RESUMEN
Parkin is an E3 ubiquitin ligase well-known for facilitating clearance of damaged mitochondria by ubiquitinating proteins on the outer mitochondrial membrane. However, knowledge of Parkin's functions beyond mitophagy is still limited. Here, we demonstrate that Parkin has functions in the nucleus and that Parkinson's disease-associated Parkin mutants, ParkinR42P and ParkinG430D, are selectively excluded from the nucleus. Further, Parkin translocates to the nucleus in response to hypoxia which correlates with increased ubiquitination of nuclear proteins. The serine-threonine kinase PINK1 is responsible for recruiting Parkin to mitochondria, but translocation of Parkin to the nucleus occurs independently of PINK1. Transcriptomic analyses of HeLa cells overexpressing wild type or a nuclear-targeted Parkin revealed that during hypoxia, Parkin contributes to both increased and decreased transcription of genes involved in regulating multiple metabolic pathways. Furthermore, a proteomics screen comparing ubiquitinated proteins in hearts from Parkin-/- and Parkin transgenic mice identified the transcription factor estrogen-related receptor α (ERRα) as a potential Parkin target. Co-immunoprecipitation confirmed that nuclear-targeted Parkin interacts with and ubiquitinates ERRα. Further analysis uncovered that nuclear Parkin increases the transcriptional activity of ERRα. Overall, our study supports diverse roles for Parkin and demonstrates that nuclear Parkin regulates transcription of genes involved in multiple metabolic pathways.
Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Hipoxia/fisiopatología , Mitofagia , Infarto del Miocardio/patología , Receptores de Estrógenos/genética , Ubiquitina-Proteína Ligasas/fisiología , Animales , Núcleo Celular/genética , Femenino , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias/patología , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Receptores de Estrógenos/metabolismo , Transcriptoma , Ubiquitinación , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Degradation of mitochondria is an important cellular quality control mechanism mediated by two distinct pathways: one involving Parkin-mediated ubiquitination and the other dependent on mitophagy receptors. It is known that mitochondria are degraded by the autophagy pathway; however, we recently reported that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Here, we have developed a protocol to isolate Rab5-positive vesicles from cells for proteomics analysis and provide additional data confirming that mitophagy regulators and mitochondrial proteins are present in these vesicles. We also demonstrate that the mitophagy receptor BNIP3 utilizes the Rab5-endosomal pathway to clear mitochondria in cells. These findings indicate that a redundancy exists in the downstream degradation pathways to ensure efficient mitochondrial clearance.
Asunto(s)
Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Mitofagia , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , RatonesRESUMEN
Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Mitocondrias/metabolismo , Mitofagia/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Apoptosis/fisiología , Autofagia/fisiología , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Línea Celular , Endosomas/ultraestructura , Femenino , Fibroblastos , Técnicas de Silenciamiento del Gen , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Mitocondrias/ultraestructura , Miocitos Cardíacos , Cultivo Primario de Células , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/fisiologíaRESUMEN
Cardiac mitochondria are responsible for generating energy in the form of ATP through oxidative phosphorylation and are crucial for cardiac function. Mitochondrial dysfunction is a major contributor to loss of myocytes and development of heart failure. Myocytes have quality control mechanisms in place to ensure a network of functional mitochondria. Damaged mitochondria are degraded by a process called mitochondrial autophagy, or mitophagy, where the organelle is engulfed by an autophagosome and subsequently delivered to a lysosome for degradation. Evidence suggests that mitophagy is important for cellular homeostasis, and reduced mitophagy leads to inadequate removal of dysfunctional mitochondria. In this review, we discuss the regulation of mitophagy and the emerging evidence of the cardioprotective role of mitophagy. We also address the prospect of therapeutically targeting mitophagy to treat patients with cardiovascular disease.