RESUMEN

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.

Asunto(s)

Canales de Calcio/metabolismo , Cerebelo/metabolismo , Cuerpo Estriado/metabolismo , Hipocampo/metabolismo , Metaloendopeptidasas/genética , Mitocondrias/metabolismo , Neuronas/metabolismo , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Calcio/metabolismo , Canales de Calcio/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Muerte Celular , Cerebelo/patología , Cuerpo Estriado/patología , Regulación de la Expresión Génica , Células HEK293 , Hipocampo/patología , Homeostasis/genética , Humanos , Transporte Iónico , Metaloendopeptidasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/patología , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Neuronas/patología , Mapeo de Interacción de Proteínas , Transducción de Señal

RESUMEN

Prohibitins form large protein and lipid scaffolds in the inner membrane of mitochondria that are required for mitochondrial morphogenesis, neuronal survival, and normal lifespan. Here, we have defined the interactome of PHB2 in mitochondria and identified DNAJC19, mutated in dilated cardiomyopathy with ataxia, as binding partner of PHB complexes. We observed impaired cell growth, defective cristae morphogenesis, and similar transcriptional responses in the absence of either DNAJC19 or PHB2. The loss of PHB/DNAJC19 complexes affects cardiolipin acylation and leads to the accumulation of cardiolipin species with altered acyl chains. Similar defects occur in cells lacking the transacylase tafazzin, which is mutated in Barth syndrome. Our experiments suggest that PHB/DNAJC19 membrane domains regulate cardiolipin remodeling by tafazzin and explain similar clinical symptoms in two inherited cardiomyopathies by an impaired cardiolipin metabolism in mitochondrial membranes.

Asunto(s)

Cardiolipinas/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Aciltransferasas , Secuencia de Aminoácidos , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Línea Celular , Células HEK293 , Humanos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Datos de Secuencia Molecular , Prohibitinas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia type 28 and spastic ataxia-neuropathy syndrome in humans; however, the pathogenic mechanism is still unclear. AFG3L2 encodes a subunit of the mitochondrial m-AAA protease, previously implicated in quality control of misfolded inner mitochondrial membrane proteins and in regulatory functions via processing of specific substrates. Here, we used a conditional Afg3l2 mouse model that allows restricted deletion of the gene in Purkinje cells (PCs) to shed light on the pathogenic cascade in the neurons mainly affected in the human diseases. We demonstrate a cell-autonomous requirement of AFG3L2 for survival of PCs. Examination of PCs prior to neurodegeneration revealed fragmentation and altered distribution of mitochondria in the dendritic tree, indicating that abnormal mitochondrial dynamics is an early event in the pathogenic process. Moreover, PCs displayed features pointing to defects in mitochondrially encoded respiratory chain subunits at early stages. To unravel the underlying mechanism, we examined a constitutive knockout of Afg3l2, which revealed a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly. We therefore propose that defective mitochondrial protein synthesis, leading to early-onset fragmentation of the mitochondrial network, is a central causative factor in AFG3L2-related neurodegeneration.

Asunto(s)

Proteasas ATP-Dependientes/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Biosíntesis de Proteínas/fisiología , Proteasas ATP-Dependientes/genética , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Supervivencia Celular , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Proteínas Mitocondriales/genética , Espasticidad Muscular/genética , Espasticidad Muscular/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Células de Purkinje , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Degeneraciones Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/metabolismo

RESUMEN

Pax5 is an essential regulator of B cell identity and function. Here, we used transgenesis and deletion mapping to identify a potent enhancer in intron 5 of the Pax5 locus. This enhancer in combination with the promoter region was sufficient to recapitulate the B lymphoid expression of Pax5. The enhancer was silenced by DNA methylation in embryonic stem cells, but became activated in multipotent hematopoietic progenitors. It contained functional binding sites for the transcription factors PU.1, IRF4, IRF8, and NF-kappaB, suggesting that these regulators contribute to sequential enhancer activation in hematopoietic progenitors and during B cell development. In contrast, the promoter region was repressed by Polycomb group proteins in non-B cells and was activated only at the onset of pro-B cell development through induction of chromatin remodeling by the transcription factor EBF1. These experiments demonstrate a stepwise activation of Pax5 in early lymphopoiesis and provide mechanistic insights into the process of B cell commitment.

Asunto(s)

Linfocitos B/inmunología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Linfopoyesis/fisiología , Factor de Transcripción PAX5 , Regiones Promotoras Genéticas , Transgenes/genética , Animales , Linfocitos B/citología , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , Citometría de Flujo , Humanos , Ratones , Datos de Secuencia Molecular , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Transactivadores/genética , Regulación hacia Arriba

RESUMEN

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).

Asunto(s)

Flavoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Apoptosis/genética , Factor Inductor de la Apoptosis , Inhibidores de Caspasas , Caspasas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Senescencia Celular/genética , Ciclofilina A/antagonistas & inhibidores , Ciclofilina A/metabolismo , ADN/genética , ADN/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , Inhibidores Enzimáticos/farmacología , Flavoproteínas/genética , Flavoproteínas/aislamiento & purificación , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Mitocondrias/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , Estrés Oxidativo/genética , Transporte de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico