RESUMEN
Production of the amyloidogenic prion protein, PrPSc, which forms infectious protein aggregates, or prions, is a key pathogenic event in prion diseases. Functional prion-like protein aggregations, such as the mitochondrial adaptor protein MAVS and the inflammasome component protein ASC, have been identified to play a protective role in viral infections in mammalian cells. In this study, to investigate if PrPSc could play a functional role against external stimuli, we infected prion-infected cells with a neurotropic influenza A virus strain, IAV/WSN. We found that prion-infected cells were highly resistant to IAV/WSN infection. In these cells, NF-κB nuclear translocation was disturbed; therefore, mitochondrial superoxide dismutase (mtSOD) expression was suppressed, and mitochondrial reactive oxygen species (mtROS) was increased. The elevated mtROS subsequently activated NLRP3 inflammasomes, leading to the suppression of IAV/WSN-induced necroptosis. We also found that prion-infected cells accumulated a portion of PrP molecules in the cytosol, and that the N-terminal potential nuclear translocation signal of PrP impeded NF-κB nuclear translocation. These results suggest that PrPSc might play a functional role in protection against viral infections by stimulating the NLRP3 inflammasome-dependent antivirus mechanism through the cytosolic PrP-mediated disturbance of NF-κB nuclear translocation, which leads to suppression of mtSOD expression and consequently upregulation of the NLRP3 inflammasome activator mtROS. IMPORTANCE: Cytosolic PrP has been detected in prion-infected cells and suggested to be involved in the neurotoxicity of prions. Here, we also detected cytosolic PrP in prion-infected cells. We further found that the nuclear translocation of NF-κB was disturbed in prion-infected cells and that the N-terminal potential nuclear translocation signal of PrP expressed in the cytosol disturbed the nuclear translocation of NF-κB. Thus, the N-terminal nuclear translocation signal of cytosolic PrP might play a role in prion neurotoxicity. Prion-like protein aggregates in other protein misfolding disorders, including Alzheimer's disease were reported to play a protective role against various environmental stimuli. We here showed that prion-infected cells were partially resistant to IAV/WSN infection due to the cytosolic PrP-mediated disturbance of the nuclear translocation of NF-κB, which consequently activated NLRP3 inflammasomes after IAV/WSN infection. It is thus possible that prions could also play a protective role in viral infections.
RESUMEN
Conformational conversion of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases. However, the conversion mechanism remains to be elucidated. Here, we generated Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which overexpress mouse PrP lacking residues 91-106. We showed that none of the mice became sick after intracerebral inoculation with RML, 22L, and FK-1 prion strains nor accumulated PrPScΔ91-106 in their brains except for a small amount of PrPScΔ91-106 detected in one 22L-inoculated mouse. However, they developed disease around 85 days after inoculation with bovine spongiform encephalopathy (BSE) prions with PrPScΔ91-106 in their brains. These results suggest that residues 91-106 are important for PrPC conversion into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions. We then narrowed down the residues 91-106 by transducing various PrP deletional mutants into RML- and 22L-infected cells and identified that PrP mutants lacking residues 97-99 failed to convert into PrPSc in these cells. Our in vitro conversion assay also showed that RML, 22L, and FK-1 prions did not convert PrPΔ97-99 into PrPScΔ97-99, but BSE prions did. We further found that PrP mutants with proline residues at positions 97 to 99 or charged residues at positions 97 and 99 completely or almost completely lost their converting activity into PrPSc in RML- and 22L-infected cells. These results suggest that the structurally flexible and noncharged residues 97-99 could be important for PrPC conversion into PrPSc following infection with RML, 22L, and FK-1 prions but not BSE prions.
