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1.
Biochim Biophys Acta Mol Basis Dis ; 1867(11): 166209, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34246750

RESUMO

Tauopathies, including Alzheimer's disease (AD), are manifested by the deposition of well-characterized amyloid aggregates of Tau protein in the brain. However, it is rather unlikely that these aggregates constitute the major form of Tau responsible for neurodegenerative changes. Currently, it is postulated that the intermediates termed as soluble oligomers, assembled on the amyloidogenic pathway, are the most neurotoxic form of Tau. However, Tau oligomers reported so far represent a population of poorly characterized, heterogeneous and unstable assemblies. In this study, to obtain the oligomers, we employed the aggregation-prone K18 fragment of Tau protein with deletion of Lys280 (K18Δ280) linked to a hereditary tauopathy. We have described a new procedure of inducing aggregation of mutated K18 which leads either to the formation of nontoxic amyloid fibrils or neurotoxic globular oligomers, depending on its phosphorylation status. We demonstrate that PKA-phosphorylated K18Δ280 oligomers are toxic to hippocampal neurons, which is manifested by loss of dendritic spines and neurites, and impairment of cell-membrane integrity leading to cell death. We also show that N1, the soluble N-terminal fragment of prion protein (PrP), protects neurons from the oligomers-induced cytotoxicity. Our findings support the hypothesis on the neurotoxicity of Tau oligomers and neuroprotective role of PrP-derived fragments in AD and other tauopathies. These observations could be useful in the development of therapeutic strategies for these diseases.


Assuntos
Neurônios/patologia , Proteínas Priônicas/metabolismo , Agregação Patológica de Proteínas/patologia , Tauopatias/patologia , Proteínas tau/metabolismo , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/patologia , Humanos , Fosforilação , Cultura Primária de Células , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Agregação Patológica de Proteínas/genética , Ligação Proteica , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tauopatias/genética , Proteínas tau/genética , Proteínas tau/isolamento & purificação
2.
Vopr Virusol ; 65(6): 326-334, 2021 Jan 07.
Artigo em Russo | MEDLINE | ID: mdl-33533229

RESUMO

The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.


Assuntos
Autopsia , Doenças Priônicas/diagnóstico , Proteínas Priônicas/genética , Doença de Emaciação Crônica/genética , Sequência de Aminoácidos/genética , Animais , Cervos/genética , Humanos , Doenças Priônicas/genética , Doenças Priônicas/patologia , Proteínas Priônicas/isolamento & purificação , Federação Russa , Doença de Emaciação Crônica/patologia
3.
Biologicals ; 67: 56-61, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32773163

RESUMO

Heparin is an anticoagulant sourced from animal tissues. In the 1990s, bovine-sourced heparin was withdrawn from the U.S. market due to a theoretical concern that the bovine spongiform encephalopathy (BSE) agent might contaminate crude heparin and spread to humans as variant Creutzfeldt-Jakob disease. Only porcine intestinal heparin is now marketed in the U.S. FDA has encouraged the reintroduction of bovine heparin. We applied a scaled-down laboratory model process to produce heparin as an active pharmaceutical ingredient (API) starting from bovine intestinal mucosa. The process consisted of two phases. To model the first phase, we applied enzymatic proteolysis, anionic resin separation and methanol precipitation of crude heparin. Bovine intestinal mucosa was spiked with BSE or scrapie agents. We assayed BSE- or scrapie-associated prion protein (PrPTSE) using the Real-Time Quaking-Induced Conversion (RT-QuIC) assay at each step. The process reduced PrPTSE by 4 log10 and 6 log10 from BSE-spiked and scrapie-spiked mucosa, respectively. To model the entire process, we spiked mucosa with scrapie agent and produced heparin API, reducing PrPTSE by 6.7 log10. The purification processes removed large amounts of PrPTSE from the final products. Heparin purification together with careful sourcing of raw materials should allow safely reintroducing bovine heparin in the U.S.


Assuntos
Produtos Biológicos/metabolismo , Encefalopatia Espongiforme Bovina/metabolismo , Heparina/isolamento & purificação , Mucosa Intestinal/metabolismo , Proteínas Priônicas/isolamento & purificação , Príons/metabolismo , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Bovinos , Contaminação de Medicamentos/prevenção & controle , Heparina/metabolismo , Heparina/farmacologia , Humanos , Proteínas Priônicas/metabolismo , Medição de Risco/métodos
4.
J Biol Chem ; 295(25): 8460-8469, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358064

RESUMO

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.


Assuntos
Encéfalo/metabolismo , Lipídeos/química , Maleatos/química , Nanoestruturas/química , Poliestirenos/química , Proteínas Priônicas/metabolismo , Animais , Cricetinae , Maleatos/síntese química , Maleatos/metabolismo , Metilaminas/química , Camundongos , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Poliestirenos/síntese química , Poliestirenos/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/isolamento & purificação , Compostos de Sulfidrila/química
5.
Eur J Med Chem ; 196: 112295, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32325366

RESUMO

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of rare neurodegenerative disorders. TSEs are characterized by the accumulation of prions (PrPSc) that represent pathological isoforms of the physiological cellular prion protein PrPC. Although the conversion of PrPC to PrPSc is still not completely understood, blocking this process may lead to develop new therapies. Here, we have generated a pharmacophore model, based on anti-prion molecules reported in literature to be effective in phenotypic assay. The model was used to conduct a virtual screen of commercial compound databases that selected a small library of ten compounds. These molecules were then screened in mouse neuroblastoma cell line chronically infected with prions (ScN2a) after excluding neurotoxicity. 1 has been identified as the therapeutic hit on the basis of the following evidence: chronic treatments of ScN2a cells using 1 eliminate PrPSc loaded in both Western blotting analysis and Real-Time Quaking-Induced Conversion (RT-QuIC) assay. We also proposed the mechanism of action of 1 by which it has the ability to bind PrPC and consequentially blocks prion conversion. Herein we describe the results of these efforts.


Assuntos
Fenotiazinas/farmacologia , Proteínas Priônicas/antagonistas & inibidores , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Camundongos , Modelos Moleculares , Estrutura Molecular , Fenotiazinas/química , Proteínas Priônicas/isolamento & purificação , Proteínas Priônicas/metabolismo , Relação Quantitativa Estrutura-Atividade
6.
FASEB J ; 34(1): 365-385, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914616

RESUMO

Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP90-231) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP90-231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP90-231 phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.


Assuntos
Amiloide/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Extração Líquido-Líquido/métodos , Doenças Priônicas/patologia , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Animais , Camundongos , Conformação de Ácido Nucleico , Doenças Priônicas/metabolismo , Proteínas Priônicas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnica de Seleção de Aptâmeros
7.
Biochem Biophys Res Commun ; 521(1): 259-264, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31635805

RESUMO

Protein misfolding diseases are a group of devastating disorders characterized by structural conversion of a soluble protein into an amyloid-like aggregate. Typically, the structural conversion occurs by misfolding of a single disease-associated protein, such as α-synuclein (αS) in Parkinson's disease, amyloid-ß in Alzheimer's disease, and prion protein (PrP) in transmissible spongiform encephalopathies (TSEs). However, accumulating evidence has implicated that cross-interactions between heterologous amyloidogenic proteins dramatically impact on amyloidogenesis and disease pathology. Here we show αS in a monomeric state can suppress amyloidogenesis of PrP in vitro. Thioflavin-T assays and transmission electron miscopy revealed that monomeric αS inhibits the nucleation step of amyloidogenesis without inhibiting the growing step. Surface plasmon resonance and co-sedimentation assays neither detected interaction between αS and monomeric PrP nor fibrillar PrP. These results suggested that αS suppress amyloidogenesis of PrP by binding to a transiently accumulated intermediate, such as a partially unfolded state. Moreover, we found that oligomeric αS, which was recently suggested to interact with PrP, also did not interact with PrP. Taken together, our study revealed a chaperon-like activity of αS against PrP amyloidogenesis, suggesting a possible involvement of αS in the pathology of TSEs.


Assuntos
Amiloidose/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Priônicas/metabolismo , alfa-Sinucleína/metabolismo , Humanos , Proteínas Priônicas/biossíntese , Proteínas Priônicas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/biossíntese , alfa-Sinucleína/isolamento & purificação
8.
Sci Rep ; 9(1): 14656, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601984

RESUMO

In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.


Assuntos
Células Dendríticas Foliculares/metabolismo , Granuloma/patologia , Doenças Priônicas/patologia , Proteínas Priônicas/metabolismo , Animais , Antígenos de Superfície/metabolismo , Modelos Animais de Doenças , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Proteínas Priônicas/toxicidade , Dobramento de Proteína , Ovinos , Baço/citologia , Tropismo
9.
J Vet Med Sci ; 81(6): 846-850, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-30982806

RESUMO

Atypical bovine spongiform encephalopathy (BSE), first identified in 2004, poses a threat due to the potential to spread the disease to cattle and other animals, including humans. Here, we estimated prion titers in various tissues of cattle infected with atypical BSE using a real-time quaking-induced conversion assay that detects amyloid seeding activity of a disease-specific prion protein, PrPSc, a major component of prions. PrPSc was detected both in and outside of nerve tissues, and some of the peripheral nerve tissues contained relatively high prion titers. Low titers of prions were also observed in masseter, jejunum, and adrenal glands. Quantitative data on prion infectivity in tissues of atypical BSE-affected cattle is useful to assess the risk of atypical BSE.


Assuntos
Encefalopatia Espongiforme Bovina , Immunoblotting/veterinária , Proteínas Priônicas/isolamento & purificação , Animais , Bovinos , Immunoblotting/métodos , Nervos Periféricos , Proteínas Priônicas/metabolismo , Príons/isolamento & purificação , Príons/patogenicidade , Distribuição Tecidual
10.
Rev. Soc. Bras. Clín. Méd ; 17(1): 31-34, jan.-mar. 2019. ilus.
Artigo em Português | LILACS | ID: biblio-1026152

RESUMO

As doenças priônicas fazem parte do grupo das síndromes de demência rapidamente progressiva com neurodegeneração. Em humanos, a doença de Creutzfeldt-Jakob é a mais prevalente. Atualmente, seu diagnóstico pode ser baseado em uma combinação do quadro clínico, ressonância magnética e eletroencefalograma com alterações típicas, juntamente da detecção de proteína 14- 3-3 no líquido cefalorraquidiano. Este relato descreve o caso de uma paciente de 74 anos, natural de Ubá (MG), admitida em um hospital da mesma cidade com quadro de demência de rápida progressão, com declínio cognitivo, ataxia cerebelar e mioclonias. No contexto clínico, aventou-se a possibilidade de doença de Creutzfeldt-Jakob e, então, foi iniciada investigação para tal, com base nos critérios diagnósticos. Também foram realizados exames para descartar a possibilidade de doenças com sintomas semelhantes. O caso foi diagnosticado como forma esporádica de doença de Creutzfeldt-Jakob. (AU)


Prion diseases are part of the rapidly progressive dementia syndromes with neurodegeneration. In humans, Creutzfeldt-Jakob disease is the most prevalent. Currently, its diagnosis may be based on a combination of clinical picture, magnetic resonance imaging, and electroencephalogram with typical changes, along with the detection of 14-3-3 protein in cerebrospinal fluid. This report describes the case of a 74-year-old woman from the city of Ubá, in the state of Minas Gerais, who was admitted to a hospital in the same city with a rapidly progressive dementia, cognitive decline, cerebellar ataxia and myoclonus. In the clinical context, the possibility of Creutzfeldt-Jakob disease was raised, and then investigation was started for this disease, based on the its diagnostic criteria. Tests have also been conducted to rule out the possibility of diseases with similar symptoms. The case was diagnosed as a sporadic form of Creutzfeldt-Jakob disease. (AU)


Assuntos
Humanos , Feminino , Idoso , Síndrome de Creutzfeldt-Jakob/diagnóstico , Transtornos da Visão , Biópsia , Imunoquímica , Espectroscopia de Ressonância Magnética , Ataxia Cerebelar/etiologia , Western Blotting , Síndrome de Creutzfeldt-Jakob/complicações , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/diagnóstico por imagem , Evolução Fatal , Demência/etiologia , Afasia Acinética/etiologia , Tontura/etiologia , Eletroencefalografia , Cérebro/patologia , Disfunção Cognitiva/etiologia , Proteínas Priônicas/isolamento & purificação , Proteínas Priônicas/líquido cefalorraquidiano , Pneumonia Associada a Assistência à Saúde , Labirintite/etiologia , Mioclonia/etiologia
11.
Vet Res ; 50(1): 6, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670087

RESUMO

Chronic wasting disease (CWD) is a fatal prion disease which infects deer, elk and moose. CWD was first described as a wasting syndrome in captive deer in Colorado and Wyoming wildlife facilities from 1967 to 1979. Currently, CWD has been reported in 26 states of the USA, three Canadian provinces, South Korea, Norway and Finland. Since human consumption of cervids is common, it is critical to determine if CWD can infect humans. Published research, including epidemiologic studies and transmission studies using animal models, including transgenic mice that express human prion protein, have suggested existence of a strong species barrier between cervid CWD and humans. In the current study, we tested CWD transmission into two additional strains of transgenic mice (tg66 and tgRM). These mice over-express human prion protein at high levels and are highly sensitive to infection by human-tropic prions. One hundred and eight mice were inoculated intracerebrally with three different sources of CWD. After long periods of observation, brain tissues from CWD-inoculated mice were screened for evidence of prion infection by RT-QuIC, immunohistochemistry (IHC) and immunoblot. No IHC or immunoblot evidence was found to suggest transmission had occurred, and most mice were negative by RT-QuIC assay. However, four mice with inconsistent positive RT-QuIC reactions were detected. The seeding activity detected in these mice may represent a low level of CWD agent, suggesting a possible transfer of CWD infection. Alternatively, these results might be due to false positive reactions or residual CWD inoculum.


Assuntos
Cervos , Proteínas Priônicas/genética , Doença de Emaciação Crônica/transmissão , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteínas Priônicas/isolamento & purificação , Doença de Emaciação Crônica/patologia
12.
J Microbiol Biotechnol ; 28(10): 1749-1759, 2018 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-30196595

RESUMO

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with >95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.


Assuntos
Proteínas Priônicas/biossíntese , Proteínas Priônicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Animais , Cromatografia Líquida , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Humanos , Corpos de Inclusão , Camundongos , Proteínas Priônicas/química , Proteínas Priônicas/isolamento & purificação , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
13.
Brain ; 141(9): 2700-2710, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29985975

RESUMO

Mammalian prions propagate by template-directed misfolding and aggregation of normal cellular prion related protein PrPC as it converts into disease-associated conformers collectively referred to as PrPSc. Mammalian species may be permissive for prion disease because these hosts have co-evolved specific co-factors that assist PrPC conformational change and prion propagation. We have tested this hypothesis by examining whether faithful prion propagation occurs in the normally PrPC-null invertebrate host Drosophila melanogaster. Ovine PrP transgenic Drosophila exposed at the larval stage to ovine scrapie showed a progressive accumulation of transmissible prions in adult flies. Strikingly, the biological properties of distinct ovine prion strains were maintained during their propagation in Drosophila. Our observations show that the co-factors necessary for strain-specific prion propagation are not unique to mammalian species. Our studies establish Drosophila as a novel host for the study of transmissible mammalian prions.


Assuntos
Proteínas de Drosophila/fisiologia , Proteínas Priônicas/fisiologia , Príons/fisiologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila melanogaster/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Doenças Priônicas/complicações , Proteínas Priônicas/biossíntese , Proteínas Priônicas/isolamento & purificação , Príons/biossíntese , Scrapie/metabolismo , Ovinos
14.
Methods Mol Biol ; 1779: 147-166, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29886532

RESUMO

Protein misfolding, aggregation, and amyloid formation is involved in a large number of diseases. Recombinantly expressed proteins to study the amyloid fibril formation process are important for mechanistic studies. We here report protocols for production, purification, and fibrillation of three different proteins commonly found in cerebral amyloid; Aß and Tau found in Alzheimer's disease, Chronic traumatic brain injury, Corticobasal degeneration, and Progressive Supranuclear Palsy and human prion protein found in Creutzfeldt-Jakob's disease. The three protocols have in common that the protein is in a pH-neutral phosphate saline buffer during fibrillation to mimic their endogenous near physiological environment.


Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Doenças Neurodegenerativas/metabolismo , Proteínas Priônicas/isolamento & purificação , Proteínas tau/isolamento & purificação , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Cromatografia por Troca Iônica , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Proteínas Priônicas/química , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas tau/química
15.
Methods Mol Biol ; 1779: 257-263, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29886538

RESUMO

A characteristic feature of transmissible spongiform encephalopathies (TSE) is the progressive accumulation of protein aggregates in the brain in a self-propagation manner. Based on this mechanism, in vitro protein amplification systems (such as real-time quaking-induced conversion (RT-QuIC)) for the detection of misfolded prion protein scrapie (PrPres) in CSF were a major step in pre-mortem diagnosis of human prion diseases. Here, we describe a protocol of the RT-QuIC assay to detect PrPres in CSF of prion disease patients. This methodology depends on prion seeds that induce misfolding and aggregation of a substrate by cycles of incubation and quaking. Besides diagnostics, further applications of the RT-QuIC appear to be promising for discrimination between different PrP subtypes or strains, understanding the mechanism of protein misfolding and pre-screening of anti-prion drugs. The technique can be further developed to be used to study characteristics of misfolded proteins in other "prion like" diseases, such as tauopathies, synucleinopathies, or amyloidopathies.


Assuntos
Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Imagem Óptica/métodos , Proteínas Priônicas/isolamento & purificação , Animais , Síndrome de Creutzfeldt-Jakob/metabolismo , Cricetinae , Corantes Fluorescentes/química , Humanos , Proteínas Priônicas/líquido cefalorraquidiano , Proteínas Priônicas/química
16.
Protein Expr Purif ; 150: 53-60, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29751084

RESUMO

Production of recombinant prion proteins is of crucial relevance in food technology (analytical standards, assay development) but also in basic research, most importantly structural biology (NMR, X-ray diffraction). Structural approaches conveniently allow for sophisticated investigation of prion disease pathogenesis, but usually require large amounts of sample material. Recently, working with recombinant prion proteins has been recategorized to biosafety levels > S1 as infectious prions may readily be generated de novo and become airborne via aerosols. Heterologous expression should therefore be established with appropriately adjusted safety precautions. We have developed a protocol for high-yield expression, purification and refolding of recombinant mammalian prion proteins at elevated biological safety levels by introducing means of abolishing aerosol formation and propagation.


Assuntos
Expressão Gênica , Proteínas Priônicas , Redobramento de Proteína , Humanos , Proteínas Priônicas/biossíntese , Proteínas Priônicas/química , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
17.
Prion ; 12(1): 63-71, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29310497

RESUMO

Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of diseases, from scrapie in sheep to chronic wasting disease (CWD) in cervids. The misfolded form of PrPC, termed PrPSc, or in this case PrPCWD, interacts with PrPC to create more PrPCWD. This process is not clearly defined but is affected by the presence and interactions of biotic and abiotic cofactors. These include nucleic acids, lipids, glycosylation, pH, and ionic character. PrPC has been shown to act as a copper-binding protein in vivo, though it also binds to other divalents as well. The significance of this action has not been conclusively elucidated. Previous reports have shown that metal binding sites occur throughout the N-terminal region of PrPC. Other cations like manganese have also been shown to affect PrPC abundance in a transcript-independent fashion. Here, we examined the ability of different divalent cations to influence the stability and in vitro conversion of two variants of PrP from elk (L/M132, 26-234). We find that copper and zinc de-stabilize PrP. We also find that PrP M132 exhibits a greater degree of divalent cation induced destabilization than L132. This supports findings that leucine at position 132 confers resistance to CWD, while M132 is susceptible. However, in vitro conversion of PrP is equally suppressed by either copper or zinc, in both L132 and M132 backgrounds. This report demonstrates the complex importance of ionic character on the PrPC folding pathway selection on the route to PrPSc formation.


Assuntos
Amiloide/metabolismo , Cobre/metabolismo , Cervos/metabolismo , Manganês/metabolismo , Proteínas Priônicas/metabolismo , Doença de Emaciação Crônica/metabolismo , Zinco/metabolismo , Animais , Cátions Bivalentes , Dicroísmo Circular , Humanos , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
18.
Protein Expr Purif ; 143: 20-27, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29031681

RESUMO

BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.


Assuntos
Bioensaio/métodos , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cricetinae , Proteínas Priônicas/genética , Proteínas Priônicas/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ovinos
19.
Methods Mol Biol ; 1658: 3-22, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28861778

RESUMO

Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrPSc and methods for production of PrPSc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrPSc for development of prion diagnostics including generation of PrPSc-specific antibody.


Assuntos
Amiloide/química , Clonagem Molecular/métodos , Corpos de Inclusão/química , Proteínas Priônicas/isolamento & purificação , Agregados Proteicos , Amiloide/biossíntese , Animais , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cricetulus , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Guanidina/química , Humanos , Camundongos , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Priônicas/biossíntese , Proteínas Priônicas/genética , Redobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
20.
Methods Mol Biol ; 1658: 35-49, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28861781

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is a powerful experimental tool for obtaining information on three-dimensional (3D) structures of proteins at atomic resolution. In inherited forms of prion diseases, misfolding of cellular prion protein, PrPC, into its pathological form, PrPSc, is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in inherited forms of prion diseases may benefit from detailed structural analysis of different human (Hu) PrP variants. Here, we describe the protocol for structure determination of HuPrP variants by NMR spectroscopy in solution that consists of preparation of NMR samples, acquisition of NMR data, NMR resonance assignments, and structure calculation.


Assuntos
Clonagem Molecular/métodos , Corpos de Inclusão/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas Priônicas/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Isótopos de Carbono , Enzimas de Restrição do DNA/química , Endopeptidases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Liofilização/métodos , Expressão Gênica , Guanidina/química , Humanos , Marcação por Isótopo/métodos , Cinética , Modelos Moleculares , Mutação , Isótopos de Nitrogênio , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Priônicas/biossíntese , Proteínas Priônicas/genética , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Redobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Termodinâmica
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