RESUMO
The conversion of cellular prion protein (PrPC) into pathogenic prion isoforms (PrPSc) and the mutation of PRNP are definite causes of prion diseases. Unfortunately, without exception, prion diseases are untreatable and fatal neurodegenerative disorders; therefore, one area of research focuses on identifying medicines that can delay the progression of these diseases. According to the concept of drug repositioning, we investigated the efficacy of the c-Abl tyrosine kinase inhibitor radotinib, which is a drug that is approved for the treatment of chronic myeloid leukemia, in the treatment of disease progression in prion models, including prion-infected cell models, Tga20 and hamster cerebellar slice culture models, and 263K scrapie-infected hamster models. Radotinib inhibited PrPSc deposition in neuronal ZW13-2 cells that were infected with the 22L or 139A scrapie strains and in cerebellar slice cultures that were infected with the 22L or 263K scrapie strains. Interestingly, hamsters that were intraperitoneally injected with the 263K scrapie strain and intragastrically treated with radotinib (100 mg/kg) exhibited prolonged survival times (159 ± 28.6 days) compared to nontreated hamsters (135 ± 9.9 days) as well as reduced PrPSc deposition and ameliorated pathology. However, intraperitoneal injection of radotinib exerted a smaller effect on the survival rate of the hamsters. Additionally, we found that different concentrations of radotinib (60, 100, and 200 mg/kg) had similar effects on survival time, but this effect was not observed after treatment with a low dose (30 mg/kg) of radotinib. Interestingly, when radotinib was administered 4 or 8 weeks after prion inoculation, the treated hamsters survived longer than the vehicle-treated hamsters. Additionally, a pharmacokinetic assay revealed that radotinib effectively crossed the blood-brain barrier. Based on our findings, we suggest that radotinib is a new candidate anti-prion drug that could possibly be used to treat prion diseases and promote the remission of symptoms.
Assuntos
Doenças Priônicas , Príons , Scrapie , Cricetinae , Animais , Ovinos , Scrapie/metabolismo , Príons/metabolismo , Proteínas PrPSc/metabolismo , Encéfalo/metabolismo , Doenças Priônicas/metabolismoRESUMO
Calsenilin modulates A-type potassium channels, regulates presenilin-mediated γ-secretase activity, and represses prodynorphin and c-fos genes expression. RhoA is involved in various cellular functions including proliferation, differentiation, migration, transcription, and regulation of the actin cytoskeleton. Although recent studies demonstrate that calsenilin can directly interact with RhoA and that RhoA inactivation is essential for neuritogenesis, it is uncertain whether there is a link between calsenilin and RhoA-regulated neuritogenesis. Here, we investigated the role of calsenilin in RhoA-regulated neuritogenesis using in vitro and in vivo systems. We found that calsenilin induced RhoA inactivation, which accompanied RhoA phosphorylation and the reduced phosphorylation levels of LIM kinase (LIMK) and cofilin. Interestingly, PC12 cells overexpressing either full-length (FL) or the caspase 3-derived C-terminal fragment (CTF) of calsenilin significantly inactivated RhoA through its interaction with RhoA and p190 Rho GTPase-activating protein (p190RhoGAP). In addition, cells expressing FL and the CTF of calsenilin had increased neurite outgrowth compared to cells expressing the N-terminal fragment (NTF) of calsenilin or vector alone. Moreover, Tat-C3 and Y27632 treatment significantly increased the percentage of neurite-bearing cells, neurite length, and the number of neurites in cells. Finally, calsenilin deficiency in the brains of calsenilin-knockout mice significantly interfered with RhoA inactivation. These findings suggest that calsenilin contributes to neuritogenesis through RhoA inactivation.
Assuntos
Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Crescimento Neuronal , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/química , Camundongos , Células PC12 , Fosforilação , Ratos , Transdução de SinaisRESUMO
Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.
