RESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Hipóxia/fisiopatologia , Mitofagia , Infarto do Miocárdio/patologia , Receptores de Estrogênio/genética , Ubiquitina-Proteína Ligases/fisiologia , Animais , Núcleo Celular/genética , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Receptores de Estrogênio/metabolismo , Transcriptoma , Ubiquitinação , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
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.
Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , CamundongosRESUMO
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.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Linhagem Celular , Endossomos/ultraestrutura , Feminino , Fibroblastos , Técnicas de Silenciamento de Genes , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Miócitos Cardíacos , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/fisiologiaRESUMO
The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.
Assuntos
Ecossistema , Espectrometria de Massas , Compostos Orgânicos/análise , Compostos Orgânicos/química , Cromatografia Líquida , Humanos , Íons/análise , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Insuficiência Cardíaca/patologia , Mitofagia , Animais , Sinalização do Cálcio , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismoRESUMO
Alcoholism has a profound impact on millions of people throughout the world. However, the ability to determine if a patient needs treatment is hindered by reliance on self-reporting and the clinician's capability to monitor the patient's response to treatment is challenged by the lack of reliable biomarkers. Using a genome-wide approach, we have previously shown that chronic alcohol use is associated with methylation changes in DNA from human cell lines. In this pilot study, we now examine DNA methylation in peripheral mononuclear cell DNA gathered from subjects as they enter and leave short-term alcohol treatment. When compared with abstinent controls, subjects with heavy alcohol use show widespread changes in DNA methylation that have a tendency to reverse with abstinence. Pathway analysis demonstrates that these changes map to gene networks involved in apoptosis. There is no significant overlap of the alcohol signature with the methylation signature previously derived for smoking. We conclude that DNA methylation may have future clinical utility in assessing acute alcohol use status and monitoring treatment response.