Asunto(s)
Enfermedades por Prión , Proteínas Priónicas , Priones , Animales , Ratones , Enfermedades por Prión/genética , Proteínas Priónicas/química , Proteínas Priónicas/genética , Priones/patogenicidad , Prolina , Isoformas de Proteínas/genética , Translocación GenéticaRESUMEN
The cellular prion protein, PrPC , is a copper-binding protein abundantly expressed in the brain, particularly by neurons, and its conformational conversion into the amyloidogenic isoform, PrPSc , plays a key pathogenic role in prion diseases. However, the role of copper binding to PrPC in prion diseases remains unclear. Here, we fed mice with a low-copper or regular diet and intracerebrally inoculated them with two different mouse-adapted RML scrapie and BSE prions. Mice with a low-copper diet developed disease significantly but only slightly later than those with a regular diet after inoculation with BSE prions, but not with RML prions, suggesting that copper could play a minor role in BSE prion pathogenesis, but not in RML prion pathogenesis. We then generated two lines of transgenic mice expressing mouse PrP with copper-binding histidine (His) residues in the N-terminal domain replaced with alanine residues, termed TgPrP(5H > A)-7342/Prnp0/0 and TgPrP(5H > A)-7524/Prnp0/0 mice, and similarly inoculated RML and BSE prions into them. Due to 2-fold higher expression of PrP(5H > A) than PrPC in wild-type (WT) mice, TgPrP(5H > A)-7524/Prnp0/0 mice were highly susceptible to these prions, compared to WT mice. However, TgPrP(5H > A)-7342/Prnp0/0 mice, which express PrP(5H > A) 1.2-fold as high as PrPC in WT mice, succumbed to disease slightly, but not significantly, later than WT mice after inoculation with RML prions, but significantly so after inoculation with BSE prions. Subsequent secondary inoculation experiments revealed that amino acid sequence differences between PrP(5H > A) and WT PrPSc created no prion transmission barrier to BSE prions. These results suggest that copper-binding His residues in PrPC are dispensable for RML prion pathogenesis but have a minor effect on BSE prion pathogenesis. Taken together, our current results suggest that copper could have a minor effect on prion pathogenesis in a strain-dependent manner through binding to His residues in the N-terminal domain of PrPC .
RESUMEN
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, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/genética , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Gripe Humana/genética , Insomnio Familiar Fatal/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Proteínas Priónicas/genética , Animales , Línea Celular Tumoral , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Síndrome de Creutzfeldt-Jakob/virología , Enfermedad de Gerstmann-Straussler-Scheinker/metabolismo , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Enfermedad de Gerstmann-Straussler-Scheinker/virología , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/patogenicidad , Gripe Humana/metabolismo , Gripe Humana/patología , Gripe Humana/virología , Insomnio Familiar Fatal/metabolismo , Insomnio Familiar Fatal/patología , Insomnio Familiar Fatal/virología , Ratones , Ratones Transgénicos , Mutación , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Conformación Proteica , Genética Inversa/métodosRESUMEN
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
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
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
The normal cellular isoform of prion protein, designated PrPC, is constitutively converted to the abnormally folded, amyloidogenic isoform, PrPSc, in prion diseases, which include Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. PrPC is a membrane glycoprotein consisting of the non-structural N-terminal domain and the globular C-terminal domain. During conversion of PrPC to PrPSc, its 2/3 C-terminal region undergoes marked structural changes, forming a protease-resistant structure. In contrast, the N-terminal region remains protease-sensitive in PrPSc. Reverse genetic studies using reconstituted PrPC-knockout mice with various mutant PrP molecules have revealed that the N-terminal domain has an important role in the normal function of PrPC and the conversion of PrPC to PrPSc. The N-terminal domain includes various characteristic regions, such as the positively charged residue-rich polybasic region, the octapeptide repeat (OR) region consisting of five repeats of an octapeptide sequence, and the post-OR region with another positively charged residue-rich polybasic region followed by a stretch of hydrophobic residues. We discuss the normal functions of PrPC, the conversion of PrPC to PrPSc, and the neurotoxicity of PrPSc by focusing on the roles of the N-terminal regions in these topics.
Asunto(s)
Enfermedades por Prión/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Animales , Humanos , Mutación , Enfermedades por Prión/genética , Proteínas Priónicas/genética , Conformación Proteica , Dominios ProteicosRESUMEN
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
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
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 photochemical conversion of 1,8a-dihydroazulene-1,1-dicarbonitrile (DHA) to vinylheptafulvene (VHF) is a positive T-type photoswitch that is well understood in solution, but has not been explored in the solid state. Upon excitation with UV light, DHA is converted into VHF in the solid state, with a distinct color change from yellow to deep-red, and retention of crystallinity. The structure of the ring-opened product was assigned to syn-VHF using variable-temperature infrared spectroscopy, and determined by X-ray photodiffraction in a crystal enriched with the product by two-photon excitation. A radical pathway becomes an observable photoreaction channel at low temperatures, and includes a strongly colored, short-lived diradical intermediate.
