RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The cluster of differentiation 147 (CD147) is identified as the signaling protein relevant importantly in various cancers, inflammations, and coronavirus disease 2019 (COVID-19) via interacting with extracellular cyclophilin A (CypA). The reduction of CD147 levels inhibits the progression of CD147-associated diseases. Thai traditional medicines (TTMs): Keaw-hom (KH), Um-ma-ruek-ka-wa-tee (UM), Chan-ta-lee-la (CT), and Ha-rak (HR) have been used as anti-pyretic and anti-respiratory syndromes caused from various conditions including cancers, inflammations, and infections. Thus, these medicines would play a crucial role in the reduction of CD147 levels. AIM OF THE STUDY: This article aimed to investigate the effects of KH, UM, CT, and HR for reducing the CD147 levels through in vitro study. Additionally, in silico study was employed to screen the active compounds reflexing the reduction of CD147 levels. MATERIALS AND METHODS: The immunofluorescent technique was used to evaluate the reduction of CD147 levels in human lung epithelial cells (BEAS-2B) stimulated with CypA for eight extracts of KH, UM, CT, and HR obtained from water decoction (D) and 70% ethanol maceration (M) including, KHD, UMD, CTD, HRD, KHM, UMM, CTM, and HRM. RESULTS: UM extracts showed the most efficiency for reduction of CD147 levels in the cytoplasm and perinuclear of BEAS-2B cells stimulated with CypA. Phenolic compounds composing polyphenols, polyphenol sugars, and flavonoids were identified as the major chemical components of UMD and UMM. Further, molecular docking calculations identified polyphenol sugars as CypA inhibitors. CONCLUSIONS: UMD and UMM are potential for reduction of CD147 levels which provide a useful information for further development of UM as potential therapeutic candidates for CD147-associated diseases such as cancers, inflammations, and COVID-19.
Assuntos
COVID-19 , Neoplasias , Humanos , Basigina/metabolismo , Medicina Tradicional Tailandesa , Simulação de Acoplamento Molecular , Ciclofilina A/química , Ciclofilina A/metabolismo , Ciclofilina A/farmacologia , Inflamação , Pulmão/metabolismo , Polifenóis , AçúcaresRESUMO
Selenium, an essential micronutrient, plays vital roles in the brain. Selenoprotein P (SELENOP), a major plasma selenoprotein, is thought to transport selenium to the brain. However, Selenop-knockout mice fed a diet containing an adequate amount of selenium shows no objective neurological dysfunction which is observed in the selenium-deficient diet-fed Selenop-knockout mice. This fact indicated that selenium from low-mass selenium-source compounds can be transported by SELENOP-independent alternative pathways to the brain. In this study, to obtain the basic information about the SELENOP-independent transport pathways, we performed ex vivo experiments in which the rat brain cell membrane fraction was analyzed to find selenium-binding and/or -interactive proteins using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several membrane proteins with the cysteine (C) thiol were found to be reactive with STS through the thiol-exchange reaction. One of the C-containing proteins in the brain cell membrane fraction was identified as peptidyl-prolyl cis-trans isomerase (PPIase) A from tryptic fragmentation experiments and database search. Among the 4 C residues in rat PPIase A, 21st C was proved to react with STS by assessment using C mutated recombinant proteins. PPIase A is ubiquitously expressed and also associates with a variety of biologically important events such as immunomodulation, intracellular signaling, transcriptional regulation and protein trafficking. Consequently, PPIase A was thought to participate in the selenium transport into the rat brain.
Assuntos
Selênio , Animais , Encéfalo , Ciclofilina A , Camundongos , Peptidilprolil Isomerase , Ratos , SelenoproteínasRESUMO
Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. Direct-acting antivirals (DAAs), including inhibitors of nonstructural proteins (NS3/4A protease, NS5A, and NS5B polymerase), represent key components of anti-HCV treatment. However, some DAAs are associated with increased drug resistance and undesired side effects. Previous reports have shown that bisamides could be a novel class of cyclophilin A (CypA) inhibitors for treating HCV as a member of combinational therapies. To fully elucidate structure-activity relationships of bisamide derivatives and find a better hit compound with diverse binding modes, 16 biamides were designed with the help of docking program. They were then synthesized using one-pot four-component Ugi reaction. 7e with selectivity index of more than 18.9 (50% effective concentration of 5.3 µM, but no cytotoxicity at 100 µM) and unique binding mode that could be dived into gatekeeper pocket was selected as a new hit compound. Surface plasmon resonance experiments revealed that 7e is able to bind to CypA with a KD of 3.66 µM. Taken together, these results suggest that 7e as a CypA inhibitor could be used as an alternative anti-HCV agent in combinational therapy in the future.
Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Ciclofilina A/antagonistas & inibidores , Desenho de Fármacos , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Amidas/síntese química , Amidas/química , Antivirais/síntese química , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Ciclofilina A/metabolismo , Relação Dose-Resposta a Droga , Hepacivirus/metabolismo , Hepatite C/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
BACKGROUND: Viruses are obligate parasites that depend on the cellular machinery of the host to regenerate and manufacture their proteins. Most antiviral drugs on the market today target viral proteins. However, the more recent strategies involve targeting the host cell proteins or pathways that mediate viral replication. This new approach would be effective for most viruses while minimizing drug resistance and toxicity. METHODS: Cytomegalovirus replication, latency, and immune response are mediated by the intermediate early protein 2, the main protein that determines the effectiveness of drugs in cytomegalovirus inhibition. This review explains how intermediate early protein 2 can modify the action of cyclosporin A, an immunosuppressive, and antiviral drug. It also links all the pathways mediated by cyclosporin A, cytomegalovirus replication, and its encoded proteins. RESULTS: Intermediate early protein 2 can influence the cellular cyclophilin A pathway, affecting cyclosporin A as a mediator of viral replication or anti-cytomegalovirus drug. CONCLUSION: Cyclosporin A has a dual function in cytomegalovirus pathogenesis. It has the immunosuppressive effect that establishes virus replication through the inhibition of T-cell function. It also has an anti-cytomegalovirus effect mediated by intermediate early protein 2. Both of these functions involve cyclophilin A pathway.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Ciclofilina A/imunologia , Ciclofilina A/metabolismo , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Antivirais/química , Ciclofilina A/antagonistas & inibidores , Infecções por Citomegalovirus/virologia , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
In this work, the relationship between cyclophilin A (CypA) and HCV prompted us to screen a series of small molecule CypA inhibitors which were previously reported by our group. Among them, compound 1, discovered as a non-immunosuppressive anti-HCV agent with an EC50 value of 0.67 µM in a virus assay, was selected for further study. Subsequent chemical modification by O-acylation led to a novel class of molecules, among which compound 25 demonstrated the most potent anti-HCV activity in the virus assay (EC50 = 0.19 µM), but low cytotoxicity and hERG cardiac toxicity. The following studies (a solution stability assay and a simple pharmacokinetic test together with a CypA enzyme inhibition assay) preliminarily indicated that 25 was a prodrug of 1. To the best of our knowledge, 25 is probably the most potent currently reported small molecule anti-HCV agent acting via the CypA inhibitory mechanism. Consequently, our study has provided a new potential small molecule for curing HCV infection.
Assuntos
Antivirais/química , Antivirais/farmacologia , Ciclofilina A/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Acilação , Antivirais/síntese química , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Hepacivirus/genética , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Estrutura Molecular , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacosRESUMO
Fragment-based drug design (FBDD) is a promising approach for the discovery and optimization of lead compounds. Despite its successes, FBDD also faces some internal limitations and challenges. FBDD requires a high quality of target protein and good solubility of fragments. Biophysical techniques for fragment screening necessitate expensive detection equipment and the strategies for evolving fragment hits to leads remain to be improved. Regardless, FBDD is necessary for investigating larger chemical space and can be applied to challenging biological targets. In this scenario, cheminformatics and computational chemistry can be used as alternative approaches that can significantly improve the efficiency and success rate of lead discovery and optimization. Cheminformatics and computational tools assist FBDD in a very flexible manner. Computational FBDD can be used independently or in parallel with experimental FBDD for efficiently generating and optimizing leads. Computational FBDD can also be integrated into each step of experimental FBDD and help to play a synergistic role by maximizing its performance. This review will provide critical analysis of the complementarity between computational and experimental FBDD and highlight recent advances in new algorithms and successful examples of their applications. In particular, fragment-based cheminformatics tools, high-throughput fragment docking, and fragment-based de novo drug design will provide the focus of this review. We will also discuss the advantages and limitations of different methods and the trends in new developments that should inspire future research.
