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1.
Am J Physiol Renal Physiol ; 318(5): F1147-F1159, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174142

RESUMO

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-ß and whether meprin-ß expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-ß knockout (ßKO) mice. Madin-Darby canine kidney cells transfected with meprin-ß cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cß, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-ß expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not ßKO mice, suggesting that meprin-ß expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cß were observed in kidney lysates for WT mice but not ßKO mice at 6 h post-IR. Proximal tubule PKA-Cß increases in WT but not ßKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-ß enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cß signaling pathway.


Assuntos
Lesão Renal Aguda/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Rim/enzimologia , Metaloendopeptidases/metabolismo , Traumatismo por Reperfusão/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Hipóxia Celular , Modelos Animais de Doenças , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Masculino , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
2.
Biomolecules ; 9(8)2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31431000

RESUMO

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.


Assuntos
Metaloendopeptidases/metabolismo , Animais , Comportamento Animal , Feminino , Masculino , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo
3.
Glia ; 67(8): 1526-1541, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30989755

RESUMO

Mitochondrial dysfunction causes neurodegeneration but whether impairment of mitochondrial homeostasis in astrocytes contributes to this pathological process remains largely unknown. The m-AAA protease exerts quality control and regulatory functions crucial for mitochondrial homeostasis. AFG3L2, which encodes one of the subunits of the m-AAA protease, is mutated in spinocerebellar ataxia SCA28 and in infantile syndromes characterized by spastic-ataxia, epilepsy and premature death. Here, we investigate the role of Afg3l2 and its redundant homologue Afg3l1 in the Bergmann glia (BG), radial astrocytes of the cerebellum that have functional connections with Purkinje cells (PC) and regulate glutamate homeostasis. We show that astrocyte-specific deletion of Afg3l2 in the mouse leads to late-onset motor impairment and to degeneration of BG, which display aberrant morphology, altered expression of the glutamate transporter EAAT2, and a reactive inflammatory signature. The neurological and glial phenotypes are drastically exacerbated when astrocytes lack both Afg31l and Afg3l2, and therefore, are totally depleted of the m-AAA protease. Moreover, mitochondrial stress responses and necroptotic markers are induced in the cerebellum. In both mouse models, targeted BG show a fragmented mitochondrial network and loss of mitochondrial cristae, but no signs of respiratory dysfunction. Importantly, astrocyte-specific deficiency of Afg3l1 and Afg3l2 triggers secondary morphological degeneration and electrophysiological changes in PCs, thus demonstrating a non-cell-autonomous role of glia in neurodegeneration. We propose that astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity in patients carrying mutations in AFG3L2, leading to a vicious circle that contributes to neuronal death.


Assuntos
Proteases Dependentes de ATP/deficiência , ATPases Associadas a Diversas Atividades Celulares/deficiência , Astrócitos/enzimologia , Cerebelo/enzimologia , Metaloendopeptidases/deficiência , Mitocôndrias/enzimologia , Doenças Neurodegenerativas/enzimologia , Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Astrócitos/patologia , Cerebelo/patologia , Modelos Animais de Doenças , Feminino , Inflamação/enzimologia , Inflamação/patologia , Masculino , Metaloendopeptidases/genética , Camundongos Transgênicos , Mitocôndrias/patologia , Doenças Neurodegenerativas/patologia , Células de Purkinje/enzimologia , Células de Purkinje/patologia
4.
EMBO Mol Med ; 11(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389680

RESUMO

Disturbances in the morphology and function of mitochondria cause neurological diseases, which can affect the central and peripheral nervous system. The i-AAA protease YME1L ensures mitochondrial proteostasis and regulates mitochondrial dynamics by processing of the dynamin-like GTPase OPA1. Mutations in YME1L cause a multi-systemic mitochondriopathy associated with neurological dysfunction and mitochondrial fragmentation but pathogenic mechanisms remained enigmatic. Here, we report on striking cell-type-specific defects in mice lacking YME1L in the nervous system. YME1L-deficient mice manifest ocular dysfunction with microphthalmia and cataracts and develop deficiencies in locomotor activity due to specific degeneration of spinal cord axons, which relay proprioceptive signals from the hind limbs to the cerebellum. Mitochondrial fragmentation occurs throughout the nervous system and does not correlate with the degenerative phenotype. Deletion of Oma1 restores tubular mitochondria but deteriorates axonal degeneration in the absence of YME1L, demonstrating that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes the observed neurological defects.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/deficiência , Metaloendopeptidases/deficiência , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Animais , Catarata/etiologia , Catarata/patologia , Modelos Animais de Doenças , GTP Fosfo-Hidrolases/metabolismo , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/patologia , Camundongos , Microftalmia/etiologia , Microftalmia/patologia , Proteínas Mitocondriais/deficiência , Medula Espinal/patologia
5.
PLoS One ; 13(10): e0205878, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30379953

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare fatal disease characterized for accelerated aging. Although the causal agent, a point mutation in LMNA gene, was identified more than a decade ago, the molecular mechanisms underlying HGPS are still not fully understood and, currently, there is no cure for the patients, which die at a mean age of thirteen. With the aim of unraveling non-previously altered molecular pathways in the premature aging process, human cell lines from HGPS patients and from healthy parental controls were studied in parallel using Next-Generation Sequencing (RNAseq) and High-Resolution Quantitative Proteomics (iTRAQ) techniques. After selection of significant proteins and transcripts and crosschecking of the results a small set of protein/transcript pairs were chosen for validation. One of those proteins, ribose-phosphate pyrophosphokinase 1 (PRPS1), is essential for nucleotide synthesis. PRPS1 loss-of-function mutants present lower levels of purine. PRPS1 protein and transcript levels are detected as significantly decreased in HGPS cell lines vs. healthy parental controls. This modulation was orthogonally confirmed by targeted techniques in cell lines and also in an animal model of Progeria, the ZMPSTE24 knock-out mouse. In addition, functional experiments through supplementation with S-adenosyl-methionine (SAMe), a metabolite that is an alternative source of purine, were done. Results indicate that SAMe has a positive effect in the proliferative capacity and reduces senescence-associated Beta-galactosidase staining of the HPGS cell lines. Altogether, our data suggests that nucleotide and, specifically, purine-metabolism, are altered in premature aging, opening a new window for the therapeutic treatment of the disease.


Assuntos
Lamina Tipo A/genética , Progéria/genética , Purinas/metabolismo , RNA Mensageiro/genética , Ribose-Fosfato Pirofosfoquinase/genética , Adulto , Animais , Linhagem Celular , Proliferação de Células , Criança , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Efeito Fundador , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lamina Tipo A/deficiência , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Progéria/tratamento farmacológico , Progéria/metabolismo , Progéria/patologia , RNA Mensageiro/metabolismo , Ribose-Fosfato Pirofosfoquinase/deficiência , S-Adenosilmetionina/farmacologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
6.
J Biol Chem ; 293(40): 15538-15555, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30139746

RESUMO

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.


Assuntos
Aspergillus fumigatus/imunologia , Complemento C3/genética , Complemento C4/genética , Complemento C5/genética , Aspergilose Pulmonar Invasiva/imunologia , Metaloendopeptidases/imunologia , Animais , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Colágeno/genética , Colágeno/imunologia , Complemento C3/metabolismo , Complemento C4/metabolismo , Complemento C5/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Imunidade Inata , Aspergilose Pulmonar Invasiva/genética , Aspergilose Pulmonar Invasiva/microbiologia , Aspergilose Pulmonar Invasiva/patologia , Lectinas/genética , Lectinas/imunologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fagocitose , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/imunologia , Esporos Fúngicos/patogenicidade
7.
Sci Transl Med ; 10(456)2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158154

RESUMO

The nuclear membrane acts as a mechanosensor that drives cellular responses following changes in the extracellular environment. Mechanically ventilated lungs are exposed to an abnormally high mechanical load that may result in clinically relevant alveolar damage. We report that mechanical ventilation in mice increased the expression of Lamin-A, a major determinant of nuclear membrane stiffness, in alveolar epithelial cells. Lamin-A expression increased and nuclear membrane compliance decreased in human bronchial epithelial cells after a mechanical stretch stimulus and in a murine model of lung injury after positive-pressure ventilation. Reducing Lamin-A maturation by depletion of the protease-encoding gene Zmpste24 preserved alveolar nuclear membrane compliance after mechanical ventilation in mice. Ventilator-induced proapoptotic gene expression changes and lung injury were reduced in mice lacking Zmpste24 compared to wild-type control animals. Similarly, treatment with the human immunodeficiency virus protease inhibitors lopinavir and ritonavir reduced the accumulation of Lamin-A at nuclear membranes and preserved nuclear membrane compliance after mechanical ventilation, mimicking the protective phenotype of Zmpste24-/- animals. These results show that the pathophysiological response to lung mechanical stretch is sensed by the nuclear membranes of lung alveolar cells, and suggest that protease inhibitors might be effective in preventing ventilator-induced lung injury.


Assuntos
Células Epiteliais Alveolares/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Mecanotransdução Celular , Membrana Nuclear/metabolismo , Respiração Artificial/efeitos adversos , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/ultraestrutura , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Humanos , Laminas/metabolismo , Lopinavir/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/ultraestrutura , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Metaloendopeptidases/deficiência , Metaloendopeptidases/metabolismo , Camundongos Endogâmicos C57BL , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/ultraestrutura , Ritonavir/farmacologia
8.
Artigo em Inglês | MEDLINE | ID: mdl-29567411

RESUMO

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.


Assuntos
Núcleo Celular/patologia , Modelos Animais de Doenças , Proteínas de Peixes/deficiência , Proteínas de Membrana/deficiência , Metaloendopeptidases/deficiência , Oryzias/genética , Progéria/patologia , Nadadeiras de Animais/enzimologia , Nadadeiras de Animais/patologia , Nadadeiras de Animais/efeitos da radiação , Animais , Animais Geneticamente Modificados , Núcleo Celular/enzimologia , Núcleo Celular/efeitos da radiação , Forma do Núcleo Celular/efeitos da radiação , Células Cultivadas , Códon sem Sentido , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Oryzias/metabolismo , Progéria/enzimologia , Progéria/genética , Tolerância a Radiação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sobrevida , Encurtamento do Telômero/efeitos da radiação
9.
Cell Death Dis ; 9(3): 304, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29467464

RESUMO

The progressive accumulation of dysfunctional mitochondria is implicated in aging and in common diseases of the elderly. To oppose this occurrence, organisms employ a variety of strategies, including the selective degradation of oxidatively damaged and misfolded mitochondrial proteins. Genetic studies in yeast indicate that the ATPase Associated with diverse cellular Activities (AAA+) family of mitochondrial proteases account for a substantial fraction of this protein degradation, but their metazoan counterparts have been little studied, despite the fact that mutations in the genes encoding these proteases cause a variety of human diseases. To begin to explore the biological roles of the metazoan mitochondrial AAA+ protease family, we have created a CRISPR/Cas9 allele of the Drosophila homolog of SPG7, which encodes an inner membrane-localized AAA+ protease known as paraplegin. Drosophila SPG7 mutants exhibited shortened lifespan, progressive locomotor defects, sensitivity to chemical and environmental stress, and muscular and neuronal degeneration. Ultrastructural examination of photoreceptor neurons indicated that the neurodegenerative phenotype of SPG7 mutants initiates at the synaptic terminal. A variety of mitochondrial defects accompanied the degenerative phenotypes of SPG7 mutants, including altered axonal transport of mitochondria, accumulation of electron-dense material in the matrix of flight muscle mitochondria, reduced activities of respiratory chain complexes I and II, and severely swollen and dysmorphic mitochondria in the synaptic terminals of photoreceptors. Drosophila SPG7 mutants recapitulate key features of human diseases caused by mutations in SPG7, and thus provide a foundation for the identification of Drosophila paraplegin substrates and strategies that could be used to ameliorate the symptoms of these diseases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Longevidade , Metaloendopeptidases/deficiência , Mitocôndrias/patologia , Músculos/patologia , Degeneração Neural/patologia , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Axônios/patologia , Comportamento Animal , Proteínas de Drosophila/genética , Drosophila melanogaster/ultraestrutura , Transporte de Elétrons , Larva , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mutação/genética , Degeneração Neural/metabolismo , Homologia de Sequência de Aminoácidos , Sinapses/patologia
10.
Am J Med Genet A ; 176(5): 1175-1179, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29341437

