RESUMEN
HIV-1 mediated neurotoxicity is thought to be associated with HIV-1 viral proteins activating astrocytes and microglia by inducing inflammatory cytokines leading to the development of HIV-associated neurocognitive disorder (HAND). In the current study, we observe how HIV-1 Nef upregulates the levels of IL-6, IP-10, and TNF-α around 6.0fold in normal human astrocytes (NHAs) compared to cell and empty vector controls. Moderate downregulation in the expression profile of inflammatory cytokines was observed due to RNA interference. Furthermore, we determine the impact of inflammatory cytokines in the upregulation of kynurenine pathway metabolites, such as indoleamine 2,3-dioxygenase (IDO), and 3-hydroxyanthranilic acid oxygenase (HAAO) in NHA, and found the same to be 3.0- and 3.2-fold, respectively. Additionally, the variation in the level of nitric oxide before and after RNA interference was significant. The upregulated cytokines and pathway-specific metabolites could be linked with the neurotoxic potential of HIV-1 Nef. Thus, the downregulation in cytokines and kynurenine metabolites observed after siRNA-Nef interference indicates the possibility of combining the RNA interference approach with current antiretroviral therapy to prevent neurotoxicity development.
Asunto(s)
Astrocitos , Infecciones por VIH , VIH-1 , Enfermedades Neuroinflamatorias , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Astrocitos/metabolismo , Astrocitos/virología , Quimiocina CXCL10/metabolismo , Citocinas/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucina-6/metabolismo , Quinurenina/metabolismo , Óxido Nítrico/metabolismo , ARN Interferente Pequeño/metabolismo , Transcriptoma , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedades Neuroinflamatorias/genética , Enfermedades Neuroinflamatorias/virología , Perfilación de la Expresión Génica , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and cerebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential applications in ALS treatment.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , 3-Hidroxiantranilato 3,4-Dioxigenasa/química , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Ácido 3-Hidroxiantranílico/metabolismo , Ácido 3-Hidroxiantranílico/farmacología , Edaravona/farmacología , Humanos , Ácido Quinolínico/metabolismoRESUMEN
BACKGROUND: The acronym VATER/VACTERL association describes the combination of at least three component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). Individuals presenting two CFs have been termed VATER/VACTERL-like. Recently, FOXF1, HSPA6, HAAO, KYNU, TRAP1, and ZIC3 have been proposed as candidate genes for VATER/VACTERL, VATER/VACTERL-like, and ARM. Re-sequencing studies identified disease-causing variants in TRAP1 and ZIC3, the contribution of other genes was not independently investigated. One affected variant carrier in FOXF1 was previously identified. Here we re-sequenced FOXF1, HSPA6, HAAO, and KYNU in 522 affected individuals. METHODS: Using molecular inversion probe (MIP) technology, re-sequencing was performed in 63 individuals with VATER/VACTERL association, 313 with VATER/VACTERL-like association, and 146 with ARM. All individuals were of European ethnicity. Variant filtering considered variants with a minor allele frequency (MAF) ≤0.01 for putative recessive disease-genes HSPA6, HAAO, and KYNU. For the putative dominant disease-gene FOXF1 we considered variants with a MAF ≤0.0001. In silico prediction tools were used for further prioritization. RESULTS: Only two variants in FOXF1 in two independently affected individuals [c.443G>T, p.(Cys148Phe); c.850T>C, p.(Tyr284His)] passed our filter criteria. One individual presented with ARM, the second presented with TE and C comprising atrial and ventricular septal defects. Sanger sequencing confirmed both variants but also their inheritance from the healthy mother. CONCLUSION: Our analysis suggests that FOXF1, HSPA6, HAAO and KYNU do not play a major role in the formation of VACTER/VACTERL phenotypes or ARM.
Asunto(s)
3-Hidroxiantranilato 3,4-Dioxigenasa , Malformaciones Anorrectales , Factores de Transcripción Forkhead , Proteínas HSP90 de Choque Térmico , Deformidades Congénitas de las Extremidades , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Canal Anal/anomalías , Malformaciones Anorrectales/genética , Esófago/anomalías , Factores de Transcripción Forkhead/genética , Proteínas HSP90 de Choque Térmico/genética , Cardiopatías Congénitas/genética , Humanos , Riñón/anomalías , Deformidades Congénitas de las Extremidades/genética , Columna Vertebral/anomalías , Tráquea/anomalíasRESUMEN
The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolved in crystallo reactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.
