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Sample records for abnormal prion protein

  1. Accumulation of abnormal prion protein in mice infected with Creutzfeldt-Jakob disease via intraperitoneal route: a sequential study.

    OpenAIRE

    Muramoto, T; Kitamoto, T.; Tateishi, J.; Goto, I.

    1993-01-01

    We immunohistochemically studied the location of abnormal prion protein in the central nervous system and visceral organs at the clinical and preclinical stages of mice infected with Creutzfeldt-Jakob disease via intraperitoneal route. Abnormal prion protein was diffusely distributed in the central nervous system. The sequential study showed that its stainings were first detected 120 days after inoculation, were found in all mice after 180 days, and were the most intense and widespread after ...

  2. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells.

    Directory of Open Access Journals (Sweden)

    Daisuke Ishibashi

    Full Text Available Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK, derived from a patient with Gerstmann-Sträussler-Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent.

  3. Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

    Science.gov (United States)

    Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

    2016-01-01

    The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

  4. Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

    Directory of Open Access Journals (Sweden)

    Maxime Belondrade

    Full Text Available The prevalence of variant Creutzfeldt-Jakob disease (vCJD in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA. This assay allowed the specific detection of minute quantities (10-8 brain dilution of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

  5. Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

    Science.gov (United States)

    Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

    2016-01-01

    The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions. PMID:26800081

  6. Early detection of abnormal prion protein in genetic human prion diseases now possible using real-time QUIC assay.

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    Kazunori Sano

    Full Text Available INTRODUCTION: The definitive diagnosis of genetic prion diseases (gPrD requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau protein in the cerebrospinal fluid (CSF, but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS. We recently developed a new in vitro amplification technology, designated "real-time quaking-induced conversion (RT-QUIC", to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined. METHOD/PRINCIPAL FINDINGS: 56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N, and 24 cases of genetic CJD (gCJD, comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%, FFI (100%, gCJD E200K (87%, and gCJD V203I (100%. On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%, FFI (0%, gCJD E200K (73%, and gCJD V203I (67%. Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI. CONCLUSION/SIGNIFICANCE: RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.

  7. The sequential development of abnormal prion protein accumulation in mice with Creutzfeldt-Jakob disease.

    OpenAIRE

    Muramoto, T; Kitamoto, T.; Tateishi, J.; Goto, I.

    1992-01-01

    The distribution and sequential development of prion protein (PrP) accumulation in the central nervous system (CNS) and non-neuronal organs of mice infected with Creutzfeldt-Jakob disease (CJD) were investigated immunohistochemically using a new pretreatment method that greatly enhanced the immunoreactivity of PrP. Prion protein accumulation in the CNS was first detected at 30 days after inoculation and then developed near the inoculation site or periventricular area, and later spread to the ...

  8. Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic?

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    Bencsik Anna A

    2010-08-01

    Full Text Available Abstract In a recent paper written by Hilbe et al (BMC vet res, 2009, the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject. Here we come back on our material as it is possible to study and demonstrate the specificity of prion immunodetection using the PET-Blot method (Paraffin Embedded Tissue - Blot. It is admitted that this method allows detecting the Proteinase K (PK resistant form of the abnormal prion protein (PrPres without any confusion with unspecific immunoreaction. We re-analysed the kidney tissue versus adrenal gland and brain samples from the same cheetah affected with TSE using this PET-Blot method. The PET-Blot analysis revealed specific PrPres detection within the brain, adrenal gland and some glomeruli of the kidney, with a complete identicalness compared to our previous detection using immunohistochemistry. In conclusion, these new data enable us to confirm with assurance the presence of specific abnormal prion protein in the adrenal gland and in the kidney of the cheetah affected with FSE. It also emphasizes the usefulness for the re-examination of any

  9. Recent progress in prion and prion-like protein aggregation

    Institute of Scientific and Technical Information of China (English)

    Chuan-Wei Yi; Wen-Chang Xu; Jie Chen; Yi Liang

    2013-01-01

    Prion diseases and prion-like protein misfolding diseases involve the accumulation of abnormally aggregated forms of the normal host proteins,such as prion protein and Tau protein.These proteins are special because of their self-duplicating and transmissible characteristics.Such abnormally aggregated proteins mainly formed in neurons,cause the neurons dysfunction,and finally lead to invariably fatal neurodegenerative diseases.Prion diseases appear not only in animals,such as bovine spongiform encephalopathy in cattle and scrapie in sheep,but also in humans,such as Creutzfeldt-Jacob disease,and even the same prion or prion-like proteins can have many different phenotypes.A lot of biological evidence has suggested that the molecular basis for different strains of prions could be hidden in protein conformations,and the misfolded proteins with conformations different from the normal proteins have been proved to be the main cause for protein aggregation.Crowded physiological environments can be imitated in vitro to study how the misfolding of these proteins leads to the diseases in vivo.In this review,we provide an overview of the existing structural information for prion and prion-like proteins,and discuss the post-translational modifications of prion proteins and the difference between prion and other infectious pathogens.We also discuss what makes a misfolded protein become an infectious agent,and show some examples of prion-like protein aggregation,such as Tau protein aggregation and superoxide dismutase 1 aggregation,as well as some cases of prion-like protein aggregation in crowded physiological environments.

  10. Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with Creutzfeldt-Jakob disease.

    OpenAIRE

    Kitamoto, T.; Muramoto, T; Mohri, S; DOH-URA, K; Tateishi, J.

    1991-01-01

    We established that follicular dendritic cells (FDCs) are the site of abnormal prion protein (PrPCJD) accumulations in lymphoid tissues from mice infected with Creutzfeldt-Jakob disease. Evidence of positive FDC staining was observed in Creutzfeldt-Jakob disease-infected mice irrespective of the inoculation route, while no such staining was seen in the control mice. We also found that the severe combined immunodeficiency mouse trait is transmittable via the intracranial route but not via the ...

  11. Membrane toxicity of abnormal prion protein in adrenal chromaffin cells of scrapie infected sheep.

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    Gillian McGovern

    Full Text Available Transmissible spongiform encephalopathies (TSEs or prion diseases are associated with accumulations of disease specific PrP (PrP(d in the central nervous system (CNS and often the lymphoreticular system (LRS. Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP(d in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP(d were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP(d accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP(d from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP(d accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP(d is at the level of plasma membranes. However, the precise nature of PrP(d-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP(d with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.

  12. Conformational conversion of prion protein in prion diseases

    Institute of Scientific and Technical Information of China (English)

    Zheng Zhou; Gengfu Xiao

    2013-01-01

    Prion diseases are a group of infectious fatal neurodegenerative diseases.The conformational conversion of a cellular prion protein (PrPC) into an abnormal misfolded isoform (PrPSc) is the key event in prion diseases pathology.Under normal conditions,the high-energy barrier separates PrPC from PrPsc isoform.However,pathogenic mutations,modifications as well as some cofactors,such as glycosaminoglycans,nucleic acids,and lipids,could modulate the conformationai conversion process.Understanding the mechanism of conformational conversion of prion protein is essential for the biomedical research and the treatment of prion diseases.Particularly,the characterization of cofactors interacting with prion protein might provide new diagnostic and therapeutic strategies.

  13. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    OpenAIRE

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  14. Prions and prion-like proteins.

    Science.gov (United States)

    Fraser, Paul E

    2014-07-18

    Prions are self-replicating protein aggregates and are the primary causative factor in a number of neurological diseases in mammals. The prion protein (PrP) undergoes a conformational transformation leading to aggregation into an infectious cellular pathogen. Prion-like protein spreading and transmission of aggregates between cells have also been demonstrated for other proteins associated with Alzheimer disease and Parkinson disease. This protein-only phenomenon may therefore have broader implications in neurodegenerative disorders. The minireviews in this thematic series highlight the recent advances in prion biology and the roles these unique proteins play in disease.

  15. Porcine prion protein amyloid.

    Science.gov (United States)

    Hammarström, Per; Nyström, Sofie

    2015-01-01

    Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

  16. Species Barrier Prevents an Abnormal Isoform of Prion Protein from Accumulating in Follicular Dendritic Cells of Mice with Creutzfeldt-Jakob Disease

    OpenAIRE

    Muramoto, Tamaki; Kitamoto, Tetsuyuki; Hoque, Mohammad Zahirul; Tateishi, Jun; Goto, Ikuo

    1993-01-01

    The accumulation of abnormal prion protein in follicular dendritic cells did not occur in mice inoculated with materials from human Creutzfeldt-Jakob disease, whereas it always occurred in mice inoculated with mouse-adapted agents, suggesting an intense expression of the species barrier in the lymphoreticular system.

  17. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

    Science.gov (United States)

    Shan, Zhifu; Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2016-07-01

    Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds. PMID:27565564

  18. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

    Science.gov (United States)

    Shan, Zhifu; Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2016-07-01

    Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.

  19. Diversity in neuroanatomical distribution of abnormal prion protein in atypical scrapie.

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    Alice Nentwig

    2007-06-01

    Full Text Available Scrapie is a transmissible spongiform encephalopathy (TSE in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrP(sc. In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K-resistant core fragment of PrP(sc distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrP(sc distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.

  20. Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein*

    Institute of Scientific and Technical Information of China (English)

    Zhiqi Song; Deming Zhao; Lifeng Yang

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathoge-nicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with spe-cific topological structure can destroy intracellular stability and contribute to prion protein pathoge-nicity. In this study, the latest molecular chaperone system associated with endoplasmic reticu-lum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular me-chanisms wil help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.

  1. Prion protein in milk.

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    Nicola Franscini

    Full Text Available BACKGROUND: Prions are known to cause transmissible spongiform encephalopathies (TSE after accumulation in the central nervous system. There is increasing evidence that prions are also present in body fluids and that prion infection by blood transmission is possible. The low concentration of the proteinaceous agent in body fluids and its long incubation time complicate epidemiologic analysis and estimation of spreading and thus the risk of human infection. This situation is particularly unsatisfactory for food and pharmaceutical industries, given the lack of sensitive tools for monitoring the infectious agent. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an adsorption matrix, Alicon PrioTrap, which binds with high affinity and specificity to prion proteins. Thus we were able to identify prion protein (PrP(C--the precursor of prions (PrP(Sc--in milk from humans, cows, sheep, and goats. The absolute amount of PrP(C differs between the species (from microg/l range in sheep to ng/l range in human milk. PrP(C is also found in homogenised and pasteurised off-the-shelf milk, and even ultrahigh temperature treatment only partially diminishes endogenous PrP(C concentration. CONCLUSIONS/SIGNIFICANCE: In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie infected sheep suffering from mastitis, the appearance of PrP(C in milk implies the possibility that milk of TSE-infected animals serves as source for PrP(Sc.

  2. Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies

    International Nuclear Information System (INIS)

    We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrPSc) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56-90, 119-127, 137-143, 143-149, 147-151, 163-169, and 219-229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrPSc. However, no mAbs reacted with protease-treated PrPSc purified from scrapie-affected mice, even when PrPSc was dispersed into a detergent-lipid protein complex, to reduce the size of PrPSc aggregates. In contrast, denaturation of PrPSc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrPSc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrPSc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrPSc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases

  3. Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein

    OpenAIRE

    Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-01-01

    Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmiss...

  4. Molecular Dynamics Studies on the Buffalo Prion Protein

    CERN Document Server

    Zhang, Jiapu

    2015-01-01

    It was reported that buffalo is a low susceptibility species resisting to TSEs (Transmissible Spongiform Encephalopathies) (same as rabbits, horses and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (in humans prion diseases are (v)CJDs, GSS, FFI, and kulu etc). It was reported that buffalo is a low susceptibility species resisting to prion diseases (as rabbits, dogs, horses). In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein, predominantly with alpha-helices, into insoluble abnormally folded infectious prions, rich in beta-sheets. This paper studies the molecular structure and structural dynamics of buffalo prion protein, in order to find out the reason why buffaloes are resistant to prion diseases. We first did molecular modeling a homology structure constructed by one mutation at residue 143 from the Nuclear Magnetic Resonanc...

  5. Evidence for oxidative damage to prion protein in prion diseases

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In prion diseases the irreversible protein structural transformation process is completed in the brains of mammals within a few months, the uniformly generated infectivity displays extraordinary resistance to inactivation, suggesting that a vital energy source is required for the production of infectious particles. Considering the high oxygen-respiration rate in the brains, prion protein oxidative damage can be the crucial factor. Both theoretical consideration of the nature of protein radical reactions and a large body of previously unraveled feature of scrapie and prion diseases have provided multiple distinct lines of compelling evidence which persuasively support a suggestion that the infectious agents may be prion (free) radicals produced from protein oxidative damage. This paper describes that scrapie prions are most likely formed from prion radicals and oxidative species-mediated sequence-specific cross-linking of benign prion proteins.

  6. Prions: Beyond a Single Protein.

    Science.gov (United States)

    Das, Alvin S; Zou, Wen-Quan

    2016-07-01

    Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a "prion." Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins-not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease. PMID:27226089

  7. Prions, protein homeostasis, and phenotypic diversity

    OpenAIRE

    Halfmann, Randal; Alberti, Simon; Lindquist, Susan

    2010-01-01

    Prions are fascinating but often misunderstood protein aggregation phenomena. The traditional association of the mammalian prion protein with disease has overshadowed a potentially more interesting attribute of prions: their ability to create protein-based molecular memories. In fungi, prions alter the relationship between genotype and phenotype in a heritable way that diversifies clonal populations. Recent findings in yeast indicate that prions might be much more common than previously real...

  8. Low Copper and High Manganese Levels in Prion Protein Plaques

    OpenAIRE

    Debbie McKenzie; Aiken, Judd M.; Johnson, Christopher J.; P.U.P.A. Gilbert; Mike Abrecht; Baldwin, Katherine L.; Robin E. Russell; Pedersen, Joel A.

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: ...

  9. Final Report for CRADA Agreement , AL-C-2006-01 with Microsens Biotechnologies: Detection of the Abnormal Prion Protein in Blood by Improving the Extraction of this Protein

    Energy Technology Data Exchange (ETDEWEB)

    Schmerr, Mary Jo

    2009-03-31

    Several conditions were examined to optimize the extraction protocol using Seprion beads for the abnormal prion protein. Different combinations of water, hexafluro-2-propanol and formic acid were used. The results of these extraction protocols showed that the magnetic beads coated with Seprion reagents were subject to degradation, themselves, when the extraction conditions that would solubilize the abnormal prion protein were used. These compounds caused interference in the immunoassay for the abnormal prion protein and rendered these protocols incompatible with the assay systems. In an attempt to overcome this problem, another approach was then used. The coated beads were used as an integral part of the assay platform. After washing away denaturing agents, the beads with the 'captured' abnormal prion were incubated directly in the immunoassay, followed by analysis by the capillary electrophoresis. When a capillary electrophoresis electro-kinetic separation was attempted, the beads disturbed the analysis making it impossible to interpret. A pressure separation method was then developed for capillary electrophoresis analysis. When 20 samples, 5 of which were positive were analyzed, the assay identified 4 of the 5 positives and had no false positives. When a larger number of samples were analyzed the results were not as good - there were false positives and false negatives. It was then observed that the amount of beads that were loaded was dependent upon how long the beads were allowed to settle before loading them into the capillary. This resulted in unacceptable variations in the results and explained that when large numbers of samples were evaluated the results were not consistent. Because the technical difficulties with using the Seprion beads could not be overcome at this time, another approach is underway that is outside of the scope of this CRADA. No further agreements have been developed. Because the results were not favorable, no manuscripts were

  10. Prion protein oligomer and its neurotoxicity

    Institute of Scientific and Technical Information of China (English)

    Pei Huang; Fulin Lian; Yi Wen; Chenyun Guo; Donghai Lin

    2013-01-01

    The prion diseases,also known as transmissible spongiform encephalopathies,are fatal neurodegenerative disorders.According to the 'protein only' hypothesis,the key molecular event in the pathogenesis of prion disease is the conformational conversion of the host-derived cellular prion protein (PrPC) into a misfolded form (scrapie PrP,prpSc).Increasing evidence has shown that the most infectious factor is the smaller subfibrillar oligomers formed by prion proteins.Both the prion oligomer and PrPSc are rich in β-sheet structure and resistant to the proteolysis of proteinase K.The prion oligomer is soluble in physiologic environments whereas PrPSc is insoluble.Various prion oligomers are formed in different conditions.Prion oligomers exhibited more neurotoxicity both in vitro and in vivo than the fibrillar forms of PrPSc,implying that prion oligomers could be potential drug targets for attacking prion diseases.In this article,we describe recent experimental evidence regarding prion oligomers,with a special focus on prion oligomer formation and its neurotoxicity.

  11. B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice

    OpenAIRE

    Montrasio, Fabio; Cozzio, Antonio; Flechsig, Eckhard; Rossi, Daniela; Klein, Michael A.; Rülicke, Thomas; Raeber, Alex J.; Vosshenrich, Christian A.J.; Proft, Juliane; Aguzzi, Adriano; Weissmann, Charles

    2001-01-01

    Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking. In spleens of wild-type mice, infectivity is associated with B and T lymphocytes and stroma but not with circulating lymphocytes. We generated transgenic prion protein knockout mice overexpressing prion protein in B lymphocytes and found that they failed to accumulate prions in spleen after i.p. inoculation. W...

  12. Relationship between magnetism and prion protein

    Directory of Open Access Journals (Sweden)

    F. Balzano

    2010-01-01

    Full Text Available The mechanism of conversion of the normal prion protein (PrPC into aggregates of its pathological conformer (PrPSc reamins unclear. The aim of this study was to evaluate the effects induced by exposure of biological samples containing PrPSC to a magnetic field induced prominent molecular changes of samples indicated by the IR spectra located in the region that contains contribution primarily from absorption of amides. This finding suggests the existence of a strong correlation between magnetism and PrPsc and supports a new hypothesis that explains the conversion of normal PrPc to abnormal isoform PrPsc.

  13. Immunopurification of pathological prion protein aggregates.

    Directory of Open Access Journals (Sweden)

    Emiliano Biasini

    Full Text Available BACKGROUND: Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP(C into a pathogenic isoform that is rich in beta-sheet structure. This conformational change may result in the formation of PrP(Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP(C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP(Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP(Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases. PRINCIPAL FINDINGS: We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP(Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP(Sc purified from prion-infected mice was able to seed misfolding of PrP(C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons. SIGNIFICANCE: The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.

  14. Low Copper and High Manganese Levels in Prion Protein Plaques

    Directory of Open Access Journals (Sweden)

    Debbie McKenzie

    2013-02-01

    Full Text Available Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs. The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1 assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2 determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM with synchrotron radiation; and (3 use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  15. Low copper and high manganese levels in prion protein plaques.

    Science.gov (United States)

    Johnson, Christopher J; Gilbert, P U P A; Abrecht, Mike; Baldwin, Katherine L; Russell, Robin E; Pedersen, Joel A; Aiken, Judd M; McKenzie, Debbie

    2013-02-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  16. Low copper and high manganese levels in prion protein plaques

    Science.gov (United States)

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  17. Recombinant Human Prion Protein Inhibits Prion Propagation in vitro

    OpenAIRE

    Jue Yuan; Yi-An Zhan; Romany Abskharon; Xiangzhu Xiao; Manuel Camacho Martinez; Xiaochen Zhou; Geoff Kneale; Jacqueline Mikol; Sylvain Lehmann; Surewicz, Witold K.; Joaquín Castilla; Jan Steyaert; Shulin Zhang; Qingzhong Kong; Petersen, Robert B.

    2013-01-01

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylin...

  18. Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

    Science.gov (United States)

    Nemecek, Julie; Nag, Nabanita; Carlson, Christina M.; Schneider, Jay R.; Heisey, Dennis M.; Johnson, Christopher J.; Asher, David M.; Gregori, Luisa

    2013-01-01

    Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrPTSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrPTSE in tissues and blood. Macaque vCJD PrPTSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrPTSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrPTSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrPTSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrPTSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrPTSE was more permissive than human PrPTSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrPTSE from brains of humans and macaques with vCJD. PrPTSE signals were reproducibly detected by Western blot in dilutions through 10-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrPTSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrPTSE in v

  19. Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

    Directory of Open Access Journals (Sweden)

    Julie Nemecek

    Full Text Available Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA to amplify abnormal prion protein (PrP(TSE from highly diluted variant Creutzfeldt-Jakob disease (vCJD-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP(TSE in tissues and blood. Macaque vCJD PrP(TSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA. Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV, a close relative of the bank vole, seeded with macaque vCJD PrP(TSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N. We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP(TSE. Meadow vole brain (170N/N PrP genotype was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP(TSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP(TSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP(TSE was more permissive than human PrP(TSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP(TSE from brains of humans and macaques with vCJD. PrP(TSE signals were reproducibly detected by Western blot in dilutions through 10⁻¹² of vCJD-infected 10% brain homogenates. This is the first report showing PrP(TSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect Pr

  20. Controlling the prion propensity of glutamine/asparagine-rich proteins.

    Science.gov (United States)

    Paul, Kacy R; Ross, Eric D

    2015-01-01

    The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.