RESUMO

A 4-year-old girl was referred to the Undiagnosed Diseases Network with a history of short stature, thin and translucent skin, macrocephaly, small hands, and camptodactyly. She had been diagnosed with possible Hallerman-Streiff syndrome. Her evaluation showed that she was mosaic for uniparental isodisomy of chromosome 1, which harbored a pathogenic c.1077dupT variant in ZMPSTE24 which predicts p.(Leu362fsX18). ZMPSTE24 is a zinc metalloproteinase that is involved in processing farnesylated proteins and pathogenic ZMPSTE24 variants cause accumulation of abnormal farnesylated forms of prelamin A. This, in turn, causes a spectrum of disease severity which is based on enzyme activity. The current patient has an intermediate form, which is a genocopy of severe Progeria.


Assuntos
Variação Biológica da População/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas de Membrana/deficiência , Metaloendopeptidases/deficiência , Fenótipo , Alelos , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Mutação , Sequenciamento Completo do Exoma
11.
Mol Med Rep ; 16(6): 8944-8952, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28990109

RESUMO

Cellular senescence is an irreversible growth arrest of cells that maintain their metabolic activities. Premature senescence can be induced by different stress factors and occurs in mouse embryonic fibroblasts (MEFs) derived from Zmpste24 metalloproteinase­deficient mice, a progeria mouse model of Hutchinson­Gilford Progeria Syndrome. Previous studies have shown that miR­342­5p, an intronic microRNA (miRNA/miR) reportedly involved in ageing associated diseases, is downregulated in Zmpste24­/­ MEFs. However, whether miR­342­5p is associated with the premature senescence phenotype of Zmpste24­/­ MEFs remains unclear. Thus, the present study investigated the effects of miR­342­5p on cellular senescence and cell proliferation in Zmpste24­/­ MEFs. The results showed that miR­342­5p overexpression ameliorated the cellular senescence phenotype to a certain extent, promoted cell proliferation and increased the G2+M cell cycle phase in Zmpste24­/­ MEFs. Nonetheless, it was difficult to observe the opposite cell phenotypes in wild­type (WT) MEFs transfected with the miR­342­5p inhibitor. Growth­arrest­specific 2 (GAS2) was identified as a target gene of miR­342­5p in Zmpste24­/­ MEFs. In addition, miR­342­5p was identified to be downregulated in WT MEFs during replicative senescence, while Gas2 was upregulated. Taken together, these findings suggest that downregulated miR­342­5p is involved in regulating cell proliferation and cell cycles in Zmpste24­/­ MEFs by suppressing GAS2 in vitro.


Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/deficiência , Metaloendopeptidases/deficiência , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Interferência de RNA , Regiões 3' não Traduzidas , Animais , Proliferação de Células , Sobrevivência Celular/genética , Senescência Celular , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Camundongos , Fenótipo
12.
Nat Genet ; 49(10): 1529-1538, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28805828

RESUMO

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.


Assuntos
Hérnia Hiatal/genética , Microcefalia/genética , Complexos Multiproteicos/genética , Mutação , Nefrose/genética , Animais , Apoptose/genética , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Movimento Celular , Citoesqueleto/ultraestrutura , Reparo do DNA/genética , Estresse do Retículo Endoplasmático/genética , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Modelos Moleculares , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Conformação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/metabolismo , Homeostase do Telômero/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
13.
Cell Death Dis ; 8(6): e2847, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569783

RESUMO

Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease that we identified as a nerve regeneration-associated molecule. The expression of DINE is upregulated in response to nerve injury in both the peripheral and central nervous systems, while its transcription is regulated by the activating transcription factor 3 (ATF3), a potent hub-transcription factor for nerve regeneration. Despite its unique hallmark of injury-induced upregulation, the physiological relevance of DINE in injured neurons has been unclear. In this study, we have demonstrated that the expression of DINE is upregulated in injured retinal ganglion cells (RGCs) in a coordinated manner with that of ATF3 after optic nerve injury, whereas DINE and ATF3 are not observed in any normal retinal cells. Recently, we have generated a mature DINE-deficient (KOTg) mouse, in which exogenous DINE is overexpressed specifically in embryonic motor neurons to avoid aberrant arborization of motor nerves and lethality after birth that occurs in the conventional DINE KO mouse. The DINE KOTg mice did not show any difference in retinal structure and the projection to brain from that of wild-type (wild type) mice under normal conditions. However, injured RGCs of DINE KOTg mice failed to regenerate even after the zymosan treatment, which is a well-known regeneration-promoting reagent. Furthermore, a DINE KOTg mouse crossed with a Atf3:BAC Tg mouse, in which green fluorescent protein (GFP) is visualized specifically in injured RGCs and optic nerves, has verified that DINE deficiency leads to regeneration failure. These findings suggest that injury-induced DINE is a crucial endopeptidase for injured RGCs to promote axonal regeneration after optic nerve injury. Thus, a DINE-mediated proteolytic mechanism would provide us with a new therapeutic strategy for nerve regeneration.


Assuntos
Fator 3 Ativador da Transcrição/genética , Metaloendopeptidases/genética , Regeneração Nervosa/genética , Traumatismos do Nervo Óptico/genética , Células Ganglionares da Retina/enzimologia , Fator 3 Ativador da Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Metaloendopeptidases/deficiência , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores/farmacologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/enzimologia , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Transdução de Sinais , Zimosan/farmacologia
14.
Mol Cell ; 64(1): 148-162, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27642048

RESUMO

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.


Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Corpo Estriado/metabolismo , Hipocampo/metabolismo , Metaloendopeptidases/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Morte Celular , Cerebelo/patologia , Corpo Estriado/patologia , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/patologia , Homeostase/genética , Humanos , Transporte de Íons , Metaloendopeptidases/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios/patologia , Mapeamento de Interação de Proteínas , Transdução de Sinais
15.
Nat Cell Biol ; 17(8): 1004-13, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26214134

RESUMO

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.


Assuntos
Envelhecimento/metabolismo , Proliferação de Células , Reprogramação Celular , Senescência Celular , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , NF-kappa B/metabolismo , Progéria/metabolismo , Fatores Etários , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Animais , Estudos de Casos e Controles , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Histona-Lisina N-Metiltransferase , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Fenótipo , Progéria/genética , Progéria/patologia , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção
17.
Biochim Biophys Acta ; 1850(7): 1368-74, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25813552

RESUMO

BACKGROUND: BMI and ADAMTS13 levels are positively correlated in man. Development of obesity is associated with angiogenesis and inflammation, and increased ADAMTS13 synthesis in the liver. METHODS: Male wild-type (WT) and ADAMTS13 deficient (Adamts13-/-) mice were kept on normal chow (SFD) or high fat diet (HFD) for 15 weeks. RESULTS: HFD feeding of WT mice resulted in significantly enhanced levels of ADAMTS13 antigen and activity as compared to SFD feeding. ADAMTS13 deficiency had no significant effect on body weight gain, subcutaneous (SC) or gonadal (GN) adipose tissue mass, or on adipocyte size. In GN fat of obese (HFD) Adamts13-/- mice, adipocyte density was higher and blood vessel density lower as compared to obese WT mice. No marked effects of genotype were observed on mRNA expression of adipogenic, endothelial, inflammatory or oxidative stress markers in adipose tissue. Analysis of metabolic parameters and of glucose and insulin tolerance did not reveal significant differences between both obese genotypes, except for higher adiponectin and cholesterol levels in obese Adamts13-/- as compared to WT mice. CONCLUSION: Our data do not support a functional role of ADAMTS13 in adiposity nor in associated angiogenesis or inflammation in mice. GENERAL SIGNIFICANCE: ADAMTS13 deficiency may cause thrombotic thrombocytopenic purpura (TTP). Obesity, which is associated with enhanced ADAMTS13 levels is nevertheless considered to be an independent risk factor for TTP. To resolve this apparent contradiction, we show that ADAMTS13 does not directly promote development of adipose tissue in a mouse model.


Assuntos
Tecido Adiposo/metabolismo , Adiposidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Metaloendopeptidases/genética , Proteína ADAMTS13 , Adiponectina/sangue , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/crescimento & desenvolvimento , Animais , Glicemia/metabolismo , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Catalase/genética , Colesterol/sangue , Citocinas/genética , Dieta Hiperlipídica/efeitos adversos , Glutationa Peroxidase/genética , Humanos , Mediadores da Inflamação/metabolismo , Leptina/sangue , Masculino , Metaloendopeptidases/deficiência , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/etiologia , Obesidade/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/genética , Superóxido Dismutase-1
18.
J Thromb Haemost ; 13(2): 283-92, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25442981

RESUMO

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. OBJECTIVES: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. METHODS: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. RESULTS: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. CONCLUSIONS: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.


Assuntos
Proteínas ADAM/genética , Plaquetas/enzimologia , Metaloendopeptidases/genética , Mutação de Sentido Incorreto , Púrpura Trombocitopênica Trombótica/genética , Proteínas ADAM/sangue , Proteínas ADAM/deficiência , Proteína ADAMTS13 , Adulto , Animais , Sítios de Ligação , Cálcio/sangue , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Células HEK293 , Homozigoto , Humanos , Metaloendopeptidases/deficiência , Camundongos Knockout , Simulação de Dinâmica Molecular , Fenótipo , Gravidez , Ligação Proteica , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/enzimologia , Transfecção
19.
Proc Natl Acad Sci U S A ; 111(34): 12396-401, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25114233

RESUMO

The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin ß-deficient mice was now found to be attached. Meprin ß is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin ß was added to the attached mucus of meprin ß-deficient mice, the mucus was detached from the epithelium. Similar to meprin ß-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin ß into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin ß to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin ß cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin ß in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Intestino Delgado/metabolismo , Metaloendopeptidases/metabolismo , Mucina-2/metabolismo , Muco/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Vida Livre de Germes/fisiologia , Intestino Delgado/microbiologia , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Knockout , Dados de Sequência Molecular , Mucina-2/química , Mucina-2/genética , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
20.
PLoS One ; 9(5): e98017, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24849253

RESUMO

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1-/- mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1-/- mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1-/- mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1-/- mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1-/- mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1-/- mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1-/- mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1-/- mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.


Assuntos
Deleção de Genes , Cirrose Hepática/genética , Cirrose Hepática/prevenção & controle , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Aminoácidos/análise , Animais , Colina/análise , Dieta Hiperlipídica/efeitos adversos , Resistência à Doença/genética , Feminino , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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