Asunto(s)
3-Hidroxiantranilato 3,4-Dioxigenasa/química , Proteínas Bacterianas/química , Cupriavidus/enzimología , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Cupriavidus/química , Cupriavidus/genética , Cinética , Lactonas/química , Lactonas/metabolismo , Modelos Moleculares , Especificidad por SustratoRESUMEN
BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine pathway (KP), is upregulated in some cancers and represents an attractive therapeutic target given its role in tumour immune evasion. However, the recent failure of an IDO inhibitor in a late phase trial raises questions about this strategy. METHODS: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject to proteomic profiling. Tissue immunohistochemistry and gene expression data were used to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. RESULTS: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme in the KP, was identified as being downregulated in RCC. Loss of QPRT expression led to increased potential for anchorage-independent growth. Gene expression, mass spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT and showed downregulation of other KP enzymes, including kynurenine 3-monoxygenase (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant maintenance or upregulation of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in the NAD+ salvage pathway. CONCLUSIONS: Widespread dysregulation of the KP is common in RCC and is likely to contribute to tumour immune evasion, carrying implications for effective therapeutic targeting of this critical pathway.
Asunto(s)
3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Carcinoma de Células Renales/genética , Citocinas/genética , Quinurenina 3-Monooxigenasa/genética , Quinurenina/genética , Nicotinamida Fosforribosiltransferasa/genética , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Quinurenina/metabolismo , Redes y Vías Metabólicas/genética , Proteómica , Escape del Tumor/genética , Escape del Tumor/inmunologíaRESUMEN
AIMS: Atherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation. METHODS AND RESULTS: In vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1ß production by macrophages. Using Ldlr-/- mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores. CONCLUSIONS: We show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.
Asunto(s)
Ácido 3-Hidroxiantranílico/metabolismo , Aterosclerosis/metabolismo , Inflamasomas/metabolismo , Lipoproteínas/sangre , Hígado/metabolismo , Macrófagos/metabolismo , Receptores de LDL/deficiencia , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenasa/antagonistas & inhibidores , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Ácido 3-Hidroxiantranílico/análogos & derivados , Ácido 3-Hidroxiantranílico/farmacología , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Humanos , Interleucina-1beta/metabolismo , Hígado/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica , Receptores de LDL/genética , Transducción de Señal , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genéticaRESUMEN
Next-generation sequencing (NGS) technologies generate thousands to millions of genetic variants per sample. Identification of potential disease-causal variants is labor intensive as it relies on filtering using various annotation metrics and consideration of multiple pathogenicity prediction scores. We have developed VPOT (variant prioritization ordering tool), a python-based command line tool that allows researchers to create a single fully customizable pathogenicity ranking score from any number of annotation values, each with a user-defined weighting. The use of VPOT can be informative when analyzing entire cohorts, as variants in a cohort can be prioritized. VPOT also provides additional functions to allow variant filtering based on a candidate gene list or by affected status in a family pedigree. VPOT outperforms similar tools in terms of efficacy, flexibility, scalability, and computational performance. VPOT is freely available for public use at GitHub (https://github.com/VCCRI/VPOT/). Documentation for installation along with a user tutorial, a default parameter file, and test data are provided.