Assuntos
Biologia Computacional/métodos , Desenho de Fármacos , Inibidores de 14-alfa Desmetilase/síntese química , Domínio Catalítico , Ciclofilina A/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Ligantes , Simulação de Acoplamento Molecular , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Receptores de Droga/química , Antagonistas do Receptor 5-HT1 de Serotonina/síntese química , Software , Relação Estrutura-AtividadeRESUMO
Cyclophilin A has attracted attention recently as a new target of anti-human immunodeficiency virus type 1 (HIV-1) drugs. However, so far no drug against HIV-1 infection exhibiting this mechanism of action has been approved. To identify new potent candidates for inhibitors, we performed in silico screening of a commercial database of more than 1,300 drug-like compounds by using receptor-based docking studies. The candidates selected from docking studies were subsequently tested using biological assays to assess anti-HIV activities. As a result, two compounds were identified as the most active. Specifically, both exhibited anti-HIV activity against viral replication at a low concentration and relatively low cytotoxicity at the effective concentration inhibiting viral growth by 50%. Further modification of these molecules may lead to the elucidation of potent inhibitors of HIV-1.
Assuntos
Fármacos Anti-HIV/farmacologia , Simulação por Computador , Ciclofilina A/metabolismo , Descoberta de Drogas , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/química , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , HIV-1/fisiologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Peso Molecular , Termodinâmica , Replicação Viral/efeitos dos fármacosRESUMO
Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.
Assuntos
Ciclofilina A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Vitamina K 3/farmacologia , Antioxidantes/farmacologia , Ciclofilina A/fisiologia , Estresse Oxidativo , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Vitamina K 3/químicaRESUMO
OBJECTIVE: To explore the action mechanism of Jiedu Quyu Zishen Recipe (JQZR) on the signal transduction of glucocorticoid receptor alpha (GRalpha) in the renal tissue of MRL/lpr mice. METHODS: Thirty MRL/lpr mice were randomly divided into three groups, i.e., the model group, the Western medicine group, and the Chinese medicine group, 10 in each. Besides, another 10 Kunming mice was taken as the normal control group. The pathological changes of the renal tissue were observed using HE staining. The expression of GRalpha was analyzed using Real-time PCR and Western blot. The effects of JQZR on the binding power of GRalpha to cyclophilin A were detected using co-immunoprecipitation. RESULTS: The renal injury degree of MRL/lpr mice in the Western medicine group and the Chinese medicine group was alleviated. Compared with the model group, the relative quantitation of GRalpha mRNA and protein expressions in the renal tissue of mice in the Western medicine group decreased, while they increased in the Chinese medicine group, showing statistical difference (P < 0.01). JQZR could significantly elevate the binding potency of GRalpha to cyclophilin A. CONCLUSION: Up-regulating the expression of GRalpha and enhancing mutual actions of GRalpha and cyclophilin A was one of JQZR's effects on improving the lesion of the renal tissue.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Rim/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Ciclofilina A/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos , Transdução de SinaisRESUMO
<p><b>OBJECTIVE</b>To explore the action mechanism of Jiedu Quyu Zishen Recipe (JQZR) on the signal transduction of glucocorticoid receptor alpha (GRalpha) in the renal tissue of MRL/lpr mice.</p><p><b>METHODS</b>Thirty MRL/lpr mice were randomly divided into three groups, i.e., the model group, the Western medicine group, and the Chinese medicine group, 10 in each. Besides, another 10 Kunming mice was taken as the normal control group. The pathological changes of the renal tissue were observed using HE staining. The expression of GRalpha was analyzed using Real-time PCR and Western blot. The effects of JQZR on the binding power of GRalpha to cyclophilin A were detected using co-immunoprecipitation.</p><p><b>RESULTS</b>The renal injury degree of MRL/lpr mice in the Western medicine group and the Chinese medicine group was alleviated. Compared with the model group, the relative quantitation of GRalpha mRNA and protein expressions in the renal tissue of mice in the Western medicine group decreased, while they increased in the Chinese medicine group, showing statistical difference (P < 0.01). JQZR could significantly elevate the binding potency of GRalpha to cyclophilin A.</p><p><b>CONCLUSION</b>Up-regulating the expression of GRalpha and enhancing mutual actions of GRalpha and cyclophilin A was one of JQZR's effects on improving the lesion of the renal tissue.</p>
Assuntos