Assuntos
Astrocitoma/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , CMP Cíclico/análogos & derivados , Desiminases de Arginina em Proteínas/biossíntese , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , CMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/fisiologia , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 3 , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs) has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC) functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO) generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS). This suppression of NO generation was independent of changes in NO synthase (NOS) enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS) transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65), and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT) phosphorylation and activator protein-1 (AP-1) transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun) were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of excessive iNOS/NO-linked immune responses.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Ornitina/análogos & derivados , Animais , Biomarcadores , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Células Dendríticas/imunologia , Dinoprostona/biossíntese , Ativação Enzimática/efeitos dos fármacos , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Ornitina/farmacologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Western Blotting , Citrulina/metabolismo , Eletroforese em Gel Bidimensional , Proteína Glial Fibrilar Ácida/análise , Humanos , Hidrolases/metabolismo , Imuno-Histoquímica , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ϵ-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.
Assuntos
Doença de Alzheimer/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Citrulina/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hidrolases/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Ácido Aminocaproico/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/imunologia , Western Blotting , Encéfalo/metabolismo , Proteínas de Transporte/imunologia , Estudos de Casos e Controles , Síndrome de Creutzfeldt-Jakob/patologia , Proteínas de Ligação a DNA/imunologia , Feminino , Lobo Frontal/metabolismo , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Fosfopiruvato Hidratase/imunologia , Plasminogênio/metabolismo , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/imunologiaRESUMO
Presenilin 1 (PS1) is a component of the γ-secretase complex that cleaves a variety of type I membrane proteins, including the ß-amyloid precursor protein (ß-APP), Notch, and neuronal (N)- and epithelial (E)-cadherins. N-cadherin is an essential adhesion molecule that forms a complex with, and is cleaved by, PS1/γ-secretase and ß-catenin in the plasma membrane. The purpose of this study was to determine whether calsenilin, a presenilin-interacting protein, has a functional role in PS1/γ-secretase-mediated N-cadherin ε-cleavage using Western blot analysis, RT-PCR, immunoprecipitation, subcellular fractionation, biotinylation, and a luciferase reporter assay in SH-SY5Y neuroblastoma cells. Here, we demonstrate that the expression of calsenilin leads to a disruption of PS1/γ-secretase-mediated ε-cleavage of N-cadherin, which results in the significant accumulation of N-cadherin C-terminal fragment 1 (Ncad/CTF1), the reduction of cytoplasmic Ncad/CTF2 release, and a deceleration of PS1-CTF delivery to the cell surface. Interestingly, we also found that the expression of calsenilin is associated with the redistribution of ß-catenin from the cell surface to a cytoplasmic pool, as well as with the negative regulation of genes that are targets of T-cell factor/ß-catenin nuclear signaling. Taken together, our findings suggest that calsenilin is a novel negative regulator of N-cadherin processing that plays an important role in ß-catenin signaling.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Presenilina-1/metabolismo , Proteínas Repressoras/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos CD/química , Caderinas/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Modelos Neurológicos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Estrutura Terciária de Proteína , Proteólise , Proteínas Repressoras/genética , Transdução de Sinais , beta Catenina/genéticaRESUMO
Although there have been several studies regarding the immunogenicity of one or two booster doses of the measles−mumps−rubella (MMR) vaccine in measles-seronegative young adults, limited data are available about how long the immune response is sustained compared with natural infection. This study included seronegative healthcare workers (HCWs) (aged 21−38 years) who received one or two doses of the measles−mumps−rubella (MMR) vaccine and HCWs with laboratory-confirmed measles infection during an outbreak in 2019. We compared neutralizing antibody titers measured using the plaque reduction neutralization (PRN) test and measles-specific immunoglobulin G (IgG) using chemiluminescent immunoassays 2 years after vaccination or infection. Among 107 HCWs with seronegative measles IgGs, the overall seroconversion rate of measles IgGs remained 82.2% (88/107), and 45.8% (49/107) of the participants had a medium (121−900) or high (>900) PRN titer after 2 years from one or two booster doses. The measles-neutralizing antibody titers of both PRN titer (ND50) and geometric mean concentration 2 years after natural infection were significantly higher than those of one or two booster doses of the MMR vaccine (p < 0.001 and p < 0.001, respectively). Our results suggest that serologic screening followed by appropriate postexposure prophylaxis can be beneficial for young HCWs without a history of natural infection especially in a measles outbreak setting, because of possible susceptibility to measles despite booster MMR vaccination 2 years ago. Long-term data about sustainable humoral immunity after one or two booster vaccination are needed based on the exact vaccination history.
