RESUMEN
Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.
Asunto(s)
Encéfalo/metabolismo , Etanolamina/farmacología , Proteínas PrPSc , Enfermedades por Prión , Animales , Línea Celular Tumoral , Ratones , Ratones Endogámicos ICR , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismoRESUMEN
Misfolding of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, which forms infectious protein aggregates, the so-called prions, is a key pathogenic event in prion diseases. No pathogens other than prions have been identified to induce misfolding of PrPC into PrPSc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrPC into PrPSc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrPSc were different from those of RML and 22L laboratory prions-evoked PrPSc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrPSc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrPC into PrPSc and formation of infectious prions.
Asunto(s)
Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/virología , Neuroblastoma/metabolismo , Neuroblastoma/virología , Proteínas Priónicas/química , Línea Celular Tumoral , Humanos , Gripe Humana/genética , Neuroblastoma/genética , Proteínas Priónicas/metabolismo , Conformación Proteica , Pliegue de ProteínaRESUMEN
Prions are infectious agents causing prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone.
Asunto(s)
Peróxido de Hidrógeno/química , Ozono/química , Priones , Acero Inoxidable , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Ratones , Ozono/farmacología , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/química , Enfermedades por Prión/etiología , Enfermedades por Prión/prevención & control , Acero Inoxidable/químicaRESUMEN
Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform, PrPSc, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrPC, designated Tg(PrP∆91-106)/Prnp0/0 mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/Prnp0/0 mice were resistant to RML, 22L and FK-1 prions, neither producing PrPSc∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrPSc∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrPSc∆91-104 after incubation with BSE-PrPSc-prions but not with RML- and 22L-PrPSc-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrPSc∆91-104 even after incubation with RML- and 22L-PrPSc-prions. These results suggest that residues 91-106 or 91-104 of PrPC are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrPC into PrPSc.
Asunto(s)
Encefalopatía Espongiforme Bovina/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Deficiencias en la Proteostasis/genética , Scrapie/genética , Eliminación de Secuencia , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Bovinos , Clonación Molecular , Susceptibilidad a Enfermedades , Encefalopatía Espongiforme Bovina/metabolismo , Encefalopatía Espongiforme Bovina/patología , Expresión Génica , Inyecciones Intraventriculares , Ratones , Ratones Transgénicos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/administración & dosificación , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Scrapie/metabolismo , Scrapie/patología , Especificidad de la EspecieRESUMEN
The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.
Asunto(s)
Virus de la Influenza A/metabolismo , Pulmón , Macrófagos , Infecciones por Orthomyxoviridae , Proteínas PrPC/metabolismo , Transducción de Señal , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/virología , Ratones , Ratones Mutantes , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/patología , Proteínas PrPC/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Conformational conversion of the cellular isoform of prion protein, designated PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an essential pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Lines of evidence indicate that the N-terminal domain, which includes the N-terminal, positively charged polybasic region and the octapeptide repeat (OR) region, is important for PrPC to convert into PrPSc after infection with prions. To further gain insights into the role of the polybasic region and the OR region in prion pathogenesis, we generated two different transgenic mice, designated Tg(PrP3K3A)/Prnp0/0 and Tg(PrP3K3A∆OR)/Prnp0/0 mice, which express PrPC with lysine residues at codons 23, 24, and 27 in the polybasic region mutated with or without a deletion of the OR region on the Prnp0/0 background, respectively, and intracerebrally inoculated them with RML and 22L prions. We show that Tg(PrP3K3A)/Prnp0/0 mice were highly resistant to the prions, indicating that lysine residues at 23, 24, and 27 could be important for the polybasic region to support prion infection. Tg(PrP3K3A∆OR)/Prnp0/0 mice also had reduced susceptibility to RML and 22L prions equivalent to Tg(PrP3K3A)/Prnp0/0 mice. The pre-OR region, including the polybasic region, of PrP3K3A∆OR, but not PrP3K3A, was unusually converted to a protease-resistant structure during conversion to PrPSc3K3A∆OR. These results suggest that, while the OR region could affect the conformation of the polybasic region during conversion of PrPC into PrPSc, the polybasic region could play a crucial role in prion pathogenesis independently of the OR region.
