RESUMEN
Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.
Asunto(s)
Aclimatación , Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Frío , ARN del Cloroplasto , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Aclimatación/genética , ARN del Cloroplasto/genética , ARN del Cloroplasto/metabolismo , Cloroplastos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Regulación de la Expresión Génica de las Plantas , Priones/metabolismo , Priones/genética , Dominios Proteicos , Empalme del ARN/genética , Separación de FasesRESUMEN
Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.
Asunto(s)
Priones , Tauopatías , Humanos , Proteínas tau/metabolismo , Tauopatías/metabolismo , Isoformas de Proteínas/metabolismo , Priones/metabolismo , Péptidos , AminoácidosRESUMEN
Since prion diseases result from infection and neurodegeneration of the central nervous system (CNS), experimental characterizations of prion strain properties customarily rely on the outcomes of intracerebral challenges. However, natural transmission of certain prions, including those causing chronic wasting disease (CWD) in elk and deer, depends on propagation in peripheral host compartments prior to CNS infection. Using gene-targeted GtE and GtQ mice, which accurately control cellular elk or deer PrP expression, we assessed the impact that peripheral or intracerebral exposures play on CWD prion strain propagation and resulting CNS abnormalities. Whereas oral and intraperitoneal transmissions produced identical neuropathological outcomes in GtE and GtQ mice and preserved the naturally convergent conformations of elk and deer CWD prions, intracerebral transmissions generated CNS prion strains with divergent biochemical properties in GtE and GtQ mice that were changed compared to their native counterparts. While CWD replication kinetics remained constant during iterative peripheral transmissions and brain titers reflected those found in native hosts, serial intracerebral transmissions produced 10-fold higher prion titers and accelerated incubation times. Our demonstration that peripherally and intracerebrally challenged Gt mice develop dissimilar CNS diseases which result from the propagation of distinct CWD prion strains points to the involvement of tissue-specific cofactors during strain selection in different host compartments. Since peripheral transmissions preserved the natural features of elk and deer prions, whereas intracerebral propagation produced divergent strains, our findings illustrate the importance of experimental characterizations using hosts that not only abrogate species barriers but also accurately recapitulate natural transmission routes of native strains.
Asunto(s)
Encéfalo , Ciervos , Priones , Enfermedad Debilitante Crónica , Animales , Enfermedad Debilitante Crónica/transmisión , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Priones/metabolismo , Priones/genética , Priones/patogenicidad , Ratones TransgénicosRESUMEN
Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.
Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Agregado de Proteínas/genética , Priones/genética , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estrés Oxidativo/genética , Amiloide/metabolismo , Oxidantes/farmacología , Oxidantes/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismoRESUMEN
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.
Asunto(s)
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
Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.
Asunto(s)
Encéfalo , Ciervos , Proteínas PrPSc , Enfermedad Debilitante Crónica , Animales , Ratones , Enfermedad Debilitante Crónica/transmisión , Encéfalo/metabolismo , Encéfalo/patología , Proteínas PrPSc/metabolismo , Conformación Proteica , Priones/metabolismo , Priones/patogenicidad , Enfermedades por Prión/transmisión , Enfermedades por Prión/patología , Enfermedades por Prión/metabolismo , Ratones TransgénicosRESUMEN
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.
Asunto(s)
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
Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Priones , Animales , Ratones , Priones/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Síndrome de Creutzfeldt-Jakob/tratamiento farmacológico , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Encéfalo/patología , Arvicolinae/metabolismoRESUMEN
Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.
