RESUMEN
Neurodegenerative diseases (NDs) such as Alzheimer's and Parkinson's disease are fatal neurological diseases that can be of idiopathic, genetic, or even infectious origin, as in the case of transmissible spongiform encephalopathies. The etiological factors that lead to neurodegeneration remain unknown but likely involve a combination of aging, genetic risk factors, and environmental stressors. Accumulating evidence hints at an association of viruses with neurodegenerative disorders and suggests that virus-induced neuroinflammation and perturbation of neuronal protein quality control can be involved in the early steps of disease development. In this review, we focus on emerging evidence for a correlation between NDs and viral infection and discuss how viral manipulations of cellular processes can affect the formation and dissemination of disease-associated protein aggregates.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedades por Prión , Virus , Envejecimiento , Crimen , HumanosRESUMEN
Transmissible spongiform encephalopathies or prion disorders are fatal infectious diseases that cause characteristic spongiform degeneration in the central nervous system. The causative agent, the so-called prion, is an unconventional infectious agent that propagates by converting the host-encoded cellular prion protein PrP into ordered protein aggregates with infectious properties. Prions are devoid of coding nucleic acid and thus rely on the host cell machinery for propagation. While it is now established that, in addition to PrP, other cellular factors or processes determine the susceptibility of cell lines to prion infection, exact factors and cellular processes remain broadly obscure. Still, cellular models have uncovered important aspects of prion propagation and revealed intercellular dissemination strategies shared with other intracellular pathogens. Here, we summarize what we learned about the processes of prion invasion, intracellular replication and subsequent dissemination from ex vivo cell models.
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Enfermedades por Prión , Priones , Animales , Sistema Nervioso Central/metabolismo , Mamíferos/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Priónicas , Priones/metabolismoRESUMEN
Neurodegenerative diseases are associated with misfolding of proteins into highly-ordered amyloid fibrils. These protein aggregates can be transmitted to other cells in which they induce aggregation of proteins of the same kind. Mechanisms of intercellular transfer include direct cell contact or transfer of aggregates within extracellular vesicles. Recent research suggests that viral proteins can increase the intercellular spreading of protein aggregation by promoting the required membrane interactions.
RESUMEN
Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.
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Fraccionamiento Celular , Fraccionamiento Químico , Vesículas Extracelulares/metabolismo , Transporte Biológico , Biomarcadores , Fraccionamiento Celular/métodos , Fraccionamiento Químico/métodos , Vesículas Extracelulares/ultraestructura , Citometría de Flujo , Humanos , Biopsia Líquida/métodos , Proteínas/metabolismoRESUMEN
Fibrous cross-ß aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-ß (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes.
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Amiloide/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Péptidos beta-Amiloides/metabolismo , Humanos , Fragmentos de Péptidos/metabolismo , Factores de Terminación de Péptidos/química , Agregado de Proteínas , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, Drosophila has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in Drosophila We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host.
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Drosophila melanogaster/genética , Enfermedades por Prión/genética , Proteínas Priónicas/genética , Animales , Animales Modificados Genéticamente , Western Blotting , Cricetinae , Drosophila melanogaster/citología , Drosophila melanogaster/efectos de los fármacos , Endopeptidasa K/metabolismo , Humanos , Locomoción/efectos de los fármacos , Ratones , Microscopía Confocal , Mutación/genética , Neurotoxinas/toxicidad , TransgenesRESUMEN
UNLABELLED: Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrP(Sc). Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrP(Sc) uptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independently of the prion strain and reduced PrP(Sc) formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrP(Sc) formation independent of the prion strain. IMPORTANCE: Recently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro. GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.
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Glicosaminoglicanos/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/fisiopatología , Animales , Transporte Biológico/fisiología , Western Blotting , Células CHO , Línea Celular , Cloratos , Cricetinae , Cricetulus , Sulfato de Dextran , Citometría de Flujo , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Enfermedades por Prión/metabolismo , Estadísticas no ParamétricasRESUMEN
Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals.
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Comunicación Celular , Citosol/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Astrocitos/citología , Técnicas de Cocultivo , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/metabolismo , Ratones , Microscopía Confocal , Enfermedades por Prión , Conformación Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
Prion diseases are fatal transmissible neurodegenerative diseases of various mammalian species. Central to these conditions is the conversion of the normal host prion protein PrP(C) into the abnormal prion conformer PrP(Sc). Mature PrP(C) is attached to the plasma membrane by a glycosylphosphatidylinositol anchor, whereas during biosynthesis and metabolism cytosolic and secreted forms of the protein may arise. The role of topological PrP(C) variants in the mechanism of prion formation and prion-induced neurotoxicity during prion disease remains undefined. In the present study we investigated whether Drosophila transgenic for ovine PrP targeted to the plasma membrane, to the cytosol or for secretion, could produce transmissible toxicity following exposure to exogenous ovine prions. Although all three topological variants of PrP were efficiently expressed in Drosophila, cytosolic PrP was conformationally distinct and required denaturation before recognition by immunobiochemical methods. Adult Drosophila transgenic for pan neuronally expressed ovine PrP targeted to the plasma membrane, to the cytosol or for secretion exhibited a decreased locomotor activity after exposure at the larval stage to ovine prions. Proteinase K-resistant PrP(Sc) was detected by protein misfolding cyclic amplification in prion-exposed Drosophila transgenic for membrane-targeted PrP. Significantly, head homogenate from all three variants of prion-exposed PrP transgenic Drosophila induced a decreased locomotor activity when transmitted to PrP recipient flies. Drosophila transgenic for PrP targeted for secretion exhibited a spontaneous locomotor defect in the absence of prion exposure that was transmissible in PrP transgenic flies. Our data are consistent with the formation of transmissible prions in PrP transgenic Drosophila.
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Membrana Celular/metabolismo , Proteínas PrPC/metabolismo , Enfermedades por Prión/metabolismo , Animales , Animales Modificados Genéticamente , Membrana Celular/genética , Membrana Celular/patología , Drosophila melanogaster , Proteínas PrPC/genética , Enfermedades por Prión/genética , Enfermedades por Prión/patología , OvinosRESUMEN
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrP(C) interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrP(C)-STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrP(C)-STI1 binding is corrupted in neuronal cell lines persistently infected with PrP(Sc), as well as in primary cultured hippocampal neurons acutely exposed to PrP(Sc). Consistent with this, high levels of eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation were found in PrP(Sc)-infected cells and in neurons acutely exposed to PrP(Sc). These data indicate that modulation of protein synthesis is critical for PrP(C)-STI1 neurotrophic functions, and point to the impairment of this process during PrP(Sc) infection as a possible contributor to neurodegeneration.
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Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/metabolismo , Priones/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular , Citoprotección , Factor 2 Eucariótico de Iniciación/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Neuritas/enzimología , Neuronas/citología , Neuronas/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas PrPSc/metabolismo , Unión Proteica , Serina-Treonina Quinasas TOR , Regulación hacia ArribaRESUMEN
Pathogenic aggregation of the protein tau is a hallmark of Alzheimer's disease and several other tauopathies. Tauopathies are characterized by the deposition of specific tau isoforms as disease-related tau filament structures. The molecular processes that determine isoform-specific deposition of tau are however enigmatic. Here we show that acetylation of tau discriminates its isoform-specific aggregation. We reveal that acetylation strongly attenuates aggregation of four-repeat tau protein, but promotes amyloid formation of three-repeat tau. We further identify acetylation of lysine 298 as a hot spot for isoform-specific tau aggregation. Solid-state NMR spectroscopy demonstrates that amyloid fibrils formed by unmodified and acetylated three-repeat tau differ in structure indicating that site-specific acetylation modulates tau structure. The results implicate acetylation as a critical regulator that guides the selective aggregation of three-repeat tau and the development of tau isoform-specific neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Humanos , Proteínas 14-3-3 , Acetilación , Proteínas tauRESUMEN
Prion-like spreading of protein misfolding is a characteristic of neurodegenerative diseases, but the exact mechanisms of intercellular protein aggregate dissemination remain unresolved. Evidence accumulates that endogenous retroviruses, remnants of viral germline infections that are normally epigenetically silenced, become upregulated in neurodegenerative diseases such as amyotrophic lateral sclerosis and tauopathies. Here we uncover that activation of endogenous retroviruses affects prion-like spreading of proteopathic seeds. We show that upregulation of endogenous retroviruses drastically increases the dissemination of protein aggregates between cells in culture, a process that can be inhibited by targeting the viral envelope protein or viral protein processing. Human endogenous retrovirus envelopes of four different clades also elevate intercellular spreading of proteopathic seeds, including pathological Tau. Our data support a role of endogenous retroviruses in protein misfolding diseases and suggest that antiviral drugs could represent promising candidates for inhibiting protein aggregate spreading.
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Esclerosis Amiotrófica Lateral , Retrovirus Endógenos , Priones , Humanos , Retrovirus Endógenos/genética , Agregado de Proteínas , AntiviralesRESUMEN
A conformational change of the cellular prion protein (PrP(c)) underlies formation of PrP(Sc), which is closely associated with pathogenesis and transmission of prion diseases. The precise conformational prerequisites and the cellular environment necessary for this post-translational process remain to be completely elucidated. At steady state, glycosylated PrP(c) is found primarily at the cell surface, whereas a minor fraction of the population is disposed of by the ER-associated degradation-proteasome pathway. However, chronic ER stress conditions and proteasomal dysfunctions lead to accumulation of aggregation-prone PrP molecules in the cytosol and to neurodegeneration. In this study, we challenged different cell lines by inducing ER stress or inhibiting proteasomal activity and analyzed the subsequent repercussion on PrP metabolism, focusing on PrP in the secretory pathway. Both events led to enhanced detection of PrP aggregates and a significant increase of PrP(Sc) in persistently prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. Remarkably, upon proteasomal impairment, an increased fraction of misfolded, fully glycosylated PrP molecules traveled through the secretory pathway and reached the plasma membrane. These findings suggest a novel pathway that possibly provides additional substrate and template necessary for prion formation when protein clearance by the proteasome is impaired.