Asunto(s)
Proteínas PrPC/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Priónicas/metabolismo , Priones/metabolismo , Animales , Lisina/metabolismo , Ratones Transgénicos , Eliminación de Secuencia/fisiologíaRESUMEN
Biopharmaceuticals represented by immunoglobulin G (IgG) are produced by the cultivation of recombinant animal cells, especially Chinese hamster ovary (CHO) cells. It is thought that the intracellular secretion process of IgG is a bottleneck in the production of biopharmaceuticals. Many studies on the regulation of endogenous secretory protein expression levels have shown improved productivity. However, these strategies have not universally improved the productivity of various proteins. A more rational and efficient establishment of high producer cells is required based on an understanding of the secretory processes in IgG producing CHO cells. In this study, a CHO cell line producing humanized IgG1, which was genetically fused with fluorescent proteins, was established to directly analyze intracellular secretion. The relationship between the amount of intracellular and secreted IgG was analyzed at the single cell level by an automated single-cell analysis and isolation system equipped with dual color fluorescent filters. The amounts of intracellular and secreted IgG showed a weak positive correlation. The amount of secreted IgG analyzed by the system showed a weak negative linear correlation with the specific growth of isolated clones. An immunofluorescent microscopy study showed that the established clones could be used to analyze the intracellular secretion bottleneck. This is the first study to report the use of fluorescent protein fusion IgG as a tool to analyze the secretion of recombinant CHO cells.
RESUMEN
The Chinese hamster ovary (CHO) cell line is the most widely used host cell for therapeutic antibody production. Although its productivity has been improved by various strategies to satisfy the growing global demand, some difficult-to-express (DTE) antibodies remain at low secretion levels. To improve the production of various therapeutic antibodies, it is necessary to determine possible rate-limiting steps in DTE antibody secretion in comparison with other high IgG producers. Here, we analyzed the protein secretion process in CHO cells producing the DTE immunoglobulin G (IgG) infliximab. The results from chase assays using a translation inhibitor revealed that infliximab secretion could be nearly completed within 2 h, at which time the cells still retained about 40% of heavy chains and 65% of light chains. Using fluorescent microscopy, we observed that these IgG chains remained in the endoplasmic reticulum and Golgi apparatus. The cells inefficiently form fully assembled heterodimer IgG by making LC aggregates, which may be the most serious bottleneck in the production of DTE infliximab compared with other IgG high producers. Our study could contribute to establish the common strategy for constructing DTE high-producer cells on the basis of rate-limiting step analysis.
RESUMEN
The production of biopharmaceutical immunoglobulin G (IgG) using cultured mammalian cells, especially Chinese hamster ovary (CHO) cells is well established and has been markedly improved through the modification of cells and cell culture engineering technologies. The establishment of high-production cell lines remains a challenge. The intracellular secretion of IgG has been investigated to identify and solve the rate-limiting steps in antibody production. However, strategies that regulate the expression of proteins that are related to antibody secretory pathway have not consistently improved their production. In this study, key features and limitations of the antibody secretion process in recombinant CHO cells were analyzed to develop more efficient approaches for establishing high-production cells. By chase assay with protein translation inhibitors, IgG secretion reached a plateau when at least 20% of IgG remained in the cells. The secretion kinetics and retention ratio of IgG varied between IgG subclasses (two types of IgG1 and an IgG3 subclass). Immunofluorescent microscopy and size exclusion chromatography showed that the remaining intracellular IgG localized mainly within the endoplasmic reticulum (ER) and less with the cis-Golgi network, despite the formation of fully assembled IgG. These results show that remaining intracellular IgG is a target for enhancing antibody secretion, even in high-production CHO cells.
Asunto(s)
Formación de Anticuerpos , Inmunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Formación de Anticuerpos/genética , Células CHO , Técnicas de Cultivo de Célula , Cricetinae , Cricetulus , Retículo Endoplásmico/metabolismo , Espacio Intracelular/metabolismo , Ingeniería Metabólica/métodos , Proteínas Recombinantes/genética , Vías Secretoras/genéticaRESUMEN
The cellular prion protein, designated PrPC, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrPC remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrPC is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrPC-null mice (Prnp0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp0/0 lungs were severely injured, with higher inflammation and higher apoptosis of epithelial cells, and contained higher reactive oxygen species (ROS) than control WT lungs. Treatment with a ROS scavenger or an inhibitor of xanthine oxidase (XO), a major ROS-generating enzyme in IAV-infected lungs, rescued Prnp0/0 mice from the lethal infection with IAV. Moreover, Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPΔOR)/Prnp0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrPC has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs. Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase, SOD1, were lower in Prnp0/0 and Tg(PrPΔOR)/Prnp0/0 lungs than in WT lungs. It is thus conceivable that PrPC functions to maintain Cu content and regulate SOD1 through the OR region in lungs, thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs. Our current results highlight the role of PrPC in protection against IAV infection, and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics.
