RESUMEN
Leber's hereditary optic neuropathy (LHON) is associated with mitochondrial DNA (mtDNA) ND mutations that are mostly homoplasmic. However, these mutations are not sufficient to explain the peculiar features of penetrance and the tissue-specific expression of the disease and are believed to be causative in association with unknown environmental or other genetic factors. Discerning between clear-cut pathogenetic variants, such as those that appear to be heteroplasmic, and less penetrant variants, such as the homoplasmic, remains a challenging issue that we have addressed here using next-generation sequencing approach. We set up a protocol to quantify MTND5 heteroplasmy levels in a family in which the proband manifests a LHON phenotype. Furthermore, to study this mtDNA haplotype, we applied the cybridization protocol. The results demonstrate that the mutations are mostly homoplasmic, whereas the suspected heteroplasmic feature of the observed mutations is due to the co-amplification of Nuclear mitochondrial Sequences.
Asunto(s)
Núcleo Celular/genética , ADN Mitocondrial/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mitocondrias/genética , Mutación/genética , Atrofia Óptica Hereditaria de Leber/genética , Adenosina Trifosfato/biosíntesis , Adolescente , Adulto , Complejo I de Transporte de Electrón/genética , Metabolismo Energético , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Linaje , Temperatura , Adulto JovenRESUMEN
This chapter covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in neurological disorders associated with complex I defects. Complex I formation and functionality in mammalian cells depends on coordinated expression of nuclear and mitochondrial genes, post-translational subunit modifications, mitochondrial import/maturation of nuclear encoded subunits, subunits interaction and stepwise assembly, and on proteolytic processing. Examples of complex I dysfunction are herein presented: homozygous mutations in the nuclear NDUFS1 and NDUFS4 genes for structural components of complex I; an autosomic recessive form of encephalopathy associated with enhanced proteolytic degradation of complex I; familial cases of Parkinson associated to mutations in the PINK1 and Parkin genes, in particular, homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex I, coexistent with mutation in the PINK1 gene. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in neurological disorders, as well as for developing therapeutical strategies.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Transporte de Electrón , Enfermedades del Sistema Nervioso/enzimología , Femenino , Humanos , Masculino , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , LinajeRESUMEN
Work is presented on the role of cAMP-dependent protein phosphorylation in post-translational processing and biosynthesis of complex I subunits in mammalian cell cultures. PKA-mediated phosphorylation of the NDUFS4 subunit of complex I promotes in cell cultures in vivo import/maturation in mitochondria of the precursor of this protein. The import promotion appears to be associated with the observed cAMP-dependent stimulation of the catalytic activity of complex I. These effects of PKA are counteracted by activation of protein phosphatase(s). PKA and the transcription factor CREB play a critical role in the biosynthesis of complex I subunits. CREB phosphorylation, by PKA and/or CaMKs, activates at nuclear and mitochondrial level a transcriptional regulatory cascade which promotes the concerted expression of nuclear and mitochondrial encoded subunits of complex I and other respiratory chain proteins.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Complejo I de Transporte de Electrón/genética , Humanos , Técnicas In Vitro , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , NADH Deshidrogenasa/química , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Subunidades de Proteína , RatasRESUMEN
This paper covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in hereditary neurological disorders associated with complex I defects. Three types of hereditary complex I dysfunction are dealt with: (i) homozygous mutations in the nuclear genes NDUFS1 and NDUFS4 of complex I, associated with mitochondrial encephalopathy; (ii) a recessive hereditary epileptic neurological disorder associated with enhanced proteolytic degradation of complex I; (iii) homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex, coexistent with mutation in the nuclear PINK1 gene in familial Parkinsonism. The genetic and biochemical data examined highlight different mechanisms by which mitochondrial bioenergetics is altered in these hereditary defects of complex I. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in sporadic neurological disorders and aging, as well as for developing therapeutical strategies.
Asunto(s)
ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/genética , Mutación , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/genética , Secuencia de Aminoácidos , Complejo I de Transporte de Electrón/química , Epilepsia/enzimología , Epilepsia/genética , Humanos , Encefalomiopatías Mitocondriales/enzimología , Encefalomiopatías Mitocondriales/genética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , NADH Deshidrogenasa/genética , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.
Asunto(s)
Encéfalo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Proteínas Mitocondriales/análisis , NADH Deshidrogenasa/metabolismo , Animales , Encéfalo/patología , Química Encefálica/fisiología , Encefalopatías Metabólicas/etiología , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/patología , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Regulación hacia Abajo , Complejo I de Transporte de Electrón/análisis , Activación Enzimática , Masculino , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , NADH Deshidrogenasa/análisis , Subunidades de Proteína/análisis , Subunidades de Proteína/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
In this study the impact of hUPF1 and hUPF2 knockdown on alternative splicing (AS) isoforms of different genes encoding subunits of respiratory chain complex I and complex IV is described. As expected, loss of both hUPF1 and hUPF2 led to impairment of nonsense-mediated mRNA decay (NMD) and accumulation of PTC-containing NMD substrates generated by both complex I and complex IV genes. The levels of some complex I splice variants, which did not contain PTC as well as the level of some complex I canonical transcripts were, however, affected only by hUPF1 knockdown. This finding confirms that NMD plays a role in the maintenance of the transcriptome integrity and reveals a specific impact of hUPF1 on the regulation of complex I genes.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Degradación de ARNm Mediada por Codón sin Sentido , Transactivadores/genética , Transcriptoma/genética , Empalme Alternativo , Codón sin Sentido , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Helicasas , Interferencia de ARN , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
AIMS: Sepsis which is the leading cause of death in intensive care units is usually related to the number and the severity of organ failure, but the mechanisms remain to be fully established. Findings of microvascular flow abnormalities, decreased oxygen consumption and elevated tissue oxygen tensions suggest that problems may lay in cellular oxygen utilization rather than in oxygen delivery per se. Several serum factors, released during sepsis syndrome, might be involved in induction of cytopathic hypoxia and increase of cellular oxidative stress. MAIN METHODS: Human fibroblast cultures were incubated 12h with 10% v/v severe septic patients' sera and measurements were carried out on cellular oxygen consumption, mitochondrial respiratory enzymes activity, H(2)O(2) generation and serum levels of cytokines/chemokines by multiplex assay. KEY FINDINGS: In fibroblast cultures a significant depression of cellular respiration and activity of mitochondrial complexes and increased H(2)O(2) production was observed after incubation with septic sera showing increased levels of TNFα, IL-1ß and IL-6. SIGNIFICANCE: During sepsis syndrome some increased cytokines might target specific mitochondrial enzymes inducing an impairment of cellular energy metabolism leading to multiple organ failure.
Asunto(s)
Sangre , Fibroblastos/citología , Mitocondrias/fisiología , Estrés Oxidativo , Sepsis/sangre , Adulto , Células Cultivadas , Medios de Cultivo , Humanos , Peróxido de Hidrógeno/metabolismo , Consumo de Oxígeno , Espectrometría de FluorescenciaRESUMEN
The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.