  1. Follicular dendritic cell-specific prion protein (PrP) expression alone is sufficient to sustain prion infection in the spleen.

    OpenAIRE

    Laura McCulloch; Brown, Karen L.; Bradford, Barry M.; John Hopkins; Mick Bailey; Klaus Rajewsky; Manson, Jean C; Mabbott, Neil A.

    2011-01-01

    Prion diseases are characterised by the accumulation of PrP(Sc), an abnormally folded isoform of the cellular prion protein (PrP(C)), in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc) accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrP(C) is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acq...

  2. Controlling the prion propensity of glutamine/asparagine-rich proteins

    OpenAIRE

    Paul, Kacy R.; Ross, Eric D.

    2015-01-01

    ABSTRACT The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discu...

  3. Prion protein self-interaction in prion disease therapy approaches

    NARCIS (Netherlands)

    Rigter, A.; Priem, J.; Langeveld, J.P.M.; Bossers, A.

    2011-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affe

  4. Yeast prions: protein aggregation is not enough.

    OpenAIRE

    Sherman, Michael Y

    2004-01-01

    Although many proteins -- both damaged and normal -- have a tendency to aggregate, only some are capable of dividing and propagating. What does it take to turn a protein aggregate into an infectious prion?

  5. Detection of type 1 prion protein in variant Creutzfeldt-Jakob disease

    NARCIS (Netherlands)

    Yull, H.M.; Ritchie, D.L.; Langeveld, J.P.M.; Zijderveld, van F.G.; Bruce, M.E.; Ironside, J.W.; Head, M.W.

    2006-01-01

    Molecular typing of the abnormal form of the prion protein (PrPSc) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrPSc molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resultin

  6. Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John

    2013-11-01

    Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

  7. Yeast prions and human prion-like proteins: sequence features and prediction methods.

    Science.gov (United States)

    Cascarina, Sean M; Ross, Eric D

    2014-06-01

    Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.

  8. Luminidependens (LD) is an Arabidopsis protein with prion behavior.

    Science.gov (United States)

    Chakrabortee, Sohini; Kayatekin, Can; Newby, Greg A; Mendillo, Marc L; Lancaster, Alex; Lindquist, Susan

    2016-05-24

    Prion proteins provide a unique mode of biochemical memory through self-perpetuating changes in protein conformation and function. They have been studied in fungi and mammals, but not yet identified in plants. Using a computational model, we identified candidate prion domains (PrDs) in nearly 500 plant proteins. Plant flowering is of particular interest with respect to biological memory, because its regulation involves remembering and integrating previously experienced environmental conditions. We investigated the prion-forming capacity of three prion candidates involved in flowering using a yeast model, where prion attributes are well defined and readily tested. In yeast, prions heritably change protein functions by templating monomers into higher-order assemblies. For most yeast prions, the capacity to convert into a prion resides in a distinct prion domain. Thus, new prion-forming domains can be identified by functional complementation of a known prion domain. The prion-like domains (PrDs) of all three of the tested proteins formed higher-order oligomers. Uniquely, the Luminidependens PrD (LDPrD) fully replaced the prion-domain functions of a well-characterized yeast prion, Sup35. Our results suggest that prion-like conformational switches are evolutionarily conserved and might function in a wide variety of normal biological processes. PMID:27114519

  9. Atypical Scrapie Prions from Sheep and Lack of Disease in Transgenic Mice Overexpressing Human Prion Protein

    OpenAIRE

    Wadsworth, Jonathan D. F.; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Martin H Groschup; Hope, James; Brandner, Sebastian; Asante, Emmanuel A.; Collinge, John

    2013-01-01

    Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue...

  10. Ex vivomammalian prions are formed of paired double helical prion protein fibrils

    OpenAIRE

    Terry, C.; Wenborn, A.; Gros, N.; Sells, J.; Joiner, S; Hosszu, L.L.P.; Tattum, M. H.; Panico, Silvia; Clare, Daniel; Collinge, J.; Saibil, Helen; Wadsworth, J D F

    2016-01-01

    Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell...

  11. Cellular Prion Protein: From Physiology to Pathology

    Directory of Open Access Journals (Sweden)

    Yutaka Kikuchi

    2012-11-01

    Full Text Available The human cellular prion protein (PrPC is a glycosylphosphatidylinositol (GPI anchored membrane glycoprotein with two N-glycosylation sites at residues 181 and 197. This protein migrates in several bands by Western blot analysis (WB. Interestingly, PNGase F treatment of human brain homogenates prior to the WB, which is known to remove the N-glycosylations, unexpectedly gives rise to two dominant bands, which are now known as C-terminal (C1 and N-terminal (N1 fragments. This resembles the β-amyloid precursor protein (APP in Alzheimer disease (AD, which can be physiologically processed by α-, β-, and γ-secretases. The processing of APP has been extensively studied, while the identity of the cellular proteases involved in the proteolysis of PrPC and their possible role in prion biology has remained limited and controversial. Nevertheless, there is a strong correlation between the neurotoxicity caused by prion proteins and the blockade of their normal proteolysis. For example, expression of non-cleavable PrPC mutants in transgenic mice generates neurotoxicity, even in the absence of infectious prions, suggesting that PrPC proteolysis is physiologically and pathologically important. As many mouse models of prion diseases have recently been developed and the knowledge about the proteases responsible for the PrPC proteolysis is accumulating, we examine the historical experimental evidence and highlight recent studies that shed new light on this issue.

  12. Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes

    OpenAIRE

    Tracy O'Connor; Nathalie Frei; Jana Sponarova; Petra Schwarz; Mathias Heikenwalder; Adriano Aguzzi

    2012-01-01

    Author Summary Prions are unique infectious agents thought to be composed entirely of an abnormal conformer of the endogenous prion protein. Prions cause a severe neurological disorder in humans and other animals known as prion disease. Though prion disease can arise spontaneously or from genetic mutations in the gene encoding the prion protein, many cases of prion disease arise due to peripheral exposure to the infectious agent. In these cases, prions must journey from the gastrointestinal t...

  13. Transition-metal prion protein attachment: Competition with copper

    Science.gov (United States)

    Hodak, Miroslav; Bernholc, Jerry

    2012-02-01

    Prion protein, PrP, is a protein capable of binding copper ions in multiple modes depending on their concentration. Misfolded PrP is implicated in a group of neurodegenerative diseases, which include ``mad cow disease'' and its human form, variant Creutzfeld-Jacob disease. An increasing amount of evidence suggests that attachment of non-copper metal ions to PrP triggers transformations to abnormal forms similar to those observed in prion diseases. In this work, we use hybrid Kohn-Sham/orbital-free density functional theory simulations to investigate copper replacement by other transition metals that bind to PrP, including zinc, iron and manganese. We consider all known copper binding modes in the N-terminal domain of PrP. Our calculations identify modes most susceptible to copper replacement and reveal metals that can successfully compete with copper for attachment to PrP.

  14. Role of Prion Protein Aggregation in Neurotoxicity

    Directory of Open Access Journals (Sweden)

    Tullio Florio

    2012-07-01

    Full Text Available In several neurodegenerative diseases, such as Parkinson, Alzheimer’s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP, the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126 and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

  15. Abnormal protein aggregationand neurodegenerativediseases

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Abnormal protein aggregation or amyloid is the major cause ofmany neurodegenerative disorders. The present review focuses on the correlation between sequence and structure features of proteins related to the diseases and abnormal protein aggregation. Recent progress has improved our knowledge on understand-ing the mechanism of amyloid formation. We suggest a nucleation model for ordered protein aggregation, which can also explain pathogenesis mechanisms of these neurodegenerative diseases in vivo.

  16. Divalent metals stabilize cellular prion proteins and alter the rate of proteinase-K dependent limited proteolysis

    Science.gov (United States)

    Background: The key biochemical event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent ca...

  17. Monitoring prion protein stability by NMR.

    Science.gov (United States)

    Julien, Olivier; Graether, Steffen P; Sykes, Brian D

    2009-01-01

    Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group of fatal neurological diseases that affect both humans and animals. At the end of the 20th century, bovine spongiform encephalopathy (BSE), better known as mad cow disease, was shown to be transmissible to humans. This resulted in considerable concern for public health and a number of questions for scientists. The first question answered was the possible source of the disease, which appears to be the prion protein (PrP). There are two major forms of this protein: the native, noninfectious form (PrP(C)), and the misfolded infectious form (PrP(Sc)). PrP(C) is mainly alpha-helical in structure, whereas PrP(Sc) aggregates into an assembly of beta-sheets, forming amyloid fibrils. Since the first solution structure of the noninfectious form of the mouse prion protein, about 30 structures of the globular portion of PrP(C) have been characterized from different organisms. However, only a few minor differences are observed when comparing one PrP(C) structure to another. The key to understanding prion formation may then be not in the structure of PrP(C), but in the mechanism underlying PrP(C) unfolding and then conversion into a misfolded fibril state. To identify the possible region(s) of PrP(C) responsible for initiating the conversion into the amyloid fibril formation, nuclear magnetic resonance (NMR) was applied to characterize the stability and structure of PrP(C) and intermediate states during the conversion from PrP(C) to PrP(Sc). Subsequently urea was used to induce unfolding, and data analysis revealed region-specific structural stabilities that may bring insights into the mechanisms underlying conversion of protein into an infectious prion. PMID:19697241

  18. Distinct patterns of spread of prion infection in brains of mice expressing anchorless or anchored forms of prion protein

    OpenAIRE

    Rangel, Alejandra; Race, Brent; Phillips, Katie; Striebel, James; Kurtz, Nancy; Chesebro, Bruce

    2014-01-01

    Background In humans and animals, prion protein (PrP) is usually expressed as a glycophosphatidylinositol (GPI)-anchored membrane protein, but anchorless PrP may be pathogenic in humans with certain familial prion diseases. Anchored PrP expressed on neurons mediates spread of prions along axons in the peripheral and central nervous systems. However, the mechanism of prion spread in individuals expressing anchorless PrP is poorly understood. Here we studied prion spread within brain of mice ex...

  19. Uptake and dynamics of infectious prion protein in the intestine.

    Science.gov (United States)

    Ano, Yasuhisa; Sakudo, Akikazu; Nakayama, Hiroyuki; Onodera, Takashi

    2009-01-01

    Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of a protease-resistant abnormal isoform of the prion protein (PrPSc), which is converted from the cellular isoform of the prion protein (PrPC). In the oral transmission of prion protein, PrPSc can invade a host body through the intestinal tract. There is only limited information available on how the infectious agent passes through one or several biological barriers before it can finally reach the brain. After oral administration, PrPSc withstands the digestive process and may be incorporated by microfold (M) cells or villous columnar epithelial cells in the intestine. After entry into the intestinal epithelium, PrPSc accumulates and is amplified in follicular dendritic cells (FDCs) within Peyer's patches and other isolated lymphoid follicles possibly by an interaction with dendritic cells or macrophages. Following accumulation in gut-associated lymphoid tissues, PrPSc is thought to move to the enteric nervous systems (ENS) by an interaction with FDCs or dendritic cells. As a result of neuroinvasion into the ENS, PrPSc spreads to the central nervous system. In addition, an epidemiological study suggested that most bovine spongiform encephalopathy cases had been exposed to the agent in the first 6 months of life. Developments of the intestinal defense and immune system may be involved in the susceptibility to infection. PMID:19275737

  20. Follicular dendritic cell-specific prion protein (PrP) expression alone is sufficient to sustain prion infection in the spleen.

    Science.gov (United States)

    McCulloch, Laura; Brown, Karen L; Bradford, Barry M; Hopkins, John; Bailey, Mick; Rajewsky, Klaus; Manson, Jean C; Mabbott, Neil A

    2011-12-01

    Prion diseases are characterised by the accumulation of PrP(Sc), an abnormally folded isoform of the cellular prion protein (PrP(C)), in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc) accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrP(C) is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrP(C) expression was specifically "switched on" or "off" only on FDC. We show that PrP(C)-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrP(C)-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrP(C) expression on FDC. PMID:22144895

  1. Follicular dendritic cell-specific prion protein (PrP expression alone is sufficient to sustain prion infection in the spleen.

    Directory of Open Access Journals (Sweden)

    Laura McCulloch

    2011-12-01

    Full Text Available Prion diseases are characterised by the accumulation of PrP(Sc, an abnormally folded isoform of the cellular prion protein (PrP(C, in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc accumulate first upon follicular dendritic cells (FDC in lymphoid tissues before spreading to the CNS. Expression of PrP(C is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrP(C expression was specifically "switched on" or "off" only on FDC. We show that PrP(C-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrP(C-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrP(C expression on FDC.

  2. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    Science.gov (United States)

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  3. Crucial Role for Prion Protein Membrane Anchoring in the Neuroinvasion and Neural Spread of Prion Infection ▿

    OpenAIRE

    Klingeborn, Mikael; Race, Brent; Meade-White, Kimberly D.; Rosenke, Rebecca; Striebel, James F.; Chesebro, Bruce

    2010-01-01

    In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in mice expressing either anchored or anchorless PrP. In contr...

  4. Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

    Science.gov (United States)

    Terry, Cassandra; Wenborn, Adam; Gros, Nathalie; Sells, Jessica; Joiner, Susan; Hosszu, Laszlo L P; Tattum, M Howard; Panico, Silvia; Clare, Daniel K; Collinge, John; Saibil, Helen R; Wadsworth, Jonathan D F

    2016-05-01

    Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP. PMID:27249641

  5. Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

    Science.gov (United States)

    Terry, Cassandra; Wenborn, Adam; Gros, Nathalie; Sells, Jessica; Joiner, Susan; Hosszu, Laszlo L P; Tattum, M Howard; Panico, Silvia; Clare, Daniel K; Collinge, John; Saibil, Helen R; Wadsworth, Jonathan D F

    2016-05-01

    Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

  6. [Functions of prion protein PrPc].

    Science.gov (United States)

    Cazaubon, Sylvie; Viegas, Pedro; Couraud, Pierre-Olivier

    2007-01-01

    It is now well established that both normal and pathological (or scrapie) isoforms of prion protein, PrPc and PrPsc respectively, are involved in the development and progression of various forms of neurodegenerative diseases, including scrapie in sheep, bovine spongiform encephalopathy (or "mad cow disease") and Creutzfeldt-Jakob disease in human, collectively known as prion diseases. The protein PrPc is highly expressed in the central nervous system in neurons and glial cells, and also present in non-brain cells, such as immune cells or epithelial and endothelial cells. Identification of the physiological functions of PrPc in these different cell types thus appears crucial for understanding the progression of prion diseases. Recent studies highlighted several major roles for PrPc that may be considered in two major domains : (1) cell survival (protection against oxidative stress and apoptosis) and (2) cell adhesion. In association with cell adhesion, distinct functions of PrPc were observed, depending on cell types : neuronal differentiation, epithelial and endothelial barrier integrity, transendothelial migration of monocytes, T cell activation. These observations suggest that PrPc functions may be particularly relevant to cellular stress, as well as inflammatory or infectious situations. PMID:17875293

  7. Insights into the physiological function of cellular prion protein

    Directory of Open Access Journals (Sweden)

    Martins V.R.

    2001-01-01

    Full Text Available Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie, appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

  8. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    Science.gov (United States)

    Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

    2009-01-01

    Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

  9. Human tonsil-derived follicular dendritic-like cells are refractory to human prion infection in vitro and traffic disease-associated prion protein to lysosomes.

    Science.gov (United States)

    Krejciova, Zuzana; De Sousa, Paul; Manson, Jean; Ironside, James W; Head, Mark W

    2014-01-01

    The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrP(TSE)) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrP(TSE) staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrP(TSE) accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrP(TSE) after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrP(TSE). PMID:24183781

  10. Prion protein induced signaling cascades in monocytes

    International Nuclear Information System (INIS)

    Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrPC), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrPC fusion proteins synthesized with a human Fc-tag. PrPC fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK1,2 and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrPC in monocytes and macrophages

  11. A systematic investigation of production of synthetic prions from recombinant prion protein.

    Science.gov (United States)

    Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K; Edgeworth, Julie A; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M; Brandner, Sebastian; Hosszu, Laszlo L P; Tattum, M Howard; Jat, Parmjit; Clarke, Anthony R; Klöhn, Peter C; Wadsworth, Jonathan D F; Jackson, Graham S; Collinge, John

    2015-12-01

    According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

  12. A naturally occurring variant of the human prion protein completely prevents prion disease.

    Science.gov (United States)

    Asante, Emmanuel A; Smidak, Michelle; Grimshaw, Andrew; Houghton, Richard; Tomlinson, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Richard-Londt, Angela; Linehan, Jacqueline M; Brandner, Sebastian; Alpers, Michael; Whitfield, Jerome; Mead, Simon; Wadsworth, Jonathan D F; Collinge, John

    2015-06-25

    Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP). A novel PrP variant, G127V, was under positive evolutionary selection during the epidemic of kuru--an acquired prion disease epidemic of the Fore population in Papua New Guinea--and appeared to provide strong protection against disease in the heterozygous state. Here we have investigated the protective role of this variant and its interaction with the common, worldwide M129V PrP polymorphism. V127 was seen exclusively on a M129 PRNP allele. We demonstrate that transgenic mice expressing both variant and wild-type human PrP are completely resistant to both kuru and classical Creutzfeldt-Jakob disease (CJD) prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to bovine spongiform encephalopathy prions to which the Fore were not exposed. Notably, mice expressing only PrP V127 were completely resistant to all prion strains, demonstrating a different molecular mechanism to M129V, which provides its relative protection against classical CJD and kuru in the heterozygous state. Indeed, this single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as deletion of the protein. Further study in transgenic mice expressing different ratios of variant and wild-type PrP indicates that not only is PrP V127 completely refractory to prion conversion but acts as a potent dose-dependent inhibitor of wild-type prion propagation.

  13. Lactoferrin from bovine colostrum regulates prolyl hydroxylase 2 activity and prevents prion protein-mediated neuronal cell damage via cellular prion protein.

    Science.gov (United States)

    Park, Y-G; Moon, J-H; Park, S-Y

    2014-08-22

    Prion disorders are associated with the conversion of normal cellular prion protein (PrPc) to the abnormal scrapie isoform of prion protein (PrPsc). Recent studies have shown that expression of normal PrPc is regulated by hypoxia-inducible factor-1 alpha (HIF-1α), and that lactoferrin increases full-length PrPc on the cell surface. Lactoferrin is an 80-kDa iron-binding glycoprotein with various biological activities, including iron-chelating ability. HIF-1α and the associated ubiquitin-proteasome pathway are regulated by HIF prolyl-hydroxylases 2 (PHD2). We hypothesized that lactoferrin regulates PHD2 expression and enzymatic activity, and the PHD2 regulation promotes HIF-1α stability and prevention of neuronal cell death mediated by prion protein (PrP) residues (106-126). Lactoferrin prevented PrP (106-126)-induced neurotoxicity by the induction of PrPc expression via promoting HIF-1α stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106-126)-induced neurotoxicity via the up-regulation of HIF-1α stability determined by PHD2 expression and enzymatic activity. These findings suggest that possible therapies such as PHD2 inhibition, or promotion of lactoferrin secretion, may have clinical benefits in neurodegenerative diseases, including prion disease. PMID:24875174

  14. A new mutation in the prion protein gene: A patient with dementia and white matter changes

    NARCIS (Netherlands)

    Van Harten, B.; Van Gool, W.A.; Van Langen, I.M.; Deekman, J.M.; Meijerink, P.H.S.; Weinstein, H.C.

    2000-01-01

    The authors describe the clinical characteristics, MRI abnormalities, and molecular findings in a patient with a novel variant of a two-octarepeat insertion mutation in the prion protein gene. This patient presented with moderately progressive dementia of presenile onset and gait ataxia. MRI showed

  15. Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo

    Directory of Open Access Journals (Sweden)

    Qingzhong Kong

    2013-07-01

    Full Text Available Prion diseases, or transmissible spongiform encephalopathies (TSEs, are associated with the conformational conversion of the cellular prion protein, PrPC, into a protease-resistant form, PrPSc. Here, we show that mutation-induced thermodynamic stabilization of the folded, α-helical domain of PrPC has a dramatic inhibitory effect on the conformational conversion of prion protein in vitro, as well as on the propagation of TSE disease in vivo. Transgenic mice expressing a human prion protein variant with increased thermodynamic stability were found to be much more resistant to infection with the TSE agent than those expressing wild-type human prion protein, in both the primary passage and three subsequent subpassages. These findings not only provide a line of evidence in support of the protein-only model of TSEs but also yield insight into the molecular nature of the PrPC→PrPSc conformational transition, and they suggest an approach to the treatment of prion diseases.

