RESUMEN
Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
Asunto(s)
Apoptosis , Endodesoxirribonucleasas/metabolismo , Neuroblastoma/patología , Neuronas/metabolismo , Enfermedad de Parkinson/patología , Sustancia Negra/patología , alfa-Sinucleína/metabolismo , Anciano , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Daño del ADN/genética , Dopamina/farmacología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Mitocondrias/metabolismo , Mitocondrias/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuronas/citología , Estrés Oxidativo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sustancia Negra/metabolismo , Células Tumorales Cultivadas , alfa-Sinucleína/genéticaRESUMEN
Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Mitocondrias/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Ciclo Celular , Citometría de Flujo , Humanos , Inmunoprecipitación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Parkinson's disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.
Asunto(s)
Proteínas Portadoras/fisiología , Muerte Celular , Proteínas del Tejido Nervioso/fisiología , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , alfa-Sinucleína/metabolismo , FosforilaciónRESUMEN
To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.
Asunto(s)
Senescencia Celular/genética , Evolución Molecular , Regulación de la Expresión Génica , Adulto , Preescolar , Bases de Datos Genéticas , Humanos , Persona de Mediana Edad , Estrés Oxidativo , Saccharomyces cerevisiae/genéticaRESUMEN
Endonuclease G is a mitochondrio-nuclear located nuclease with dual-vital and lethal-functions. Besides its role in apoptosis execution, we have recently shown that depletion of endonuclease G leads to necrotic cell death in yeast. Here, we present further mechanistic elucidation of endonuclease G's vital functions. The deletion of the yeast Endonuclease G gene causes the complete elimination of tetraploid cells during exponential growth. Consistently, conditional knockdown of mammalian endonuclease G selectively kills tetraploid but not diploid clones of the human HCT116 colon carcinoma cell line. We conclude that endonuclease G is important for the viability of polyploid mammalian and yeast cells.
Asunto(s)
Muerte Celular/fisiología , Endodesoxirribonucleasas/metabolismo , Genes Fúngicos/fisiología , Poliploidía , ADN de Hongos/metabolismo , Endodesoxirribonucleasas/genética , Eliminación de Gen , Células HCT116 , Humanos , ARN Interferente Pequeño/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , TransfecciónRESUMEN
Endonuclease G (EndoG) is located in mitochondria yet translocates into the nucleus of apoptotic cells during human degenerative diseases. Nonetheless, a direct involvement of EndoG in cell-death execution remains equivocal, and the mechanism for mitochondrio-nuclear translocation is not known. Here, we show that the yeast homolog of EndoG (Nuc1p) can efficiently trigger apoptotic cell death when excluded from mitochondria. Nuc1p induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factor. Instead, the permeability transition pore, karyopherin Kap123p, and histone H2B interact with Nuc1p and are required for cell death upon Nuc1p overexpression, suggesting a pathway in which mitochondrial pore opening, nuclear import, and chromatin association are successively involved in EndoG-mediated death. Deletion of NUC1 diminishes apoptotic death when mitochondrial respiration is increased but enhances necrotic death when oxidative phosphorylation is repressed, pointing to dual--lethal and vital--roles for EndoG.