RESUMEN
Cytomegalovirus (CMV) infection poses risks to newborns, necessitating effective therapies. Given that the damage includes both viral infection of brain cells and immune system-related damage, here we investigate the involvement of cellular prion protein (PrP), which plays vital roles in neuroprotection and immune regulation. Using a murine model, we show the role of PrP in tempering neonatal T cell immunity during CMV infection. PrP-null mice exhibit enhanced viral control through elevated virus-specific CD8 T cell responses, leading to reduced viral titers and pathology. We further unravel the molecular mechanisms by showing CMV-induced upregulation followed by release of PrP via the metalloproteinase ADAM10, impairing CD8 T cell response specifically in neonates. Additionally, we confirm PrP downregulation in human CMV (HCMV)-infected fibroblasts, underscoring the broader relevance of our observations beyond the murine model. Furthermore, our study highlights how PrP, under the stress of viral pathogenesis, reveals its impact on neonatal immune modulation.
Asunto(s)
Animales Recién Nacidos , Linfocitos T CD8-positivos , Infecciones por Citomegalovirus , Citomegalovirus , Ratones Noqueados , Animales , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Citomegalovirus/inmunología , Humanos , Ratones , Linfocitos T CD8-positivos/inmunología , Femenino , Fibroblastos/metabolismo , Fibroblastos/virología , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Proteína ADAM10/metabolismo , Proteína ADAM10/genéticaRESUMEN
The cellular prion protein (PrPC) plays many roles in the developing and adult brain. In addition, PrPC binds to several amyloids in oligomeric and prefibrillar forms and may act as a putative receptor of abnormal misfolded protein species. The role of PrPC in tau seeding and spreading is not known. In the present study, we have inoculated well-characterized sarkosyl-insoluble fractions of sporadic Alzheimer's disease (sAD) into the brain of adult wild-type mice (Prnp+/+), Prnp0/0 (ZH3 strain) mice, and mice over-expressing the secreted form of PrPC lacking their GPI anchor (Tg44 strain). Phospho-tau (ptau) seeding and spreading involving neurons and oligodendrocytes were observed three and six months after inoculation. 3Rtau and 4Rtau deposits from the host tau, as revealed by inoculating Mapt0/0 mice and by using specific anti-mouse and anti-human tau antibodies suggest modulation of exon 10 splicing of the host mouse Mapt gene elicited by exogenous sAD-tau. However, no tau seeding and spreading differences were observed among Prnp genotypes. Our results show that PrPC does not affect tau seeding and spreading in vivo.
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Enfermedad de Alzheimer , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Ratones , Humanos , Encéfalo/metabolismo , Encéfalo/patología , Proteínas PrPC/metabolismo , Proteínas PrPC/genética , Ratones Transgénicos , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Sarcosina/análogos & derivados , Sarcosina/farmacología , Neuronas/metabolismo , Neuronas/patología , Modelos Animales de EnfermedadRESUMEN
INTRODUCTION: Gerstmann-Sträussler-Scheinker disease (GSS) is an autosomal-dominant inherited prion disease most often associated with the human prion protein gene (PRNP)-P102L mutation. Although patients manifest considerable phenotypic heterogeneity, the involvement of the nigrostriatal system has not been well-studied. METHODS: We performed dopamine transporter single-photon emission computed tomography (DAT-SPECT) using 123I-ioflupane to investigate the nigrostriatal system function in nine patients with the PRNP-P102L mutation. We also examined the pathological findings in another patient whose predominant feature was ataxia and who died 5 years after disease onset. RESULTS: Striatum uptake of 123I-ioflupane indicated by specific binding ratio (SBR) values was significantly reduced in two patients. The DAT-SPECT examination was performed 6 months after disease onset in one of these patients who manifested rapidly developing cognitive decline mimicking Creutzfeldt-Jakob disease. DAT-SPECT was also performed 9 years after disease onset in another patient who manifested the conventional features of GSS involving ataxia and dementia in the initial phase but showed akinetic mutism at the examination time. Another patient examined 2 years after disease onset who predominantly manifested ataxia showed marginally abnormal SBR values. An autopsy case showed moderate neuronal loss in the substantia nigra, and the degree of neuronal loss was similar in most other parts of the brain. CONCLUSION: Nigrostriatal system involvement may occur in patients with GSS associated with the PRNP-P102L mutation, even though parkinsonism is not the predominant feature.