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Retículo Endoplásmico/metabolismo , Proteínas PrPC/metabolismo , Enfermedades por Prión/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estrés Fisiológico , Respuesta de Proteína Desplegada , Animales , Línea Celular Tumoral , Retículo Endoplásmico/genética , Glicosilación , Ratones , Proteínas PrPC/genética , Enfermedades por Prión/genética , Complejo de la Endopetidasa Proteasomal/genética , Transporte de Proteínas/genéticaRESUMEN
Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the ß-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.
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Priones/química , Priones/metabolismo , Animales , Línea Celular , Dicroismo Circular , Disulfuros/síntesis química , Disulfuros/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Mutación , Priones/genética , Priones/ultraestructuraRESUMEN
Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained soluble, addition of fibrils of bacterially produced Sup35NM to the medium efficiently induced appearance of phenotypically and biochemically distinct NM-HA aggregates that were inherited by daughter cells. Importantly, NM-HA aggregates also were infectious to recipient mammalian cells expressing soluble NM-HA and, to a lesser extent, to yeast. The fact that the yeast Sup35NM domain can propagate as a prion in neuroblastoma cells strongly argues that cellular mechanisms support prion-like inheritance in the mammalian cytosol.
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Neuroblastoma/patología , Enfermedades por Prión/transmisión , Priones/biosíntesis , Proteínas de Saccharomyces cerevisiae/efectos adversos , Animales , Ratones , Técnicas de Sonda Molecular , Factores de Terminación de Péptidos , Priones/efectos adversos , Células Tumorales CultivadasRESUMEN
Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.
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Amiloide/metabolismo , Agregación Patológica de Proteínas/patología , ARN/metabolismo , Proteínas tau/metabolismo , Amiloide/ultraestructura , Técnicas Biosensibles , Humanos , Resonancia Magnética Nuclear Biomolecular , ARN/ultraestructura , ARN de Hongos/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Proteínas tau/aislamiento & purificación , Proteínas tau/ultraestructuraRESUMEN
Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.
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Enzima Convertidora de Angiotensina 2/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Agregación Patológica de Proteínas/virología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Adulto , Anciano , Encéfalo/patología , Estudios de Casos y Controles , Línea Celular , Endocitosis , Femenino , Humanos , Microscopía Intravital , Masculino , Persona de Mediana Edad , Priones/metabolismo , Agregación Patológica de Proteínas/patología , Pliegue de Proteína , Proteínas tau/metabolismoRESUMEN
Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.
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Replicación del ADN/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Modelos Genéticos , Complejo de Reconocimiento del Origen/genética , Línea Celular Tumoral , Humanos , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Complejo de Reconocimiento del Origen/metabolismoRESUMEN
The cellular prion protein (PrP(c)) is a glycosyl-phosphatidylinositol (GPI)-anchored protein trafficking in the secretory and endocytic pathway and localized mainly at the plasma membrane. Conversion of PrP(c) into its pathogenic isoform PrP(Sc) is associated with pathogenesis and transmission of prion diseases. Intramolecular cleavage in the middle, the extreme C-terminal part or within the GPI anchor and shedding of PrP(c) modulate this conversion process by reducing the substrate for prion formation. These phenomena provide similarities with the processing of amyloid precursor protein in Alzheimer's disease. Sorting nexins are a family of proteins with important functions in protein trafficking. In this study, we investigated the role of the newly described sorting nexin 33 (SNX33) in trafficking and processing of PrP(c). We found that overexpression of SNX33 in neuronal and non-neuronal cell lines resulted in increased shedding of full-length PrP(c) from the plasma membrane and modulated the rate of PrP(c) endocytosis. This was paralleled by reduction of PrP(Sc) formation in persistently and newly infected cells. Using deletion mutants, we demonstrate that production of PrP fragment N1 is not influenced by SNX33. Our data provide new insights into the cellular mechanisms of PrP(c) shedding and show how this can affect cellular PrP(Sc) conversion.
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Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Priones/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/fisiología , Amiloide/química , Animales , Biotinilación , Encéfalo/metabolismo , Proteínas Portadoras/química , Línea Celular , Línea Celular Tumoral , Endocitosis , Eliminación de Gen , Humanos , Ratones , Modelos Biológicos , Enfermedades por Prión/metabolismo , Priones/metabolismo , Transporte de Proteínas , Nexinas de Clasificación , Proteínas de Transporte Vesicular/químicaRESUMEN
Prion diseases are neurodegenerative diseases associated with the accumulation of a pathogenic isoform of the host-encoded prion protein. The cellular responses to prion infection are not well defined. By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis.