RESUMEN
BACKGROUND: Non-syndromic hereditary optic neuropathy (HON) has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families. The objective of the present study was to identify the molecular cause of non-syndromic LHON-like disease in siblings born to non-consanguineous parents of French origin. METHODS: We used a combination of genetic analysis (gene mapping and whole-exome sequencing) in a multiplex family of non-syndromic HON and of functional analyses in patient-derived cultured skin fibroblasts and the yeast Yarrowia lipolytica. RESULTS: We identified compound heterozygote NDUFS2 disease-causing mutations (p.Tyr53Cys; p.Tyr308Cys). Studies using patient-derived cultured skin fibroblasts revealed mildly decreased NDUFS2 and complex I abundance but apparently normal respiratory chain activity. In the yeast Y. lipolytica ortholog NUCM, the mutations resulted in absence of complex I and moderate reduction in nicotinamide adenine dinucleotide-ubiquinone oxidoreductase activity, respectively. CONCLUSIONS: Biallelism for NDUFS2 mutations causing severe complex I deficiency has been previously reported to cause Leigh syndrome with optic neuropathy. Our results are consistent with the view that compound heterozygosity for severe and hypomorphic NDUFS2 mutations can cause non-syndromic HON. This observation suggests a direct correlation between the severity of NDUFS2 mutations and that of the disease and further support that there exist a genetic overlap between non-syndromic and syndromic HON due to defective mitochondrial function.
Asunto(s)
Mutación/genética , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Estudios de Casos y Controles , Bovinos , Secuencia Conservada/genética , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/genética , Femenino , Fibroblastos/metabolismo , Haplotipos/genética , Heterocigoto , Humanos , Masculino , Mitocondrias/genética , Proteínas Mutantes/metabolismo , NADH Deshidrogenasa/química , Oftalmoscopía , Linaje , Fenotipo , Tomografía de Coherencia Óptica , Yarrowia/metabolismoRESUMEN
Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 µg/100 µl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 µg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Macrófagos/metabolismo , Neoplasias Experimentales/patología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Carcinógenos/toxicidad , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Metilcolantreno/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.
Asunto(s)
Catepsina B/metabolismo , Hipoxia de la Célula/genética , Macrófagos/metabolismo , Estabilidad del ARN/genética , Tristetraprolina/metabolismo , Catepsina B/inmunología , Línea Celular , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Tristetraprolina/inmunologíaRESUMEN
The large membrane protein complexes of mitochondrial oxidative phosphorylation are composed of central subunits that are essential for their bioenergetic core function and accessory subunits that may assist in regulation, assembly or stabilization. Although sequence conservation is low, a significant proportion of the accessory subunits is characterized by a common single transmembrane (STMD) topology. The STMD signature is also found in subunits of other membrane protein complexes. We hypothesize that the general function of STMD subunits is to organize the hydrophobic subunits of large membrane protein complexes in specialized environments like the inner mitochondrial membrane.
Asunto(s)
Proteínas de la Membrana/química , Complejos Multiproteicos/química , Secuencia de Aminoácidos , Animales , Bovinos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/genética , Membranas Mitocondriales/química , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación Oxidativa , Estructura Terciaria de Proteína , Subunidades de Proteína , Homología Estructural de Proteína , Yarrowia/química , Yarrowia/genéticaRESUMEN
Cytochrome b is a pivotal protein subunit of the cytochrome bc(1) complex and forms the ubiquinol oxidation site in the enzyme that is generally thought to be the primary site where electrons are aberrantly diverted from the enzyme, reacting with oxygen to form superoxide anion. In addition, recent studies have shown that mutations in cytochrome b can substantially increase rates of oxygen radical formation by the bc(1) complex. It would, thus, be advantageous to be able to manipulate cytochrome b by mutagenesis of the cytochrome b gene to better understand the role of cytochrome b in oxygen radical formation. Cytochrome b is encoded in the mitochondrial genome in eukaryotic cells, and introduction of point mutations into the gene is generally cumbersome because of the tedious screening process for positive clones. In addition, previously it has been especially difficult to introduce point mutations that lead to loss of respiratory function, as might be expected of mutations that markedly enhance oxygen radical formation. To more efficiently introduce amino acid changes into cytochrome b we have devised a method for mutagenesis of the Saccharomyces cerevisiae mitochondrial cytochrome b gene that uses a recoded ARG8 gene as a "placeholder" for the wild-type b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory-competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on nonfermentable substrates. If the mutated cytochrome b is nonfunctional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)).
