RESUMO
ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described ß-secretase to generate Aß peptides in Alzheimer's disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aß peptides generation is the metalloproteinase meprin ß, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin ß expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b-/-). We examined levels of canonical and truncated Aß species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b-/-. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aß1-40 and 1-42 levels are reduced in APP/lon mice when meprin ß is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aß2-x peptide deposition is decreased in APP/lon × Mep1b-/- mice. Importantly, loss of meprin ß improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin ß within the amyloidogenic pathway and Aß production in vivo.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Aprendizagem , Transtornos da Memória/patologia , Metaloendopeptidases/deficiência , Idoso , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/patologia , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Metaloendopeptidases/metabolismo , Camundongos Knockout , Peptídeos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Nardilysin (NRDC) has been shown to be involved in post-translational histone modifications, in addition to enhancement in ectodomain shedding of membrane-anchored protein, which play significant roles in various pathophysiology, including glucose homeostasis, inflammatory diseases and cancer. The present study sought to determine roles of NRDC in the liver on lipid and lipoprotein metabolism. We established liver-specific NRDC deficient mice by use of NRD1 floxed mice and albumin promoter-Cre recombinase (Cre) transgenic mice, and found that their serum low-density lipoprotein (LDL) cholesterol levels were significantly lower than those in control littermate mice. In the liver, LDL receptor (LDLR) mRNA expression was significantly upregulated, while inducible degrader of LDLR (IDOL) and microsomal triglyceride transfer protein (MTP) mRNA expression was significantly downregulated, in liver-specific NRDC deficient mice. Hepatic cell-surface LDLR expression levels were significantly elevated and serum pro-protein convertase subtilisin-kexin type 9 (PCSK9) levels were significantly reduced in mice with hepatic NRDC deficiency. In cultured hepatocytes, NRDC deficiency significantly reduced secreted PCSK9 and increased cell-surface LDLR expression. On the other hand, NRDC overexpression in cultured hepatocytes significantly increased secreted PCSK9 and lowered cell-surface LDLR expression. Thus, NRDC in murine hepatocytes appears to play key roles in cholesterol homeostasis, although the precise molecular mechanisms remain to be determined.
Assuntos
LDL-Colesterol/sangue , Hepatócitos/metabolismo , Fígado/metabolismo , Metaloendopeptidases/deficiência , Animais , Células Cultivadas , Masculino , Metaloendopeptidases/genética , Camundongos Transgênicos , Pró-Proteína Convertase 9/sangue , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
Several progeroid disorders are caused by deficiency in the endoprotease ZMPSTE24 which leads to accumulation of prelamin A at the nuclear envelope. ZMPSTE24 cleaves prelamin A twice: at the third carboxyl-terminal amino acid following farnesylation of a -CSIM motif; and 15 residues upstream to produce mature lamin A. The carboxyl-terminal cleavage can also be performed by RAS-converting enzyme 1 (RCE1) but little is known about the importance of this cleavage for the ability of prelamin A to cause disease. Here, we found that knockout of RCE1 delayed senescence and increased proliferation of ZMPSTE24-deficient fibroblasts from a patient with non-classical Hutchinson-Gilford progeria syndrome (HGPS), but did not influence proliferation of classical LMNA-mutant HGPS cells. Knockout of Rce1 in Zmpste24-deficient mice at postnatal week 4-5 increased body weight and doubled the median survival time. The absence of Rce1 in Zmpste24-deficient fibroblasts did not influence nuclear shape but reduced an interaction between prelamin A and AKT which activated AKT-mTOR signaling and was required for the increased proliferation. Prelamin A levels increased in Rce1-deficient cells due to a slower turnover rate but its localization at the nuclear rim was unaffected. These results strengthen the idea that the presence of misshapen nuclei does not prevent phenotype improvement and suggest that targeting RCE1 might be useful for treating the rare progeroid disorders associated with ZMPSTE24 deficiency.
Assuntos
Genes ras/genética , Proteínas de Membrana/deficiência , Metaloendopeptidases/deficiência , Progéria/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , FenótipoRESUMO
Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc-/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc-/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75NTR , Ngfr) is increased in Nrdc-/- LV due to the reduced ectodomain shedding of p75NTR . Importantly, the reduction of p75NTR rescued the abnormal innervation phenotype of Nrdc-/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc-/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75NTR .