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Cuerpo Estriado , Enfermedad de Gerstmann-Straussler-Scheinker , Mutación , Proteínas Priónicas , Priones , Sustancia Negra , Tomografía Computarizada de Emisión de Fotón Único , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/patología , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Enfermedad de Gerstmann-Straussler-Scheinker/diagnóstico por imagen , Nortropanos , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Priones/genética , Priones/metabolismo , Sustancia Negra/diagnóstico por imagen , Sustancia Negra/patología , Sustancia Negra/metabolismoRESUMEN
Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.
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Proteínas Qa-SNARE , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Animales , Ratones , Humanos , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/química , Neuronas/metabolismo , Agregado de Proteínas , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/genéticaRESUMEN
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.
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Citosol , Inflamasomas , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Especies Reactivas de Oxígeno , Animales , Citosol/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratones , Humanos , Mitocondrias/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Línea Celular , Superóxido Dismutasa/metabolismo , Priones/metabolismo , Proteínas Priónicas/metabolismo , NecroptosisRESUMEN
Misfolding of the prion protein is linked to multiple neurodegenerative diseases. A better understanding of the process requires the identification and structural characterization of intermediate conformations via which misfolding proceeds. In this study, three conserved aromatic residues (Tyr168, Phe174, and Tyr217) located in the C-terminal domain of mouse PrP (wt moPrP) were mutated to Ala. The resultant mutant protein, 3A moPrP, is shown to adopt a molten globule (MG)-like native conformation. Hydrogen-deuterium exchange studies coupled with mass spectrometry revealed that for 3A moPrP, the free energy gap between the MG-like native conformation and misfolding-prone partially unfolded forms is reduced. Consequently, 3A moPrP misfolds in native conditions even in the absence of salt, unlike wt moPrP, which requires the addition of salt to misfold. 3A moPrP misfolds to a ß-rich dimer in the absence of salt, which can rapidly form an oligomer upon the addition of salt. In the presence of salt, 3A moPrP misfolds to a ß-rich oligomer about a thousand-fold faster than wt moPrP. Importantly, the misfolded structure of the dimer is similar to that of the salt-induced oligomer. Misfolding to oligomer seems to be induced at the level of the dimeric unit by monomer-monomer association, and the oligomer grows by accretion of misfolded dimeric units. Additionally, it is shown that the conserved aromatic residues collectively stabilize not only monomeric protein, but also the structural core of the ß-rich oligomers. Finally, it is also shown that 3A moPrP misfolds much faster to amyloid-fibrils than does the wt protein.
Asunto(s)
Proteínas Priónicas , Pliegue de Proteína , Multimerización de Proteína , Animales , Ratones , Proteínas Priónicas/química , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Conformación Proteica , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , MutaciónRESUMEN
Most cases of human prion disease arise due to spontaneous misfolding of WT or mutant prion protein, yet recapitulating this event in animal models has proven challenging. It remains unclear whether spontaneous prion generation can occur within the mouse lifespan in the absence of protein overexpression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knockin mice that express the misfolding-prone bank vole prion protein (BVPrP). While mice expressing WT BVPrP (I109 variant) remained free from neurological disease, a subset of mice expressing BVPrP with mutations (D178N or E200K) causing genetic prion disease developed progressive neurological illness. Brains from spontaneously ill knockin mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant BVPrP. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP-knockin mice exhibited prion seeding activity and transmitted disease to mice expressing WT BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical before and after transmission, suggesting that both mutations guide the formation of a similar atypical prion strain. These findings imply that knockin mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action.