Asunto(s)
Interfaz Usuario-Computador , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Algoritmos , Bases de Datos Genéticas , Cardiopatías/congénito , Cardiopatías/genética , Humanos , Polimorfismo Genético , Secuenciación del ExomaRESUMEN
PURPOSE: We evaluated the association of hypospadias and 17 susceptibility loci previously identified by a European genome-wide association study in a cohort of Japanese patients. We also examined the expression of candidate genes in male mouse embryos to determine the possible underlying mechanisms of this disease. MATERIALS AND METHODS: We enrolled 169 Japanese patients (mean age at surgery 3.7 years) who underwent repair of hypospadias. Genotyping of 17 single nucleotide polymorphisms was performed using a multiplex polymerase chain reaction invader assay. We also performed in situ hybridization to determine whether candidate genes were expressed in the male genital tubercle during embryonic development of the external genitalia in mice. RESULTS: Single nucleotide polymorphism rs3816183 of HAAO was significantly associated with susceptibility to hypospadias in general (p = 0.0019) and to anterior/middle hypospadias (p = 0.0283) and posterior hypospadias (p = 0.0226), while single nucleotide polymorphism rs6499755 of IRX6 showed an association with susceptibility to anterior/middle hypospadias (p = 0.0472). In mouse embryos there was no significant upregulation of Haao expression in the developing male external genitalia. Irx3 and Irx5, which are linked to Irx6 within the IrxB cluster, were expressed in the mesenchyme remote from the urethral plate epithelium during the critical embryonic period for masculinization. Irx6 was expressed in the ectodermal epithelium, demonstrating prominent dorsal ectodermal expression without expression in the ventral ectoderm adjacent to the urethral plate during the same period. CONCLUSIONS: Genetic variations of HAAO and IRX6 influence susceptibility to hypospadias in the Japanese population. Further research is needed to clarify the mechanism by which variations in these genes contribute to the pathogenesis of hypospadias.
Asunto(s)
3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hipospadias/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Adolescente , Animales , Pueblo Asiatico/genética , Niño , Preescolar , Ectodermo/metabolismo , Embrión de Mamíferos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Recién Nacido , Japón , Masculino , Ratones , Ratones Endogámicos ICR , Organogénesis/genética , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Uretra/crecimiento & desarrolloRESUMEN
The pathway of tryptophan (Trp)-nicotinamide is very important nutritionally because a vitamin nicotinamide is biosynthesized from an amino acid Trp. Until we started studying the factors that affect the Trp-nicotinamide conversion rate, little data existed. Data obtained from TDO (Trp 2,3-dioxygenase)-KO (knock-out) mice have revealed that mice can biosynthesize a necessary amount of nicotinamide from Trp by indoleamine 2,3-dioxygenase (IDO) even when TDO is lacking. It has also been shown that 3-hydroxyanthranilic acid is a key intermediate. Urine upper metabolites such as kynurenic acid and xanthurenic acid originate from non-hepatic tissues but not from the liver. Data obtained from quinolinic acid phosphoribosyltransferase (QPRT)-KO mice indicated that the Trpâquinolinic acid conversion ratio was 6%. Urine quinolinic acid levels and the conversion ratio of Trp to nicotinamide were the same between hetero and wild mice. These findings indicate that QPRT is not the rate-limiting enzyme in the conversion. Thus, the limiting factors in the conversion of Trp to nicotinamide are the amounts of 3-hydroxyanthranilic acid and quinolinic acid in the liver and the activity of liver 3-hydroxyanthranilic acid 3,4-dioxygenase. Studies on factors have shown that conversion of Trp to nicotinamide is increased by adequate intake of good quality protein, and adequate intake of unsaturated fatty acids and starch. However, conversion was decreased by deficient niacin, vitamin B2, or vitamin B6, excessive intake of protein, saturated fatty acids, or glucose and fructose, or intake of protein with low Trp content, and insufficient mineral intake.