Animais , Camundongos , Ciclofilina A , Metabolismo , Medicamentos de Ervas Chinesas , Farmacologia , Rim , Metabolismo , Patologia , Nefrite Lúpica , Metabolismo , Patologia , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos , Receptores de Glucocorticoides , Metabolismo , Transdução de SinaisRESUMO
Uncaria rhynchophylla (Miq) Jack (UR) is a traditional Chinese herb and is used for the treatment of convulsive disorders, including epilepsy. Our previous study has shown that UR, as well as its major component rhynchophylline (RH), has an anticonvulsive effect and this effect is closely related to its scavenging activities of oxygen free radicals. The purpose of the present study was to investigate the effect of (UR) on the expression of proteins using a proteomics analysis in Sprague-Dawley (SD) rats with kainic acid (KA)-induced epileptic seizures. We profiled the differentially expressed proteins on two-dimensional electrophoresis (2-DE) maps derived from the frontal cortex and hippocampus of rat brain tissue 24 hours after KA-induced epileptic seizures. The results indicated that macrophage migration inhibitory factor (MIF) and cyclophilin A were under expressed in frontal cortex by an average of 0.19- and 0.23-fold, respectively. In the frontal cortex, MIF and cyclophilin A were significantly decreased in the KA group and these decreases were confirmed by the Western blots. However, in the hippocampus, only cyclophilin A was significantly decreased in the KA group. In addition, in real-time quantitative PCR (Q-PCR), MIF and cyclophilin A gene expressions were also significantly under expressed in the frontal cortex, and only the cyclophilin A gene was also significantly under expressed in the hippocampus in the KA group. These under expressions of MIF and cyclophilin A could be overcome by the treatment of UR and RH. In conclusion, the under expressions of MIF and cyclophilin A in the frontal cortex and hippocampus in KA-treated rats, which were overcome by both UR and UH treatment, suggesting that both MIF and cyclophilin A at least partly participate in the anticonvulsive effect of UR.
Assuntos
Encéfalo/efeitos dos fármacos , Ciclofilina A/metabolismo , Epilepsia/metabolismo , Expressão Gênica/efeitos dos fármacos , Fatores Inibidores da Migração de Macrófagos/metabolismo , Extratos Vegetais/farmacologia , Uncaria , Animais , Western Blotting , Encéfalo/metabolismo , Ciclofilina A/genética , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Ácido Caínico , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Fitoterapia , Extratos Vegetais/uso terapêutico , Proteômica , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/tratamento farmacológico , Convulsões/etiologia , Convulsões/metabolismo , Regulação para CimaRESUMO
The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin-proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross-sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS-driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization.
Assuntos
Leucina/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/patologia , Atrofia Muscular/prevenção & controle , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Administração Oral , Animais , Ciclofilina A/genética , Suplementos Nutricionais , Regulação Enzimológica da Expressão Gênica , Elevação dos Membros Posteriores , Histocitoquímica , Insulina/sangue , Leucina/administração & dosagem , Leucina/farmacologia , Masculino , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/patologia , Atrofia Muscular/sangue , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genéticaRESUMO
Copper dyshomeostasis is responsible for the neurological symptoms observed in the genetically inherited copper-dependent disorders (e.g., Menkes' and Wilson's diseases), but it has been also shown to have an important role in neurodegenerative diseases such as Alzheimer disease, prion diseases, Parkinson's disease and amyotrophic lateral sclerosis. It is widely accepted that increased extracellular copper levels contribute to neuronal pathogenic process by increasing the production of dangerous radical oxygen species, but the existence of other molecular mechanisms explaining copper neurotoxicity has not been investigated yet. By using a cellular model based on hypothalamic GN11 cultured neurons exposed to copper supplementation and by analysing the cell conditioned media, we try here to identify new molecular events explaining the association between extracellular copper accumulation and neuronal damages. We show here that increased extracellular copper levels produce a wide complex of alterations in the neuronal extracellular environment. In particular, copper affects the secretion of molecules involved in the protection of neurons against oxidative stress, such as cyclophilin A (CypA), or of molecules capable of shifting neuronal cells towards a pro-inflammatory state, such as IL-1alpha, IL-12, Rantes, neutrophil gelatinase-associated lipocalin (NGAL) and secreted protein acidic and rich in cysteine (SPARC). Copper pro-inflammatory properties have been confirmed by using primary neurons.
