RESUMO
BACKGROUND AND HYPOTHESIS: Using a mouse model of MPA with microvascular lesion with a clone (VasSF) of recombinant single chain fragments of the variable region of human IgG, we previously showed that vasculitis-associated apolipoprotein A2 (VAP2) may be a therapeutic target for vasculitis. The present study estimated the target molecules for VasSF and the association between VAP2 and cytokine levels in patient sera in terms of microvascular lesion severity. METHODS: Sera and clinical information were collected from patients with microscopic polyangiitis and granulomatosis with polyangiitis (MPA/GPA) and infectious disease. Neutrophil counts, levels of C-reactive protein (CRP), creatinine, total cholesterol associated with microvascular lesion, HDL cholesterol, low-density lipoprotein cholesterol, triglycerides, glomerular filtration rate (eGFR), and cytokines were estimated. Serum VAP2 signals were determined with Western blotting. RESULTS: VasSF bound to a 24â¯kDa molecule in the serum of active MPA/GPA patients. Anti-AP2 antibody also bound with the same 24â¯kDa molecule, named VAP2, because of size difference from normal APOA2. The VAP2 signal was significantly stronger in the active-disease group but significantly weakened in remission. The signal correlated positively with eGFR but not with the Birmingham Vasculitis Activity Score, CRP, MPO-ANCA, or PR3-ANCA levels. It correlated negatively with MPO activity, IL-16, MIF, and IL-1Ra. Moreover, VasSF bound to a 17â¯kDa molecule in the remission phase. CONCLUSION: The 24â¯kDa VAP2 molecule may be associated with neutrophil functions because of its inverse correlation with MPO activity, IL-16, MIF, and IL-1Ra, suggesting that VAP2-APOA1 formation in HDL triggers microvascular injury. VasSF may reverse the injury by removing APOA1-VAP2 heterodimers from peripheral blood vessels.
RESUMO
BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated vasculitis is a pauci-immune disease with the inflammation of the small blood vessels. The efficacies of antibody drugs for induction therapies of vasculitis vary among cases. Here, we developed a novel clone of a single chain Fv region (ScFv) with vasculitis-specific therapeutic potential. MATERIALS AND METHODS: The clone, termed VasSF, was selected from our Escherichia coli expression library of recombinant human ScFv based on the therapeutic efficacy in an SCG/Kj mouse model of MPO-ANCA-associated vasculitis (MAAV), such as improvement of the urinary score and decreased crescent formation in glomeruli, granulomatous in lung, MPO-ANCA biomarkers, the anti-moesin antibody, and some cytokine levels. RESULTS: We identified vasculitis-associated apolipoprotein A-II (VAP2) as a target molecule of the clone and confirmed the independently-established VAP2 antibodies were also therapeutic in SCG/Kj mice. In MAAV, MPO-ANCA and cytokines stimulate neutrophils by facilitating heterodimer formation of VAP2 with apolipoprotein A-I in HDL. CONCLUSION: VasSF would constitute a novel antibody drug for vasculitis by suppressing the heterodimer formation of the apolipoproteins.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Anticorpos de Cadeia Única/uso terapêutico , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).
Assuntos
Neuroblastoma/metabolismo , Doenças Priônicas/metabolismo , Príons/química , Príons/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Proteínas Priônicas , Príons/genética , Alinhamento de SequênciaRESUMO
Transmissible spongiform encephalopathies are infectious and neurodegenerative disorders that cause neural deposition of aggregates of the disease-associated form of PrP(Sc). PrP(Sc) reproduces by recruiting and converting the cellular PrP(C), and ScN2a cells support PrP(Sc) propagation. We found that incubation of ScN2a cells with a fibril peptide named P9, which comprises an intrinsic sequence of residues 167-184 of mouse PrP(C), significantly reduced the amount of PrP(Sc) in 24 hr. P9 did not affect the rates of synthesis and degradation of PrP(C). Interestingly, immunofluorescence analysis showed that the incubation of ScN2a cells with P9 induced colocalization of the accumulation of PrP with cathepsin D-positive compartments, whereas the accumulation of PrP in the cells without P9 colocalized mainly with lysosomal associated membrane proteins (LAMP)-1-positive compartments but rarely with cathepsin D-positive compartments in perinuclear regions. Lysosomal enzyme inhibitors attenuated the anti-PrP(Sc) activity; however, a proteasome inhibitor did not impair P9 activity. In addition, P9 neither promoted the ubiquitination of cellular proteins nor caused the accumulation of LC3-II, a biochemical marker of autophagy. These results indicate that P9 promotes PrP(Sc) redistribution from late endosomes to lysosomes, thereby attaining PrP(Sc) degradation.