Asunto(s)
Citocromos b/metabolismo , Mitocondrias/enzimología , Mutación Puntual , Especies Reactivas de Oxígeno/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Ciclooxigenasa 2/genética , Cartilla de ADN , Intrones , Plásmidos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismoRESUMEN
The mitochondrial cytochrome bc(1) complex is an essential respiratory enzyme in oxygen-utilizing eukaryotic cells. Its core subunit, cytochrome b, contains two sites, center P and center N, that participate in the electron transfer activity of the bc(1) complex and that can be blocked by specific inhibitors. In yeast, there are various point mutations that confer inhibitor resistance at center P or center N. However, there are no yeast strains in which the bc(1) complex is resistant to both center P and center N inhibitors. We attempted to create such strains by crossing yeast strains with inhibitor-resistant mutations at center P with yeast strains with inhibitor-resistant mutations at center N. Characterization of yeast colonies emerging from the cross revealed that there were multiple colonies resistant against either inhibitor alone but that the mutational changes were ineffective when combined and when the yeast were grown in the presence of both inhibitors. Inhibitor titrations of bc(1) complex activities in mitochondrial membranes from the various yeast mutants showed that a mutation that confers resistance to an inhibitor at center P, when combined with a mutation that confers resistance to an inhibitor at center N, eliminates or markedly decreases the resistance conferred by the center N mutation. These results indicate that there is a pathway for structural communication between the two active sites of cytochrome b and open new possibilities for the utilization of center N as a potential drug target.
Asunto(s)
Citocromos b/metabolismo , Farmacorresistencia Fúngica/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Dominio Catalítico/fisiología , Cruzamientos Genéticos , Citocromos b/antagonistas & inhibidores , Citocromos b/genética , Citocromos c1/antagonistas & inhibidores , Citocromos c1/genética , Citocromos c1/metabolismo , Farmacorresistencia Fúngica/genética , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.
Asunto(s)
Citocromos b/genética , Citocromos b/metabolismo , Mutagénesis , Mutación/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Medios de Cultivo , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Fermentación , Genes Fúngicos , Vectores Genéticos , Intrones/genética , Mitocondrias/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Transaminasas/metabolismoRESUMEN
We have compared the efficacy of inhibition of the cytochrome bc1 complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC50 approximately 10 nM, while the IC50 for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc1 complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc1 complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites.
Asunto(s)
Antimicina A/análogos & derivados , Benzaldehídos/farmacología , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antimicina A/farmacología , Bovinos , Complejo III de Transporte de Electrones/genética , Mitocondrias Cardíacas/enzimología , Datos de Secuencia Molecular , Paracoccus denitrificans/enzimología , Piridonas/farmacología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.
Asunto(s)
Benzaldehídos/farmacología , Complejo III de Transporte de Electrones/química , Mutación , Saccharomyces cerevisiae/metabolismo , Antraquinonas , Antifúngicos/farmacología , Antimicina A/análogos & derivados , Antimicina A/farmacología , Benzoquinonas/química , Mapeo Cromosómico , Medios de Cultivo , ADN Mitocondrial/metabolismo , Complejo III de Transporte de Electrones/genética , Mitocondrias/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Piridonas/farmacologíaRESUMEN
Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2-detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin-like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1-cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.
Asunto(s)
Antioxidantes/metabolismo , Catalasa/metabolismo , Citosol/metabolismo , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Catalasa/química , Catalasa/genética , Cartilla de ADN , ADN Protozoario/genética , Peróxido de Hidrógeno/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Estrés Oxidativo , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Toxoplasma/genéticaRESUMEN
The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian 'bloodstream form' trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.