RESUMO
Mitochondrial dynamics continually maintain cell survival and bioenergetics through mitochondrial quality control processes (fission, fusion, and mitophagy). Aberrant mitochondrial quality control has been implicated in the pathogenic mechanism of various human diseases, including cancer, cardiac dysfunction, and neurological disorders, such as Alzheimer's disease, Parkinson's disease, and prion disease. However, the mitochondrial dysfunction-mediated neuropathological mechanisms in prion disease are still uncertain. Here, we used both in vitro and in vivo scrapie-infected models to investigate the involvement of mitochondrial quality control in prion pathogenesis. We found that scrapie infection led to the induction of mitochondrial reactive oxygen species (mtROS) and the loss of mitochondrial membrane potential (ΔΨm), resulting in enhanced phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 and its subsequent translocation to the mitochondria, which was followed by excessive mitophagy. We also confirmed decreased expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes and reduced ATP production by scrapie infection. In addition, scrapie-infection-induced aberrant mitochondrial fission and mitophagy led to increased apoptotic signaling, as evidenced by caspase 3 activation and poly (ADP-ribose) polymerase cleavage. These results suggest that scrapie infection induced mitochondrial dysfunction via impaired mitochondrial quality control processes followed by neuronal cell death, which may have an important role in the neuropathogenesis of prion diseases.
Assuntos
Mitocôndrias , Neurônios , Doenças Priônicas , Animais , Humanos , Camundongos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Mitofagia/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Doenças Priônicas/patologia , Príons/efeitos adversos , Príons/metabolismo , Scrapie/metabolismo , Scrapie/patologiaRESUMO
Peptidylarginine deiminases (PADs)-mediated post-translational citrullination processes play key roles in protein functions and structural stability through the conversion of arginine to citrulline in the presence of excessive calcium concentrations. In brain, PAD2 is abundantly expressed and can be involved in citrullination in disease. Recently, we have reported pathological characterization of PAD2 and citrullinated proteins in scrapie-infected mice, but the implication of protein citrullination in the pathophysiology in human prion disease is not clear. In the present study, we explored the molecular and biological involvement of PAD2 and the pathogenesis of citrullinated proteins in frontal cortex of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found increased expression of PAD2 in reactive astrocytes that also contained increased levels of citrullinated proteins. In addition, PAD activity was significantly elevated in patients with sCJD compared to controls. From two-dimensional gel electrophoresis and MALDI-TOF mass analysis, we found various citrullinated candidates, including cytoskeletal and energy metabolism-associated proteins such as vimentin, glial fibrillary acidic protein, enolase, and phosphoglycerate kinase. Based on these findings, our investigations suggest that PAD2 activation and aberrant citrullinated proteins could play a role in pathogenesis and have value as a marker for the postmortem classification of neurodegenerative diseases.
Assuntos
Citrulina/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Lobo Frontal/metabolismo , Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Idoso , Idoso de 80 Anos ou mais , Astrócitos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Síndrome de Creutzfeldt-Jakob/patologia , Ativação Enzimática/fisiologia , Feminino , Imunofluorescência , Lobo Frontal/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para CimaRESUMO
Peptidylarginine deiminases (PADs), which are a group of posttranslational modification enzymes, are involved in protein citrullination (deimination) by the conversion of peptidylarginine to peptidylcitrulline in a calcium concentration-dependent manner. Among the PADs, PAD2 is widely distributed in various tissues and is the only type that is expressed in brain. To elucidate the involvement of protein citrullination by PAD2 in the pathogenesis of brain-specific prion diseases, we examined the profiles of citrullinated proteins using the brains of scrapie-infected mice as a prion disease model. We found that, compared with controls, increased levels of citrullinated proteins of various molecular weights were detected in different brain sections of scrapie-infected mice. In support of this data, expression levels of PAD2 protein as well as its enzyme activity were significantly increased in brain sections of scrapie-infected mice, including hippocampus, brain stem, and striatum. Additionally, the expression levels of PAD2 mRNA were increased during scrapie infection. Moreover, PAD2 immunoreactivity was increased in scrapie-infected brains, with staining detected primarily in reactive astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, various citrullinated proteins were identified in the brains of scrapie-infected mice, including glial fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This study suggests that accumulated citrullinated proteins and abnormal activation of PAD2 may function in the pathogenesis of prion diseases and serve as potential therapeutic targets.
Assuntos
Encéfalo/enzimologia , Encéfalo/patologia , Citrulina/metabolismo , Hidrolases/genética , Proteínas PrPSc/patogenicidade , Proteínas/metabolismo , Regulação para Cima/genética , Animais , Astrócitos/enzimologia , Eletroforese em Gel Bidimensional , Imunofluorescência , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Proteína Glial Fibrilar Ácida/química , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/química , Proteína Básica da Mielina/metabolismo , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/metabolismo , Transporte Proteico , Desiminases de Arginina em Proteínas , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Myelin basic protein (MBP) citrullination by peptidylarginine deiminase (PAD) enzymes leads to incomplete protein-lipid bilayer interactions and vulnerability to proteolytic enzymes, resulting in disorganization of the myelin sheath in the central nervous system. Therefore, citrullinated MBP (citMBP) has been suggested as a hallmark of demyelination, but how citMBP is implicated in prion diseases remains unknown. For the first time, we developed mouse monoclonal anti-citMBP IgG1 (clones 1B8, 1H1, and 3C6) and IgM (clone 3G5) antibodies that recognize human citMBP at its R25, R122, and R130 residues and at its C-terminal region (or the corresponding sites in mouse MBP), respectively. Using a biochemical, immunohistochemical, and immunogold-silver staining for electron microscopy techniques, we found that MBP residue R23 (corresponding to human R25) was specifically citrullinated, was stained as intense punctae in the corpus callosum, the striatum, and the cerebellar white matter, and was predominantly localized in disorganized myelin in the brains of scrapie-infected mice. In the brains of Creutzfeldt-Jakob disease (CJD) patients, MBP residues R25, R122, and R130 were markedly citrullinated and were stained as fibrils and punctae. In particular, white matter regions, such as the midbrain and the medulla, exhibited high levels of citMBP compared to other regions. However, the high levels of citMBP were not correlated with PAD2 expression. The clone 3G5 recognized significantly increased expression of the 18.5 kDa and/or 21.5 kDa variants of MBP in prion disease. Our findings suggest that significantly increased levels of citMBP may reflect demyelinating neuropathology, and that these newly developed antibodies may be useful for identifying demyelination.