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Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Ratones Transgénicos , Enfermedades por Prión , Proteínas Priónicas , Animales , Ratones , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Enfermedades por Prión/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Mutación Missense , Humanos , Arvicolinae/genética , Arvicolinae/metabolismo , Sustitución de Aminoácidos , Priones/genética , Priones/metabolismo , Pliegue de ProteínaRESUMEN
Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region. This exon is homologous to exon 2 in nonprimate species but contains a start codon that would yield an upstream open reading frame with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.
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Exones , Proteínas Priónicas , Sitios de Empalme de ARN , Humanos , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Empalme del ARN , Intrones , Regulación de la Expresión Génica , Animales , Priones/metabolismo , Priones/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/genética , Regiones no Traducidas 5'RESUMEN
The spontaneous aggregation of infectious or misfolded forms of prion protein is known to be responsible for neurotoxicity in brain cells, which ultimately leads to the progression of prion disorders. Bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease (CJD) in humans are glaring examples in this regard. Square-planar complexes with labile ligands and indole-based compounds are found to be efficiently inhibitory against protein aggregation. Herein, we report the synthesis of an indole-based cyclometallated palladium complex. The ligand and complex were characterized by various spectroscopic techniques such as UV-visible, NMR, IR, and HRMS. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with PrP106-126 was studied using UV-visible spectroscopy, CD spectroscopy, MALDI-TOF MS, and molecular docking. The inhibition effects of the complex on the PrP106-126 aggregation, fibrillization and amyloid formation phenomena were analysed through the ThT assay, CD, TEM and AFM. The effect of the complex on the aggregation process of PrP106-126 was determined kinetically through the ThT assay. The complex presented high binding affinity with the peptide and influenced the peptide's conformation and aggregation in different modes of binding. Furthermore, the MTT assay on neuronal HT-22 cells showed considerable protective properties of the complex against PrP106-126-mediated cytotoxicity. These findings suggest that the compound influences peptide aggregation in different ways, and the anti-aggregation action is primarily associated with the metal's physicochemical properties and the reactivity rather than the ligand. As a result, we propose that this compound be investigated as a potential therapeutic molecule in metallopharmaceutical research to treat prion disease (PD).
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Complejos de Coordinación , Indoles , Paladio , Agregado de Proteínas , Paladio/química , Paladio/farmacología , Humanos , Indoles/química , Indoles/farmacología , Agregado de Proteínas/efectos de los fármacos , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Proteínas Priónicas/antagonistas & inhibidores , PrionesRESUMEN
Protein aggregation in the form of amyloid fibrils has long been associated with the onset and development of various amyloidoses, including Alzheimer's, Parkinson's or prion diseases. Recent studies of their fibril formation process have revealed that amyloidogenic protein cross-interactions may impact aggregation pathways and kinetic parameters, as well as the structure of the resulting aggregates. Despite a growing number of reports exploring this type of interaction, they only cover just a small number of possible amyloidogenic protein pairings. One such pair is between two neurodegeneration-associated proteins: the pro-inflammatory S100A9 and prion protein, which are known to co-localize in vivo. In this study, we examined their cross-interaction in vitro and discovered that the fibrillar form of S100A9 modulated the aggregation pathway of mouse prion protein 89-230 fragment, while non-aggregated S100A9 also significantly inhibited its primary nucleation process. These results complement previous observations of the pro-inflammatory protein's role in amyloid aggregation and highlight its potential role against neurodegenerative disorders.
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Amiloide , Calgranulina B , Proteínas Priónicas , Agregado de Proteínas , Calgranulina B/metabolismo , Calgranulina B/química , Animales , Ratones , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Amiloide/metabolismo , Amiloide/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/química , CinéticaRESUMEN
Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.
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Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide , Enfermedades Neurodegenerativas , Humanos , Proteína ADAM10/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Proteínas Priónicas/metabolismo , Proteínas de la Membrana/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , AnticuerposRESUMEN
Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrPSc, a pathological form of the host's cellular prion protein (PrPC), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrPSc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrPC (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.
