RESUMEN
The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Priónicas/metabolismo , Priones/metabolismo , Animales , Arvicolinae , Sistema Nervioso Central/patología , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Ratones Transgénicos , Enfermedades por Prión/patología , Estructura Terciaria de Proteína , Deficiencias en la Proteostasis/patologíaRESUMEN
Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.
Asunto(s)
Enfermedades por Prión , Priones , Animales , Priones/metabolismo , Proteínas Priónicas/genética , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Mamíferos/metabolismo , Pliegue de ProteínaRESUMEN
Coronaviruses (CoVs) of genera α, ß, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Ratones , Humanos , SARS-CoV-2/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Pulmón/metabolismo , ARN MensajeroRESUMEN
Among transmissible spongiform encephalopathies or prion diseases affecting humans, sporadic forms such as sporadic Creutzfeldt-Jakob disease are the vast majority. Unlike genetic or acquired forms of the disease, these idiopathic forms occur seemingly due to a random event of spontaneous misfolding of the cellular PrP (PrPC) into the pathogenic isoform (PrPSc). Currently, the molecular mechanisms that trigger and drive this event, which occurs in approximately one individual per million each year, remain completely unknown. Modelling this phenomenon in experimental settings is highly challenging due to its sporadic and rare occurrence. Previous attempts to model spontaneous prion misfolding in vitro have not been fully successful, as the spontaneous formation of prions is infrequent and stochastic, hindering the systematic study of the phenomenon. In this study, we present the first method that consistently induces spontaneous misfolding of recombinant PrP into bona fide prions within hours, providing unprecedented possibilities to investigate the mechanisms underlying sporadic prionopathies. By fine-tuning the Protein Misfolding Shaking Amplification method, which was initially developed to propagate recombinant prions, we have created a methodology that consistently produces spontaneously misfolded recombinant prions in 100% of the cases. Furthermore, this method gives rise to distinct strains and reveals the critical influence of charged surfaces in this process.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Priones , Humanos , Imagen por Resonancia Magnética , TemblorRESUMEN
Coronaviruses (CoVs) have the largest genome among RNA viruses and store large amounts of information without genome integration as they replicate in the cell cytoplasm. The replication of the virus is a continuous process, whereas the transcription of the subgenomic mRNAs is a discontinuous one, involving a template switch, which resembles a high frequency recombination mechanism that may favor virus genome variability. The origin of the three deadly human CoVs SARS-CoV, MERS-CoV and SARS-CoV-2 are zoonotic events. SARS-CoV-2 has incorporated in its spike protein a furine proteolytic site that facilitates the activation of the virus in any tissue, making this CoV strain highly polytropic and pathogenic. Using MERS-CoV as a model, a propagation-deficient RNA replicon was generated by removing E protein gene (essential for viral morphogenesis and involved in virulence), and accessory genes 3, 4a, 4b and 5 (responsible for antagonism of the innate immune response) to attenuate the virus: MERS-CoV-Δ[3,4a,4b,5,E]. This RNA replicon is strongly attenuated and elicits sterilizing protection after a single immunization in transgenic mice with the receptor for MERS-CoV, making it a promising vaccine candidate for this virus and an interesting platform for vector-based vaccine development. A strategy could be developed for the design of RNA replicon vaccines for other human pathogenic coronaviruses.
RESUMEN
Transmissible spongiform encephalopathies or prion diseases are rapidly progressive neurodegenerative diseases, the clinical manifestation of which can resemble other promptly evolving neurological maladies. Therefore, the unequivocal ante-mortem diagnosis is highly challenging and was only possible by histopathological and immunohistochemical analysis of the brain at necropsy. Although surrogate biomarkers of neurological damage have become invaluable to complement clinical data and provide more accurate diagnostics at early stages, other neurodegenerative diseases show similar alterations hindering the differential diagnosis. To solve that, the detection of the pathognomonic biomarker of disease, PrPSc, the aberrantly folded isoform of the prion protein, could be used. However, the amounts in easily accessible tissues or body fluids at pre-clinical or early clinical stages are extremely low for the standard detection methods. The solution comes from the recent development of in vitro prion propagation techniques, such as Protein Misfolding Cyclic Amplification (PMCA) and Real Time-Quaking Induced Conversion (RT-QuIC), which have been already applied to detect minute amounts of PrPSc in different matrixes and make early diagnosis of prion diseases feasible in a near future. Herein, the most relevant tissues and body fluids in which PrPSc has been detected in animals and humans are being reviewed, especially those in which cell-free prion propagation systems have been used with diagnostic purposes.
Asunto(s)
Proteínas PrPSc/metabolismo , Enfermedades por Prión/diagnóstico , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Humanos , Proteínas PrPSc/química , Enfermedades por Prión/metabolismoRESUMEN
Transmissible spongiform encephalopathies (TSEs) are a group of invariably fatal neurodegenerative disorders. The causal agent is an aberrantly folded isoform (PrPSc or prion) of the endogenous prion protein (PrPC) which is neurotoxic and amyloidogenic and induces misfolding of its physiological counterpart. The intrinsic physical characteristics of these infectious proteinaceous pathogens makes them highly resistant to the vast majority of physicochemical decontamination procedures used typically for standard disinfection. This means prions are highly persistent in contaminated tissues, the environment (surfaces) and, of great concern, on medical and surgical instruments. Traditionally, decontamination procedures for prions are tested on natural isolates coming from the brain of infected individuals with an associated high heterogeneity resulting in highly variable results. Using our novel ability to produce highly infectious recombinant prions in vitro we adapted the system to enable recovery of infectious prions from contaminated materials. This method is easy to perform and, importantly, results in highly reproducible propagation in vitro. It exploits the adherence of infectious prion protein to beads of different materials allowing accurate and repeatable assessment of the efficacy of disinfectants of differing physicochemical natures to eliminate infectious prions. This method is technically easy, requires only a small shaker and a standard biochemical technique and could be performed in any laboratory.
RESUMEN
The misfolding of the cellular prion protein (PrPC) into the disease-associated isoform (PrPSc) and its accumulation as amyloid fibrils in the central nervous system is one of the central events in transmissible spongiform encephalopathies (TSEs). Due to the proteinaceous nature of the causal agent the molecular mechanisms of misfolding, interspecies transmission, neurotoxicity and strain phenomenon remain mostly ill-defined or unknown. Significant advances were made using in vivo and in cellula models, but the limitations of these, primarily due to their inherent complexity and the small amounts of PrPSc that can be obtained, gave rise to the necessity of new model systems. The production of recombinant PrP using E. coli and subsequent induction of misfolding to the aberrant isoform using different techniques paved the way for the development of cell-free systems that complement the previous models. The generation of the first infectious recombinant prion proteins with identical properties of brain-derived PrPSc increased the value of cell-free systems for research on TSEs. The versatility and ease of implementation of these models have made them invaluable for the study of the molecular mechanisms of prion formation and propagation, and have enabled improvements in diagnosis, high-throughput screening of putative anti-prion compounds and the design of novel therapeutic strategies. Here, we provide an overview of the resultant advances in the prion field due to the development of recombinant PrP and its use in cell-free systems.