Assuntos
Anticorpos Monoclonais/imunologia , Sistema Nervoso Central/patologia , Citrulinação , Doenças Desmielinizantes/metabolismo , Proteína Básica da Mielina/metabolismo , Doenças Priônicas/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Scrapie/imunologia , Scrapie/patologia , Substância Branca/metabolismo , Substância Branca/patologiaRESUMO
Hepatic stellate cells (HSCs) play pivotal roles in hepatic fibrosis as they synthesize glial fibrillary acidic protein (GFAP), which is increased in activated HSCs. GFAP-expressing HSCs and myofibroblasts accumulate in and around hepatic fibrosis lesions. Peptidylarginine deiminase 2 (PAD2) is responsible for the citrullination of GFAP (cit-GFAP). However, the involvement of PAD2 and cit-GFAP in hepatic fibrosis remains unclear. To determine the expression of PAD2 and cit-GFAP in hepatic fibrosis, C57BL/6 mice underwent bile duct ligation (BDL) or a sham operation. In BDL livers, the expression of PAD2 and its enzyme activity were significantly increased compared with controls. In addition, PAD2-postitive cells were rarely observed in only the portal vein and the small bile duct in sham-operated livers, whereas an increased number of PAD2-positive cells were detected in the bile duct and Glisson's sheath in BDL livers. Interestingly, PAD2 was colocalized with α-SMA-positive cells and CK19-positive cells in BDL livers, indicating upregulated PAD2 in activated HSCs and portal fibroblasts of the livers of BDL mice. We also found that citrullinated proteins were highly accumulated in the livers of BDL mice compared with controls. Moreover, the expression level of GFAP and the amount of cit-GFAP were higher in BDL livers than in control livers. In correlation with PAD2 localization, cit-GFAP was observed in α-SMA-positive and CK19-positive cells in the livers of BDL mice. These results suggest that the increased expression and activation of PAD2 along with increased citrullinated proteins, specifically cit-GFAP, may play important roles in the pathogenesis of hepatic fibrosis.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , Actinas/metabolismo , Animais , Ductos Biliares/lesões , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Citrulinação , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Veia Porta/metabolismo , Veia Porta/patologia , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas/metabolismo , Distribuição AleatóriaRESUMO
Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.
Assuntos
Citocromos c/química , Citocromos c/metabolismo , Ácido Peroxinitroso/química , Animais , Cianetos/metabolismo , Estabilidade Enzimática , Fluoresceínas/metabolismo , Oxirredução , Peroxidases/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Superóxidos/metabolismo , Tirosina/análogos & derivados , Tirosina/síntese químicaRESUMO
Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.
Assuntos
Conexina 43/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Scrapie/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Membrana Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Sistema de Sinalização das MAP Quinases/fisiologia , Mesocricetus , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer's disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.
Assuntos
Doenças Neurodegenerativas/patologia , Fosfolipase D/metabolismo , Doenças Priônicas/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/enzimologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doenças Neurodegenerativas/enzimologia , Fosfolipase D/líquido cefalorraquidiano , Doenças Priônicas/enzimologiaRESUMO
Cl-amidine, which is a small-molecule inhibitor of PAD, has therapeutic potential for inflammation-mediated diseases. However, little is known regarding the manner by which PAD inhibition by Cl-amidine regulates inflammatory conditions. Here, we investigated the effects of PAD inhibition by Cl-amidine on the functioning of DCs, which are pivotal immune cells that mediate inflammatory diseases. When DC maturation was induced by TLR agonists, reduced cytokine levels (IL-6, IL-1ß, and IL-12p70) were observed in Cl-amidine-treated DCs. Cl-amidine-treated, LPS-activated DCs exhibited alterations in their mature and functional statuses with up-regulated antigen uptake, down-regulated CD80, and MHC molecules. In addition, Cl-amidine-treated DCs dysregulated peptide-MHC class formations. Interestingly, the decreased cytokines were independent of MAPK/NF-κB signaling pathways and transcription levels, indicating that PAD inhibition by Cl-amidine may be involved in post-transcriptional steps of cytokine production. Transmission electron microscopy revealed morphotypical changes with reduced dendrites in the Cl-amidine-treated DCs, along with altered cellular compartments, including fragmented ERs and the formation of foamy vesicles. Furthermore, in vitro and in vivo Cl-amidine treatments impaired the proliferation of naïve CD4(+) and CD8(+) T cells. Overall, our findings suggest that Cl-amidine has therapeutic potential for treating inflammation-mediated diseases.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/imunologia , Hidrolases/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Ornitina/análogos & derivados , Receptores Toll-Like/agonistas , Animais , Antígeno B7-1/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Citocinas/imunologia , Células Dendríticas/patologia , Feminino , Hidrolases/imunologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , NF-kappa B/farmacologia , Ornitina/farmacologia , Desiminases de Arginina em Proteínas , Receptores Toll-Like/imunologiaRESUMO
The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.