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Ciervos , Enfermedad Debilitante Crónica , Animales , Enfermedad Debilitante Crónica/transmisión , Enfermedad Debilitante Crónica/metabolismo , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Ratones Transgénicos , Noruega , Marcación de Gen , Priones/metabolismo , Priones/genética , Priones/patogenicidadRESUMEN
Objective: To investigate the effects and possible mechanisms of caffeic acid phenethyl ester (CAPE) on the replication, amplification, and fibre formation of prions (PrPSc). Methods: The CCK8 assay was used to detect the cell viability of the prion-infected cell model SMB-S15 after CAPE treatment for 3 days and 7 days and the maximum safe concentration of CAPE for SMB-S15 was obtained. The cells were treated with a concentration within a safe range, and the content of PrPSc in the cells before and after CAPE treatment was analyzed by western blot. Protein misfolding cycle amplification (PMCA) and western blot were used to assess changes in PrPSc level in amplification products following CAPE treatment. Real-time-quaking induced conversion assay (RT-QuIC) technology was employed to explore the changes in fibril formation before and after CAPE treatment. The binding affinity between CAPE and murine recombinant full-length prion protein was determined using a molecular interaction assay. Results: CCK8 cell viability assay results demonstrated that treatment with 1 µmol/L CAPE for 3 and 7 days did not exhibit statistically significant differences in cell viability compared to the control group (all P<0.05). However, when the concentration of CAPE exceeded 1 µmol/L, a significant reduction in cell viability was observed in cells treated with CAPE for 3 and 7 days, compared to the control group (all P<0.05). Thus, 1 µmol/L was determined as the maximum safe concentration of CAPE treatment for SMB-S15 cells. The western blot results revealed that treatment with CAPE for both 3 and 7 days led to a detectable reduction in the levels of PrPSc in SMB-S15 cells (all P<0.05). The products of PMCA experiments were assessed using western blot. The findings revealed a significant decrease in the levels of PrPSc (relative grey value) in the PMCA amplification products of adapted-strains SMB-S15, 139A, and ME7 following treatment with CAPE, as compared to the control group (all P<0.05). The RT-QuIC experimental results demonstrated a reduction in fibril formation (as indicated by ThT peak values) in CAPE-treated mouse-adapted strains 139A, ME7, and SMB-S15, as well as in SMB-S15 cells infected with prions. Furthermore, CAPE exhibited varying degrees of inhibition towards different seed fibrils formation, with statistically significant differences observed (all P<0.05). Notably, CAPE exhibited a more pronounced inhibitory effect on ME7 seed fibrils. Molecular interaction analyses demonstrated significant binding between CAPE and murine recombinant prion protein, and the association constant was (2.92±0.41)×10-6 mol/L. Conclusions: CAPE inhibits PrPSc replication, amplification, and fibril formation in vitro possibly due to specific interactions with the prion protein at the molecular level.
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Ácidos Cafeicos , Alcohol Feniletílico , Animales , Ácidos Cafeicos/farmacología , Ratones , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/farmacología , Supervivencia Celular/efectos de los fármacos , Proteínas PrPSc/metabolismo , Priones , Línea Celular , Proteínas Priónicas/metabolismoRESUMEN
Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid-onset dementia and death. However, elimination of endogenous PrP halts prion disease progression. In this study, we describe Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates endogenous DNA methyltransferases, thereby reducing transgene size and cytotoxicity. When delivered to the mouse brain by systemic injection of adeno-associated virus (AAV), Prnp-targeted CHARM ablates PrP expression across the brain. Furthermore, we have temporally limited editor expression by implementing a kinetically tuned self-silencing approach. CHARM potentially represents a broadly applicable strategy to suppress pathogenic proteins, including those implicated in other neurodegenerative diseases.