  16. Normal modes of prion proteins: from native to infectious particle.

    Science.gov (United States)

    Samson, Abraham O; Levitt, Michael

    2011-03-29

    Prion proteins (PrP) are the infectious agent in transmissible spongiform encephalopathies (i.e., mad cow disease). To be infectious, prion proteins must undergo a conformational change involving a decrease in α-helical content along with an increase in β-strand content. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two α-helices by one and two residues, as well as an extension of two β-strands by three residues each, which could instigate the conformational change. The conformational change occurs in a region that is compatible with immunological studies, and it is observed more frequently in mutant prions that are prone to conversion than in wild-type prions because of differences in their starting structures, which are amplified through normal modes. These findings are valuable for our comprehension of the conversion mechanism associated with the conformational change in prion proteins. PMID:21338080

  17. Effects of Solution Chemistry and Aging Time on Prion Protein Adsorption and Replication of Soil-Bound Prions

    OpenAIRE

    Samuel E Saunders; Yuan, Qi; Bartz, Jason C.; Bartelt-Hunt, Shannon

    2011-01-01

    Prion interactions with soil may play an important role in the transmission of chronic wasting disease (CWD) and scrapie. Prions are known to bind to a wide range of soil surfaces, but the effects of adsorption solution chemistry and long-term soil binding on prion fate and transmission risk are unknown. We investigated HY TME prion protein (PrPSc) adsorption to soil minerals in aqueous solutions of phosphate buffered saline (PBS), sodium chloride, calcium chloride, and deionized water using ...

  18. Generic amyloidogenicity of mammalian prion proteins from species susceptible and resistant to prions.

    Science.gov (United States)

    Nyström, Sofie; Hammarström, Per

    2015-01-01

    Prion diseases are lethal, infectious diseases associated with prion protein (PrP) misfolding. A large number of mammals are susceptible to both sporadic and acquired prion diseases. Although PrP is highly conserved and ubiquitously expressed in all mammals, not all species exhibit prion disease. By employing full length recombinant PrP from five known prion susceptible species (human, cattle, cat, mouse and hamster) and two species considered to be prion resistant (pig and dog) the amyloidogenicity of these PrPs has been delineated. All the mammalian PrPs, even from resistant species, were swiftly converted from the native state to amyloid-like structure when subjected to a native condition conversion assay. The PrPs displayed amyloidotypic tinctorial and ultrastructural hallmarks. Self-seeded conversion of the PrPs displayed significantly decreased lag phases demonstrating that nucleation dependent polymerization is a dominating mechanism in the fibrillation process. Fibrils from Aβ1-40, Aβ1-42, Lysozyme, Insulin and Transthyretin did not accelerate conversion of HuPrP whereas fibrils from HuPrP90-231 and HuPrP121-231 as well as full length PrPs of all PrPs efficiently seeded conversion showing specificity of the assay requiring the C-terminal PrP sequence. Our findings have implications for PrP misfolding and could have ramifications in the context of prion resistant species and silent carriers.

  19. Computational Studies of the Structural Stability of Rabbit Prion Protein Compared to Human and Mouse Prion Proteins

    CERN Document Server

    Zhang, Jiapu

    2011-01-01

    Prion diseases are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. The neurodegenerative diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Str$\\ddot{a}$ussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or 'mad-cow' disease) and chronic wasting disease in cattle belong to prion diseases. By now there have not been some effective therapeutic approaches to treat all these prion diseases. Dogs, rabbits and horses were reported to be resistant to prion diseases. By the end of year 2010 all the NMR structures of dog, rabbit and horse prion proteins (X-ray for rabbits too) had been finished to release into protein data bank. Thus, at this moment it is very worth studying the NMR and X-ray molecular structures of horse, dog and rabbit prion proteins to obtain insights into their immunity prion diseases. The author found that dog and horse prion proteins have sta...

  20. "Protein-only" or "virino" in prion diseases?

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    60-year prion and scrapie research has led to a dilemma in understanding the unknown aetiology of the infectious neurodegenerative disorders with intriguing features. Current progress and dilemma in prion research are briefly but critically reviewed. Instead of providing a comprehensive coverage of the research history, attentions in this view are drawn toward both the major breakthrough in the advancement of protein-only hypothesis, and the puzzle why this hypothesis has not been fully accepted. In order to resolve the prion enigma in neuroscience, it is suggested that both technical and concept barriers remain to be crossed. Since prion research is a multi-interdisciplinary subject, this view is intended to both facilitate a better understanding of prion phenomenon by more scientists in natural science, and invite scientists outside the fields of molecular genetics and protein science for collaboration.

  1. Insights into prion protein function from atomistic simulations

    OpenAIRE

    Hodak, Miroslav; Bernholc, Jerzy

    2010-01-01

    Computer simulations are a powerful tool for studies of biological systems. They have often been used to study prion protein (PrP), a protein responsible for neurodegenerative diseases, which include “mad cow disease” in cattle and Creutzfeldt-Jacob disease in humans. An important aspect of the prion protein is its interaction with copper ion, which is thought to be relevant for PrP’s yet undetermined function and also potentially play a role in prion diseases. For studies of copper attachmen...

  2. Morphological and Functional Abnormalities in Mitochondria Associated with Synaptic Degeneration in Prion Disease

    OpenAIRE

    Sisková, Zuzana; Mahad, Don Joseph; Pudney, Carianne; Campbell, Graham; Cadogan, Mark; Asuni, Ayodeji; O'Connor, Vincent; Perry, Victor Hugh

    2010-01-01

    Synaptic and dendritic pathology is a well-documented component of prion disease. In common with other neurodegenerative diseases that contain an element of protein misfolding, little is known about the underlying mechanisms of synaptic degeneration. In particular, in prion disease the relationship between synaptic malfunction, degeneration, and mitochondria has been neglected. We investigated a wide range of mitochondrial parameters, including changes in mitochondrial density, inner membrane...

  3. The structural stability of wild-type horse prion protein.

    Science.gov (United States)

    Zhang, Jiapu

    2011-10-01

    Prion diseases (e.g. Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE or 'mad-cow' disease) and chronic wasting disease (CWD) in cattles) are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches or medications to treat all these prion diseases. Rabbits, dogs, and horses are the only mammalian species reported to be resistant to infection from prion diseases isolated from other species. Recently, the β2-α2 loop has been reported to contribute to their protein structural stabilities. The author has found that rabbit prion protein has a strong salt bridge ASP177-ARG163 (like a taut bow string) keeping this loop linked. This paper confirms that this salt bridge also contributes to the structural stability of horse prion protein. Thus, the region of β2-α2 loop might be a potential drug target region. Besides this very important salt bridge, other four important salt bridges GLU196-ARG156-HIS187, ARG156-ASP202 and GLU211-HIS177 are also found to greatly contribute to the structural stability of horse prion protein. Rich databases of salt bridges, hydrogen bonds and hydrophobic contacts for horse prion protein can be found in this paper. PMID:21875155

  4. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

    Science.gov (United States)

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

    2014-07-01

    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

  5. Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion.

    Science.gov (United States)

    Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP(Sc)). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP(Sc) results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP(Sc). However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP(Sc) can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.

  6. Molecular dynamics studies on the buffalo prion protein.

    Science.gov (United States)

    Zhang, Jiapu; Wang, Feng; Chatterjee, Subhojyoti

    2016-01-01

    It was reported that buffalo is a low susceptibility species resisting to transmissible spongiform encephalopathies (TSEs) (same as rabbits, horses, and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (except for rabbits, dogs, horses, and buffalo), manifesting as scrapie in sheep and goats; bovine spongiform encephalopathy (BSE or "mad-cow" disease) in cattle; chronic wasting disease in deer and elk; and Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and Kulu in humans etc. In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP(C)), predominantly with α-helices, into insoluble abnormally folded infectious prions (PrP(Sc)), rich in β-sheets. In this article, we studied the molecular structure and structural dynamics of buffalo PrP(C) (BufPrP(C)), in order to understand the reason why buffalo is resistant to prion diseases. We first did molecular modeling of a homology structure constructed by one mutation at residue 143 from the NMR structure of bovine and cattle PrP(124-227); immediately we found that for BufPrP(C)(124-227), there are five hydrogen bonds (HBs) at Asn143, but at this position, bovine/cattle do not have such HBs. Same as that of rabbits, dogs, or horses, our molecular dynamics studies also revealed there is a strong salt bridge (SB) ASP178-ARG164 (O-N) keeping the β2-α2 loop linked in buffalo. We also found there is a very strong HB SER170-TYR218 linking this loop with the C-terminal end of α-helix H3. Other information, such as (i) there is a very strong SB HIS187-ARG156 (N-O) linking α-helices H2 and H1 (if mutation H187R is made at position 187, then the hydrophobic core of PrP(C) will be exposed (L.H. Zhong (2010). Exposure of hydrophobic core in human prion protein pathogenic mutant H187R. Journal of

  7. Assessing proteinase K resistance of fish prion proteins in a scrapie-infected mouse neuroblastoma cell line.

    Science.gov (United States)

    Salta, Evgenia; Kanata, Eirini; Ouzounis, Christos A; Gilch, Sabine; Schätzl, Hermann; Sklaviadis, Theodoros

    2014-11-13

    The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrP(C), into an aberrant and partially proteinase K resistant isoform, PrP(Sc). Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK), as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs.

  8. Effects of solution chemistry and aging time on prion protein adsorption and replication of soil-bound prions.

    Directory of Open Access Journals (Sweden)

    Samuel E Saunders

    Full Text Available Prion interactions with soil may play an important role in the transmission of chronic wasting disease (CWD and scrapie. Prions are known to bind to a wide range of soil surfaces, but the effects of adsorption solution chemistry and long-term soil binding on prion fate and transmission risk are unknown. We investigated HY TME prion protein (PrP(Sc adsorption to soil minerals in aqueous solutions of phosphate buffered saline (PBS, sodium chloride, calcium chloride, and deionized water using western blotting. The replication efficiency of bound prions following adsorption in these solutions was also evaluated by protein misfolding cyclic amplification (PMCA. Aging studies investigated PrP(Sc desorption and replication efficiency up to one year following adsorption in PBS or DI water. Results indicate that adsorption solution chemistry can affect subsequent prion replication or desorption ability, especially after incubation periods of 30 d or longer. Observed effects were minor over the short-term (7 d or less. Results of long-term aging experiments demonstrate that unbound prions or prions bound to a diverse range of soil surfaces can readily replicate after one year. Our results suggest that while prion-soil interactions can vary with solution chemistry, prions bound to soil could remain a risk for transmitting prion diseases after months in the environment.

  9. New insights into structural determinants of prion protein folding and stability.

    Science.gov (United States)

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  10. Insights into prion protein function from atomistic simulations.

    Science.gov (United States)

    Hodak, Miroslav; Bernholc, Jerzy

    2010-01-01

    Computer simulations are a powerful tool for studies of biological systems. They have often been used to study prion protein (PrP), a protein responsible for neurodegenerative diseases, which include "mad cow disease" in cattle and Creutzfeldt-Jacob disease in humans. An important aspect of the prion protein is its interaction with copper ion, which is thought to be relevant for PrP's yet undetermined function and also potentially play a role in prion diseases. for studies of copper attachment to the prion protein, computer simulations have often been used to complement experimental data and to obtain binding structures of Cu-PrP complexes. This paper summarizes the results of recent ab initio calculations of copper-prion protein interactions focusing on the recently discovered concentration-dependent binding modes in the octarepeat region of this protein. In addition to determining the binding structures, computer simulations were also used to make predictions about PrP's function and the role of copper in prion diseases. The results demonstrate the predictive power and applicability of ab initio simulations for studies of metal-biomolecular complexes. PMID:20118658

  11. Peroxymonosulfate Rapidly Inactivates the Disease-Associated Prion Protein.

    Science.gov (United States)

    Chesney, Alexandra R; Booth, Clarissa J; Lietz, Christopher B; Li, Lingjun; Pedersen, Joel A

    2016-07-01

    Prions, the etiological agents in transmissible spongiform encephalopathies, exhibit remarkable resistance to most methods of inactivation that are effective against conventional pathogens. Prions are composed of pathogenic conformers of the prion protein (PrP(TSE)). Some prion diseases are transmitted, in part, through environmental routes. The recalcitrance of prions to inactivation may lead to a persistent reservoir of infectivity that contributes to the environmental maintenance of epizootics. At present, few methods exist to remediate prion-contaminated land surfaces. Here we conducted a proof-of-principle study to examine the ability of peroxymonosulfate to degrade PrP(TSE). We find that peroxymonosulfate rapidly degrades PrP(TSE) from two species. Transition-metal-catalyzed decomposition of peroxymonosulfate to produce sulfate radicals appears to enhance degradation. We further demonstrate that exposure to peroxymonosulfate significantly reduced PrP(C) to PrP(TSE) converting ability as measured by protein misfolding cyclic amplification, used as a proxy for infectivity. Liquid chromatography-tandem mass spectrometry revealed that exposure to peroxymonosulfate results in oxidative modifications to methionine and tryptophan residues. This study indicates that peroxymonosulfate may hold promise for decontamination of prion-contaminated surfaces. PMID:27247993

  12. Protease-resistant prions selectively decrease Shadoo protein.

    Directory of Open Access Journals (Sweden)

    Joel C Watts

    2011-11-01

    Full Text Available The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C into PrP(Sc, a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho, a protein that resembles the flexibly disordered N-terminal domain of PrP(C, were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc. Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc. Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc during prion disease.

  13. Prion protein and susceptibility to kainate-induced seizures

    OpenAIRE

    Striebel, James F.; Race, Brent; Chesebro, Bruce

    2013-01-01

    Prion protein (PrP) is a cell surface glycoprotein which is required for susceptibility to prion infection and disease. However, PrP is expressed in many different cell types located in numerous organs. Therefore, in addition to its role in prion diseases, PrP may have a large variety of other biological functions involving the nervous system and other systems. We recently showed that susceptibility to kainate-induced seizures differed in Prnp−/− and Prnp+/+ mice on the C57BL/10SnJ background...

  14. Persistence of pathogenic prion protein during simulated wastewater treatment processes

    Science.gov (United States)

    Hinckley, G.T.; Johnson, C.J.; Jacobson, K.H.; Bartholomay, C.; Mcmahon, K.D.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

    2008-01-01

    Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP TSE) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. ?? 2008 American Chemical Society.

  15. Molecular dynamics studies on the NMR and X-ray structures of rabbit prion proteins.

    Science.gov (United States)

    Zhang, Jiapu; Zhang, Yuanli

    2014-02-01

    Prion diseases, traditionally referred to as transmissible spongiform encephalopathies (TSEs), are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species, manifesting as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE or mad-cow disease) in cattle, chronic wasting disease in deer and elk, and Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and kulu in humans, etc. These neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP(C)) into insoluble abnormally folded infectious prions (PrP(Sc)), and the conversion of PrP(C) to PrP(Sc) is believed to involve conformational change from a predominantly α-helical protein to one rich in β-sheet structure. Such a conformational change may be amenable to study by molecular dynamics (MD) techniques. For rabbits, classical studies show that they have a low susceptibility to be infected by PrP(Sc), but recently it was reported that rabbit prions can be generated through saPMCA (serial automated Protein Misfolding Cyclic Amplification) in vitro and the rabbit prion is infectious and transmissible. In this paper, we first do a detailed survey on the research advances of rabbit prion protein (RaPrP) and then we perform MD simulations on the NMR and X-ray molecular structures of rabbit prion protein wild-type and mutants. The survey shows to us that rabbits were not challenged directly in vivo with other known prion strains and the saPMCA result did not pass the test of the known BSE strain of cattle. Thus, we might still look rabbits as a prion resistant species. MD results indicate that the three α-helices of the wild-type are stable under the neutral pH environment (but under low pH environment the three α-helices have been unfolded into β-sheets), and the three α-helices of the mutants (I214V and S173N) are unfolded into rich β-sheet structures under

  16. Green fluorescent protein as a reporter of prion protein folding

    Directory of Open Access Journals (Sweden)

    Dalton Kevin

    2006-08-01

    Full Text Available Abstract Background The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP, as a method to examine the refolding of the amino terminal domain of mouse prion protein. Results Fusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence. Conclusion Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

  17. Peroxiredoxin 6 promotes upregulation of the prion protein (PrP in neuronal cells of prion-infected mice

    Directory of Open Access Journals (Sweden)

    Wagner Wibke

    2012-12-01

    Full Text Available Abstract Background It has been widely established that the conversion of the cellular prion protein (PrPC into its abnormal isoform (PrPSc is responsible for the development of transmissible spongiform encephalopathies (TSEs. However, the knowledge of the detailed molecular mechanisms and direct functional consequences within the cell is rare. In this study, we aimed at the identification of deregulated proteins which might be involved in prion pathogenesis. Findings Apolipoprotein E and peroxiredoxin 6 (PRDX6 were identified as upregulated proteins in brains of scrapie-infected mice and cultured neuronal cell lines. Downregulation of PrP gene expression using specific siRNA did not result in a decrease of PRDX6 amounts. Interestingly, selective siRNA targeting PRDX6 or overexpression of PRDX6 controlled PrPC and PrPSc protein amounts in neuronal cells. Conclusions Besides its possible function as a novel marker protein in the diagnosis of TSEs, PDRX6 represents an attractive target molecule in putative pharmacological intervention strategies in the future.

  18. Mapping functional prion-prion protein interaction sites using prion protein based peptide-arrays

    NARCIS (Netherlands)

    Rigter, A.; Priem, J.; Timmers-Parohi, D.; Langeveld, J.; Bossers, A.

    2009-01-01

    Protein-protein interactions are at the basis of most if not all biological processes in living cells. Therefore, adapting existing techniques or developing new techniques to study interactions between proteins are of importance in elucidating which amino acid sequences contribute to these interacti

  19. A novel mutation (G114V) in the prion protein gene in a family with inherited prion disease.

    Science.gov (United States)

    Rodriguez, M-M; Peoc'h, K; Haïk, S; Bouchet, C; Vernengo, L; Mañana, G; Salamano, R; Carrasco, L; Lenne, M; Beaudry, P; Launay, J-M; Laplanche, J-L

    2005-04-26

    Inherited prion diseases are characterized by mutations in the PRNP gene encoding the prion protein (PrP). We report a novel missense mutation in the PRNP gene (resulting in a G114V mutation in PrP) in members of a Uruguayan family with clinical and histopathologic features of prion disease. Affected individuals were characterized by an early age at onset, initial neuropsychiatric symptoms, late dementia with prominent pyramidal and extrapyramidal symptoms, and long disease duration.

  20. Unusual cerebral vascular prion protein amyloid distribution in scrapie-infected transgenic mice expressing anchorless prion protein

    OpenAIRE

    Rangel, Alejandra; Race, Brent; Klingeborn, Mikael; Striebel, James; Chesebro, Bruce

    2013-01-01

    Background In some prion diseases, misfolded aggregated protease-resistant prion protein (PrPres) is found in brain as amyloid, which can cause cerebral amyloid angiopathy. Small diffusible precursors of PrPres amyloid might flow with brain interstitial fluid (ISF), possibly accounting for the perivascular and intravascular distribution of PrPres amyloid. We previously reported that PrPres amyloid in scrapie-infected transgenic mice appeared to delay clearance of microinjected brain ISF trace...

  1. Strain-dependent profile of misfolded prion protein aggregates.

    Science.gov (United States)

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration. PMID:26877167

  2. Strain-dependent profile of misfolded prion protein aggregates.

    Science.gov (United States)

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

  3. What Makes a Protein Sequence a Prion?

    Science.gov (United States)

    Sabate, Raimon; Rousseau, Frederic; Schymkowitz, Joost; Ventura, Salvador

    2015-01-01

    Typical amyloid diseases such as Alzheimer's and Parkinson's were thought to exclusively result from de novo aggregation, but recently it was shown that amyloids formed in one cell can cross-seed aggregation in other cells, following a prion-like mechanism. Despite the large experimental effort devoted to understanding the phenomenon of prion transmissibility, it is still poorly understood how this property is encoded in the primary sequence. In many cases, prion structural conversion is driven by the presence of relatively large glutamine/asparagine (Q/N) enriched segments. Several studies suggest that it is the amino acid composition of these regions rather than their specific sequence that accounts for their priogenicity. However, our analysis indicates that it is instead the presence and potency of specific short amyloid-prone sequences that occur within intrinsically disordered Q/N-rich regions that determine their prion behaviour, modulated by the structural and compositional context. This provides a basis for the accurate identification and evaluation of prion candidate sequences in proteomes in the context of a unified framework for amyloid formation and prion propagation. PMID:25569335

  4. Enhancement of phagocytotic activity by prion protein in PrP-deficient macrophage cells.

    Science.gov (United States)

    Uraki, Ryuta; Sakudo, Akikazu; Ando, Saeko; Kitani, Hiroshi; Onodera, Takashi

    2010-10-01

    Macrophages, especially follicular dendritic cells, contribute to the pathogenesis of prion diseases by accumulating an abnormal isoform of prion protein (PrPSc), which is converted from the cellular isoform of prion protein (PrPC). As information on the function of PrPC in macrophages is limited, we have established a prion protein (PrP) gene (Prnp)-deficient macrophage cell line from the bone marrow of ZrchI Prnp-/- mice. These cells expressed macrophage specific proteins (F4/80 and MOMA-2) and displayed phagocytotic properties. The Prnp-/- macrophage cell line (MplZ) showed shorter pseudopodium extension and less phagocytotic activity than a Prnp+/+ macrophage cell line (MWF). In addition, the MplZ cells were more sensitive to serum deprivation than the MWF cells and underwent apoptotic cell death in these conditions. These findings suggest that PrPC enhances the incorporation of materials possibly including PrPSc and decreases the sensitivity of cells to oxidative stress, which may be induced by PrPSc accumulation. PMID:20818492

  5. Prions

    Directory of Open Access Journals (Sweden)

    W. Bodemer

    2016-09-01

    BSE is always unknown. Telemetry revealed a shift in sleep–wake cycles early on, long before behavioral changes or clinical symptoms appeared. Pathology confirmed non-neuronal tissue as hidden places where prions exist. The rhesus model also allowed first comparative studies of epigenetic modifications on RNA in peripheral blood and brain tissue collected from uninfected and prion-infected animals. To conclude, our studies clearly demonstrated that this model is valid since progression to disease is almost identical to human CJD.

