RESUMO
Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.
Assuntos
Aldeído Oxirredutases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Anemia de Fanconi/metabolismo , Formaldeído/toxicidade , Glutationa/metabolismo , Aldeído Oxirredutases/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Dano ao DNA , Modelos Animais de Doenças , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Formaldeído/metabolismo , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Oxirredução , Estresse OxidativoRESUMO
El síndrome de Sjogren-Larsson se caracteriza por retardo mental, ictiosis congènita y diplejía o cuadriplejía espástica. El defecto primario en este síndrome es la mutación del gen ALDH3A2, que codifica la enzima aldehído deshidrogenasa grasa y causa una deficiencia enzimática que produce una acumulación de alcoholes y aldehídos grasos en los tejidos que comprometen la integridad de la membrana celular, cuyos efectos pueden observarse en la piel, los ojos y el sistema nervioso central. El diagnóstico se realiza por medio de la cuantificación de la actividad de la enzima. Se describe el caso de una paciente con signos clínicos patognomónicos del síndrome de Sjogren-Larsson, cuyo diagnóstico se realizó por medio de la cuantificación de la actividad enzimática en un cultivo de fibroblastos. Además, tomando en cuenta el árbol genealógico de la paciente, se realizó el estudio en los padres y un hermano con signos sugestivos del síndrome de Sjogren-Larsson.
Sjogren-Larsson syndrome is characterized by congenital ichthyosis, mental retardation and spastic diplegia or quadriplegia. The primary defect in this syndrome is mutation of ALDH3A2 gen that codes for the fatty aldehyde dehydrogenase. Deficiency of this enzyme causes an accumulation of fatty alcohols and fatty aldehydes, leading to altered cell-membrane integrity. Skin, eyes, and the central nervous system are affected latter. The diagnosis is carried out through the cuantification of the enzyme activity.
Assuntos
Humanos , Feminino , Criança , Síndrome de Sjogren-Larsson/diagnóstico , Aldeído Oxirredutases/genética , Síndrome de Sjogren-Larsson/genética , Fibroblastos/enzimologia , MutaçãoRESUMO
Sjogren-Larsson syndrome is characterized by congenital ichthyosis, mental retardation and spastic diplegia or quadriplegia. The primary defect in this syndrome is mutation of ALDH3A2 gen that codes for the fatty aldehyde dehydrogenase. Deficiency of this enzyme causes an accumulation of fatty alcohols and fatty aldehydes, leading to altered cell-membrane integrity. Skin, eyes, and the central nervous system are affected latter. The diagnosis is carried out through the cuantification of the enzyme activity. This case report describes the diagnosis of a clinical syndrome with symptoms of Sjogren-Larsson syndrome by the quantification of the enzymatic activity in a culture of fibroblasts. Also, taking into account the genealogy of the patient, the study was conducted in the parents and a brother with signs suggestive of Sjogren-Larsson syndrome.
El síndrome de Sjogren-Larsson se caracteriza por retardo mental, ictiosis congènita y diplejía o cuadriplejía espástica. El defecto primario en este síndrome es la mutación del gen ALDH3A2, que codifica la enzima aldehído deshidrogenasa grasa y causa una deficiencia enzimática que produce una acumulación de alcoholes y aldehídos grasos en los tejidos que comprometen la integridad de la membrana celular, cuyos efectos pueden observarse en la piel, los ojos y el sistema nervioso central. El diagnóstico se realiza por medio de la cuantificación de la actividad de la enzima. Se describe el caso de una paciente con signos clínicos patognomónicos del síndrome de Sjogren-Larsson, cuyo diagnóstico se realizó por medio de la cuantificación de la actividad enzimática en un cultivo de fibroblastos. Además, tomando en cuenta el árbol genealógico de la paciente, se realizó el estudio en los padres y un hermano con signos sugestivos del síndrome de Sjogren-Larsson.