RESUMEN
Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP region of amino acids 23 to 50 including a positively charged amino acid cluster and weakly interacted with the PrP octarepeat peptide region of residues 51 to 90. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that of control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis, even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N terminus of PrP, but the pathophysiological roles of the PrP-melanin interaction remain unclear.IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors, such as metal ions, lipids, nucleic acids, antiprion compounds, and several proteins, including abnormal PrP in prion disease and the Aß oligomer in Alzheimer's disease. In the present study, melanin, a main determinant of skin color, was newly found to interact with this N-terminal region and inhibits abnormal PrP formation in prion-infected cells. However, the data for prion infection in mice lacking melanin production suggest that melanin is not associated with the prion disease mechanism, although the incidence of prion disease is reportedly much higher in white people than in black people. Thus, the roles of the PrP-melanin interaction remain to be further elucidated, but melanin might be a useful competitive tool for evaluating the functions of other ligands at the N-terminal region.
Asunto(s)
Melaninas/metabolismo , Enfermedades por Prión/prevención & control , Priones/metabolismo , Animales , Línea Celular , Melaninas/administración & dosificación , Ratones , Neuronas/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Unión Proteica , Mapeo de Interacción de Proteínas , Análisis de SupervivenciaRESUMEN
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
BACKGROUND: The QRS duration does not always reflect the left ventricular (LV) activation delay in patients with ventricular conduction disturbances. The R-wave offset in left chest leads may more closely reflect the LV activation delay than the QRS offset. METHODS: We evaluated 138 cases with left bundle branch block (LBBB, n=11), right BBB (RBBB, n=38), non-specific intraventricular conduction disturbance (n=11), narrow QRS (<120ms, n=56) and right ventricular pacing (n=22). Cases with right axis deviation (120 to 270 degrees) were excluded. The intervals from the QRS onset to the V-waves in coronary sinus bipolar electrograms (QCS) were measured, and the longest interval was defined as the QCSmax. In the 12-lead electrocardiogram, the interval from the QRS onset to the R-wave offset (QR) was measured and then averaged in leads I-aVL, II-III-aVF, V1-V2, V3-V4 and V5-V6. RESULTS: Significant correlations (p<0.05) were found between QCSmax and QR in I-aVL (r=0.83), II-III-aVF (r=0.51) and V5-V6 (r=0.86) in cases with a normal axis (0 to 90 degrees, n=64); and I-aVL (r=0.90), II-III-aVF (r=0.31) and V5-V6 (r=0.69) in cases with left axis deviation (-45 to -89 degrees, n=52). Overall, the QRS duration was also correlated with QCSmax (r=0.72, p<0.001); however, this correlation was weaker than the correlation between QCSmax and QR in I-aVL (r=0.89, p<0.001) due to disparities in RBBB (p<0.001). CONCLUSIONS: The interval from the QRS onset to R-wave offset in the left chest leads reflects the degree of LV activation delay regardless of differences in QRS duration and morphology.
Asunto(s)
Bloqueo de Rama/diagnóstico , Bloqueo de Rama/fisiopatología , Seno Coronario/fisiopatología , Electrocardiografía/métodos , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/fisiopatología , Anciano , Algoritmos , Bloqueo de Rama/complicaciones , Acoplamiento Excitación-Contracción , Femenino , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Masculino , Contracción Miocárdica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Disfunción Ventricular Izquierda/etiologíaRESUMEN
One of the most inevitable limitations of any material that is exposed to mechanical impact is that they are inexorably prone to mechanical damage, such as cracking, denting, gouging, or wearing. To confront this challenge, the field of polymers has developed materials that are capable of autonomous self-healing and recover their macroscopic integrity similar to biological organisms. However, the study of this phenomenon has mostly remained within the soft materials community and has not been explored by solid-state organic chemists. The first evidence of self-healing in a molecular crystal is now presented using crystals of dipyrazolethiuram disulfide. The crystals were mildly compressed and the degree of healing was found to be 6.7 %. These findings show that the self-healing properties can be extended beyond mesophasic materials and applied towards the realm of ordered solid-state compounds.