  6. Prions Ex Vivo: What Cell Culture Models Tell Us about Infectious Proteins

    OpenAIRE

    Sybille Krauss; Ina Vorberg

    2013-01-01

    Prions are unconventional infectious agents that are composed of misfolded aggregated prion protein. Prions replicate their conformation by template-assisted conversion of the endogenous prion protein PrP. Templated conversion of soluble proteins into protein aggregates is also a hallmark of other neurodegenerative diseases. Alzheimer's disease or Parkinson's disease are not considered infectious diseases, although aggregate pathology appears to progress in a stereotypical fashion reminiscent...

  7. Regional brain metabolite abnormalities in inherited prion disease and asymptomatic gene carriers demonstrated in vivo by quantitative proton magnetic resonance spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Waldman, A.D.; Cordery, R.J.; Godbolt, A.; Rossor, M.N. [University College London, Dementia Research Group, Department of Neurodegenerative Disease, Institute of Neurology, London (United Kingdom); Imperial College of Science, Technology and Medicine, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, London (United Kingdom); MacManus, D.G. [University College London, NMR Research Unit, Department of Clinical Neurology, Institute of Neurology, London (United Kingdom); Collinge, J. [University College London, MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, London (United Kingdom)

    2006-06-15

    Inherited prion diseases are caused by mutations in the gene which codes for prion protein (PrP), leading to proliferation of abnormal PrP isomers in the brain and neurodegeneration; they include Gerstmann-Straeussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). We studied two patients with symptomatic inherited prion disease (P102L) and two pre-symptomatic P102L gene carriers using quantitative magnetic resonance spectroscopy (MRS). Short echo time spectra were acquired from the thalamus, caudate region and frontal white matter, metabolite levels and ratios were measured and z-scores calculated for individual patients relative to age-matched normal controls. MRS data were compared with structural magnetic resonance imaging. One fCJD case had generalised atrophy and showed increased levels of myo-inositol (MI) in the thalamus (z=3.7). The other had decreased levels of N-acetylaspartate (z=4) and diffuse signal abnormality in the frontal white matter. Both asymptomatic gene carriers had normal imaging, but increased frontal white matter MI (z=4.3, 4.1), and one also had increased MI in the caudate (z=5.3). Isolated MI abnormalities in asymptomatic gene carriers are a novel finding and may reflect early glial proliferation, prior to significant neuronal damage. MRS provides potential non-invasive surrogate markers of early disease and progression in inherited prion disease. (orig.)

  8. Regional brain metabolite abnormalities in inherited prion disease and asymptomatic gene carriers demonstrated in vivo by quantitative proton magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Inherited prion diseases are caused by mutations in the gene which codes for prion protein (PrP), leading to proliferation of abnormal PrP isomers in the brain and neurodegeneration; they include Gerstmann-Straeussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). We studied two patients with symptomatic inherited prion disease (P102L) and two pre-symptomatic P102L gene carriers using quantitative magnetic resonance spectroscopy (MRS). Short echo time spectra were acquired from the thalamus, caudate region and frontal white matter, metabolite levels and ratios were measured and z-scores calculated for individual patients relative to age-matched normal controls. MRS data were compared with structural magnetic resonance imaging. One fCJD case had generalised atrophy and showed increased levels of myo-inositol (MI) in the thalamus (z=3.7). The other had decreased levels of N-acetylaspartate (z=4) and diffuse signal abnormality in the frontal white matter. Both asymptomatic gene carriers had normal imaging, but increased frontal white matter MI (z=4.3, 4.1), and one also had increased MI in the caudate (z=5.3). Isolated MI abnormalities in asymptomatic gene carriers are a novel finding and may reflect early glial proliferation, prior to significant neuronal damage. MRS provides potential non-invasive surrogate markers of early disease and progression in inherited prion disease. (orig.)

  9. Yeast prion architecture explains how proteins can be genes

    Science.gov (United States)

    Wickner, Reed

    2013-03-01

    Prions (infectious proteins) transmit information without an accompanying DNA or RNA. Most yeast prions are self-propagating amyloids that inactivate a normally functional protein. A single protein can become any of several prion variants, with different manifestations due to different amyloid structures. We showed that the yeast prion amyloids of Ure2p, Sup35p and Rnq1p are folded in-register parallel beta sheets using solid state NMR dipolar recoupling experiments, mass-per-filament-length measurements, and filament diameter measurements. The extent of beta sheet structure, measured by chemical shifts in solid-state NMR and acquired protease-resistance on amyloid formation, combined with the measured filament diameters, imply that the beta sheets must be folded along the long axis of the filament. We speculate that prion variants of a single protein sequence differ in the location of these folds. Favorable interactions between identical side chains must hold these structures in-register. The same interactions must guide an unstructured monomer joining the end of a filament to assume the same conformation as molecules already in the filament, with the turns at the same locations. In this way, a protein can template its own conformation, in analogy to the ability of a DNA molecule to template its sequence by specific base-pairing. Bldg. 8, Room 225, NIH, 8 Center Drive MSC 0830, Bethesda, MD 20892-0830, wickner@helix.nih.gov, 301-496-3452

  10. Prion protein amyloidosis with divergent phenotype associated with two novel nonsense mutations in PRNP

    NARCIS (Netherlands)

    C. Jansen (Casper); P. Parchi (Piero); S. Capellari (Sabina); A.J. Vermeij (Ad); P. Corrado (Patrizia); F. Baas (Frank); R. Strammiello (Rosario); W.A. van Gool (Willem); J.C. van Swieten; A.J.M. Rozemuller (Annemieke)

    2010-01-01

    textabstractStop codon mutations in the gene encoding the prion protein (PRNP) are very rare and have thus far only been described in two patients with prion protein cerebral amyloid angiopathy (PrP-CAA). In this report, we describe the clinical, histopathological and pathological prion protein (PrP

  11. Prion protein accumulation in lipid rafts of mouse aging brain.

    Directory of Open Access Journals (Sweden)

    Federica Agostini

    Full Text Available The cellular form of the prion protein (PrP(C is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrP(C. In old mice, this change favors PrP(C accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrP(C translocation into detergent-resistant membranes (DRMs, we looked at PrP(C compartmentalization in hippocampi from acid sphingomyelinase (ASM knockout (KO mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrP(C in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases.

  12. Curcumin Reduces Amyloid Fibrillation of Prion Protein and Decreases Reactive Oxidative Stress

    OpenAIRE

    Raymond Chung; Cheng-I Lee; Chi-Fen Lin; Cheng-Ping Jheng; Kun-Hua Yu

    2013-01-01

    Misfolding and aggregation into amyloids of the prion protein (PrP) is responsible for the development of fatal transmissible neurodegenerative diseases. Various studies on curcumin demonstrate promise for the prevention of Alzheimer’s disease and inhibition of PrPres accumulation. To evaluate the effect of curcumin on amyloid fibrillation of prion protein, we first investigated the effect of curcumin on mouse prion protein (mPrP) in a cell-free system. Curcumin reduced the prion fibril forma...

  13. Concentration-dependent Cu(II) binding to prion protein

    Science.gov (United States)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  14. Characterization of variant Creutzfeldt-Jakob disease prions in prion protein-humanized mice carrying distinct codon 129 genotypes.

    Science.gov (United States)

    Takeuchi, Atsuko; Kobayashi, Atsushi; Ironside, James W; Mohri, Shirou; Kitamoto, Tetsuyuki

    2013-07-26

    To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.

  15. Normal Modes of Prion Proteins: From Native to Infectious particle◊

    OpenAIRE

    Samson, Abraham O.; Levitt, Michael

    2011-01-01

    Prion proteins (PrP) are the infectious agent in transmissible spongiform encephalopathies (i.e. mad cow disease). To be infectious, prion proteins must undergo a conformational change involving a decrease of α-helical content along with an increase of β-strand structure. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two α-helices by one and two residues, as well as an extension of two β-strands by three residues each whic...

  16. Inherited prion disease A117V is not simply a proteinopathy but produces prions transmissible to transgenic mice expressing homologous prion protein.

    Directory of Open Access Journals (Sweden)

    Emmanuel A Asante

    Full Text Available Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP gene (PRNP and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP(Sc, pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP ((CtmPrP. Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP(Sc was demonstrated in the brains of recipient transgenic mice. This PrP(Sc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of (CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.

  17. Humic substances interfere with detection of pathogenic prion protein

    Science.gov (United States)

    Smith, Christen B.; Booth, Clarissa J.; Wadzinski, Tyler J.; Legname, Giuseppe; Chappell, Rick; Johnson, Christopher J.; Pedersen, Joel A.

    2014-01-01

    Studies examining the persistence of prions (the etiological agent of transmissible spongiform encephalopathies) in soil require accurate quantification of pathogenic prion protein (PrPTSE) extracted from or in the presence of soil particles. Here, we demonstrate that natural organic matter (NOM) in soil impacts PrPTSE detection by immunoblotting. Methods commonly used to extract PrPTSE from soils release substantial amounts of NOM, and NOM inhibited PrPTSE immunoblot signal. The degree of immunoblot interference increased with increasing NOM concentration and decreasing NOM polarity. Humic substances affected immunoblot detection of prion protein from both deer and hamsters. We also establish that after interaction with humic acid, PrPTSE remains infectious to hamsters inoculated intracerebrally, and humic acid appeared to slow disease progression. These results provide evidence for interactions between PrPTSE and humic substances that influence both accurate measurement of PrPTSE in soil and disease transmission.

  18. Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

    Science.gov (United States)

    Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

    2015-01-01

    Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

  19. Prions

    Directory of Open Access Journals (Sweden)

    J. M. Godoy

    1991-06-01

    Full Text Available Os autores se propõem a revisar alguns aspectos básicos sobre os prions, alertando sobre a possível participação destes na etiologia de algumas enfermidades degenerativas do sistema nervoso.

  20. Variation in the prion protein sequence in Dutch goat breeds

    NARCIS (Netherlands)

    Windig, J.J.; Hoving, R.A.H.; Priem, J.; Bossers, A.; Keulen, van L.J.M.; Langeveld, J.P.M.

    2016-01-01

    Scrapie is a neurodegenerative disease occurring in goats and sheep. Several haplotypes of the prion protein increase resistance to scrapie infection and may be used in selective breeding to help eradicate scrapie. In this study, frequencies of the allelic variants of the PrP gene are determined

  1. Cell-biological aspects of the prion protein in transgenic Xenopus intermediate pituitary cells

    NARCIS (Netherlands)

    Rosmalen, J.W.G. van

    2007-01-01

    In mammals, prions are the causative agents of various neurodegenerative diseases (e.g. scrapie, mad cow and Creutzfeldt-Jakob disease) in which the three-dimensional structure of the normal cellular form of the prion protein (PrPC) is misfolded into the infectious scrapie form (PrPSc or prion). In

  2. Chronic Lymphocytic Inflammation Specifies the Organ Tropism of Prions

    Science.gov (United States)

    Heikenwalder, Mathias; Zeller, Nicolas; Seeger, Harald; Prinz, Marco; Klöhn, Peter-Christian; Schwarz, Petra; Ruddle, Nancy H.; Weissmann, Charles; Aguzzi, Adriano

    2005-02-01

    Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1+ cells expressing the normal cellular prion protein PrPC. By contrast, inflamed organs of mice lacking lymphotoxin-α or its receptor did not accumulate the abnormal isoform PrPSc, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.

  3. Yeast prions are useful for studying protein chaperones and protein quality control.

    Science.gov (United States)

    Masison, Daniel C; Reidy, Michael

    2015-01-01

    Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.

  4. Prions in Variably Protease-Sensitive Prionopathy: An Update

    NARCIS (Netherlands)

    Zou, W.Q.; Gambetti, P.; Xiao, X.; Yuan, J.; Langeveld, J.P.M.; Pirisinu, L.

    2013-01-01

    Human prion diseases, including sporadic, familial, and acquired forms such as Creutzfeldt-Jakob disease (CJD), are caused by prions in which an abnormal prion protein (PrPSc) derived from its normal cellular isoform (PrPC) is the only known component. The recently-identified variably protease-sensi

  5. Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

    NARCIS (Netherlands)

    Meade-White, K.; Race, B.; Trifilo, M.; Bossers, A.; Favara, C.; Lacasse, R.; Miller, M.; Williams, E.; Oldstone, M.; Race, R.; Chesebro, B.

    2007-01-01

    Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer

  6. Helicobacter pylori upregulates prion protein expression in gastric mucosa: A possible link to prion disease

    Institute of Scientific and Technical Information of China (English)

    Peter C Konturek; Karolina Bazela; Vitaliy Kukharskyy; Michael Bauer; Eckhart G Hahn; Detlef Schuppan

    2005-01-01

    AIM: Pathological prion protein (PrPSC) is responsible for the development of transmissible spongiform encephalopathies (TSE). While PrPc enters the organism via the oral route, less data is available to know about its uptake and the role of gastrointestinal inflammation on the expression of prion precursor PrPc, which is constitutively expressed in the gastric mucosa.METHODS: We studied PrPc expression in the gastric mucosa of 10 Helicobacter pylori-positive patients before and after successful H pylori eradication compared to non-infected controls using RT-PCR and Western blotting.The effect of central mediators of gastric inflammation,i.e., gastrin, prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) on PrPc expression was analyzed in gastric cell lines.RESULTS: PrPc expression was increased in H pyloriinfection compared with non-infected controls and decreased to normal after successful eradication. Gastrin,PGE2, and IL-1β dose-dependently upregulated PrPc in gastric cells, while TNF-α had no effect.CONCLUSION: H pylori infection leads to the upregulation of gastric PrPc expression. This can be linked to H pylori induced hypergastrinemia and increased mucosal PGE2 and IL-1β synthesis.H pylori creates a milieu for enhanced propagation of prions in the gastrointestinal tract.

  7. A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein

    OpenAIRE

    Edskes, Herman K.; Wickner, Reed B.

    2000-01-01

    Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt–Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required f...

  8. Prion protein degradation by lichens of the genus Cladonia

    Science.gov (United States)

    Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.

    2012-01-01

    It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

  9. The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion

    Science.gov (United States)

    Giachin, Gabriele; Mai, Phuong Thao; Tran, Thanh Hoa; Salzano, Giulia; Benetti, Federico; Migliorati, Valentina; Arcovito, Alessandro; Longa, Stefano Della; Mancini, Giordano; D'Angelo, Paola; Legname, Giuseppe

    2015-10-01

    The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

  10. Prion protein: structural features and related toxicity

    Institute of Scientific and Technical Information of China (English)

    Ping Ping Hu; Cheng Zhi Huang

    2013-01-01

    Transmissible spongiform encephalopathies,or prion diseases,is a group of infectious neurodegenerative disorders.The conformational conversion from cellular form (PrPC) to disease-causing isoform (PrPSc) is considered to be the most important and remarkable event in these diseases,while accumulation of PrPSc is thought to be the main reason for cell death,inflammation and spongiform degeneration observed in infected individuals.Although these rare but unique neurodegenerative disorders have attracted much attention,there are still many questions that remain to be answered.Knowledge of the scrapie agent structures and the toxic species may have significance for understanding the causes of the diseases,and could be helpful for rational design of novel therapeutic and diagnostic methods.In this review,we summarized the available experimental evidence concerning the relationship among the structural features,aggregation status of misfolded PrP and related neurotoxicity in the course of prion diseases development.In particular,most data supports the idea that the smaller oligomeric PrPSc aggregates,rather than the mature amyloid fibers,exhibit the highest toxicity to the host.

  11. Role of autophagy in prion protein-induced neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Hao Yao; Deming Zhao; Sher Hayat Khan; Lifeng Yang

    2013-01-01

    Prion diseases,characterized by spongiform degeneration and the accumulation of misfolded and aggregated PrPSc in the central nervous system,are one of fatal neurodegenerative and infectious disorders of humans and animals.In earlier studies,autophagy vacuoles in neurons were frequently observed in neurodegenerative diseases such as Alzheimer's,Parkinson's,and Huntington's diseases as well as prion diseases.Autophagy is a highly conserved homeostatic process by which several cytoplasmic components (proteins or organelles) are sequestered in a doublemembrane-bound vesicle termed 'autophagosome' and degraded upon their fusion with lysosome.The pathway of intercellular self-digestion at basal physiological levels is indispensable for maintaining the healthy status of tissues and organs.In case of prion infection,increasing evidence indicates that autophagy has a crucial ability of eliminating pathological PrPSc accumulated within neurons.In contrast,autophagy dysfunction in affected neurons may contribute to the formation of spongiform changes.In this review,we summarized recent findings about the effect of mammalian autophagy in neurodegenerative disorders,particularly in prion diseases.We also summarized the therapeutic potential of some small molecules (such as lithium,rapamycin,Sirtuin 1 and resveratrol) targets to mitigate such diseases on brain function.Furthermore,we discussed the controversial role of autophagy,whether it mediates neuronal toxicity or serves a protective function in neurodegenerative disorders.

  12. Peripheral prion disease pathogenesis is unaltered in the absence of sialoadhesin (Siglec-1/CD169)

    OpenAIRE

    Bradford, Barry; Crocker, P R; Mabbott, Neil

    2014-01-01

    Prions are a unique group of pathogens, which are considered to comprise solely of an abnormally folded isoform of the cellular prion protein. The accumulation and replication of prions within secondary lymphoid organs is important for their efficient spread from the periphery to the brain where they ultimately cause neurodegeneration and death. Mononuclear phagocytes (MNP) play key roles in prion disease pathogenesis. Some MNP appear to facilitate the propagation of prions to and within lymp...

  13. 朊病毒蛋白Prion的研究进展%Progresses on prion proteins

    Institute of Scientific and Technical Information of China (English)

    林东海; 文祎

    2011-01-01

    Transmissible spongiform encephalopathies are a family of fatal neurodegenerative disorders. The pathogen has been identified as the abnormal isoform of the host-derived prion protein. The molecular mechanism of the conformational conversion of the prion protein is unclear so far. However, much valuable information closely related to the conformational change has been obtained by means of physical, chemical and biological techniques.Recent progresses on three-dimensional structures, dynamics, unfolding and intermolecular interactions of prion proteins are reviewed herein, together with novel results about the solution structure and dynamics of the rabbit prion protein achieved from our laboratory.%传染性海绵状脑病是一类致死性的神经系统退行性疾病,其发病机制与朊病毒蛋白prion的异常构象有关.迄今为止,prion致病的构象变化机制依然是一个未解之谜.国内外研究人员从不同切入点着手,采用物理、化学、生物等多种学科的技术手段,发现并积累了大量与prion构象转变相关的有价值的信息.本文综述了近年来人们在prion蛋白的三维结构、动力学、去折叠以及相互作用等方面取得的研究进展,并简要地介绍了本实验室在兔prion蛋白溶液结构和动力学方面的研究工作.

  14. Identification of a Protein that Purifies with the Scrapie Prion

    Science.gov (United States)

    Bolton, David C.; McKinley, Michael P.; Prusiner, Stanley B.

    1982-12-01

    Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.

  15. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Science.gov (United States)

    Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

    2014-07-01

    Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

  16. Strain Specific Resistance to Murine Scrapie Associated with a Naturally Occurring Human Prion Protein Polymorphism at Residue 171

    OpenAIRE

    Striebel, James F.; Race, Brent; Meade-White, Kimberly D.; LaCasse, Rachel; Chesebro, Bruce

    2011-01-01

    Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion diseas...

  17. Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171.

    OpenAIRE

    Striebel, James F.; Brent Race; Meade-White, Kimberly D.; Rachel LaCasse; Bruce Chesebro

    2011-01-01

    Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion diseas...

  18. Intramolecular versus intermolecular disulfide bonds in prion proteins.

    Science.gov (United States)

    Welker, Ervin; Raymond, Lynne D; Scheraga, Harold A; Caughey, Byron

    2002-09-01

    Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.