Assuntos
Cobre/metabolismo , Cobre/farmacologia , Citocinas/metabolismo , Neurônios/efeitos dos fármacos , Oligoelementos/metabolismo , Oligoelementos/farmacologia , Proteínas de Fase Aguda/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/química , Ciclofilina A/metabolismo , Relação Dose-Resposta a Droga , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Hipotálamo/citologia , Lipocalina-2 , Lipocalinas/metabolismo , Camundongos , Osteonectina/metabolismo , Mapeamento de Peptídeos , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrofotometria AtômicaRESUMO
Acupuncture is frequently used as an alternative therapy for Parkinson's disease (PD), and it attenuates dopaminergic (DA) neurodegeneration in the substantia nigra (SN) in PD animal models. Using proteomic analysis, we investigated whether acupuncture alters protein expression in the SN to favor attenuation of neuronal degeneration. In C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day), intraperitoneal (i.p.) for 5 days, 2 or 100 Hz electroacupuncture (EA) was applied at the effective and specific acupoint, GB34, once a day for 12 consecutive days from the first MPTP treatment. Both treatments in MPTP mice led to restoration of behavioral impairment and rescued tyrosine hydroxylase (TH)-positive DA neurodegeneration. Using peptide fingerprinting MS, we identified changes in 22 proteins in the SN following MPTP treatment, and nine of these proteins were normalized by EA. They were involved in cell death regulation, inflammation, or restoration from damage. The levels of cyclophilin A (CypA), which is a neuroprotective agent, were unchanged by MPTP treatment but were increased in MPTP-EA mice. These results suggest that acupoint GB34-specific EA changes protein expression profiles in the SN in favor of DA neuronal survival in MPTP-treated mice, and that EA treatment may be an effective therapy for PD patients.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Eletroacupuntura , Doença de Parkinson/terapia , Proteoma/metabolismo , Pontos de Acupuntura , Análise de Variância , Animais , Fator Neurotrófico Derivado do Encéfalo , Ciclofilina A/metabolismo , Dopaminérgicos/farmacologia , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Camundongos , Modelos Animais , Neurônios , Mapeamento de Peptídeos , Proteoma/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Inactivating PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome) mutations cause X-linked hypophosphatemia in humans and mice (Hyp) through overproduction of fibroblast growth factor 23 (FGF23) a phosphaturic factor, by osteocytes. Matrix extracellular phosphoglycoprotein (MEPE) is also elevated in Hyp and other hypophosphatemic disorders. In addition, the administration of an ASARM (acidic serine-aspartate rich MEPE-associated motif) peptide derived from MEPE causes phosphaturia and inhibits bone mineralization in mice, suggesting that MEPE also plays a role in phosphate homeostasis. Since recent studies found that MEPE binds specifically to PHEX in vitro, we tested the effect of recombinant-MEPE and its ASARM peptide on PHEX enzyme activity in vitro and FGF23 expression in bone marrow stromal cell cultures ex vivo. We found that both recombinant MEPE and synthetic phosphorylated ASARM peptide (ASARM-PO(4)) inhibit PHEX enzyme activities in an in vitro fluorescent-quenched PHEX enzyme activity assay. The ASARM-PO(4) peptide inhibits PHEX enzyme activity in a dose-dependent manner with a K(i) of 128 nM and V(max-i) of 100%. Recombinant MEPE also inhibits PHEX activity (K(i) = 2 nM and V(max-i) = 26%). Long-term bone marrow stromal cell cultures supplemented with 10 microM ASARM-PO(4) peptide resulted in significant elevation of FGF23 transcripts and inhibition of mineralization. These findings suggest that MEPE inhibits mineralization and PHEX activity and leads to increased FGF23 production. The resulting coordination of mineralization and release of a phosphaturic factor by MEPE may serve a physiological role in regulating systemic phosphate homeostasis to meet the needs for bone mineralization.
Assuntos
Células da Medula Óssea/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Raquitismo Hipofosfatêmico Familiar/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X , Glicoproteínas/farmacologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/farmacologia , Animais , Células da Medula Óssea/patologia , Células Cultivadas , Ciclofilina A/genética , Relação Dose-Resposta a Droga , Raquitismo Hipofosfatêmico Familiar/patologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/análise , Fatores de Crescimento de Fibroblastos/genética , Homeostase , Camundongos , Camundongos Knockout , Endopeptidase Neutra Reguladora de Fosfato PHEX/antagonistas & inibidores , Fosfatos/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. Disruption of normal imprinting leads to abnormal embryogenesis, certain inherited diseases, and is associated with various cancers. In the context of screening for the gene(s) responsible for the alteration of phenotype in cyclophilin A knockdown (CypA-KD) P19 cells, we observed a silent paternally expressed gene, Peg3. Treatment of CypA-KD P19 cells with the DNA demethylating agent 5-aza-dC reversed the silencing of Peg3 biallelically. Genomic bisulfite sequencing and methylation-specific PCR revealed DNA hypermethylation in CypA-KD P19 cells, as the normally unmethylated paternal allele acquired methylation that resulted in biallelic methylation of Peg3. Chromatin immunoprecipitation assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 and MBD2 binding on the cytosine-guanine dinucleotide (CpG) islands of Peg3. Our results indicate that DNA hypermethylation on the paternal allele and allele-specific acquisition of histone methylation leads to silencing of Peg3 in CypA-KD P19 cells. This study is the first demonstration of the epigenetic function of CypA in protecting the paternal allele of Peg3 from DNA methylation and inactive histone modifications.
