RESUMO
Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na(+)/Ca(2+) exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.
Assuntos
Apoptose , Cálcio/metabolismo , Células-Tronco Fetais/enzimologia , Mitocôndrias/enzimologia , Neurônios/enzimologia , Transtornos Parkinsonianos/enzimologia , Proteínas Quinases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Células Cultivadas , Citosol/metabolismo , Metabolismo Energético , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/patologia , Células-Tronco Fetais/efeitos da radiação , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Homeostase , Humanos , Potencial da Membrana Mitocondrial , Mesencéfalo/embriologia , Mesencéfalo/enzimologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/efeitos da radiação , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , NADPH Oxidases/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/efeitos da radiação , Oxirredução , Estresse Oxidativo , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Fatores de Tempo , Raios UltravioletaRESUMO
In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson's disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinson's disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinson's disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinson's disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.
Assuntos
Proteínas Mitocondriais/fisiologia , Doença de Parkinson/etiologia , Proteínas Quinases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Sítios de Ligação/genética , Encéfalo/patologia , Linhagem Celular , Ativação Enzimática/genética , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , MAP Quinase Quinase Quinase 3/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Modelos Biológicos , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Proteínas Quinases/genética , Serina Endopeptidases/genética , Transdução de SinaisRESUMO
The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition.
Assuntos
Adesão Celular/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Neoplasias/genética , Neurofibromina 2/genética , Proteínas Supressoras de Tumor/genética , Proliferação de Células/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Inibição de Contato/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Neoplasias/patologia , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Transdução de Sinais/genéticaRESUMO
The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.
Assuntos
Corpo Estriado/embriologia , Corpo Estriado/enzimologia , Serina Endopeptidases/fisiologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Corpo Estriado/anormalidades , DNA/genética , Feminino , Marcação de Genes , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Neurônios/patologia , Transtornos Parkinsonianos/embriologia , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/genética , Fenótipo , Gravidez , Proteínas/metabolismo , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets.
Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Apoptose , Proliferação de Células , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Transporte Proteico , Receptor ErbB-2/imunologia , Receptor ErbB-3/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Complexo CD3/metabolismo , Imunoglobulina G/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
PURPOSE: To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H(2)O(2)-induced oxidative stress in human RPE, in the Ccl2(-)(/)(-)Cx3cr1(-)(/)(-) double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice. METHODS: Oxidative stress was induced in ARPE-19 cells by 1 mM H(2)O(2) for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice. RESULTS: H(2)O(2)-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H(2)O(2)-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi(-)(/)(-) photoreceptors and RPE. CONCLUSIONS: These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.
Assuntos
Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Degeneração Retiniana/enzimologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Apoptose , Western Blotting , Receptor 1 de Quimiocina CX3C , Caspase 3 , Linhagem Celular , Sobrevivência Celular , Quimiocina CCL2/genética , Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Proteínas Mitocondriais/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirimidinonas/farmacologia , Interferência de RNA , Receptores de Quimiocinas/genética , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tionas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
The cellular generation of toxic metabolites and subsequent detoxification failure can cause the uncontrolled accumulation of these metabolites in cells, leading to cellular dysfunction. Amyloid-beta protein (Abeta), a normal metabolite of neurons, tends to form toxic oligomeric structures that cause neurodegeneration. It is unclear how healthy neurons control the levels of intracellular oligomeric Abeta in order to avoid neurodegeneration. Using immunochemical and biochemical studies, we show that the Abeta-binding serine protease Omi is a stress-relieving heat-shock protein that protects neurons against neurotoxic oligomeric Abeta. Through its PDZ domain, Omi binds preferentially to neurotoxic oligomeric forms of Abeta rather than non-toxic monomeric forms to detoxify oligomeric Abeta by disaggregation. This specific interaction leads not only to mutual detoxification of the pro-apoptotic activity of Omi and Abeta-induced neurotoxicity, but also to a reduction of neurotoxic-Abeta accumulation. The neuroprotective role of Omi is further supported by its upregulation during normal neurogenesis and neuronal maturation in mice, which could be in response to the increase in the generation of oligomeric Abeta during these processes. These findings provide novel and important insights into the detoxification pathway of intraneuronal oligomeric Abeta in mammals and the protective roles of Omi in neurodegeneration, suggesting a novel therapeutic target in neurodegenerative diseases.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Proteínas de Choque Térmico/metabolismo , Proteínas Mitocondriais/metabolismo , Serina Endopeptidases/metabolismo , Estresse Fisiológico , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Proteínas de Choque Térmico/genética , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Estrutura Quaternária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.
Assuntos
Sobrevivência Celular/fisiologia , Dopamina/metabolismo , Mitocôndrias/fisiologia , Neurônios/fisiologia , Proteínas Quinases/fisiologia , Animais , Western Blotting , Células Cultivadas , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neurônios/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra- and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor-limiting conditions. E4-ORF1 mimics growth factor signaling by activating PI3-kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4-ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4-ORF4 protein phosphatase 2A-binding domain. We also show that mTOR activation is required for efficient S-phase entry, independently of E2F activation, in adenovirus-infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.