  19. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    Science.gov (United States)

    Millhauser, Glenn L.

    2007-05-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

  20. The Role of Functional Prion-Like Proteins in the Persistence of Memory.

    Science.gov (United States)

    Si, Kausik; Kandel, Eric R

    2016-01-01

    Prions are a self-templating amyloidogenic state of normal cellular proteins, such as prion protein (PrP). They have been identified as the pathogenic agents, contributing to a number of diseases of the nervous system. However, the discovery that the neuronal RNA-binding protein, cytoplasmic polyadenylation element-binding protein (CPEB), has a prion-like state that is involved in the stabilization of memory raised the possibility that prion-like proteins can serve normal physiological functions in the nervous system. Here, we review recent experimental evidence of prion-like properties of neuronal CPEB in various organisms and propose a model of how the prion-like state may stabilize memory. PMID:27037416

  1. Transmission of Atypical Bovine Prions to Mice Transgenic for Human Prion Protein

    OpenAIRE

    Béringue, Vincent; Herzog, Laëtitia; Reine, Fabienne; Le Dur, Annick; Casalone, Cristina; Vilotte, Jean-Luc; Laude, Hubert

    2008-01-01

    To assess risk for cattle-to-human transmission of prions that cause uncommon forms of bovine spongiform encephalopathy (BSE), we inoculated mice expressing human PrP Met129 with field isolates. Unlike classical BSE agent, L-type prions appeared to propagate in these mice with no obvious transmission barrier. H-type prions failed to infect the mice.

  2. Molecular modeling of the conformational dynamics of the cellular prion protein

    Science.gov (United States)

    Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

    2014-03-01

    Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

  3. Prions mediated neurodegenerative disorders.

    Science.gov (United States)

    Huang, W-J; Chen, W-W; Zhang, X

    2015-11-01

    Prions are unprecedented infectious pathogens that are devoid of nucleic acid and cause a group of rare and invariably fatal neurodegenerative disorders, affecting approximately 1 person per 1 million inhabitants annually worldwide. These disorders include Creutzfeld-Jacob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, fatal insomnia (FI), and variable protease-sensitive prionopathy (VPSPr), all of which involve a conformational change of the normal cellular prion protein (PrPC) into the abnormal scrapie prion protein (PrPSc) through a posttranslational process during which PrPc acquires high β-sheet content. This structural change is accompanied by profound changes in the physicochemical properties of PrPC, rendering the molecule resistant to proteolysis. The conformational change of PrPC can occur due to either spontaneous conversion, dominant mutations in the prion protein (PRNP) gene encoding PrPC, or infection with pathogenic isoform PrPsc from exogenous sources. There is general agreement that PrPC serves as a substrate for conversion to abnormal PrPSc. This latter multiplies exponentially and aggregates in the brain, forming deposits that are associated with the neurodegenerative changes. Although the understanding of the primary causes of prion-induced neurodegeneration is still limited, propagation of PrPSc and neurotoxic signaling seem to interplay in pathogenic process of prions. Here, we review recent findings that have provided fresh insights into this process, and present an overview of incidence, causes and spectrum of related disorders.

  4. Evolutionary Implications of Metal Binding Features in Different Species’ Prion Protein: An Inorganic Point of View

    OpenAIRE

    Diego La Mendola; Enrico Rizzarelli

    2014-01-01

    Prion disorders are a group of fatal neurodegenerative conditions of mammals. The key molecular event in the pathogenesis of such diseases is the conformational conversion of prion protein, PrPC, into a misfolded form rich in β-sheet structure, PrPSc, but the detailed mechanistic aspects of prion protein conversion remain enigmatic. There is uncertainty on the precise physiological function of PrPC in healthy individuals. Several evidences support the notion of its role in copper homeostasis...

  5. Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains.

    Science.gov (United States)

    Orrú, Christina D; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

    2015-06-01

    Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

  6. Molecular dynamics studies on the structural stability of wild-type dog prion protein.

    Science.gov (United States)

    Zhang, Jiapu; Liu, David D W

    2011-06-01

    Prion diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or 'mad-cow' disease) and chronic wasting disease in cattle are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches to treat all these prion diseases. In 2008, canine mammals including dogs (canis familials) were the first time academically reported to be resistant to prion diseases (Vaccine 26: 2601-2614 (2008)). Thus, it is very worth studying the molecular structures of dog prion protein to obtain insights into the immunity of dogs to prion diseases. This paper studies the molecular structural dynamics of wild-type dog prion protein. The comparison analyses with rabbit prion protein show that the dog prion protein has stable molecular structures whether under neutral or low pH environments. We also find that the salt bridges such as D177-R163 contribute to the structural stability of wild-type rabbit prion protein under neutral pH environment. PMID:21469747

  7. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    OpenAIRE

    Millhauser, Glenn L.

    2007-01-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat d...

  8. Sialylation of prion protein controls the rate of prion amplification, the cross-species barrier, the ratio of PrPSc glycoform and prion infectivity.

    Directory of Open Access Journals (Sweden)

    Elizaveta Katorcha

    2014-09-01

    Full Text Available The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP(C into the disease-associated, transmissible form (PrP(Sc. Pr(PC is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP(C and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrP(C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP(Sc glycoform ratio. The current study demonstrates that undersialylated PrP(C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb at the expense of oversialylated PrP(C. As a result, PMCAb-derived PrP(Sc was less sialylated than brain-derived PrP(Sc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP(C using sialidase was found to increase the rate of PrP(Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP(C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP(C was also found to control PrP(Sc glycoform ratio. A decrease in Pr(PC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP(Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP(C differed from that of spleen-derived PrP(C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP(C, suggesting that Neu1 is not responsible for desialylation of Pr

  9. Progress in the development of therapeutic antibodies targeting prion proteins and β-amyloid peptides

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Prion diseases and Alzheimer’s disease (AD) are characterized by protein misfolding, and can lead to dementia. However, prion diseases are infectious and transmissible, while AD is not. The similarities and differences between these diseases have led researchers to perform comparative studies. In the last 2 decades, progress has been made in immunotherapy using anti-prion protein and anti-β-amyloid antibodies. In this study, we review new ideas and strategies for therapeutic antibodies targeting prion diseases and AD through conformation dependence.

  10. Prion Protein-specific antibodies that detect multiple TSE Agents with high sensitivity

    NARCIS (Netherlands)

    McCutcheon, S.; Langeveld, J.P.M.; Tan, B.C.; Gill, A.C.; Wolf, de C.A.; Martin, S.; Gonzalez, L.; Alibhai, J.; Alejo Blanco, A.R.; Campbell, L.; Hunter, N.; Houston, E.F.

    2014-01-01

    This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning re

  11. High CJD infectivity remains after prion protein is destroyed.

    Science.gov (United States)

    Miyazawa, Kohtaro; Emmerling, Kaitlin; Manuelidis, Laura

    2011-12-01

    The hypothesis that host prion protein (PrP) converts into an infectious prion form rests on the observation that infectivity progressively decreases in direct proportion to the decrease of PrP with proteinase K (PK) treatment. PrP that resists limited PK digestion (PrP-res, PrP(sc)) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique transmissible spongiform encephalopathy (TSE) agent strains. Recently, a PK sensitive form of PrP has been proposed as the prion. Thus we re-evaluated total PrP (sensitive and resistant) and used a cell-based assay for titration of infectious particles. A keratinase (NAP) known to effectively digest PrP was compared to PK. Total PrP in FU-CJD infected brain was reduced to ≤0.3% in a 2 h PK digest, yet there was no reduction in titer. Remaining non-PrP proteins were easily visualized with colloidal gold in this highly infectious homogenate. In contrast to PK, NAP digestion left 0.8% residual PrP after 2 h, yet decreased titer by >2.5 log; few residual protein bands remained. FU-CJD infected cells with 10× the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 log). Extreme PK digestions were needed to reduce cell PrP to report on maximal PrP destruction and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles.

  12. Casein kinase Ⅱ interacts with prion protein in vitro and forms complex with na-tive prion protein in vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase Ⅱ (CK2) is a ubiquitously expressed and evolutiouarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2α and CK2β complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2α and glutathione S-transferase-histidine-CK2β were expressed in Escherichia coll. The interaction between CK2 and PrP was evaluated with co-immunoprecipi-tation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2α, but not with CK2β. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2α subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2α subunit. The domain responsible for interacting with CK2α was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2α (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.

  13. Classifying prion and prion-like phenomena.

    Science.gov (United States)

    Harbi, Djamel; Harrison, Paul M

    2014-01-01

    The universe of prion and prion-like phenomena has expanded significantly in the past several years. Here, we overview the challenges in classifying this data informatically, given that terms such as "prion-like", "prion-related" or "prion-forming" do not have a stable meaning in the scientific literature. We examine the spectrum of proteins that have been described in the literature as forming prions, and discuss how "prion" can have a range of meaning, with a strict definition being for demonstration of infection with in vitro-derived recombinant prions. We suggest that although prion/prion-like phenomena can largely be apportioned into a small number of broad groups dependent on the type of transmissibility evidence for them, as new phenomena are discovered in the coming years, a detailed ontological approach might be necessary that allows for subtle definition of different "flavors" of prion / prion-like phenomena.

  14. Clusterin expression in follicular dendritic cells associated with prion protein accumulation.

    Science.gov (United States)

    Sasaki, K; Doh-ura, K; Ironside, Jw; Mabbott, N; Iwaki, T

    2006-08-01

    Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis. PMID:16767691

  15. Prion pathogenesis and secondary lymphoid organs (SLO): Tracking the SLO spread of prions to the brain

    OpenAIRE

    Mabbott, N A

    2012-01-01

    Prion diseases are subacute neurodegenerative diseases that affect humans and a range of domestic and free-ranging animal species. These diseases are characterized by the accumulation of PrP (Sc) , an abnormally folded isoform of the cellular prion protein (PrP (C) ), in affected tissues. The pathology during prion disease appears to occur almost exclusively within the central nervous system. The extensive neurodegeneration which occurs ultimately leads to the death of the host. An intriguing...

  16. Abbreviated incubation times for human prions in mice expressing a chimeric mouse–human prion protein transgene

    OpenAIRE

    Korth, Carsten; Kaneko, Kiyotoshi; Groth, Darlene; Heye, Norbert; Telling, Glenn; Mastrianni, James; Parchi, Piero; Gambetti, Pierluigi; Will, Robert; Ironside, James; Heinrich, Cornelia; Tremblay, Patrick; Stephen J DeArmond; Prusiner, Stanley B.

    2003-01-01

    Transgenic (Tg) mouse lines that express chimeric mouse–human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt–Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening ...

  17. Crossing species barrier by PrPSc replication in vitro generates new infectious prions

    OpenAIRE

    Castilla, Joaquín; Gonzalez-Romero, Dennisse; Saá, Paula; Morales, Rodrigo; Castro, Jorge; Soto, Claudio

    2008-01-01

    Prions are unconventional infectious agents composed exclusively by the misfolded prion protein (PrPSc), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrPC). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here we report the generation of novel infectious prions by inter-species transmission of PrPSc misfolding in vitro. Hamster PrPC misfolded by m...

  18. Polymorphisms in the prion protein gene and in the doppel gene increase susceptibility for Creutzfeldt-Jakob disease

    NARCIS (Netherlands)

    Croes, EA; Alizadeh, BZ; Bertoli-Avella, AM; Rademaker, T; Vergeer-Drop, J; Dermaut, B; Houwing-Duistermaat, JJ; Wientjens, DPWM; Hofman, A; Van Broeckhoven, C; van Duijn, CM

    2004-01-01

    The prion protein gene (PRNP) plays a central role in the origin of Creutzfeldt - Jakob disease (CJD), but there is growing interest in other polymorphisms that may be involved in CJD. Polymorphisms upstream of PRNP that may modulate the prion protein production as well as polymorphisms in the prion

  19. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    International Nuclear Information System (INIS)

    A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of ∼50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers

  20. Fatal transmissible amyloid encephalopathy: a new type of prion disease associated with lack of prion protein membrane anchoring.

    Directory of Open Access Journals (Sweden)

    Bruce Chesebro

    2010-03-01

    Full Text Available Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres. PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen, a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI. Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer's disease.

  1. Modulation and elimination of yeast prions by protein chaperones and co-chaperones

    OpenAIRE

    Reidy, Michael; Masison, Daniel C.

    2011-01-01

    The yeast system has provided considerable insight into the biology of amyloid and prions. Here we focus on how alterations in abundance or function of protein chaperones and co-chaperones affect propagation of yeast prions. In spite of a considerable amount of information, a clear understanding of the molecular mechanisms underlying these effects remains wanting.

  2. Genetic variation of the prion protein gene (PRNP) in alpaca (Vicugna pacos)

    Science.gov (United States)

    Transmissible spongiform encephalopathies (TSE) are caused by accumulation of a misfolded form of the prion protein (PrP). The normal cellular isoform of PrP is produced by the prion gene (PRNP) and is highly expressed in the central nervous system. Currently, there is an absence of information rega...

  3. Mathematical models for the diffusion magnetic resonance signal abnormality in patients with prion diseases

    Directory of Open Access Journals (Sweden)

    Matteo Figini

    2015-01-01

    Full Text Available In clinical practice signal hyperintensity in the cortex and/or in the striatum on magnetic resonance (MR diffusion-weighted images (DWIs is a marker of sporadic Creutzfeldt–Jakob Disease (sCJD. MR diagnostic accuracy is greater than 90%, but the biophysical mechanisms underpinning the signal abnormality are unknown. The aim of this prospective study is to combine an advanced DWI protocol with new mathematical models of the microstructural changes occurring in prion disease patients to investigate the cause of MR signal alterations. This underpins the later development of more sensitive and specific image-based biomarkers. DWI data with a wide a range of echo times and diffusion weightings were acquired in 15 patients with suspected diagnosis of prion disease and in 4 healthy age-matched subjects. Clinical diagnosis of sCJD was made in nine patients, genetic CJD in one, rapidly progressive encephalopathy in three, and Gerstmann–Sträussler–Scheinker syndrome in two. Data were analysed with two bi-compartment models that represent different hypotheses about the histopathological alterations responsible for the DWI signal hyperintensity. A ROI-based analysis was performed in 13 grey matter areas located in affected and apparently unaffected regions from patients and healthy subjects. We provide for the first time non-invasive estimate of the restricted compartment radius, designed to reflect vacuole size, which is a key discriminator of sCJD subtypes. The estimated vacuole size in DWI hyperintense cortex was in the range between 3 and 10 µm that is compatible with neuropathology measurements. In DWI hyperintense grey matter of sCJD patients the two bi-compartment models outperform the classic mono-exponential ADC model. Both new models show that T2 relaxation times significantly increase, fast and slow diffusivities reduce, and the fraction of the compartment with slow/restricted diffusion increases compared to unaffected grey matter of

  4. The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis

    OpenAIRE

    David DONALDSON; Else, Kathryn J.; Mabbott, Neil

    2015-01-01

    ABSTRACT Prion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy s...

  5. Naturally prion resistant mammals: a utopia?

    Science.gov (United States)

    Fernández-Borges, Natalia; Chianini, Francesca; Eraña, Hasier; Vidal, Enric; Eaton, Samantha L; Pintado, Belén; Finlayson, Jeanie; Dagleish, Mark P; Castilla, Joaquín

    2012-01-01

    Each known abnormal prion protein (PrP (Sc) ) is considered to have a specific range and therefore the ability to infect some species and not others. Consequently, some species have been assumed to be prion disease resistant as no successful natural or experimental challenge infections have been reported. This assumption suggested that, independent of the virulence of the PrP (Sc) strain, normal prion protein (PrP (C) ) from these 'resistant' species could not be induced to misfold. Numerous in vitro and in vivo studies trying to corroborate the unique properties of PrP (Sc) have been undertaken. The results presented in the article "Rabbits are not resistant to prion infection" demonstrated that normal rabbit PrP (C) , which was considered to be resistant to prion disease, can be misfolded to PrP (Sc) and subsequently used to infect and transmit a standard prion disease to leporids. Using the concept of species resistance to prion disease, we will discuss the mistake of attributing species specific prion disease resistance based purely on the absence of natural cases and incomplete in vivo challenges. The BSE epidemic was partially due to an underestimation of species barriers. To repeat this error would be unacceptable, especially if present knowledge and techniques can show a theoretical risk. Now that the myth of prion disease resistance has been refuted it is time to re-evaluate, using the new powerful tools available in modern prion laboratories, whether any other species could be at risk. PMID:22954650

  6. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Directory of Open Access Journals (Sweden)

    Julia M Harris

    2014-07-01

    Full Text Available Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion

  7. Cell type-specific neuroprotective activity of untranslocated prion protein.

    Directory of Open Access Journals (Sweden)

    Elena Restelli

    Full Text Available BACKGROUND: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. PRINCIPAL FINDINGS: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. SIGNIFICANCE: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

  8. Cooperative binding modes of Cu(II) in prion protein

    Science.gov (United States)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  9. Analysis of factors that modulate the toxicity of the yeast prion protein Rnq1

    OpenAIRE

    Li, Zhiyuan

    2016-01-01

    Prions are infectious proteins that form transmissible, self-propagating amyloids that convert protein from its normal state into the prion state. The accumulation of amyloid is the causative agent of several neurodegenerative diseases, for instance, Huntington’s disease, which is caused by a polyglutamine expansion in the huntingtin (Htt) protein. In this study, a yeast-based Huntington’s disease model was created to investigate the mechanism of amyloid toxicity and how nuclear genes modulat...

  10. A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein.

    Science.gov (United States)

    Massignan, Tania; Cimini, Sara; Stincardini, Claudia; Cerovic, Milica; Vanni, Ilaria; Elezgarai, Saioa R; Moreno, Jorge; Stravalaci, Matteo; Negro, Alessandro; Sangiovanni, Valeria; Restelli, Elena; Riccardi, Geraldina; Gobbi, Marco; Castilla, Joaquín; Borsello, Tiziana; Nonno, Romolo; Biasini, Emiliano

    2016-01-01

    Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrP(C) may provide an opportunity to overcome these problems. PrP(C) ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrP(C), and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrP(C)-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrP(C)-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrP(C) may produce a dual effect of blocking prion replication and inhibiting PrP(C)-mediated toxicity. PMID:26976106

  11. Role of cellular prion proteins in the function of macrophages and dendritic cells.

    Science.gov (United States)

    Nitta, Kayako; Sakudo, Akikazu; Masuyama, Jun; Xue, Guangai; Sugiura, Katsuaki; Onodera, Takashi

    2009-01-01

    The cellular isoform of prion proteins (PrPC) is expressed in hematopoietic stem cells, granulocytes, T and B lymphocyte natural killer cells, platelets, monocytes, dendritic cells, and follicular dendritic cells, which may act as carrier cells for the spread of its abnormal isoform (PrPSc) before manifesting transmissible spongiform encephalopathies (TSEs). In particular, macrophages and dendritic cells seem to be involved in the replication of PrPSc after ingestion. In addition, information on the role of PrPC during phagocytotic activity in these cells has been obtained. A recent study showed that resident macrophages from ZrchI PrP gene (Prnp)-deficient (Prnp-/-) mice show augmented phagocytotic activity compared to Prnp+/+ counterparts. In contrast, our study suggests that Rikn Prnp-/- peritoneal macrophages show pseudopodium extension arrest and up-regulation of phagocytotic activity compared to Prnp+/+ cells. Although reports regarding phagocytotic activity in resident and peritoneal macrophages are inconsistent between ZrchI and Rikn Prnp-/- mice, it seems plausible that PrPC in macrophages could contribute to maintain the immunological environment. This review will introduce the recent progress in understanding the functions of PrPC in macrophages and dendritic cells under physiological conditions and its involvement in the pathogenesis of prion diseases. PMID:19275736

  12. A survey and a molecular dynamics study on the (central) hydrophobic region of prion proteins.

    Science.gov (United States)

    Zhang, Jiapu; Wang, Feng

    2014-01-01

    Prion diseases which are serious neurodegenerative diseases that affect humans and animals occur in various of species. Unlike many other neurodegenerative diseases affected by amyloid, prion diseases can be highly infectious. Prion diseases occur in many species. In humans, prion diseases include the fatal human neurodegenerative diseases such as Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia (FFI), Gerstmann-Strussler-Scheinker syndrome (GSS) and Kuru etc. In animals, prion diseases are related to the bovine spongiform encephalopathy (BSE or 'mad-cow' disease) in cattle, the chronic wasting disease (CWD) found in deer and elk, and scrapie seen in sheep and goats, etc. More seriously, the fact that transmission of the prion diseases across the species barrier to other species such as humans has caused a major public health concern worldwide. For example, the BSE in Europe, the CWD in North America, and variant CJDs (vCJDs) in young people of UK. Fortunately, it is discovered that the hydrophobic region of prion proteins (PrP) controls the formation of diseased prions (PrP(Sc)), which provide some clues in control of such diseases. This article provides a detailed survey of recent studies with respect to the PrP hydrophobic region of human PrP(110-136) using molecular dynamics studies. PMID:25373387

  13. Surface charge of polyoxometalates modulates polymerization of the scrapie prion protein

    OpenAIRE

    Wille, Holger; Shanmugam, Maheswaran; Murugesu, Muralee; Ollesch, Julian; Stubbs, Gerald; Long, Jeffrey R.; Safar, Jiri G.; Prusiner, Stanley B

    2009-01-01

    Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrPSc. N-terminally truncated PrPSc, denoted PrP 27–30, retains infectivity and polymerizes into rods with the ultrastructural and tinctorial properties of amyloid. We report here that some polyoxometalates (POMs) favor polymerization of PrP 27-30 into prion rods, whereas other POMs promote assembly of the protein into 2D crystals. Antibodies reacting with epitopes in denatured PrP 27-30 also ...