Assuntos
Coração/inervação , Metaloendopeptidases/genética , Receptor de Fator de Crescimento Neural/genética , Sistema Nervoso Simpático/metabolismo , Animais , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Bradicardia/genética , Bradicardia/fisiopatologia , Células Cultivadas , Ecocardiografia , Coração/fisiopatologia , Frequência Cardíaca/genética , Frequência Cardíaca/fisiologia , Metaloendopeptidases/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Células PC12 , Ratos , Receptor de Fator de Crescimento Neural/deficiência , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/fisiopatologiaRESUMO
Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.
Assuntos
Proteínas de Bactérias/imunologia , Metaloendopeptidases/imunologia , Fosfotransferases (Aceptor do Grupo Carboxila)/imunologia , Serina Endopeptidases/imunologia , Úlcera Cutânea/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Quimiocina CCL4/genética , Quimiocina CCL4/imunologia , Feminino , Regulação da Expressão Gênica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-1alfa/genética , Interleucina-1alfa/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Fosfotransferases (Aceptor do Grupo Carboxila)/deficiência , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Pele/imunologia , Pele/microbiologia , Pele/patologia , Úlcera Cutânea/genética , Úlcera Cutânea/microbiologia , Úlcera Cutânea/patologia , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
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
Injúria Renal Aguda/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Rim/enzimologia , Metaloendopeptidases/metabolismo , Traumatismo por Reperfusão/enzimologia , Injúria Renal Aguda/genética , Injúria 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 SinaisRESUMO
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 , FenótipoRESUMO
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/patologiaRESUMO
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/metabolismoRESUMO
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/farmacologiaRESUMO
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 , FicolinasRESUMO
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 do ExomaRESUMO
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 , FenótipoRESUMO
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 , Poro de Transição de Permeabilidade Mitocondrial , Neurônios/patologia , Mapeamento de Interação de Proteínas , Transdução de SinaisRESUMO
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 , Glutationa Peroxidase GPX1RESUMO
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 AminoácidosRESUMO
Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.
Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Hidroximetilglutaril-CoA Redutases/metabolismo , Metaloendopeptidases/metabolismo , Proteólise/efeitos dos fármacos , Esteróis/farmacologia , Animais , Sequência de Bases , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Glicosilação/efeitos dos fármacos , Humanos , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Tripsina/metabolismoRESUMO
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/metabolismoRESUMO
Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A-accumulating) and Zmpste24-proficient (mature lamin A-containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target.
Assuntos
Neoplasias/patologia , Proteínas Nucleares/metabolismo , Progéria/metabolismo , Progéria/patologia , Precursores de Proteínas/metabolismo , Envelhecimento/patologia , Animais , Biomarcadores/metabolismo , Carcinogênese/patologia , Feminino , Humanos , Lamina Tipo A , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Metaloendopeptidases/deficiência , Metaloendopeptidases/metabolismo , Camundongos , Mosaicismo , Invasividade Neoplásica , Neoplasias/metabolismo , FenótipoRESUMO
INTRODUCTION: Loss of adult stem cell function during aging contributes to impaired tissue regeneration. Here, we tested the aging-related decline in regeneration potential of adult stem cells residing in the skeletal muscle. METHODS: We isolated muscle-derived stem/progenitor cells (MDSPCs) from progeroid Zmpste24-deficient mice (Zmpste24(-/-)) with accelerated aging phenotypes to investigate whether mutation in lamin A has an adverse effect on muscle stem/progenitor cell function. RESULTS: Our results indicate that MDSPCs isolated from Zmpste24(-/-) mice show reduced proliferation and myogenic differentiation. In addition, Zmpste24(-/-) MDSPCs showed impaired muscle regeneration, with a limited engraftment potential when transplanted into dystrophic muscle, compared with wild-type (WT) MDSPCs. Exposure of progeroid Zmpste24(-/-) MDSPCs to WT MDSPCs rescued the myogenic differentiation defect in vitro. CONCLUSIONS: These results demonstrate that adult stem/progenitor cell dysfunction contributes to impairment of tissue regeneration and suggest that factors secreted by functional cells are indeed important for the therapeutic effect of adult stem cells.