Assuntos
Lisossomos/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Proteína PrP 27-30/síntese química , Proteína PrP 27-30/imunologia , Proteínas PrPSc/metabolismo , Sequência de Aminoácidos , Animais , Catepsina D/análise , Endossomos/química , Proteínas de Membrana Lisossomal/análise , Lisossomos/química , Camundongos , Microscopia Confocal , Alinhamento de SequênciaRESUMO
A new screening method was developed to detect bovine spongiform encephalopathy (BSE). This method is advantageous because it has a simpler and safer protocol than commercial kits. A new device was developed for this method; it was named the BioMasher, to homogenize brain tissue by passing it through a porous rigid polypropylene filter. In this system, a purification step was eliminated in the sample preparation. Thus, the time needed for sample pretreatment is substantially shortened, and the risk of infection during sample processing is effectively reduced. Monoclonal antibodies to prion protein were created and used to construct a sensitive sandwich enzyme-linked immunosorbent assay system. The sensitivity of this assay kit using frozen BSE-positive brain is comparable or more sensitive than commercial kits. Moreover, the detection sensitivity for deteriorated samples, which were kept at 37 degrees C for 1 day, is 10- to 30-fold more sensitive than a commercial kit.
Assuntos
Química Encefálica , Encefalopatia Espongiforme Bovina/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Príons/análise , Animais , Anticorpos Monoclonais/isolamento & purificação , Bovinos , Filtração/métodos , Camundongos , Príons/imunologia , Príons/isolamento & purificação , Sensibilidade e Especificidade , Fatores de TempoRESUMO
In prion diseases, the normal cellular form of prion protein (PrP(C)) is converted into the disease-associated isoforms (PrP(Sc)) which accumulate in the infected tissues. Although the precise mechanism of this conversion remains unsolved, drugs of various categories have been reported to reduce the accumulation of PrP(Sc) in prion-infected cultured cells. We here show that AY-9944 (a 7-dehydrocholesterol reductase inhibitor) and U18666A (a 24-dehydrocholesterol reductase inhibitor) prevent PrP(Sc) from accumulating in prion-infected mouse neuroblastoma cells (ScN2a), with an ED50 of about 0.5 microM and 10 nM, respectively. In order to evaluate the efficacy of these two inhibitors in vivo, C57BL/6J mice inoculated with mouse-adapted scrapie-prion received repetitive intraperitoneal injections of U18666A (10 mg/kg) or a mixture of U18666A (10 mg/kg) and AY-9944 (12 mg/kg). By contrast to the potent anti-prion effects observed in ScN2a cells, the in vivo trial was abortive with neither drug halting the progression of the disease.
Assuntos
Androstenos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Príons/efeitos dos fármacos , Scrapie/tratamento farmacológico , Scrapie/mortalidade , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/farmacologia , Androstenos/administração & dosagem , Androstenos/química , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Neuroblastoma/virologia , Taxa de Sobrevida , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/administração & dosagem , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/químicaRESUMO
Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.
Assuntos
Hepacivirus/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Proteínas do Core Viral/metabolismo , Linhagem Celular , HumanosRESUMO
Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.