  14. Molecular dynamics simulations on structural conformations of rhodopsin and prion proteins

    International Nuclear Information System (INIS)

    Molecular dynamics simulations were performed to investigate the structural conformation of the rhodopsin and prion proteins. We have estimated the effect of specific disease-related amino acid mutations on the dynamics and conformational changes

  15. Prions in Yeast

    OpenAIRE

    Liebman, Susan W; Chernoff, Yury O.

    2012-01-01

    The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in unde...

  16. Prion protein detection in serum using micromechanical resonator arrays.

    Science.gov (United States)

    Varshney, Madhukar; Waggoner, Philip S; Montagna, Richard A; Craighead, Harold G

    2009-12-15

    Prion proteins that have transformed from their normal cellular counterparts (PrP(c)) into infectious form (PrP(res)) are responsible for causing progressive neurodegenerative diseases in numerous species, such as bovine spongiform encephalopathy (BSE) in cattle (also known as mad cow disease), scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans. Due to a possible link between BSE and CJD it is highly desirable to develop non-invasive and ante mortem tests for the detection of prion proteins in bovine samples. Such ante mortem tests of all cows prior to slaughter will help to prevent the introduction of PrP(res) into the human food supply. Furthermore, detection of PrP(res) in donated blood will also help to prevent the transmission of CJD among humans through blood transfusion. In this study, we have continued development of a micromechanical resonator array that is capable of detecting PrP(c) in bovine blood serum. The sensitivity of the resonators for the detection of PrP(c) is further enhanced by the use of secondary mass labels. A pair of antibodies is used in a sandwich immunoassay format to immobilize PrP(c) on the surface of resonators and attach nanoparticles as secondary mass labels to PrP(c). Secondary mass labeling is optimized in terms of incubation time to maximize the frequency shifts that correspond to the presence of PrP(c) on the surface of resonators. Our results show that a minimum of 200 pg mL(-1) of PrP(c) in blood serum can be detected using micromechanical resonator arrays. PMID:19836525

  17. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  18. Influence of prion strain on prion protein adsorption to soil in a competitive matrix.

    Science.gov (United States)

    Saunders, Samuel E; Bartz, Jason C; Bartelt-Hunt, Shannon L

    2009-07-15

    It is likely that the soil environment serves as a stable reservoir of infectious chronic wasting disease (CWD) and scrapie prions, as well as a potential reservoir of bovine spongiform encephalopathy (BSE, or "mad cow" disease). Prion adsorption to soil may play an important role in prion mobility, proteolysis, and infectivity. Differences in PrP environmental fate are possible due to the strain- and species-dependent structure of PrP(Sc). Kinetic and isothermal studies of PrP adsorption to sand and two whole soils were conducted using HY and DY TME-infected hamster, uninfected hamster, and CWD-infected elk brain homogenates as competitive PrP sources. The role of the N-terminus in PrP adsorption was also investigated. We report strain and species differences in PrP adsorption to soil over time and as a function of aqueous concentration, indicating that the fate of prions in the environment may vary with the prion strain and species infected. Our data also provide evidence that the N-terminal region of PrP enhances adsorption to clay but may hinder adsorption to sand. PrP adsorption was maximal at an intermediate aqueous concentration, most likely due to the competitive brain homogenate matrix in which it enters the soil environment. PMID:19708348

  19. Prion protein NMR structure and species barrier for prion diseases

    OpenAIRE

    Billeter, Martin; Riek, Roland; Wider, Gerhard; Hornemann, Simone; Glockshuber, Rudi; Wüthrich, Kurt

    1997-01-01

    The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amin...

  20. Evolutionary Implications of Metal Binding Features in Different Species’ Prion Protein: An Inorganic Point of View

    Directory of Open Access Journals (Sweden)

    Diego La Mendola

    2014-05-01

    Full Text Available Prion disorders are a group of fatal neurodegenerative conditions of mammals. The key molecular event in the pathogenesis of such diseases is the conformational conversion of prion protein, PrPC, into a misfolded form rich in β-sheet structure, PrPSc, but the detailed mechanistic aspects of prion protein conversion remain enigmatic. There is uncertainty on the precise physiological function of PrPC in healthy individuals. Several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ mainly through a domain composed by four to five repeats of eight amino acids. In addition to mammals, PrP homologues have also been identified in birds, reptiles, amphibians and fish. The globular domain of protein is retained in the different species, suggesting that the protein carries out an essential common function. However, the comparison of amino acid sequences indicates that prion protein has evolved differently in each vertebrate class. The primary sequences are strongly conserved in each group, but these exhibit a low similarity with those of mammals. The N-terminal domain of different prions shows tandem amino acid repeats with an increasing amount of histidine residues going from amphibians to mammals. The difference in the sequence affects the number of copper binding sites, the affinity and the coordination environment of metal ions, suggesting that the involvement of prion in metal homeostasis may be a specific characteristic of mammalian prion protein. In this review, we describe the similarities and the differences in the metal binding of different species’ prion protein, as revealed by studies carried out on the entire protein and related peptide fragments.

  1. Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

    Science.gov (United States)

    The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

  2. MAD COW DISEASE: New Recruits for French Prion Research.

    Science.gov (United States)

    Casassus, B

    2000-12-01

    As panic over "mad cow disease" engulfs France and threatens to spread to other countries in Western Europe, French research minister Roger-Gérard Schwartzenberg last week unveiled detailed plans for spending $27 million the government has earmarked for prion disease research in 2001. Next year's budget for studying prions--infectious, abnormal proteins linked to bovine spongiform encephalopathy and its human form, variant Creutzfeldt-Jakob disease--will triple France's current prion research spending. PMID:17798203

  3. Crystallographic Studies of Prion Protein (PrP) Segments Suggest How Structural Changes Encoded by Polymorphism at Residue 129 Modulate Susceptibility to Human Prion Disease

    Energy Technology Data Exchange (ETDEWEB)

    Apostol, Marcin I.; Sawaya, Michael R.; Cascio, Duilio; Eisenberg, David (UCLA)

    2010-09-23

    A single nucleotide polymorphism (SNP) in codon 129 of the human prion gene, leading to a change from methionine to valine at residue 129 of prion protein (PrP), has been shown to be a determinant in the susceptibility to prion disease. However, the molecular basis of this effect remains unexplained. In the current study, we determined crystal structures of prion segments having either Met or Val at residue 129. These 6-residue segments of PrP centered on residue 129 are 'steric zippers,' pairs of interacting {beta}-sheets. Both structures of these 'homozygous steric zippers' reveal direct intermolecular interactions between Met or Val in one sheet and the identical residue in the mating sheet. These two structures, plus a structure-based model of the heterozygous Met-Val steric zipper, suggest an explanation for the previously observed effects of this locus on prion disease susceptibility and progression.

  4. All quiet on the neuronal front: NMDA receptor inhibition by prion protein

    OpenAIRE

    Steele, Andrew D.

    2008-01-01

    The normal function of the prion protein (PrP)—the causative agent of mad cow or prion disease—has long remained out of reach. Deciphering PrP's function may help to unravel the complex chain of events triggered by PrP misfolding during prion disease. In this issue of the JCB, an exciting paper (Khosravani, H., Y. Zhang, S. Tsutsui, S. Hameed, C. Altier, J. Hamid, L. Chen, M. Villemaire, Z. Ali, F.R. Jirik, and G.W. Zamponi. 2008. J. Cell Biol. 181:551–565) connects diverse observations regar...

  5. Study on interaction between microtubule associated protein tau and prion protein

    Institute of Scientific and Technical Information of China (English)

    HAN Jun; ZHOU Wei; DONG Xiaoping; ZHANG Jin; YAO Hailan; WANG Xiaofan; LI Feng; CHEN Lan; GAO Chen; GAO Jianmei; NIE Kai

    2006-01-01

    Microtubule-associated protein tau is considered to play roles in many neurodegenerative diseases including some transmissible spongiform encephalopathies. To address the possible molecular linkage of prion protein (PrP) and tau, a GST-fusion segment of human tau covering the three-repeat region and various PrP segments was used in the tests of GST pull-down and immunoprecipitation. We found tau protein interacted with various style prion proteins such as native prion protein (PrPC) or protease-resistant isoform (prpSc). Co-localization signals of tau and PrP were found in the CHO cell tranfected with both PrP and tau gene. The domain of interaction with tau was located at N-terminal of PrP (residues 23 to 91). The evidence of molecular interactions between PrP and tau protein highlights a potential role of tau in the biological function of PrP and the pathogenesis of TSEs.

  6. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  7. Prions and the blood and immune systems.

    Science.gov (United States)

    Mabbott, Neil; Turner, Marc

    2005-04-01

    Prion diseases take a number of forms in animals and humans. They are caused by conformational change in widely expressed prion protein leading to the formation of intracellular aggregates. Although the main focus of disease is the central nervous system, it is known that involvement of the immune system occurs in peripherally transmitted disease in particular. Animal experiments suggest that in some prion diseases follicular dendritic cells in the germinal centers are a major site of initial accumulation, and that abnormal prion protein and infectivity are detectable in peripheral lymphoid tissue from the earliest phase of disease. This raises the possibility that in a human peripherally transmitted prion disease like variant Creutzfeldt-Jakob disease, further transmission could occur through blood or tissue products or contamination of surgical instrumentation. Indeed two recent reports confirm that this disease has been transmitted by blood, raising significant public health concerns. PMID:15820951

  8. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    Science.gov (United States)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  9. Trafficking and degradation pathways in pathogenic conversion of prions and prion-like proteins in neurodegenerative diseases.

    Science.gov (United States)

    Victoria, Guiliana Soraya; Zurzolo, Chiara

    2015-09-01

    Several neurodegenerative diseases such as transmissible spongiform encephalopathies, Alzheimer's and Parkinson's diseases are caused by the conversion of cellular proteins to a pathogenic conformer. Despite differences in the primary structure and subcellular localization of these proteins, which include the prion protein, α-synuclein and amyloid precursor protein (APP), striking similarity has been observed in their ability to seed and convert naïve protein molecules as well as transfer between cells. This review aims to cover what is known about the intracellular trafficking of these proteins as well as their degradation mechanisms and highlight similarities in their movement through the endocytic pathway that could contribute to the pathogenic conversion and seeding of these proteins which underlies the basis of these diseases.

  10. Superoxide dismutase activity of Cu-bound prion protein

    Science.gov (United States)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2009-03-01

    Misfolding of the prion protein, PrP, has been linked to a group of neurodegenerative diseases, including the mad cow disease in cattle and the Creutzfeldt-Jakob disease in humans. The normal function of PrP is still unknown, but it was found that the PrP can efficiently bind Cu(II) ions. Early experiments suggested that Cu-PrP complex possesses significant superoxide dismutase (SOD) activity, but later experiments failed to confirm it and at present this issue remains unresolved. Using a recently developed hybrid DFT/DFT method, which combines Kohn-Sham DFT for the solute and its first solvation shells with orbital-free DFT for the remainder of the solvent, we have investigated SOD activity of PrP. The PrP is capable of incorporating Cu(II) ions in several binding modes and our calculations find that each mode has a different SOD activity. The highest activity found is comparable to those of well-known SOD proteins, suggesting that the conflicting experimental results may be due to different bindings of Cu(II) in those experiments.

  11. Combined copper/zinc attachment to prion protein

    Science.gov (United States)

    Hodak, Miroslav; Bernholc, Jerry

    2013-03-01

    Misfolding of prion protein (PrP) is responsible for diseases such as ``mad-cow disease'' in cattle and Creutzfeldt-Jacob in humans. Extensive experimental investigation has established that this protein strongly interacts with copper ions, and this ability has been linked to its still unknown function. Attachment of other metal ions (zinc, iron, manganese) have been demonstrated as well, but none of them could outcompete copper. Recent finding, however, indicates that at intermediate concentrations both copper and zinc ions can attach to the PrP at the octarepeat region, which contains high affinity metal binding sites. Based on this evidence, we have performed density functional theory simulations to investigate the combined Cu/Zn attachment. We consider all previously reported binding modes of copper at the octarepeat region and examine a possibility simultaneous Cu/Zn attachment. We find that this can indeed occur for only one of the known binding sites, when copper changes its coordination mode to allow for attachment of zinc ion. The implications of the simultaneous attachment on neural function remain to be explored.

  12. Differential stability of the bovine prion protein upon urea unfolding

    Science.gov (United States)

    Julien, Olivier; Chatterjee, Subhrangsu; Thiessen, Angela; Graether, Steffen P; Sykes, Brian D

    2009-01-01

    Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrPC; and the misfolded, infectious, and proteinase K-resistant form, PrPSc. The C-terminal domain of PrPC is mainly α-helical in structure, whereas PrPSc in known to aggregate into an assembly of β-sheets, forming amyloid fibrils. To identify the regions of PrPC potentially involved in the initial steps of the conversion to the infectious conformation, we have used high-resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrPC (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz 1H NMR spectra reveals region-specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native β-sheet of PrPC is a primary step in the urea-induced unfolding process, while strong hydrophobic interactions between helices α1 and α3, and between α2 and α3, stabilize these regions even at very high concentrations of urea. PMID:19693935

  13. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    DEFF Research Database (Denmark)

    Jen, Angela; Parkyn, Celia J; Mootoosamy, Roy C;

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low-density lipop...

  14. Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

    Directory of Open Access Journals (Sweden)

    Zi Yang

    Full Text Available Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q/asparagine (N-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+] or [URE3] prions. We explore in detail the events leading to the loss (curing of [PSI(+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+].

  15. Lichens: unexpected anti-prion agents?

    Science.gov (United States)

    Rodriguez, Cynthia M.; Bennett, James P.; Johnson, Christopher J.

    2012-01-01

    The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than 20 lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.

  16. Expression of the Prion Protein Family Member Shadoo Causes Drug Hypersensitivity That Is Diminished by the Coexpression of the Wild Type Prion Protein.

    Science.gov (United States)

    Nyeste, Antal; Bencsura, Petra; Vida, István; Hegyi, Zoltán; Homolya, László; Fodor, Elfrieda; Welker, Ervin

    2016-02-26

    The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N terminus, specifically the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPΔHD and PrPΔCR) when coexpressed. The prion protein family member Doppel, which possesses a three-dimensional fold similar to the C-terminal part of PrP, is also harmful to neuronal and other cells in various models, a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that, contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPΔCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrPs with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer disease.

  17. Conformational change in hamster scrapie prion protein (PrP27-30) associated with proteinase K resistance and prion infectivity.

    Science.gov (United States)

    Suzuki, Sachiko Y; Takata, Masuhiro; Teruya, Kenta; Shinagawa, Morikazu; Mohri, Shirou; Yokoyama, Takashi

    2008-02-01

    The scrapie prion protein (PrP27-30) is a crucial component of the prion and is responsible for its transmissibility. Structural information on this protein is limited because it is insoluble and shows aggregated properties. In this study, PrP27-30 was effectively dispersed using sonication under the weak alkaline condition. Subsequently, the small PrP27-30 aggregates were subjected to different pH, heat, and denaturing conditions. The loss of proteinase K (PK) resistance of PrP27-30 and prion infectivity were monitored along with spectroscopic changes. Prion inactivation could not be achieved by the loss of PK resistance alone; a significant loss of the PrP27-30 amyloid structure, which was represented by a decrease in thioflavin T fluorescence, was required for the loss of transmissibility.

  18. Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    Science.gov (United States)

    Nichols, TA; Pulford, Bruce; Wyckoff, A Christy; Meyerett, Crystal; Michel, Brady; Gertig, Kevin; Hoover, Edward A; Jewell, Jean E; Telling, Glenn C

    2009-01-01

    Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles.1–4 CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces.5–7 Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 × 10−7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 × 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission. PMID:19823039

  19. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

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    Victoria A Lawson

    Full Text Available BACKGROUND: The accumulation of protease resistant conformers of the prion protein (PrP(res is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. METHODOLOGY/PRINCIPAL FINDING: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS from the PrP(C substrate was found to specifically prevent PrP(res formation seeded by mouse derived PrP(Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res formation, while having no effect on PrP(res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. CONCLUSIONS/SIGNIFICANCE: Cofactor requirements for PrP(res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

  20. Prion protein gene frequencies in three Sicilian dairy sheep populations

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    Santo Caracappa

    2010-01-01

    Full Text Available The objective of this paper was to investigate the prion protein (PrP genotype and haplotype frequencies in three Sicilian dairy sheep populations. The three populations were: (1 1096 Valle del Belice animals, (2 1143 Comisana animals, and (3 1771 individuals from 5 flocks with scrapie outbreaks, in which the animals were crossbreds derived from indigenous Sicilian dairy breeds. PrP genotypes are described for the three codons 136 (Alanine or Valine; A, V, 154 (Histidine or Arginine; H, R, and 171 (Glutamine, Arginine or Histidine; Q, R, H which represent polymorphisms known to be linked with scrapie susceptibility. The Valle del Belice haplotype frequencies were 32.3% ARR, 6.5% AHQ, 1.0% ARH, 58.8% ARQ, and 1.4% VRQ. The Comisana frequencies were 39.4% ARR, 2.9% AHQ, 2.9% ARH, 50.9% ARQ, and 3.9% VRQ. In the flocks with scrapie outbreaks the frequencies were 32.8% ARR, 2.4% AHQ, 1.7% ARH, 59.1% ARQ, and 3.9% VRQ. In all three populations ARQ and ARR were the most frequent haplotypes. Multiple generations of strong selection will be needed to fixate the most resistant ARR haplotype.

  1. Regulation of embryonic cell adhesion by the prion protein.

    Directory of Open Access Journals (Sweden)

    Edward Málaga-Trillo

    2009-03-01

    Full Text Available Prion proteins (PrPs are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1 mediates Ca(+2-independent homophilic cell adhesion and signaling; and (2 modulates Ca(+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin-based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

  2. Correlation between prion protein gene codon 129 polymorphism and late-onset Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Hairong Qian; Luning Wang; Xiaokun Qi; Jianwei Liu; Jing Liu; Ling Ye; Hengge Xie; Wei Wang; Feng Qiu

    2009-01-01

    BACKGROUND:Studies addressing the correlation between prion protein gene codon 129 polymorphism,Alzheimer's disease,and cognitive disorders have mainly focused on Caucasians.However,prion protein gene codon 129 polymorphism is thought to also affect the Chinese Han and Wei populations.OBJECTIVE:To analyze the differences of prion protein gene codon 129 distribution among the elderly Chinese Han,East Asian,and Caucasian populations,and to study the correlation between prion protein gene codon 129 distribution and late-onset Alzheimer's disease.DESIGN,TIME AND SETTING:A gene polymorphism analysis was performed in the Institute of Geriatrics,General Hospital of Chinese PLA between January 2006 and January 2007.PARTICIPANTS:A total of 152 elderly Chinese Han people were selected from the Beijing Troop Cadre's Sanitarium.Among them,60 patients with late-onset Alzheimer's disease,with a mean age of (82±7) years (range 67-94 years) and disease course of (5.9±4.4) years,comprising 44 males with a mean age of (83±7) years and 16 females with a mean age of (78±7) years,were selected for the case group.An additional 92 healthy elderly subjects,with a mean of (76±9) years (range 60-94 years),comprising 76 males with a mean age of (77±9) years and 16 females with a mean age of (70±8) years,were selected for the control group.There were no significant differences in age and gender between the two groups (P>0.05).METHODS:DNA was extracted from peripheral blood leukocytes using routine phenol/chloroform methodology.Prion protein gene codon 129 polymorphism and ApoE polymorphism were measured using PCR-restriction fragment length polymorphism.The ApoEε allele was considered the standard for analyzing correlations between prion protein gene codon 129 polymorphism and late-onset Alzheimer's disease.MAIN OUTCOME MEASURES:Prion protein gene codon 129 distribution;correlation between genotypic frequency and allele frequency of prion protein gene codon 129 with Alzheimer

  3. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    Directory of Open Access Journals (Sweden)

    Stefanie A.G. Black

    2014-08-01

    Full Text Available Although it is well established that misfolding of the cellular prion protein (PrPC into the beta-sheet-rich, aggregated scrapie conformation (PrPSc causes a variety of transmissible spongiform encephalopathies (TSEs, the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Abeta peptides, suggesting a role for PrPC in Alzheimer’s disease. Our recent findings suggest that Abeta peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for Alzheimer’s disease and other neurodegenerative disorders involving dysfunction of PrPC.