Assuntos
Ciclofilina A/química , Metilação de DNA , Epigênese Genética , Histonas/química , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Alelos , Animais , Imunoprecipitação da Cromatina , Ilhas de CpG , DNA Complementar/metabolismo , Inativação Gênica , Impressão Genômica , Humanos , Fatores de Transcrição Kruppel-Like , Lisina/química , CamundongosRESUMO
Allergic symptoms in sensitized individuals are caused by proteins named allergens. We report here the cloning and the production of the cyclophilin Bet v 7, one of the birch pollen allergens. Recombinant Bet v 7 was produced in bacteria and used to raise a rabbit anti-Bet v 7 antiserum. With this antiserum we detected cyclophilin A in several pollen species and we demonstrated immunological cross-reactivity among those plant cyclophilins A by immunoblot and ELISA inhibition experiments. However, we could not detect cyclophilins in extracts of animal or mould origin with our anti-Bet v 7 antiserum. By inhibition experiments with purified mould cyclophilins, we confirmed the absence of cross-reactivity between plant cyclophilins and non-plant cyclophilins. In addition, our results indicate that the level of immunological cross-reactivity correlates with the level of sequence identity among the cyclophilin A family. This allowed us to define the plant cyclophilin A sub-family as being immunologically distinct, which might have implications at the clinical level in the allergy practice.
Assuntos
Alérgenos/genética , Betula/genética , Ciclofilina A/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Alérgenos/imunologia , Animais , Anticorpos/química , Antígenos de Plantas , Betula/química , Betula/imunologia , Ciclofilina A/química , Ciclofilina A/imunologia , Fungos/química , Fungos/genética , Fungos/imunologia , Humanos , Hipersensibilidade/imunologia , Folhas de Planta/química , Folhas de Planta/imunologia , Proteínas de Plantas/imunologia , Pólen/genética , Pólen/imunologia , Coelhos , Especificidade da EspécieRESUMO
Cyclophilin A (CypA) is a member of cyclophilins, a family of the highly homologous peptidyl prolyl cis-trans isomerases (PPIases), which can bind to cyclosporin A (CsA). CypA plays critical roles in various biological processes, including protein folding, assembly, transportation, regulation of neuron growth, and HIV replication. The discovery of CypA inhibitor is now of a great special interest in the treatment of immunological disorders. In this study, a series of novel small molecular CypA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. The SPECS_1 database containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA. After SPR-based binding affinity assay, 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-molar level (compounds 1-15). Seven compounds were selected as the starting point for the further structure modification in considering binding activity, synthesis difficulty, and structure similarity. We thus synthesized 40 new small molecular compounds (1-6, 15, 16a-q, 17a-d, and 18a-l), and four of which (compounds 16b, 16h, 16k, and 18g) showed high CypA PPIase inhibition activities with IC50s of 2.5-6.2 microM. Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities against the proliferation of spleen cells.
Assuntos
Ciclofilina A/antagonistas & inibidores , Inibidores Enzimáticos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Ciclofilina A/química , Ciclofilina A/metabolismo , Bases de Dados como Assunto , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Modelos Moleculares , Estrutura Molecular , Valor Preditivo dos Testes , Conformação Proteica , Baço/citologia , Baço/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacosRESUMO
The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.
Assuntos
Proteínas de Transporte/fisiologia , Neuroglia/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Imunoprecipitação da Cromatina , Cromatografia em Gel , Clonagem Molecular , Ciclofilina A/química , Primers do DNA/química , Enzimas Reparadoras do DNA , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Dados de Sequência Molecular , Neurônios/metabolismo , Proteínas Nucleares , Oligonucleotídeos/química , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Fatores de Processamento de RNA , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Spliceossomos/metabolismo , Fatores de Tempo , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.