Asunto(s)
Proteínas PrPC/metabolismo , Proteínas Priónicas/metabolismo , Animales , Encéfalo/patología , Cobre/metabolismo , Susceptibilidad a Enfermedades/metabolismo , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Proteínas PrPC/fisiología , Enfermedades por Prión/metabolismo , Proteínas Priónicas/farmacología , Priones/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Background: Proteolytic processing of the prion protein (PrP
Asunto(s)
Proteína ADAM10/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Proteínas Priónicas/metabolismo , Animales , RatonesRESUMEN
Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrPC into PrPSc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/Prnp0/0 mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrPScΔOR in their brains. We show here that Tg(PrPΔOR)/Prnp0/0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrPScΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrPC into PrPSc after infection with BSE prions. However, Tg(PrPΔOR)/Prnp0/0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrPScΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/Prnp0/0 mice than PrPSc in control wild-type mice. Taken together, these results indicate that the OR region of PrPC could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions.IMPORTANCE Structure-function relationship studies of PrPC conformational conversion into PrPSc are worthwhile to understand the mechanism of the conversion of PrPC into PrPSc We show here that, by inoculating Tg(PrPΔOR)/Prnp0/0 mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrPC into PrPSc after infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrPScΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrPScΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrPC into PrPSc after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions.
Asunto(s)
Susceptibilidad a Enfermedades , Encefalopatía Espongiforme Bovina/fisiopatología , Proteínas PrPC/química , Proteínas PrPC/fisiología , Enfermedades por Prión/fisiopatología , Priones/patogenicidad , Animales , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/prevención & control , Humanos , Ratones , Ratones Transgénicos , Oligopéptidos/química , Oligopéptidos/genética , Proteínas PrPC/genética , Enfermedades por Prión/prevención & control , Priones/química , Priones/genética , Eliminación de SecuenciaRESUMEN
Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform of prion protein, PrPSc, which leads to marked accumulation of PrPSc in brains, is a key pathogenic event in prion diseases, a group of fatal neurodegenerative disorders caused by prions. However, the exact mechanism of PrPSc accumulation in prion-infected neurons remains unknown. We recently reported a novel cellular mechanism to support PrPSc accumulation in prion-infected neurons, in which PrPSc itself promotes its accumulation by evading the cellular inhibitory mechanism, which is newly identified in our recent study. We showed that the VPS10P sorting receptor sortilin negatively regulates PrPSc accumulation in prion-infected neurons, by interacting with PrPC and PrPSc and trafficking them to lysosomes for degradation. However, PrPSc stimulated lysosomal degradation of sortilin, disrupting the sortilin-mediated degradation of PrPC and PrPSc and eventually evoking further accumulation of PrPSc in prion-infected neurons. These findings suggest a positive feedback amplification mechanism for PrPSc accumulation in prion-infected neurons.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Priones/metabolismo , Animales , Humanos , Lisosomas/metabolismo , Ratones , Proteínas PrPC/metabolismo , Transporte de ProteínasRESUMEN
Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Priones/metabolismo , Animales , Lisosomas/metabolismo , Ratones , Neuronas/metabolismo , Enfermedades por Prión/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
The N-terminal polybasic region of the normal prion protein, PrPC, which encompasses residues 23-31, is important for prion pathogenesis by affecting conversion of PrPC into the pathogenic isoform, PrPSc. We previously reported transgenic mice expressing PrP with residues 25-50 deleted in the PrP-null background, designated as Tg(PrP∆preOR)/Prnp 0/0 mice. Here, we produced two new lines of Tg(PrP∆preOR)/Prnp 0/0 mice, each expressing the mutant protein, PrP∆preOR, 1.1 and 1.6 times more than PrPC in wild-type mice, and subsequently intracerebrally inoculated RML and 22L prions into them. The lower expresser showed slightly reduced susceptibility to RML prions but not to 22L prions. The higher expresser exhibited enhanced susceptibility to both prions. No prion transmission barrier was created in Tg(PrP∆preOR)/Prnp 0/0 mice against full-length PrPSc. PrPSc∆preOR accumulated in the brains of infected Tg(PrP∆preOR)/Prnp 0/0 mice less than PrPSc in control wild-type mice, although lower in RML-infected Tg(PrP∆preOR)/Prnp 0/0 mice than in 22L-infected mice. Prion infectivity in infected Tg(PrP∆preOR)/Prnp 0/0 mice was also lower than that in wild-type mice. These results indicate that deletion of residues 25-50 only slightly affects prion susceptibility, the conversion of PrPC into PrPSc, and prion infectivity in a strain-specific way. PrP∆preOR retains residues 23-24 and lacks residues 25-31 in the polybasic region. It is thus conceivable that residues 23-24 rather than 25-31 are important for the polybasic region to support prion pathogenesis. However, other investigators have reported that residues 27-31 not 23-24 are important to support prion pathogenesis. Taken together, the polybasic region might support prion pathogenesis through multiple sites including residues 23-24 and 27-31.
Asunto(s)
Enfermedades por Prión , Proteínas Priónicas/metabolismo , Secuencia de Aminoácidos , Animales , Susceptibilidad a Enfermedades , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Priónicas/genética , Secuencias Repetitivas de Aminoácido , Eliminación de SecuenciaRESUMEN
Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.
Asunto(s)
Enfermedades por Prión/metabolismo , Priones/genética , Priones/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Animales , Encéfalo/metabolismo , Cricetinae , Susceptibilidad a Enfermedades , Masculino , Ratones , Ratones Noqueados , Enfermedades por Prión/genética , Proteínas Priónicas , Priones/química , Proteínas Recombinantes de Fusión/químicaRESUMEN
Soluble insulin receptor (sIR), the ectodomain of IR, has been detected in human plasma, and its concentration parallels that of blood glucose in patients with diabetes. IR has a pivotal role in glucose homeostasis and diabetes development; therefore, cleavage of IR promoted by hyperglycemia is involved in insulin resistance and glucose toxicity. To elucidate the physiology of sIR, we developed an in vitro model mimicking the changes in sIR levels in plasma from patients with diabetes. Among four human cell lines that expressed IR, spontaneous cleavage of IR occurred only in HepG2 cells. The molecular characteristics of sIR derived from HepG2 cells were similar to those of sIR detected in human plasma. The concentration of sIR in the medium did not differ between basal and high-glucose conditions in the initial 24-h period, but increasing the duration of pre-stimulation (>48 h) led to a significant increase in sIR levels in cells exposed to high glucose. Additionally, glucose-dependent increment of sIR was reversible in this model. These results are consistent with the observation of plasma sIR in patients with diabetes. Using this model, O-linked N-acetylglucosamine modification was determined to be involved in high-glucose-induced IR cleavage. A calcium-dependent protease was shown to cleave IR extracellularly. These findings show that this in vitro model could be useful for determining the molecular mechanism underlying IR cleavage.
Asunto(s)
Glucosa/farmacología , Proteolisis/efectos de los fármacos , Receptor de Insulina/metabolismo , Acetilglucosamina/metabolismo , Acilación/efectos de los fármacos , Glucemia/metabolismo , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Diabetes Mellitus/sangre , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Modelos Biológicos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Péptido Hidrolasas/metabolismo , Interferencia de ARN , Receptor de Insulina/sangre , Factores de TiempoRESUMEN
The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.
Asunto(s)
Endosomas/patología , Proteínas de la Membrana/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Animales , Membrana Celular/metabolismo , Membrana Celular/patología , Endocitosis , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/patología , Humanos , Proteínas de la Membrana/análisis , Ratones , Priones/análisis , Priones/metabolismo , Transporte de ProteínasRESUMEN
Conformational conversion of normal cellular prion protein PrP(C) into pathogenic PrP(Sc) is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP(C), insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP(Sc) is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP(C) and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP(Sc) accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.
Asunto(s)
Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Priones/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Biotina/metabolismo , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Endocitosis , Endosomas/metabolismo , Humanos , Insulina/metabolismo , Masculino , Ratones , Modelos Biológicos , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Transporte de Proteínas , Receptor de Insulina/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismoRESUMEN
Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.