/metabolismo , Citratos/metabolismo , Isocitratos/metabolismo , Oxo-Ácido-Liasas/metabolismo , Propionatos/metabolismo , Ácido Pirúvico/metabolismo , Salmonella enterica/enzimología , Aconitato Hidratasa/genética , Ácido Aconítico/metabolismo , Catálisis , Ciclo del Ácido Cítrico/genética , Mutagénesis Sitio-Dirigida , Oxo-Ácido-Liasas/genética , Salmonella enterica/genética , Salmonella enterica/crecimiento & desarrollo , Salmonella typhimurium/enzimologíaRESUMEN
Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.
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Lisina/biosíntesis , Lisina/genética , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacología , ADN de Hongos/análisis , Suplementos Dietéticos , Farmacorresistencia Microbiana , Fermentación , Genes Fúngicos , Concentración de Iones de Hidrógeno , Cariotipificación , Lisina/farmacología , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Ploidias , Inhibidores de la Síntesis de la Proteína/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Esporas FúngicasRESUMEN
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase has been expressed in an active form in Escherichia coli and purified to homogeneity. Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system. After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg. The purified enzyme is stable when stored in 20% glycerol at -80 degrees C. The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins. The enzyme exhibits a Km = 20 microM for (S)-HMG-CoA. Divalent cations (Mg2+ and Mn2+) markedly stimulate the enzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans. The activator constant, K(a), for Mg2+ (6.9 mM) is 3 orders of magnitude greater than that for Mn2+ (2.0 microM). While EDTA does not affect activity, o-phenanthroline treatment markedly inhibits the enzyme. In contrast, m-phenanthroline is ineffective, suggesting that the ortho isomer's effect is attributable to chelation of a tightly bound metal ion. Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper. In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10. When the growth medium is supplemented with 1 mM CuSO4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit. Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Oxo-Ácido-Liasas/metabolismo , Pseudomonas/enzimología , Western Blotting , Cationes Bivalentes , Cromatografía por Intercambio Iónico , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Estabilidad de Enzimas , Escherichia coli/genética , Calor , Metales/metabolismo , Oxo-Ácido-Liasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría AtómicaRESUMEN
Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.