RESUMEN
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).
Asunto(s)
Neuroblastoma/metabolismo , Enfermedades por Prión/metabolismo , Priones/química , Priones/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Proteínas Priónicas , Priones/genética , Alineación de SecuenciaRESUMEN
AIMS: Because benefits of cardiac resynchronization therapy (CRT) appear to be less favourable in non-left bundle branch block (LBBB) patients, this prospective longitudinal study tested the hypothesis that QRS morphology and echocardiographic mechanical dyssynchrony were associated with long-term outcome after CRT. METHODS AND RESULTS: Two-hundred and seventy-eight consecutive New York Heart Association class III and IV CRT patients with QRS ≥120 ms and ejection fraction ≤35% were studied. The pre-specified primary endpoint was death, heart transplant, or left ventricular assist device over 4 years. Dyssynchrony assessed before CRT included interventricular mechanical delay (IVMD) and speckle-tracking radial strain using pre-specified cut-offs for each. Of 254 with baseline quantitative echocardiographic data available, 128 had LBBB, 81 had intraventricular conduction delay (IVCD), and 45 had right bundle branch block (RBBB). Radial dyssynchrony was observed in 85% of the patients with LBBB, 59% with IVCD*, and 40% with RBBB* (*P < 0.01 vs. LBBB). Of 248 (98%) with follow-up, LBBB patients had a significantly more favourable long-term survival than non-LBBB patients. However, non-LBBB patients with dyssynchrony had a more favourable event-free survival than those without dyssynchrony: radial dyssynchrony hazard ratio 2.6, 95% confidence interval (CI) 1.47-4.53 (P = 0.0008) and IVMD hazard ratio 4.9, 95% CI 2.60-9.16 (P = 0.0007). Right bundle branch block patients who lacked dyssynchrony had the least favourable outcome. CONCLUSION: Non-LBBB patients with dyssynchrony had a more favourable long-term survival than non-LBBB patients who lacked dyssynchrony. Mechanical dyssynchrony and QRS morphology are associated with outcome following CRT.
Asunto(s)
Bloqueo de Rama/terapia , Terapia de Resincronización Cardíaca , Disfunción Ventricular Izquierda/terapia , Anciano , Análisis de Varianza , Bloqueo de Rama/fisiopatología , Ecocardiografía Doppler/métodos , Electrocardiografía , Femenino , Insuficiencia Cardíaca/terapia , Trasplante de Corazón/estadística & datos numéricos , Humanos , Estimación de Kaplan-Meier , Masculino , Estudios Prospectivos , Volumen Sistólico/fisiología , Resultado del Tratamiento , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Evaluation of longitudinal strain (LS) from two-dimensional echocardiography is useful for global and regional left ventricular (LV) dysfunction assessment. We determined whether the LS reflects contraction process in patients with asynchronous LV activation. We studied 144 patients with an ejection fraction ≤ 35%, who had left bundle branch block (LBBB, n = 42), right ventricular apical (RVA) pacing (n = 34), LV basal- or mid-lateral pacing (n = 23), and no conduction block (Narrow-QRS, n = 45). LS distribution maps were constructed using 3 standard apical views. The times from the QRS onset-to-early systolic positive peak (Q-EPpeak) and late systolic negative peak (Q-LNpeak) were measured to determine the beginning and end of contractions in each segment. Negative strain in LBBB initially appeared in the septum and basal-lateral contracted late. In RVA and LV pacing, the contracted area enlarged centrifugally from the pacing site. Narrow-QRS showed few regional differences in strain during the systolic period. The Q-EPpeak and Q-LNpeak exhibited similar sequences characterized by septum to basal-lateral via the apical regions in LBBB, apical to basal regions in RVA pacing, and lateral to a relatively large delayed contracted area between the apical- and basal-septum in LV pacing. Differences in Q-LNpeaks between the apical and basal segments in delayed contracted wall were 107 ± 30 ms in LBBB, 133 ± 46 ms in RVA pacing, and 37 ± 20 ms in LV pacing (p < 0.05, between QRS groups). Specific LV contraction processes were demonstrated by evaluating the LS distribution and time-to-peak strain. These evaluations may have potential to estimate the activation sequence in patients with asynchronous LV activation.