Assuntos
Aldeído Oxirredutases/genética , Síndrome de Sjogren-Larsson/diagnóstico , Criança , Feminino , Fibroblastos/enzimologia , Humanos , Mutação , Síndrome de Sjogren-Larsson/genéticaRESUMO
Rhodococcus opacus PD630 accumulates significant amounts of triacylglycerols (TAG), but is not able to de novo synthesize wax esters (WE) from structural unrelated carbon sources, such as gluconate. In this study, strain PD630 was engineered to produce WE by heterologous expression of maqu_2220 gene, which encodes a fatty acyl-CoA reductase for the production of fatty alcohols in Marinobacter hydrocarbonoclasticus. Recombinant cells produced ca. 46% of WE and 54% of TAG (of total WE+TAG) from gluconate compared with the wild type, which produced 100% of TAG. Cell growth was not affected by the heterologous expression of MAQU_2220. Several saturated and monounsaturated WE species were produced by cells, with C18:C16, C16:C16 and C16:C18 as main species. The fatty acid composition of WE fraction in PD630maqu_2220 was enriched with C16:0, C18:0, whereas C16:0, C18:0 and C18:1 predominated in the TAG fraction. Significant amounts of WE and TAG were accumulated by PD630maqu_2220 from whey, an inexpensive waste material from dairy industries, without affecting cell biomass production. This is the first report on WE synthesis by R. opacus from gluconate, which demonstrates that lipid metabolism of this bacterium is flexible enough to assimilate heterologous components for the production of new lipid derivatives with industrial interest.
Assuntos
Ésteres/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Ceras/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Indústria de Laticínios , Escherichia coli/genética , Resíduos Industriais , Engenharia Metabólica , Triglicerídeos/metabolismo , Soro do Leite/química , Soro do Leite/metabolismoRESUMO
Exogenous supply of nitric oxide (NO) increases drought tolerance in sugarcane plants. However, little is known about the role of NO produced by plants under water deficit. The aim of this study was to test the hypothesis that drought-tolerance in sugarcane is associated with NO production and metabolism, with the more drought-tolerant genotype presenting higher NO accumulation in plant tissues. The sugarcane genotypes IACSP95-5000 (drought-tolerant) and IACSP97-7065 (drought-sensitive) were submitted to water deficit by adding polyethylene glycol (PEG-8000) in nutrient solution to reduce the osmotic potential to -0.4 MPa. To evaluate short-time responses to water deficit, leaf and root samples were taken after 24 h under water deficit. The drought-tolerant genotype presented higher root extracellular NO content, which was accompanied by higher root nitrate reductase (NR) activity as compared to the drought-sensitive genotype under water deficit. In addition, the drought-tolerant genotype had higher leaf intracellular NO content than the drought-sensitive one. IACSP95-5000 exhibited decreases in root S-nitrosoglutathione reductase (GSNOR) activity under water deficit, suggesting that S-nitrosoglutathione (GSNO) is less degraded and that the drought-tolerant genotype has a higher natural reservoir of NO than the drought-sensitive one. Those differences in intracellular and extracellular NO contents and enzymatic activities were associated with higher leaf hydration in the drought-tolerant genotype as compared to the sensitive one under water deficit.
Assuntos
Secas , Óxido Nítrico/metabolismo , Saccharum/metabolismo , Saccharum/fisiologia , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genótipo , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , S-Nitrosoglutationa/metabolismoRESUMO
Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.
Assuntos
Aldeído Oxirredutases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Óxido Nítrico/metabolismo , Aldeído Oxirredutases/antagonistas & inibidores , Aldeído Oxirredutases/genética , Animais , Diferenciação Celular , Fusão Celular , Embrião de Galinha , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , S-Nitrosoglutationa/metabolismo , S-Nitrosotióis/metabolismo , S-Nitrosotióis/farmacologia , Transdução de Sinais , Guanilil Ciclase Solúvel/genética , Guanilil Ciclase Solúvel/metabolismo , Guanilil Ciclase Solúvel/farmacologia , Tionucleotídeos/farmacologia , Triazenos/farmacologiaRESUMO
Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M-1s-1, respectively), while the rate constant of peroxynitrite reduction was (0.6-1.4) ×106M-1s-1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106 and 1×106M-1s-1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.
Assuntos
Aldeído Oxirredutases/química , Proteínas de Bactérias/química , Ácidos Graxos/química , Mycobacterium tuberculosis/química , Peroxirredoxinas/química , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/enzimologia , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian-South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding.
Assuntos
Aldeído Oxirredutases/genética , Transtorno do Espectro Autista/genética , Exoma , Fatores de Transcrição Forkhead/genética , Receptores do Ácido Retinoico/genética , Tretinoína/metabolismo , Adulto , Aldeído Oxirredutases/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Sequência de Bases , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Estudos de Coortes , Colômbia , Embrião de Mamíferos , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Linhagem , Testes Psicológicos , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta , Transdução de SinaisAssuntos
Anormalidades do Olho/genética , Fatores de Transcrição Forkhead/genética , Mutação , Aldeído Oxirredutases/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Fator 3 de Diferenciação de Crescimento/genética , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit.