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1.
PLoS One ; 8(7): e71081, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23936256

RESUMO

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP(C)) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP(Sc)). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP(Sc) in prion-infected brain homogenates as an initiating seed to convert PrP(C) and trigger the self-propagation of PrP(Sc) over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP(Sc). More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP(Sc) seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP(Sc). However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.


Assuntos
Lipídeos/química , Príons/química , Redobramento de Proteína , RNA/química , Proteínas Recombinantes/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Detergentes/química , Feminino , Camundongos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPC/ultraestrutura , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Proteínas PrPSc/ultraestrutura , Príons/metabolismo , Príons/ultraestrutura , Proteólise , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Solubilidade
2.
PLoS One ; 7(1): e30872, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22295118

RESUMO

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.


Assuntos
Coinfecção , Vírus da Leucemia Murina de Friend/fisiologia , Proteínas PrPSc/metabolismo , Doenças Priônicas/virologia , Animais , Células Dendríticas Foliculares/metabolismo , Células Dendríticas Foliculares/virologia , Suscetibilidade a Doenças , Exossomos/metabolismo , Exossomos/virologia , Período de Incubação de Doenças Infecciosas , Camundongos , Células NIH 3T3 , Baço/imunologia
3.
Proteomics ; 11(19): 3853-65, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21805638

RESUMO

Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, ß-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn and ß-sheet structures attributed to PrP(Sc) . The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the α-helical, loop/turn and ß-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself.


Assuntos
Proteínas PrPSc/isolamento & purificação , Doenças Priônicas/diagnóstico , Proteínas/isolamento & purificação , Proteômica/métodos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cricetinae , Ferritinas/isolamento & purificação , Ferritinas/metabolismo , Humanos , Camundongos , Proteínas PrPSc/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Espectrometria de Massas em Tandem/métodos
4.
PLoS Pathog ; 6(12): e1001217, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21152012

RESUMO

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.


Assuntos
Determinação de Ponto Final/métodos , Ensaios de Triagem em Larga Escala/normas , Príons/análise , Amiloide/análise , Animais , Encéfalo , Líquido Cefalorraquidiano/química , Cricetinae , Cervos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Métodos , Vison , Estrutura Secundária de Proteína , Ovinos
5.
Proteomics ; 10(15): 2858-69, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20518029

RESUMO

Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation of an aggregated isoform of the prion protein (PrP). This pathological isoform, termed PrP(Sc), appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein cofactors may be involved in TSE pathogenesis or whether there are PrP(Sc)-associated proteins which help to determine TSE strain-specific disease phenotypes. We enriched PrP(Sc) from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray LC-MS/MS. These samples were compared with "mock" PrP(Sc) preparations from uninfected brains. PrP was the major component of the infected samples and ferritin was the most abundant impurity. Mock enrichments contained no detectable PrP but did contain a significant amount of ferritin. Of the total proteins identified, 32% were found in both mock and infected samples. The similarities between PrP(Sc) samples from 22L and Chandler TSE strains suggest that the non-PrP(Sc) protein components found in standard enrichment protocols are not strain specific.


Assuntos
Encéfalo/patologia , Proteínas PrPSc/metabolismo , Príons/metabolismo , Proteômica , Scrapie/metabolismo , Animais , Camundongos , Proteínas PrPSc/isolamento & purificação , Proteínas Priônicas , Príons/isolamento & purificação
6.
J Mol Biol ; 340(4): 731-8, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15223316

RESUMO

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Fator de Transcrição AraC , Arabinose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Dimerização , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Maleabilidade , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/isolamento & purificação , Solubilidade , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação
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