Assuntos
Encéfalo/patologia , Citrulina/metabolismo , Hidrolases/metabolismo , Doenças Priônicas/enzimologia , Doenças Priônicas/patologia , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Humanos , Doenças Priônicas/metabolismo , Príons/metabolismo , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Proteínas/metabolismoRESUMO
The failure of Mycobacterium bovis BCG as a TB vaccine against TB reactivation suggests that latency-associated proteins should be included in alternative TB vaccine development. Further, antigens known to generate protective immunity against the strong Th1 stimulatory response to reactivated TB should be included in novel vaccine design. Recent studies have emphasized the importance of Rpfs from Mycobacterium tuberculosis in the reactivation process and cellular immunity. However, little is known about how RpfB mediates protective immunity against M. tuberculosis. Here, we investigated the functional roles and signaling mechanisms of RpfB in DCs and its implications in the development of T cell immunity. DCs treated with RpfB displayed features of mature and functional status, with elevated expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF-α, IL-1ß, IL-6, and IL-12p70). Activation of DCs was mediated by direct binding of RpfB to TLR4, followed by MyD88/TRIF-dependent signaling to MAPKs and NF-κB signaling pathways. Specifically, we found that the RpfB G5 domain is the most important part in RpfB binding to TLR4. RpfB-treated DCs effectively polarized naïve CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2. Importantly, RpfB induced the expansion of memory CD4(+)/CD8(+)CD44(high)CD62L(low) T cells in the spleen of M. tuberculosis-infected mice. Our data suggest that RpfB regulates innate immunity and activates adaptive immunity through TLR4, a finding that may help in the design of more effective vaccines.
Assuntos
Proteínas de Bactérias/metabolismo , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Morte Celular , Diferenciação Celular/imunologia , Citocinas/metabolismo , Células Dendríticas/citologia , Endotoxinas/metabolismo , Ativação Enzimática , Feminino , Memória Imunológica , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Testes de Neutralização , Fenótipo , Ligação Proteica , Reprodutibilidade dos Testes , Deleção de Sequência/genética , Transdução de Sinais/imunologia , Células Th1/citologia , Receptor 2 Toll-Like/metabolismoRESUMO
Peptidylarginine deiminase (PAD) and citrullinated proteins have emerged as key molecules in various human diseases, but detailed subcellular localizations of PAD2 and citrullinated proteins are poorly mapped in brain under normal and pathologic conditions. We performed subcellular fractionation and electron microscopic analysis using brains of normal and scrapie-infected mice. Peptidylarginine deiminase 2 was abundantly present in cytosol and weakly in microsomal and mitochondrial fractions and expression in these fractions was higher in brains of scrapie-infected mice. Despite relatively low PAD2 expression, in microsomal and mitochondrial fractions, citrullinated proteins were present at high levels in these fractions in scrapie-infected brains. Surprisingly, increased PAD2 expression and accumulated citrullinated proteins were also found in nuclear fractions in scrapie-infected brains. By electron microscopy, PAD2 and citrullinated proteins in scrapie-infected brains were widely distributed in most cellular compartments including mitochondria, endoplasmic reticulum, glial filaments, nuclei, and Golgi apparatus in astrocytes and hippocampal neurons. Taken together, we report for the first time the nuclear localization of PAD2 and the detailed subcellular localization of PAD2 and of citrullinated proteins in scrapie-infected brains. Our findings suggest that different subcellular compartmentalization of PAD2 and citrullinated proteins may have different physiological roles in normal and neurodegenerative conditions.