Asunto(s)
Ciervos , Priones , Enfermedad Debilitante Crónica , Animales , Ratones , Priones/metabolismo , Enfermedad Debilitante Crónica/metabolismo , Ciervos/metabolismo , SueloRESUMEN
Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative disease pathogenesis. Phagocytic glia are responsible for regulating the load of pathological proteins in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. A forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings uncover new mechanisms that enhance our understanding of the beneficial and harmful effects of phagocytic glia in HD and other neurodegenerative diseases.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de Drosophila , Drosophila , Proteína Huntingtina , Enfermedad de Huntington , Neuroglía , Animales , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Enfermedad de Huntington/genética , Neuroglía/metabolismo , Neuroglía/patología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Femenino , Masculino , Fagocitosis/fisiología , Lisosomas/metabolismo , Fagosomas/metabolismo , Animales Modificados Genéticamente , Priones/metabolismo , Priones/genética , Neuronas/metabolismoRESUMEN
Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.
Asunto(s)
Proteínas PrPSc , Pliegue de Proteína , Animales , Pliegue de Proteína/efectos de los fármacos , Proteínas PrPSc/metabolismo , Proteínas PrPSc/química , Ratones , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/metabolismo , Priones/metabolismoRESUMEN
Chaperone-mediated autophagy (CMA), a proteolytic system contributing to the degradation of intracellular proteins in lysosomes, is upregulated in tumors for pro-tumorigenic and pro-survival purposes. In this study, bioinformatics analysis revealed the co-occurrence of upregulated CMA and PD-L1 accumulation in metastatic melanoma with adaptive immune resistance (AIR) to anti-PD1 treatment, suggesting the potential therapeutic effects of rewiring CMA for PD-L1 degradation. Furthermore, this co-occurrence is attributed to IFN-γ-mediated compensatory up-regulation of PD-L1 and CMA, accompanied by enhanced macropinocytosis. Drawing inspiration from the cellular uptake of prions via macropinocytosis, a prion-like chemical inducer of proximity called SAP was engineered using self-assembly of the designed chiral peptide PHA. By exploiting sensitized macropinocytosis, SAP clandestinely infiltrates tumor cells and subsequently disintegrates into PHA, which reprograms CMA by inducing PD-L1 close to HSPA8. SAP degrades PD-L1 in a CMA-dependent manner and effectively restores the anti-tumor immune response in both allografting and Hu-PDX melanoma mouse models with AIR while upholding a high safety profile. Collectively, the reported SAP not only presents an immune reactivation strategy with clinical translational potential for overcoming AIR in cutaneous melanomas but serves as a reproducible example of precision-medicine-guided drug development that fully leverages specific cellular indications in pathological states.
Asunto(s)
Autofagia Mediada por Chaperones , Melanoma , Priones , Ratones , Animales , Antígeno B7-H1/metabolismo , Melanoma/metabolismo , Priones/metabolismo , Lisosomas/metabolismoRESUMEN
BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.
Asunto(s)
Encéfalo , Síndrome de Creutzfeldt-Jakob , Cabras , Ratones Transgénicos , Priones , Scrapie , Zoonosis , Animales , Síndrome de Creutzfeldt-Jakob/transmisión , Síndrome de Creutzfeldt-Jakob/patología , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Scrapie/transmisión , Scrapie/patología , Ratones , Zoonosis/transmisión , Encéfalo/patología , Encéfalo/metabolismo , Ovinos , Bovinos , Priones/metabolismo , Fenotipo , Bazo/patología , Encefalopatía Espongiforme Bovina/transmisión , Encefalopatía Espongiforme Bovina/patología , Encefalopatía Espongiforme Bovina/metabolismo , Enfermedades de las Cabras/transmisión , Enfermedades de las Cabras/patología , Modelos Animales de EnfermedadRESUMEN
Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.
Asunto(s)
Proteínas Fúngicas , Dominios Proteicos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Candida albicans/metabolismo , Priones/metabolismo , Priones/química , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Separación de Fases , Proteínas de Unión al ADNRESUMEN
BACKGROUND: Prion diseases, also known as transmissible spongiform encephalopathies (TSEs) remain one of the deleterious disorders, which have affected several animal species. Polymorphism of the prion protein (PRNP) gene majorly determines the susceptibility of animals to TSEs. However, only limited studies have examined the variation in PRNP gene in different Nigerian livestock species. Thus, this study aimed to identify the polymorphism of PRNP gene in Nigerian livestock species (including camel, dog, horse, goat, and sheep). We sequenced the open reading frame (ORF) of 65 camels, 31 village dogs and 12 horses from Nigeria and compared with PRNP sequences of 886 individuals retrieved from public databases. RESULTS: All the 994 individuals were assigned into 162 haplotypes. The sheep had the highest number of haplotypes (n = 54), and the camel had the lowest (n = 7). Phylogenetic tree further confirmed clustering of Nigerian individuals into their various species. We detected five non-synonymous SNPs of PRNP comprising of G9A, G10A, C11G, G12C, and T669C shared by all Nigerian livestock species and were in Hardy-Weinberg Equilibrium (HWE). The amino acid changes in these five non-synonymous SNP were all "benign" via Polyphen-2 program. Three SNPs G34C, T699C, and C738G occurred only in Nigerian dogs while C16G, G502A, G503A, and C681A in Nigerian horse. In addition, C50T was detected only in goats and sheep. CONCLUSION: Our study serves as the first to simultaneously investigate the polymorphism of PRNP gene in Nigerian livestock species and provides relevant information that could be adopted in programs targeted at breeding for prion diseases resistance.
Asunto(s)
Enfermedades por Prión , Priones , Scrapie , Animales , Caballos/genética , Ovinos/genética , Perros , Priones/genética , Priones/metabolismo , Proteínas Priónicas/genética , Polimorfismo de Nucleótido Simple , Ganado/genética , Sistemas de Lectura Abierta , Filogenia , Camelus/genética , Enfermedades por Prión/genética , Enfermedades por Prión/veterinaria , Cabras/genética , Cabras/metabolismo , Scrapie/genéticaRESUMEN
Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.
Asunto(s)
Organoides , Priones , Enfermedad Debilitante Crónica , Enfermedad Debilitante Crónica/transmisión , Humanos , Priones/metabolismo , Animales , Encéfalo/patología , GenotipoRESUMEN
Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 µm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, ß-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.
Asunto(s)
Diferenciación Celular , Enfermedades por Prión , Esferoides Celulares , Esferoides Celulares/metabolismo , Ratones , Animales , Diferenciación Celular/fisiología , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Línea Celular , Técnicas de Cultivo de Célula/métodos , Neuronas/metabolismo , Técnicas de Cultivo Tridimensional de Células/métodos , Priones/metabolismoRESUMEN
AIM: CH1641 was discovered in 1970 as a scrapie isolate that was unlike all other classical strains of scrapie isolated so far. We performed bio-assays of CH1641 in mice in order to further characterise this specific isolate. METHODS: We inoculated the original CH1641 isolate into ovine and bovine prion protein (PrP) transgenic mice as well as wild-type mice. In addition, we performed cross- and back passages between the various mouse lines to examine if one identical prion strain was isolated in all mouse lines or whether multiple prion strains exist in CH1641. RESULTS: We report the first successful transmission of CH1641 to wild-type RIII mice and via RIII mice to wild-type VM mice. Unexpectedly, analysis of the protease-resistant prion protein (PrPres ) in wild-type mice showed a classical scrapie banding pattern differing from the banding pattern of the original CH1641 isolate. Cross- and back passages of CH1641 between the various mouse lines confirmed that the same prion strain had been isolated in all mouse lines. CONCLUSIONS: The CH1641 isolate consists of a single prion strain but its molecular banding pattern of PrPres differs between wild-type mice and PrP transgenic mice. Consequently, molecular banding patterns of PrPres should be used with caution in strain typing since they do not solely depend on the properties of the prion strain but also on the host prion protein.
Asunto(s)
Priones , Scrapie , Ratones , Animales , Bovinos , Ovinos , Priones/metabolismo , Scrapie/metabolismo , Proteínas Priónicas/genética , Proteínas PrPSc/metabolismo , Ratones TransgénicosRESUMEN
Both wild-type and mutant tau proteins can misfold into prions and self-propagate in the central nervous system of animals and people. To extend the work of others, we investigated the molecular basis of tau prion-mediated neurodegeneration in transgenic (Tg) rats expressing mutant human tau (P301S); this line of Tg rats is denoted Tg12099. We used the rat Prnp promoter to drive the overexpression of mutant tau (P301S) in the human 0N4R isoform. In Tg12099(+/+) rats homozygous for the transgene, ubiquitous expression of mutant human tau resulted in the progressive accumulation of phosphorylated tau inclusions, including silver-positive tangles in the frontal cortices and limbic system. Signs of central nervous system dysfunction were found in terminal Tg12099(+/+) rats exhibiting severe neurodegeneration and profound atrophy of the amygdala and piriform cortex. The greatest increases in tau prion activity were found in the corticolimbic structures. In contrast to the homozygous Tg12099(+/+) rats, we found lower levels of mutant tau in the hemizygous rats, resulting in few neuropathologic changes up to 2 years of age. Notably, these hemizygous rats could be infected by intracerebral inoculation with recombinant tau fibrils or precipitated tau prions from the brain homogenates of sick, aged homozygous Tg12099(+/+) rats. Our studies argue that the regional propagation of tau prions and neurodegeneration in the Tg12099 rats resembles that found in human primary tauopathies. These findings seem likely to advance our understanding of human tauopathies and may lead to effective therapeutics for Alzheimer's disease and other tau prion disorders.
Asunto(s)
Encéfalo , Ratas Transgénicas , Proteínas tau , Animales , Proteínas tau/metabolismo , Proteínas tau/genética , Humanos , Ratas , Encéfalo/patología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Priones/metabolismo , Priones/genética , Tauopatías/patología , Tauopatías/metabolismo , Tauopatías/genética , Degeneración Nerviosa/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , MutaciónRESUMEN
BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor that exhibits resistance to current treatment, making the identification of novel therapeutic targets essential. In this context, cellular prion protein (PrPC) stands out as a potential candidate for new therapies. Encoded by the PRNP gene, PrPC can present increased expression levels in GBM, impacting cell proliferation, growth, migration, invasion and stemness. Nevertheless, the exact molecular mechanisms through which PRNP/PrPC modulates key aspects of GBM biology remain elusive. METHODS: To elucidate the implications of PRNP/PrPC in the biology of this cancer, we analyzed publicly available RNA sequencing (RNA-seq) data of patient-derived GBMs from four independent studies. First, we ranked samples profiled by bulk RNA-seq as PRNPhigh and PRNPlow and compared their transcriptomic landscape. Then, we analyzed PRNP+ and PRNP- GBM cells profiled by single-cell RNA-seq to further understand the molecular context within which PRNP/PrPC might function in this tumor. We explored an additional proteomics dataset, applying similar comparative approaches, to corroborate our findings. RESULTS: Functional profiling revealed that vesicular dynamics signatures are strongly correlated with PRNP/PrPC levels in GBM. We found a panel of 73 genes, enriched in vesicle-related pathways, whose expression levels are increased in PRNPhigh/PRNP+ cells across all RNA-seq datasets. Vesicle-associated genes, ANXA1, RAB31, DSTN and SYPL1, were found to be upregulated in vitro in an in-house collection of patient-derived GBM. Moreover, proteome analysis of patient-derived samples reinforces the findings of enhanced vesicle biogenesis, processing and trafficking in PRNPhigh/PRNP+ GBM cells. CONCLUSIONS: Together, our findings shed light on a novel role for PrPC as a potential modulator of vesicle biology in GBM, which is pivotal for intercellular communication and cancer maintenance. We also introduce GBMdiscovery, a novel user-friendly tool that allows the investigation of specific genes in GBM biology.