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Hígado/metabolismo , Niacinamida/biosíntesis , Triptófano/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Ácido 3-Hidroxiantranílico/metabolismo , Animales , Dieta , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ácido Quinurénico/metabolismo , Estado Nutricional , Pentosiltransferasa/metabolismo , Ácido Quinolínico/metabolismo , Deficiencia de Vitamina B/complicaciones , Xanturenatos/metabolismoRESUMEN
Tryptophan is metabolized primarily via the kynurenine pathway (KP), which involves several enzymes, including indoleamine 2,3-dioxygenase, tryptophan 2,3 dioxygenase (TDO), kynurenine aminotransferases (KATs), kynurenine monooxygenase (KMO) etc. The majority of metabolites are neuroactive: some of them, such as kynurenic acid, show neuroprotective effects, while others contribute to free radical production, leading to neurodegeneration. Imbalance of the pathway is assumed to contribute to the development of several neurodegenerative diseases, psychiatric disorders, migraine and multiple sclerosis. Our aim was to summarize published data on genetic alterations of enzymes involved in the KP leading to disturbances of the pathway that can be related to different diseases. To achieve this, a PubMed literature search was performed for publications on genetic alterations of the KP enzymes upto April 2017. Several genetic alterations of the KP have been identified and have been proposed to be associated with diseases. Here we must emphasize that despite the large number of recognized genetic alterations, the number of firmly established causal relations with specific diseases is still small. The realization of this by those interested in the field is very important and finding such connections should be a major focus of related research. Polymorphisms of the genes encoding the enzymes of the KP have been associated with autism, multiple sclerosis and schizophrenia, and were shown to affect the immune response of patients with bacterial meningitis, just to mention a few. To our knowledge, this is the first comprehensive review of the genetic alterations of the KP enzymes. We believe that the identification of genetic alterations underlying diseases has great value regarding both treatment and diagnostics in precision medicine, as this work can promote the understanding of pathological mechanisms, and might facilitate medicinal chemistry approaches to substitute missing components or correct the disturbed metabolite balance of KP.
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Quinurenina/metabolismo , Mutación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Arilformamidasa/genética , Arilformamidasa/metabolismo , Carboxiliasas/genética , Carboxiliasas/metabolismo , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina 3-Monooxigenasa/genética , Quinurenina 3-Monooxigenasa/metabolismo , Redes y Vías Metabólicas/genética , Transaminasas/genética , Transaminasas/metabolismo , Triptófano/metabolismo , Triptófano Oxigenasa/genética , Triptófano Oxigenasa/metabolismoRESUMEN
AIM: Poststroke depression (PSD), which occurs in approximately one-third of stroke survivors, is clinically important because of its association with slow functional recovery and increased mortality. In addition, the underlying pathophysiological mechanisms are still poorly understood. METHODS: We used a mouse model of PSD to examine the neurobiological mechanisms of PSD and the beneficial effects of aripiprazole, an atypical antipsychotic drug. PSD was induced in mice by combining middle cerebral artery occlusion (MCAO) with spatial restraint stress. The body weight, sucrose preference, and forced swim tests were performed at 5, 7, and 9 weeks and the Morris water maze test at 10 weeks after completing MCAO and spatial restraint stress. RESULTS: Mice subjected to MCAO and spatial restraint stress showed significant depressive-like behavior in the sucrose preference test and forced swim test as well as cognitive impairment in the Morris water maze test. The PSD-like phenotype was accompanied by an indoleamine 2,3-dioxygenase 1 (IDO1) expression increase in the nucleus accumbens, hippocampus, and hypothalamus, but not in the striatum. Furthermore, the increased IDO1 levels were localized in Iba-1(+) cells but not in NeuN(+) or GFAP(+) cells, indicating that microglia-induced IDO1 expression was prominent in the PSD mouse brain. Moreover, 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), quinolinic acid (QUIN), and reactive oxygen species (ROS) were significantly increased in the nucleus accumbens, hippocampus, and hypothalamus of PSD mice. Importantly, a 2-week aripiprazole (1 mg/kg, per os) regimen, which was initiated 1 day after MCAO, ameliorated depressive-like behavior and impairment of cognitive functions in PSD mice that was accompanied by downregulation of IDO1, HAAO, QUIN, and ROS. CONCLUSIONS: Our results suggest that the IDO1-dependent neurotoxic kynurenine metabolism induced by microglia functions in PSD pathogenesis. The beneficial effect of aripiprazole on depressive-like behavior and cognitive impairment may be mediated by inhibition of IDO1, HAAO, QUIN, and ROS.
Asunto(s)
Isquemia Encefálica/complicaciones , Trastorno Depresivo/etiología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/metabolismo , Síndromes de Neurotoxicidad/etiología , Estrés Psicológico/complicaciones , Accidente Cerebrovascular/complicaciones , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Animales , Isquemia Encefálica/fisiopatología , Trastorno Depresivo/metabolismo , Trastorno Depresivo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Ácido Quinolínico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Restricción Física , Aprendizaje Espacial , Accidente Cerebrovascular/fisiopatologíaRESUMEN
BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).
Asunto(s)
3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Anomalías Congénitas/genética , Suplementos Dietéticos , Hidrolasas/genética , NAD/deficiencia , Niacina/uso terapéutico , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Canal Anal/anomalías , Animales , Anomalías Congénitas/prevención & control , Modelos Animales de Enfermedad , Esófago/anomalías , Femenino , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/prevención & control , Humanos , Hidrolasas/metabolismo , Riñón/anomalías , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/prevención & control , Masculino , Ratones , Ratones Noqueados , Mutación , NAD/biosíntesis , NAD/genética , Análisis de Secuencia de ADN , Columna Vertebral/anomalías , Tráquea/anomalíasRESUMEN
3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O2 to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation. Here, the first crystal structure of human 3HAO with the native iron bound in its active site is presented, together with an additional structure with zinc (a known inhibitor of human 3HAO) bound in the active site. The metal-binding environment is examined both structurally and via inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectroscopy (XRF) and electron paramagnetic resonance spectroscopy (EPR). The studies identified Met35 as the source of potential new interactions with substrates and inhibitors, which may prove useful in future therapeutic efforts.
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3-Hidroxiantranilato 3,4-Dioxigenasa/química , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Hierro/química , Hierro/metabolismo , Modelos Moleculares , Níquel/química , Níquel/metabolismo , Unión Proteica , Conformación Proteica , Zinc/química , Zinc/metabolismoRESUMEN
While upregulation of 2,3-dioxygenase (IDO) accompanied by degradation of tryptophan along the kynurenine pathway have been reported to exert antimicrobial effects against a wide range of infectious agents, its role in the replication of influenza A virus remains uncertain. We performed experiments using influenza A/WSN/33 virus infection of mouse fibroblast cell-line (NIH-3T3) to study the effects of IDO on viral replication. Influenza infection resulted in prominent elevations of transcripts encoding IDO, interferon (IFN)-ß, and segment 8 of the virus in NIH-3T3 cells. Introduction of siRNA targeted against IDO followed by infection resulted in further increased levels of viral RNA without altering IFN-ß expression. Inhibition of IDO during the infection also resulted in reduction of virus-driven upregulation of 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), but not kynurenine 3-monooxygenase (KMO), which are enzymes downstream in the kynurenine pathway. Thus, induction of IDO appears to contribute to limiting replication of the WSN/33 strain of influenza A virus in murine NIH-3T3 cells.
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Indolamina-Pirrol 2,3,-Dioxigenasa/farmacología , Virus de la Influenza A/efectos de los fármacos , Infecciones por Orthomyxoviridae/enzimología , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Animales , Antivirales/farmacología , Regulación Viral de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Virus de la Influenza A/fisiología , Interferón beta/genética , Ratones , Células 3T3 NIH , Infecciones por Orthomyxoviridae/virología , Triptófano/metabolismoRESUMEN
The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single cellular sources but not multicellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches. It is shown that the first metal presented preferentially binds to the catalytic site rather than the rubredoxin-like site, which selectively binds iron when the catalytic site is occupied. Furthermore, an iron ion bound to the rubredoxin-like site is readily delivered to an empty catalytic site of metal-free HAO via an intermolecular transfer mechanism. Through the use of metal analysis and catalytic activity measurements, we show that a downstream metabolic intermediate can selectively remove the catalytic iron. As the prokaryotic HAO is often crucial for cell survival, there is a need for ensuring its activity. These results suggest that the rubredoxin-like site is a possible auxiliary iron source to the catalytic center when it is lost during catalysis in a pathway with metabolic intermediates of metal-chelating properties. A spare tire concept is proposed based on this biochemical study, and this concept opens up a potentially new functional paradigm for iron-sulfur centers in iron-dependent enzymes as transient iron binding and shuttling sites to ensure full metal loading of the catalytic site.
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3-Hidroxiantranilato 3,4-Dioxigenasa/química , Proteínas Bacterianas/química , Burkholderiaceae/enzimología , Hierro/química , Secuencias de Aminoácidos , Catálisis , Dominio Catalítico , Humanos , Saccharomyces cerevisiae/enzimología , Relación Estructura-ActividadRESUMEN
3-Hydroxyanthranilate 3,4-dioxygenase () is a non-heme iron dependent enzyme. It catalyses the cleavage of the benzene ring of 3-hydroxyanthranilic acid (3-Ohaa), an intermediate in the kynurenine pathway, and therefore represents a potential target in treating numerous disorders related to the concentration of quinolinic acid (QUIN), the kynurenine pathway product, in tissues. The stability and behaviour of the enzyme in nearly physiological conditions, studied by the empirical molecular modelling methods enabled us to determine the influence of several, for the enzyme activity relevant, point mutations (Arg43Ala, Arg95Ala and Glu105Ala) on the protein structure, particularly on the active site architecture and the metal ion environment, as well as on the substrate, 3-Ohaa, binding. Besides, the water population of the active site, and the protein flexibility as well as the amino acid residues interaction networks relevant for the enzyme activity were determined for the 3-Ohaa complexes with the native and mutated enzyme variants. Finally, using the hybrid quantum-mechanics/molecular-mechanics (QM/MM) calculations the catalysed 3-Ohaa oxidation into 2-amino-3-carboxymuconic acid semialdehyde was elucidated.
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3-Hidroxiantranilato 3,4-Dioxigenasa/química , Biología Computacional , Modelos Moleculares , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Sitios de Unión , Catálisis , Biología Computacional/métodos , Humanos , Hierro/química , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
The conversion of tryptophan (Trp) â nicotinamide (Nam) is an important pathway for supplying vitamin niacin. We reported the following two phenomena: (1) severe food restriction led to an increase in the Trp â Nam conversion compared with free-access control group; (2) the conversion of Trp â Nam is also increased by vitamin B1 deficiency compared with free-access control group. The present study was done to clarify whether or not a true reason about an increase in the Trp â Nam conversion is a vitamin B1 deficiency or severe food restriction. The present results showed that vitamin B1 deficiency suppressed the increased conversion of Trp â Nam induced by severe food restriction, probably by suppressing 3-hydroxyanthranilic acid 3,4-dioxygenase protein synthesis in liver.
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Privación de Alimentos , Hígado/metabolismo , Niacinamida/metabolismo , Tiamina/metabolismo , Triptófano/metabolismo , Deficiencia de Vitamina B/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenasa/biosíntesis , Ácido 3-Hidroxiantranílico/metabolismo , Animales , Hígado/patología , Masculino , Niacina/metabolismo , Biosíntesis de Proteínas , Ratas , Ratas Wistar , Deficiencia de Vitamina B/patologíaAsunto(s)
Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Ácido Quinolínico/síntesis química , 3-Hidroxiantranilato 3,4-Dioxigenasa/antagonistas & inhibidores , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Enfermedades del Sistema Nervioso Central/patología , Inhibidores Enzimáticos/síntesis química , Humanos , Ácido Quinurénico/química , Ácido Quinurénico/metabolismo , Ácido Quinolínico/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismoRESUMEN
3-Hydroxyanthranilic acid 3,4-dioxygenase (3-HAO) is the enzyme responsible for the production of the neurotoxic tryptophan metabolite quinolinic acid (QUIN). Elevated brain levels of QUIN are observed in several neurodegenerative diseases, but pharmacological investigation on its role in the pathogenesis of these conditions is difficult because only one class of substrate-analogue 3-HAO inhibitors, with poor chemical stability, has been reported so far. Here we describe the design, synthesis, and biological evaluation of a novel class of chemically stable inhibitors based on the 2-aminonicotinic acid 1-oxide nucleus. After the preliminary in vitro evaluation of newly synthesized compounds using brain tissue homogenate, we selected the most active inhibitor and showed its ability to acutely reduce the production of QUIN in the rat brain in vivo. These findings provide a novel pharmacological tool for the study of the mechanisms underlying the onset and propagation of neurodegenerative diseases.
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3-Hidroxiantranilato 3,4-Dioxigenasa/antagonistas & inhibidores , Encéfalo/metabolismo , Óxidos N-Cíclicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Ácido Quinolínico/metabolismo , Animales , Encéfalo/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Ácidos Nicotínicos/farmacología , RatasRESUMEN
To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile ß amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.