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Encéfalo , Metilación de ADN , Dependovirus , Silenciador del Gen , Histonas , Proteínas Priónicas , Animales , Humanos , Ratones , Encéfalo/metabolismo , Dependovirus/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Histonas/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , TransgenesRESUMEN
Prion-like proteins (PrLPs) have emerged as beneficial molecules with implications in adaptive responses. These proteins possess a conserved prion-like domain (PrLD) which is an intrinsically disordered region capable of adopting different conformations upon perceiving external stimuli. Owing to changes in protein conformation, functional characteristics of proteins harboring PrLDs get altered thereby, providing a unique mode of protein-based regulation. Since PrLPs are ubiquitous in nature and involved in diverse functions, through this study, we aim to explore the role of such domains in yet another important physiological process viz. plant-microbe interactions to get insights into the mechanisms dictating cross-kingdom interactions. We have evaluated the presence and functions of PrLPs in 18 different plant-associated fungi of agricultural importance to unravel their role in plant-microbe interactions. Of the 241,997 proteins scanned, 3,820 (~ 1.6%) were identified as putative PrLPs with pathogenic fungi showing significantly higher PrLP density than their beneficial counterparts. Further, through GO enrichment analysis, we could predict several PrLPs from pathogenic fungi to be involved in virulence and formation of stress granules. Notably, PrLPs involved in (retro)transposition were observed exclusively in pathogenic fungi. We even analyzed publicly available data for the expression alterations of fungal PrLPs upon their interaction with their respective hosts which revealed perturbation in the levels of some PrLP-encoding genes during interactions with plants. Overall, our work sheds light into the probable role of prion-like candidates in plant-fungi interaction, particularly in context of pathogenesis, paving way for more focused studies for validating their role.
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Proteínas Fúngicas , Hongos , Plantas , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Plantas/microbiología , Hongos/genética , Hongos/metabolismo , Hongos/patogenicidad , Simulación por Computador , Enfermedades de las Plantas/microbiología , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/química , Priones/metabolismo , Priones/genética , Priones/química , Virulencia , Interacciones Huésped-PatógenoRESUMEN
Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer's disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrPC) during disease progression. That is why, by means of cross studies of miRNAs up-regulated in AD with in silico identification of potential miRNAs-binding to 3'UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3'UTR-PRNP, and second, we analyzed the levels of PrPC expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR-519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non-AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3'UTR in vitro and promotes downregulation of PrPC. Moreover, miR-519a-3p was found to be up-regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.
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Regiones no Traducidas 3' , Enfermedad de Alzheimer , Biomarcadores , MicroARNs , Proteínas Priónicas , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/diagnóstico , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Biomarcadores/metabolismo , Regiones no Traducidas 3'/genética , Femenino , Anciano , Masculino , Anciano de 80 o más Años , Proteínas PrPC/metabolismo , Proteínas PrPC/genéticaRESUMEN
Liquid-liquid phase separation (LLPS) of the mammalian prion protein is mainly driven by its intrinsically disordered N-terminal domain (N-PrP). However, the specific intermolecular interactions that promote LLPS remain largely unknown. Here, we used extensive mutagenesis and comparative analyses of evolutionarily distant PrP species to gain insight into the relationship between protein sequence and phase behavior. LLPS of mouse PrP is dependent on two polybasic motifs in N-PrP that are conserved in all tetrapods. A unique feature of mammalian N-PrP is the octarepeat domain with four histidines that mediate binding to copper ions. We now show that the octarepeat is critical for promoting LLPS and preventing the formation of PrP aggregates. Amphibian N-PrP, which contains the polybasic motifs but lacks a repeat domain and histidines, does not undergo LLPS and forms nondynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating the phase transition of PrP. This activity of the octarepeat domain was neither dependent on the four highly conserved histidines nor on copper binding. Instead, the regularly spaced tryptophan residues were critical for regulating LLPS, presumably via cation-π interactions with the polybasic motifs. Our study reveals a novel role for the tryptophan residues in the octarepeat in controlling phase transition of PrP and indicates that the ability of mammalian PrP to undergo LLPS has evolved with the octarepeat in the intrinsically disordered domain but independently of the histidines.
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Cobre , Histidina , Proteínas Priónicas , Dominios Proteicos , Animales , Ratones , Secuencias de Aminoácidos , Cobre/metabolismo , Cobre/química , Histidina/metabolismo , Histidina/química , Separación de Fases , Proteínas Priónicas/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/genéticaRESUMEN
Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.
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Proteínas Priónicas , alfa-Sinucleína , Proteínas tau , Proteínas tau/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/análisis , Humanos , Proteínas Priónicas/metabolismo , Animales , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Priones/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismoRESUMEN
Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.