Assuntos
Hepacivirus/fisiologia , Antígenos da Hepatite C/genética , Antígenos da Hepatite C/isolamento & purificação , Metabolismo dos Lipídeos , Proteínas do Core Viral/genética , Proteínas do Core Viral/isolamento & purificação , Linhagem Celular , RNA Helicases DEAD-box , Hepacivirus/patogenicidade , Humanos , Fígado/virologia , Proteômica/métodos , RNA Helicases/metabolismo , Replicação ViralRESUMO
Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. It is an inner nuclear membrane protein and phosphorylated in a cell cycle-dependent manner. However, the means of phosphorylation of emerin are poorly understood. We investigated the regulation mechanism for the binding of emerin to chromatin, focusing on its cell cycle-dependent phosphorylation in a Xenopus egg cell-free system. It was shown that emerin dissociates from chromatin depending on mitotic phosphorylation of the former, and this plays a critical role in the dissociation of emerin from barrier-to-autointegration factor (BAF). Then, we analyzed the mitotic phosphorylation sites of emerin. Emerin was strongly phosphorylated in an M-phase Xenopus egg cell-free system, and five phosphorylated sites, Ser49, Ser66, Thr67, Ser120, and Ser175, were identified on analysis of chymotryptic and tryptic emerin peptides using a phosphopeptide-concentrating system coupled with a Titansphere column, which specifically binds phosphopeptides, and tandem mass spectrometry sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at Ser175 regulates the dissociation of emerin from BAF.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Timopoietinas/metabolismo , Animais , Ciclo Celular , Sistema Livre de Células , Cromatina/química , Cromatina/metabolismo , Quimotripsina/química , Citosol/metabolismo , Glutationa Transferase/metabolismo , Humanos , Espectrometria de Massas , Mitose , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutação , Peptídeos/química , Fosfopeptídeos/química , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Serina/química , Espectrometria de Massas por Ionização por Electrospray , Tripsina/química , XenopusRESUMO
Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication. In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated. When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrPSc) replication without reducing the viability of the cells. It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC50) = 0.20 microM, 99% effective concentration (EC99) = 0.86 microM vs. EC50 = 0.45 microM, EC99 = 1.5 microM. Besides, no apparent cell damage was observed at the concentration of up to 4.3 microM (100 micrograms/ml). In combination treatment with 0.43 microM (10 micrograms/ml) of rPrP-Q218K, EC99 of quinacrine was decreased from 1.5 microM to 0.5 microM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine. Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy. Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist.
Assuntos
Proteínas PrPSc/metabolismo , Proteínas Recombinantes/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Códon/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Genes Dominantes , Glicosilfosfatidilinositóis/genética , Camundongos , Mutagênese Sítio-Dirigida , Proteínas PrPSc/genética , Conformação Proteica , Estrutura Quaternária de Proteína/genética , Proteínas Recombinantes/genéticaRESUMO
The effects of Soto's 'beta-sheet breaker peptide' and its polymer on PrPSc formation in ScN2a cells were investigated. Surface plasmon resonance study indicated that direct binding between PrP(81-145) and the 'beta-sheet breaker peptide' is not specific and may not play a major role in the inhibition of PrPSc formation.
Assuntos
Biopolímeros/metabolismo , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Proteínas PrPSc/patogenicidade , Príons/metabolismo , Sequência de Aminoácidos , Animais , Biopolímeros/química , Camundongos , Dados de Sequência Molecular , Neuroblastoma/patologia , Fragmentos de Peptídeos/química , Peptídeos/química , Príons/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated by p300 and PCAF in vivo and in vitro. In this study we identified, by mass spectrometry, two lysine residues in the DNA binding domain (DBD), Lys-75 and Lys-78, to be the major acetylation sites in IRF-2. Although acetylation of IRF-2 did not alter DNA binding activity in vitro, mutation of Lys-75 diminished the IRF-2-dependent activation of histone H4 promoter activity. Acetylation of IRF-2 and IRF-2-stimulated H4 promoter activity were inhibited by the adenovirus E1A, indicating the involvement of p300/CBP. Mutation of Lys-78, a residue conserved throughout the IRF family members, led to the abrogation of DNA binding activity independently of acetylation. H4 is transcribed only in rapidly growing cells and its promoter activity is dependent on cell growth. Consistent with a role for acetylated IRF-2 in cell growth control, IRF-2 was acetylated only in growing NIH 3T3 cells, but not in growth-arrested counterparts. Chromatin immunoprecipitation assays showed that IRF-2 interacted with p300 and bound to the endogenous H4 promoter only in growing cells, although the levels of total IRF-2 were comparable in both growing and growth-arrested cells. These results indicate that IRF-2 is acetylated in a cell growth-dependent manner, which enables it to contribute to transcription of cell growth-regulated promoters.