  4. Contributions of the Prion Protein Sequence, Strain, and Environment to the Species Barrier.

    Science.gov (United States)

    Sharma, Aditi; Bruce, Kathryn L; Chen, Buxin; Gyoneva, Stefka; Behrens, Sven H; Bommarius, Andreas S; Chernoff, Yury O

    2016-01-15

    Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-β-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to the transmission of prions between two species with divergent sequences, termed a species barrier. Here we study the relative effects of protein sequence, seed conformation, and environment on the species barrier strength and specificity for the yeast prion protein Sup35p from three closely related species of the Saccharomyces sensu stricto group; namely, Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces paradoxus. Through in vivo plasmid shuffle experiments, we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than by the cellular environment. In vitro data confirm that the kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance with their positions in the Hofmeister series, as observed previously for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, whereas anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Therefore, our work shows that the protein sequence and the conformation variant (strain) of the prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro.

  5. Contributions of the Prion Protein Sequence, Strain, and Environment to the Species Barrier.

    Science.gov (United States)

    Sharma, Aditi; Bruce, Kathryn L; Chen, Buxin; Gyoneva, Stefka; Behrens, Sven H; Bommarius, Andreas S; Chernoff, Yury O

    2016-01-15

    Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-β-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to the transmission of prions between two species with divergent sequences, termed a species barrier. Here we study the relative effects of protein sequence, seed conformation, and environment on the species barrier strength and specificity for the yeast prion protein Sup35p from three closely related species of the Saccharomyces sensu stricto group; namely, Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces paradoxus. Through in vivo plasmid shuffle experiments, we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than by the cellular environment. In vitro data confirm that the kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance with their positions in the Hofmeister series, as observed previously for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, whereas anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Therefore, our work shows that the protein sequence and the conformation variant (strain) of the prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro. PMID:26565023

  6. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    Science.gov (United States)

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  7. Computational Studies of the Structural Stability of Rabbit Prion Protein Compared to Human and Mouse Prion Proteins

    OpenAIRE

    Zhang, Jiapu

    2011-01-01

    Prion diseases are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. The neurodegenerative diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Str$\\ddot{a}$ussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or 'mad-cow' disease) and chronic wasting disease in cattle belong to prion diseases. By now there have not been some effective therapeut...

  8. Prions in Variably Protease-Sensitive Prionopathy: An Update

    OpenAIRE

    Laura Pirisinu; Jan Langeveld; Jue Yuan; Xiangzhu Xiao; Wen-Quan Zou; Pierluigi Gambetti

    2013-01-01

    Human prion diseases, including sporadic, familial, and acquired forms such as Creutzfeldt-Jakob disease (CJD), are caused by prions in which an abnormal prion protein (PrPSc) derived from its normal cellular isoform (PrPC) is the only known component. The recently-identified variably protease-sensitive prionopathy (VPSPr) is characterized not only by an atypical clinical phenotype and neuropathology but also by the deposition in the brain of a peculiar PrPSc. Like other forms of human prion ...

  9. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    Science.gov (United States)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  10. Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host

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    Warner Richard

    2009-02-01

    Full Text Available Abstract Background In order to study the sites of uptake and mechanisms of dissemination of scrapie prions in the natural host under controlled conditions, lambs aged 14 days and homozygous for the VRQ allele of the PrP gene were infected by the oral route. Infection occurred in all lambs with a remarkably short and highly consistent incubation period of approximately 6 months. Challenge of lambs at approximately eight months of age resulted in disease in all animals, but with more variable incubation periods averaging significantly longer than those challenged at 14 days. This model provides an excellent system in which to study the disease in the natural host by virtue of the relatively short incubation period and close resemblance to natural infection. Results Multiple sites of prion uptake were identified, of which the most important was the Peyer's patch of the distal ileum. Neuroinvasion was detected initially in the enteric nervous system prior to infection of the central nervous system. At end stage disease prion accumulation was widespread throughout the entire neuraxis, but vacuolar pathology was absent in most animals that developed disease at 6–7 months of age. Conclusion Initial spread of detectable PrP was consistent with drainage in afferent lymph to dependent lymph nodes. Subsequent accumulation of prions in lymphoid tissue not associated with the gut is consistent with haematogenous spread. In addition to macrophages and follicular dendritic cells, prion containing cells consistent with afferent lymph dendritic cells were identified and are suggested as a likely vehicle for carriage of prions from initial site of uptake to the lymphoreticular system, and as potential carriers of prion protein in blood. It is apparent that spongiform change, the characteristic lesion of scrapie and other prion diseases, is not responsible for the clinical signs in sheep, but may develop in an age dependent manner.

  11. Dividing roles of prion protein in staurosporine-mediated apoptosis.

    Science.gov (United States)

    Zhang, Ying; Qin, Kefeng; Wang, Jianwei; Hung, Tao; Zhao, Richard Y

    2006-10-20

    Prion protein (PrPC) is a normal cellular glycoprotein that is expressed in almost all tissues including the central nervous system. Much attention has been focused on this protein because conversion of the normal PrPC to the diseased form (PrPSc) plays an essential role in transmissible spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jakob disease. In spite of the extensive effort, the normal physiological function of PrPC remains elusive. Emerging evidence suggests that PrPC plays a protective role against cellular stresses including apoptosis induced by various pro-apoptotic agents such as Bax and staurosporine (STS), however, other reports showed overexpression of PrPC enhances STS-mediated apoptosis. In this study, we took a different approach by depleting endogenous PrPC using specific interfering RNA technique and compared the depleting and overproducing effects of PrPC on STS-induced apoptosis in neuro-2a (N2a) cells. We demonstrate here that down-regulation of PrPC sensitizes N2a cells to STS-induced cytotoxicity and apoptosis. The enhanced apoptosis induced by STS was shown by increased DNA fragmentation, immunoreactivity of Bax, and caspase-3 cleavage. We also showed that overproduction of PrPC had little or no effect on STS-mediated DNA fragmentation in N2a cells but it augments STS-mediated apoptosis in HEK293 cells, suggesting a cell line-specific effect. In addition, the inhibitory effect of PrPC on STS-mediated cellular stress appears to be modulated in part through induction of cell cycle G2 accumulation. Together, our data suggest that physiological level of endogenous PrPC plays a protective role against STS-mediated cellular stress. Loss of this protection could render cells more prone to cellular insults such as STS. PMID:16950206

  12. Dynamically-expressed prion-like proteins form a cuticle in the pharynx of Caenorhabditis elegans

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    Julia B. George-Raizen

    2014-10-01

    Full Text Available In molting animals, a cuticular extracellular matrix forms the first barrier to infection and other environmental insults. In the nematode Caenorhabditis elegans there are two types of cuticle: a well-studied collagenous cuticle lines the body, and a poorly-understood chitinous cuticle lines the pharynx. In the posterior end of the pharynx is the grinder, a tooth-like cuticular specialization that crushes food prior to transport to the intestine for digestion. We here show that the grinder increases in size only during the molt. To gain molecular insight into the structure of the grinder and pharyngeal cuticle, we performed a microarray analysis to identify mRNAs increased during the molt. We found strong transcriptional induction during the molt of 12 of 15 previously identified abu genes encoding Prion-like (P glutamine (Q and asparagine (N rich PQN proteins, as well as 15 additional genes encoding closely related PQN proteins. abu/pqn genes, which we name the abu/pqn paralog group (APPG genes, were expressed in pharyngeal cells and the proteins encoded by two APPG genes we tested localized to the pharyngeal cuticle. Deleting the APPG gene abu-14 caused abnormal pharyngeal cuticular structures and knocking down other APPG genes resulted in abnormal cuticular function. We propose that APPG proteins promote the assembly and function of a unique cuticular structure. The strong developmental regulation of the APPG genes raises the possibility that such genes would be identified in transcriptional profiling experiments in which the animals' developmental stage is not precisely staged.

  13. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    International Nuclear Information System (INIS)

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrPC. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrPC at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  14. Toward unfolding the prion misfolding mystery: protein free radical chemistry in transmissible spongiform encephalopathies

    International Nuclear Information System (INIS)

    Owing to the high oxygen-respiration in the brain of mammals, oxidative damage to prion protein has been suggested to be an additional factor. A large body of intriguing features of scrapie and prion diseases have provided multiple lines of indirect chemistry evidence, suggesting that the infectious agents may be putative forms of sequence-specific prion radicals (SSPR) and/or their immediate precursors in the transmissible spongiform encephalopathies (TSE). Here a molecular mechanism corresponding to the self-replication of scrapie protein mediated by prion free-radical processes, consonant with 'protein-only' hypotheses is proposed. This new theory may not only aid our understanding of the occurrence of prions, but also provides new insight into the possible chemistry principles underlying the neutrodegenerative disorders. It is anticipated that future studies based on this suggestion and chemistry principles of genetic diseases may allow us to determine an effective approach to stop mad cow disease and its human version, new variant of Creutzfeldt-Jakob disease (v CJD)

  15. Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPSc. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

  16. Octapeptide repeat insertions in the prion protein gene and early onset dementia

    NARCIS (Netherlands)

    E.A. Croes (Esther); J.J. Houwing-Duistermaat (Jeanine); K. Sleegers (Kristel); F. Forey; B. van Harten; M. Cruts (Marc); C.M. van Duijn (Cock); J. Theuns (Jessie); J.C. van Swieten; C. van Broeckhoven (Christine)

    2004-01-01

    textabstractOBJECTIVES: The most common familial early onset dementia mutations are found in the genes involved in Alzheimer's disease; the amyloid precursor protein (APP) and the presenilin 1 and 2 (PSEN1 and 2) genes; the prion protein gene (PRNP) may be involved. METHODS: Following identification

  17. Stress-dependent proteolytic processing of the actin assembly protein Lsb1 modulates a yeast prion.

    Science.gov (United States)

    Ali, Moiez; Chernova, Tatiana A; Newnam, Gary P; Yin, Luming; Shanks, John; Karpova, Tatiana S; Lee, Andrew; Laur, Oskar; Subramanian, Sindhu; Kim, Dami; McNally, James G; Seyfried, Nicholas T; Chernoff, Yury O; Wilkinson, Keith D

    2014-10-01

    Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments. PMID:25143386

  18. Chimeric elk/mouse prion proteins in transgenic mice

    OpenAIRE

    Tamguney, G; Giles, K; Oehler, A.; Johnson, NL; DeArmond, SJ; Prusiner, SB

    2013-01-01

    Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). ...

  19. Variation in the prion protein sequence in Dutch goat breeds.

    Science.gov (United States)

    Windig, J J; Hoving, R A H; Priem, J; Bossers, A; van Keulen, L J M; Langeveld, J P M

    2016-10-01

    Scrapie is a neurodegenerative disease occurring in goats and sheep. Several haplotypes of the prion protein increase resistance to scrapie infection and may be used in selective breeding to help eradicate scrapie. In this study, frequencies of the allelic variants of the PrP gene are determined for six goat breeds in the Netherlands. Overall frequencies in Dutch goats were determined from 768 brain tissue samples in 2005, 766 in 2008 and 300 in 2012, derived from random sampling for the national scrapie surveillance without knowledge of the breed. Breed specific frequencies were determined in the winter 2013/2014 by sampling 300 breeding animals from the main breeders of the different breeds. Detailed analysis of the scrapie-resistant K222 haplotype was carried out in 2014 for 220 Dutch Toggenburger goats and in 2015 for 942 goats from the Saanen derived White Goat breed. Nine haplotypes were identified in the Dutch breeds. Frequencies for non-wild type haplotypes were generally low. Exception was the K222 haplotype in the Dutch Toggenburger (29%) and the S146 haplotype in the Nubian and Boer breeds (respectively 7 and 31%). The frequency of the K222 haplotype in the Toggenburger was higher than for any other breed reported in literature, while for the White Goat breed it was with 3.1% similar to frequencies of other Saanen or Saanen derived breeds. Further evidence was found for the existence of two M142 haplotypes, M142 /S240 and M142 /P240 . Breeds vary in haplotype frequencies but frequencies of resistant genotypes are generally low and consequently selective breeding for scrapie resistance can only be slow but will benefit from animals identified in this study. The unexpectedly high frequency of the K222 haplotype in the Dutch Toggenburger underlines the need for conservation of rare breeds in order to conserve genetic diversity rare or absent in other breeds. PMID:26991480

  20. A comparative molecular dynamics study on thermostability of human and chicken prion proteins

    International Nuclear Information System (INIS)

    To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrPC and CkPrPC), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrPC is comparable with that of CkPrPC, which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrPC

  1. Detecting and discriminating among pathogenic protein conformers(prions), using mass spectrometry-based and antibody-based approaches(Abstract)

    Science.gov (United States)

    A set of fatal neurological diseases that includes scrapie and chronic wasting disease (CWD) are caused by a pathological protein referred to as a prion (PrPSc). A prion propagates an infection by converting a normal cellular protein (PrPC) into a prion. Unlike viral, bacterial, or fungal pathogens,...

  2. Sheep scrapie susceptibility-linked polymorphisms do not modulate the initial binding of cellular to disease-associated prion protein prior to conversion

    NARCIS (Netherlands)

    Rigter, A.; Bossers, A.

    2005-01-01

    Conversion of the host-encoded protease-sensitive cellular prion protein (PrPC) into the scrapie-associated protease-resistant isoform (PrPSc) of prion protein (PrP) is the central event in transmissible spongiform encephalopathies or prion diseases. Differences in transmissibility and susceptibilit

  3. All quiet on the neuronal front: NMDA receptor inhibition by prion protein.

    Science.gov (United States)

    Steele, Andrew D

    2008-06-01

    The normal function of the prion protein (PrP)--the causative agent of mad cow or prion disease--has long remained out of reach. Deciphering PrP's function may help to unravel the complex chain of events triggered by PrP misfolding during prion disease. In this issue of the JCB, an exciting paper (Khosravani, H., Y. Zhang, S. Tsutsui, S. Hameed, C. Altier, J. Hamid, L. Chen, M. Villemaire, Z. Ali, F.R. Jirik, and G.W. Zamponi. 2008. J. Cell Biol. 181:551-565) connects diverse observations regarding PrP into a coherent framework whereby PrP dampens the activity of an N-methyl-D-aspartate (NMDA) receptor (NMDAR) subtype and reduces excitotoxic lesions. The findings of this study suggest that understanding the normal function of proteins associated with neurodegenerative disease may elucidate the molecular pathogenesis. PMID:18504309

  4. All quiet on the neuronal front: NMDA receptor inhibition by prion protein.

    Science.gov (United States)

    Steele, Andrew D

    2008-05-01

    The normal function of the prion protein (PrP)-the causative agent of mad cow or prion disease-has long remained out of reach. Deciphering PrP's function may help to unravel the complex chain of events triggered by PrP misfolding during prion disease. In this issue of the JCB, an exciting paper (Khosravani, H., Y. Zhang, S. Tsutsui, S. Hameed, C. Altier, J. Hamid, L. Chen, M. Villemaire, Z. Ali, F.R. Jirik, and G.W. Zamponi. 2008. J. Cell Biol. 181:551-565) connects diverse observations regarding PrP into a coherent framework whereby PrP dampens the activity of an N-methyl-d-aspartate (NMDA) receptor (NMDAR) subtype and reduces excitotoxic lesions. The findings of this study suggest that understanding the normal function of proteins associated with neurodegenerative disease may elucidate the molecular pathogenesis.

  5. Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

    Science.gov (United States)

    Russo, F.; Johnson, C.J.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

    2009-01-01

    Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml-1 (PrPTSE:MnO2=1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

  6. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  7. Myiasis as a risk factor for prion diseases in humans.

    Science.gov (United States)

    Lupi, O

    2006-10-01

    Prion diseases are transmissible spongiform encephalopathies of humans and animals. The oral route is clearly associated with some prion diseases, according to the dissemination of bovine spongiform encephalopathy (BSE or mad cow disease) in cattle and kuru in humans. However, other prion diseases such as scrapie (in sheep) and chronic wasting disease (CWD) (in cervids) cannot be explained in this way and are probably more associated with a pattern of horizontal transmission in both domestic and wild animals. The skin and mucous membranes are a potential target for prion infections because keratinocytes and lymphocytes are susceptible to the abnormal infective isoform of the prion protein. Iatrogenic transmission of Creutzfeldt-Jakob disease (CJD) was also recognized after corneal transplants in humans and scrapie was successfully transmitted to mice after ocular instillation of infected brain tissue, confirming that these new routes could also be important in prion infections. Some ectoparasites have been proven to harbour prion rods in laboratory experiments. Prion rods were identified in both fly larvae and pupae; adult flies are also able to express prion proteins. The most common causes of myiasis in cattle and sheep, closely related animals with previous prion infections, are Hypoderma bovis and Oestrus ovis, respectively. Both species of flies present a life cycle very different from human myiasis, as they have a long contact with neurological structures, such as spinal canal and epidural fat, which are potentially rich in prion rods. Ophthalmomyiases in humans is commonly caused by both species of fly larvae worldwide, providing almost direct contact with the central nervous system (CNS). The high expression of the prion protein on the skin and mucosa and the severity of the inflammatory response to the larvae could readily increase the efficiency of transmission of prions in both animals and humans. PMID:16987255

  8. Prion Protein Self Interactions; a gateway to novel therapeutic strategies?

    NARCIS (Netherlands)

    Rigter, A.; Langeveld, J.P.M.; Zijderveld, van F.G.; Bossers, A.

    2010-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders and include among others Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. The central event in disease development in TSEs is the refol

  9. Disease-associated prion protein in neural and lymphoid tissues of mink (Mustela vison) inoculated with transmissible mink encephalopathy

    Science.gov (United States)

    Transmissible mink encephalopathy (TME) is a prion disorder of farmed raised mink. As with the other transmissible spongiform encephalopathies, the disorder is associated with accumulation of the misfolded prion protein in the brain and an invariably fatal outcome. TME outbreaks have been rare but...

  10. A distinct proteinase K resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

    NARCIS (Netherlands)

    Bouzalas, I.; Lörtscher, F.; Dovas, C.; Oevermann, A.; Langeveld, J.P.M.; Papanastassopoulou, M.; Papadopoulos, O.; Zurbriggen, A.; Seuberlich, T.

    2011-01-01

    Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrPres

  11. [Prion neuroinfections].

    Science.gov (United States)

    Tichý, J

    2004-01-01

    Prion neuroinfections represent an emergent problem namely after the epidemic brake out of the bovine spongiform encephalopathies, which culminated in 1993. Isolated cases can still emerge. The next case was the discovery of a new variant of Creutzfeldt-Jakob disease in the comparatively young persons with proved alimentary infection route--about 140 cases has been described. Physiological prions are glycoproteins, affixed to the rafts of cell membranes (namely in neurons) by glycosylphosphatidylinositol anchor. They act as signalling molecules and participate in the cooper metabolism. Despite the intensive study in many prestigious laboratories, the way how the pathogenic isoform of prions, given by the prevalence of beta-conformations in the polypeptide chain develops has not been proved yet. Pathogenic isoforms are formed intracellulary after the precedent endocytosis. Aggregations of abnormal prion molecules build up prion amyloid deposits in the form of fibrils or plaques. The loss of neurons brings about the neurological symptoms, which are not directly related to the amounts of amyloid deposits. Characteristic findings of spongiosis and the immunohistochemical evidence of protease-resistant prions belong to the only reliable tests. Reliable intravital test in the blood or urine is not yet available. All forms of human and animal transmissible spongiform encephalopathies develop only in genetically sensitive individuals. A brief overview of clinical forms and some biochemical and genetic aspects of the disease are given.

  12. Prion pathogenesis and secondary lymphoid organs (SLO): tracking the SLO spread of prions to the brain.

    Science.gov (United States)

    Mabbott, Neil A

    2012-01-01

    Prion diseases are subacute neurodegenerative diseases that affect humans and a range of domestic and free-ranging animal species. These diseases are characterized by the accumulation of PrP (Sc), an abnormally folded isoform of the cellular prion protein (PrP (C)), in affected tissues. The pathology during prion disease appears to occur almost exclusively within the central nervous system. The extensive neurodegeneration which occurs ultimately leads to the death of the host. An intriguing feature of the prion diseases, when compared with other protein-misfolding diseases, is their transmissibility. Following peripheral exposure, some prion diseases accumulate to high levels within lymphoid tissues. The replication of prions within lymphoid tissue has been shown to be important for the efficient spread of disease to the brain. This article describes recent progress in our understanding of the cellular mechanisms that influence the propagation of prions from peripheral sites of exposure (such as the lumen of the intestine) to the brain. A thorough understanding of these events will lead to the identification of important targets for therapeutic intervention, or alternatively, reveal additional processes that influence disease susceptibility to peripherally-acquired prion diseases.

  13. Cloning and expression of prion protein encoding gene of flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; SUN Xiuqin; ZHANG Jinxing; ZAN Jindong

    2008-01-01

    The prion protein (PrP) encoding gene of flounder (Paralichthys olivaceus) was cloned.It was not interrupted by an intron.This gene has two promoters in its 5' upstream,indicating that its transcription may be intensive,and should have an important function.It was expressed in all 14 tissues tested,demonstrating that it is a house-keeping gene.Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  14. Controllable synthesis of polyoxometalates nanocubes and their specific interactions with prion proteins

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    It is found that Keggin-type of polyoxometalates nanocubes could be formed in aqueous medium under the adjustment of dimethylformamide (DMF),and could be changed from solid nanocubes to hollow ones with increasing temperature. Further investigations show that this Keggin-type of polyoxometalates nanocubes can specifically interact with prion protein,and the light scattering signals are en-hanced in proportion to the content of prion in the range of 3.36―840 ng·mL-1.

  15. COMPOSITE PEPTIDE COMPOUNDS FOR DIAGNOSIS AND TREATMENT OF DISEASES CAUSED BY PRION PROTEINS

    DEFF Research Database (Denmark)

    2004-01-01

    that they together form a non-linear sequence which mimics the tertiary structure of one or more PrPSc-specific epitopes as evidenced by the test described herein. The use of such conjugates as immunogens for the production of antibodies that specifically bind to the pathogenic form of a prion protein is revealed...

  16. Immunohistochemical detection and distribution of prion protein in a goat with natural scrapie.

    Science.gov (United States)

    Valdez, Reginald A; Rock, Matthew J; Anderson, Anne K; O'Rourke, Katherine I

    2003-03-01

    Formalin-fixed, paraffin-embedded tissue sections from a 3-year-old female Angora goat suffering from clinical scrapie were immunostained after hydrated autoclaving using a monoclonal antibody (mAb, F99/97.6.1; IgG1) specific for a conserved epitope on the prion protein. Widespread and prominent deposition of the scrapie isoform of the prion protein (PrPSc) was observed in the brain, brainstem, spinal cord, retina, postganglionic neurons associated with parasympathetic ganglia of myenteric and submucosal plexuses, Peyer's patches, peripheral lymph nodes, and pharyngeal and palatine tonsils. The goat was homozygous for PrP alleles encoding 5 octapeptide repeat sequences in the N-terminal region of the prion protein and isoleucine at codon 142, a genotype associated with high susceptibility and short incubation times in goats. The results of this study indicate that mAb F99/97.6.1 is useful for detection of PrPSc deposition, and this is a specific and reliable immunohistochemical adjunct to histopathology for diagnosis of natural caprine scrapie, although precise determination of the diagnostic sensitivity and specificity of the assay as a diagnostic test for scrapie in goats will require examination of a sufficiently large sample size. As with ovine scrapie, prion protein is widely distributed in the central and peripheral nervous systems, gastrointestinal tract, and lymphoid tissues in natural caprine scrapie. PMID:12661726

  17. Prion Protein M129V Polymorphism Affects Retrieval-Related Brain Activity

    Science.gov (United States)

    Buchmann, Andreas; Mondadori, Christian R. A.; Hanggi, Jurgen; Aerni, Amanda; Vrticka, Pascal; Luechinger, Roger; Boesiger, Peter; Hock, Christoph; Nitsch, Roger M.; de Quervain, Dominique J.-F.; Papassotiropoulos, Andreas; Henke, Katharina

    2008-01-01

    The prion protein Met129Val polymorphism has recently been related to human long-term memory with carriers of either the 129[superscript MM] or the 129[superscript MV] genotype recalling 17% more words than 129[superscript VV] carriers at 24 h following learning. Here, we sampled genotype differences in retrieval-related brain activity at 30 min…

  18. Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine or cervid prion protein.

    Science.gov (United States)

    Madsen-Bouterse, Sally A; Schneider, David A; Zhuang, Dongyue; Dassanayake, Rohana P; Balachandran, Aru; Mitchell, Gordon B; O'Rourke, Katherine I

    2016-09-01

    Development of mice expressing either ovine (Tg338) or cervid (TgElk) prion protein (PrP) have aided in characterization of scrapie and chronic wasting disease (CWD), respectively. Experimental inoculation of sheep with CWD prions has demonstrated the potential for interspecies transmission but, infection with CWD versus classical scrapie prions may be difficult to differentiate using validated diagnostic platforms. In this study, mouse bioassay in Tg338 and TgElk was utilized to evaluate transmission of CWD versus scrapie prions from small ruminants. Mice (≥5 per homogenate) were inoculated with brain homogenates from clinically affected sheep or goats with naturally acquired classical scrapie, white-tailed deer with naturally acquired CWD (WTD-CWD) or sheep with experimentally acquired CWD derived from elk (sheep-passaged-CWD). Survival time (time to clinical disease) and attack rates (brain accumulation of protease resistant PrP, PrPres) were determined. Inoculation with classical scrapie prions resulted in clinical disease and 100 % attack rates in Tg338, but no clinical disease at endpoint (>300 days post-inoculation, p.i.) and low attack rates (6.8 %) in TgElk. Inoculation with WTD-CWD prions yielded no clinical disease or brain PrPres accumulation in Tg338 at endpoint (>500 days p.i.), but rapid onset of clinical disease (~121 days p.i.) and 100 % attack rate in TgElk. Sheep-passaged-CWD resulted in transmission to both mouse lines with 100 % attack rates at endpoint in Tg338 and an attack rate of ~73 % in TgElk with some culled due to clinical disease. These primary transmission observations demonstrate the potential of bioassay in Tg338 and TgElk to help differentiate possible infection with CWD versus classical scrapie prions in sheep and goats.

  19. Structural Determinants of Phenotypic Diversity and Replication Rate of Human Prions

    OpenAIRE

    Safar, Jiri G; Xiangzhu Xiao; Kabir, Mohammad E.; Shugui Chen; Chae Kim; Tracy Haldiman; Yvonne Cohen; Wei Chen; Cohen, Mark L.; Surewicz, Witold K.

    2015-01-01

    Author Summary Sporadic Creutzfeldt-Jakob disease (sCJD) represents ~90% of all human prion diseases worldwide. This neurodegenerative disease, which is transmissible and invariably fatal, is characterized by variable progression rates and remarkable diversity of clinical and pathological traits. The infectious sCJD prions propagating the pathology mainly in the brain are assemblies of abnormally folded isoform (PrPSc) of a host-encoded prion protein (PrPC). The structure and replication mech...

  20. A novel expression system for production of soluble prion proteins in E. coli

    OpenAIRE

    Abskharon Romany NN; Ramboarina Stephanie; El Hassan Hassan; Gad Wael; Apostol Marcin I; Giachin Gabriele; Legname Giuseppe; Steyaert Jan; Messens Joris; Soror Sameh H; Wohlkonig Alexandre

    2012-01-01

    Abstract Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Su...

  1. The sensitive [SWI+] prion: New perspectives on yeast prion diversity

    OpenAIRE

    Hines, Justin K; Craig, Elizabeth A

    2011-01-01

    Yeast prions are heritable protein-based genetic elements which rely on molecular chaperone proteins for stable transmission to cell progeny. Within the past few years, five new prions have been validated and 18 additional putative prions identified in Saccharomyces cerevisiae. The exploration of the physical and biological properties of these “nouveau prions” has begun to reveal the extent of prion diversity in yeast. We recently reported that one such prion, [SWI+], differs from the best st...

  2. Development of techniques in magnetic resonance and structural studies of the prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Bitter, Hans-Marcus L.

    2000-07-01

    Magnetic resonance is the most powerful analytical tool used by chemists today. Its applications range from determining structures of large biomolecules to imaging of human brains. Nevertheless, magnetic resonance remains a relatively young field, in which many techniques are currently being developed that have broad applications. In this dissertation, two new techniques are presented, one that enables the determination of torsion angles in solid-state peptides and proteins, and another that involves imaging of heterogenous materials at ultra-low magnetic fields. In addition, structural studies of the prion protein via solid-state NMR are described. More specifically, work is presented in which the dependence of chemical shifts on local molecular structure is used to predict chemical shift tensors in solid-state peptides with theoretical ab initio surfaces. These predictions are then used to determine the backbone dihedral angles in peptides. This method utilizes the theoretical chemicalshift tensors and experimentally determined chemical-shift anisotropies (CSAs) to predict the backbone and side chain torsion angles in alanine, leucine, and valine residues. Additionally, structural studies of prion protein fragments are described in which conformationally-dependent chemical-shift measurements were made to gain insight into the structural differences between the various conformational states of the prion protein. These studies are of biological and pathological interest since conformational changes in the prion protein are believed to cause prion diseases. Finally, an ultra-low field magnetic resonance imaging technique is described that enables imaging and characterization of heterogeneous and porous media. The notion of imaging gases at ultra-low fields would appear to be very difficult due to the prohibitively low polarization and spin densities as well as the low sensitivities of conventional Faraday coil detectors. However, Chapter 5 describes how gas imaging

  3. Towards unfolding the prion misfolding mystery: Protein free radical chemistry in transmissible spongiform encephalopathies

    Institute of Scientific and Technical Information of China (English)

    YANG Chi-Ming

    2003-01-01

    Owing to the high oxygen-respiration in the brain of mammals, oxidative damage to prion protein hasbeen suggested to be an additional factor. A large body of intriguing features of scrapie and prion diseases haveprovided multiple lines of indirect chemistry evidence, suggesting that the infectious agents may be putative forms ofsequence-specific prion radicals (SSPR) and/or their immediate precursors in the transmissible spongiform encepha-lopathies (TSE). Here a molecular mechanism corresponding to the self-replication of scrapie protein mediated byprion free-radical processes, consonant with "protein-only" hypotheses is proposed. This new theory may not onlyaid our understanding of the occurrence of prions, but also provides new insight into the possible chemistry principlesunderlying the neurodegenerative disorders. It is anticipated that future studies based on this suggestion and chem-istry principles of genetic diseases may allow us to determine an effective approach to stop mad cow disease and itshuman version, new variant of Creutzfeldt-Jakob disease (v CJD).

  4. LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein

    DEFF Research Database (Denmark)

    Parkyn, Celia J; Vermeulen, Esmeralda G M; Mootoosamy, Roy C;

    2008-01-01

    The trafficking of normal cellular prion protein (PrP(C)) is believed to control its conversion to the altered conformation (designated PrP(Sc)) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrP(C) on neurons is rapidly and consti......The trafficking of normal cellular prion protein (PrP(C)) is believed to control its conversion to the altered conformation (designated PrP(Sc)) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrP(C) on neurons is rapidly...... required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrP(C) in biosynthetic compartments, with a concomitant lowering of surface PrP(C), suggesting that LRP1 expedites the trafficking of PrP(C) to the neuronal surface. PrP(C) and LRP1 can be co......-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrP(C) binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 microM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface...

  5. A prion primer

    OpenAIRE

    Cashman, N R

    1997-01-01

    By biological and medical criteria, prions are infectious agents; however, many of their properties differ profoundly from those of conventional microbes. Prions are "encoded" by alterations in protein conformation rather than in nucleic acid or amino acid sequence. New epidemic prion diseases (bovine spongiform encephalopathy and new variant Creutzfeldt-Jakob disease) have recently emerged under the active surveillance of the modern world. The risk of contracting prion disease from blood pro...

  6. Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine and cervid prion protein

    Science.gov (United States)

    Identifying transmissible spongiform encephalopathy (TSE) reservoirs that could lead to disease re-emergence is imperative to U.S. scrapie eradication efforts. Transgenic mice expressing the cervid (TgElk) or ovine (Tg338) prion protein have aided characterization of chronic wasting disease (CWD) an...

  7. Molecular Dynamics Studies on the Structural Stability of Wild-Type Rabbit Prion Protein: Surface Electrostatic Charge Distributions

    CERN Document Server

    Zhang, Jiapu

    2011-01-01

    Prion diseases cover a large range of neurodegenerative diseases in humans and animals, which are invariably fatal and highly infectious. By now there have not been some effective therapeutic approaches or medications to treat all prion diseases. Fortunately, numerous experimental experiences have showed that rabbits are resistant to infection from prion diseases isolated from other species, and recently the molecular structures of rabbit prion protein and its mutants were released into protein data bank. Prion diseases are "protein structural conformational" diseases. Thus, in order to reveal some secrets of prion diseases, it is amenable to study rabbits by techniques of the molecular structure and its dynamics. Wen et al. (PLoS One 5(10) e13273 (2010), Journal of Biological Chemistry 285(41) 31682-31693 (2010)) reported the surface of NMR RaPrPC(124-228) molecular snapshot has a large land of continuous positive charge distribution, which contributes to the structural stability of rabbit prion protein. Thi...

  8. Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification.

    Science.gov (United States)

    Moudjou, Mohammed; Chapuis, Jérôme; Mekrouti, Mériem; Reine, Fabienne; Herzog, Laetitia; Sibille, Pierre; Laude, Hubert; Vilette, Didier; Andréoletti, Olivier; Rezaei, Human; Dron, Michel; Béringue, Vincent

    2016-01-01

    Prions are formed of misfolded assemblies (PrP(Sc)) of the variably N-glycosylated cellular prion protein (PrP(C)). In infected species, prions replicate by seeding the conversion and polymerization of host PrP(C). Distinct prion strains can be recognized, exhibiting defined PrP(Sc) biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrP(Sc) assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrP(C) glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrP(C) species of interest as substrate. Applying the technique to PrP(C) glycosylation mutants expressing cells revealed that neither PrP(C) nor PrP(Sc) glycoform stoichiometry was instrumental to PrP(Sc) formation and strainness perpetuation. Our study supports the view that strain properties, including PrP(Sc) glycotype are enciphered within PrP(Sc) structural backbone, not in the attached glycans. PMID:27384922

  9. Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt–Jakob disease: a case report

    Directory of Open Access Journals (Sweden)

    Rodríguez-Martínez Ana B

    2012-10-01

    Full Text Available Abstract Introduction The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation A 74-year-old Caucasian woman showed a sporadic Creutzfeldt–Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient’s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt–Jakob disease. This highlights the

  10. A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits

    OpenAIRE

    1994-01-01

    The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt- Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. ...

  11. Prion protein insertional mutations increase aggregation propensity but not fiber stability

    Directory of Open Access Journals (Sweden)

    True Heather L

    2008-03-01

    Full Text Available Abstract Background Mutations in the PRNP gene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI+] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI+] and convert from [psi-] to [PSI+] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivo and in vitro. Results Investigation of the chimeric proteins in vitro reveals that repeat-expansions increase aggregation propensity and that the kinetics of fiber formation depends on the number of repeats. The fiber formation reactions are promiscuous in that the chimeric protein containing 14 repeats can readily cross-seed fiber formation of proteins that have the wild type number of repeats. Morphologically, the amyloid fibers formed by repeat-expanded proteins associate with each other to form large clumps that were not as prevalent in fibers formed by proteins containing the wild type number of repeats. Despite the increased aggregation propensity and lateral association of the repeat expanded proteins, there was no corresponding increase in the stability of the fibers formed. Therefore, we predict that the differences in fibers formed with different repeat lengths may not be due to

  12. An Engineered PrPsc-like Molecule from the Chimera of Mammalian Prion Protein and Yeast Ure2p Prion-inducing Domain

    Institute of Scientific and Technical Information of China (English)

    Shao-Man YIN; Man-Sun SY; Po TIEN

    2004-01-01

    Production of the pathogenic prion isoform prpsc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentβ-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors ofPrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

  13. An Engineered PrPsc-like Molecule from the Chimera of Mammalian Prion Protein and Yeast Ure2p Prion-inducing Domain

    Institute of Scientific and Technical Information of China (English)

    Shao-ManYIN; Man-SunSY; PoTIEN

    2004-01-01

    Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentB-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPse and will be helpful for further understanding the mechanism of conformational conversion process.

  14. In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

    Science.gov (United States)

    Johnson, Christopher J.; Morawski, A.R.; Carlson, C.M.; Chang, H.

    2013-01-01

    Background: Transmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown. Results: We acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R 154Q171). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R 154Q171genotype sheep. Conclusions: Our results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including

  15. A survey and a molecular dynamics study on the (central) hydrophobic region of prion proteins

    CERN Document Server

    Zhang, Jiapu

    2014-01-01

    Prion diseases are invariably fatal neurodegenerative diseases that affect humans and animals. Unlike most other amyloid forming neurodegenerative diseases, these can be highly infectious. Prion diseases occur in a variety of species. They include the fatal human neurodegenerative diseases Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, the bovine spongiform encephalopathy (BSE or 'mad-cow' disease) in cattle, the chronic wasting disease (CWD) in deer and elk, and scrapie in sheep and goats, etc. Transmission across the species barrier to humans, especially in the case of BSE in Europe, CWD in North America, and variant CJDs (vCJDs) in young people of UK, is a major public health concern. Fortunately, scientists reported that the (central) hydrophobic region of prion proteins (PrP) controls the formation of diseased prions. This article gives a detailed survey on PrP hydrophobic region and does molecular dynamics studies of human PrP(110-136...

  16. Chaperoning prions: the story unfolds

    OpenAIRE

    O'Connor, David; Jones, Gary

    2006-01-01

    Prions are infectious proteins that are responsible for a number of mammalian degenerative diseases. The discovery of prions in yeast has allowed detailed genetic analysis to be carried out to identify cellular factors involved in prion propagation. It is now clear that a complex relationship exists between molecular chaperones and prion propagation. Prions may actually have evolved to exploit the cell's chaperone machinery to ensure their own propaga...

  17. Yeast prions: structure, biology, and prion-handling systems.

    Science.gov (United States)

    Wickner, Reed B; Shewmaker, Frank P; Bateman, David A; Edskes, Herman K; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E

    2015-03-01

    A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants.

  18. Smart protein biogate as a mediator to regulate competitive host-guest interaction for sensitive ratiometric electrochemical assay of prion

    Science.gov (United States)

    Yu, Peng; Zhang, Xiaohua; Zhou, Jiawan; Xiong, Erhu; Li, Xiaoyu; Chen, Jinhua

    2015-11-01

    A novel competitive host-guest strategy regulated by protein biogate was developed for sensitive and selective analysis of prion protein. The methylene blue (MB)-tagged prion aptamer (MB-Apt) was introduced to the multiwalled carbon nanotubes-β-cyclodextrins (MWCNTs-β-CD) composites-modified glassy carbon (GC) electrode through the host-guest interaction between β-CD and MB. In the absence of prion, MB-Apt could be displaced by ferrocenecarboxylic acid (FCA) due to its stronger binding affinity to β-CD, resulting in a large oxidation peak of FCA. However, in the presence of prion, the specific prion-aptamer interaction drove the formation of protein biogate to seal the cavity of β-CD, which hindered the guest displacement of MB by FCA and resulted in the oxidation peak current of MB (IMB) increased and that of FCA (IFCA) decreased. The developed aptasensor showed good response towards the target (prion protein) with a low detection limit of 160 fM. By changing the specific aptamers, this strategy could be easily extended to detect other proteins, showing promising potential for extensive applications in bioanalysis.

  19. Detection of Prion Proteins and TSE Infectivity in the Rendering and Biodiesel Manufacture Processes

    Energy Technology Data Exchange (ETDEWEB)

    Brown, R.; Keller, B.; Oleschuk, R. [Queen' s University, Kingston, Ontario (Canada)

    2007-03-15

    This paper addresses emerging issues related to monitoring prion proteins and TSE infectivity in the products and waste streams of rendering and biodiesel manufacture processes. Monitoring is critical to addressing the knowledge gaps identified in 'Biodiesel from Specified Risk Material Tallow: An Appraisal of TSE Risks and their Reduction' (IEA's AMF Annex XXX, 2006) that prevent comprehensive risk assessment of TSE infectivity in products and waste. The most important challenge for monitoring TSE risk is the wide variety of sample types, which are generated at different points in the rendering/biodiesel production continuum. Conventional transmissible spongiform encephalopathy (TSE) assays were developed for specified risk material (SRM) and other biological tissues. These, however, are insufficient to address the diverse sample matrices produced in rendering and biodiesel manufacture. This paper examines the sample types expected in rendering and biodiesel manufacture and the implications of applying TSE assay methods to them. The authors then discuss a sample preparation filtration, which has not yet been applied to these sample types, but which has the potential to provide or significantly improve TSE monitoring. The main improvement will come from transfer of the prion proteins from the sample matrix to a matrix compatible with conventional and emerging bioassays. A second improvement will come from preconcentrating the prion proteins, which means transferring proteins from a larger sample volume into a smaller volume for analysis to provide greater detection sensitivity. This filtration method may also be useful for monitoring other samples, including wash waters and other waste streams, which may contain SRM, including those from abattoirs and on-farm operations. Finally, there is a discussion of emerging mass spectrometric methods, which Prusiner and others have shown to be suitable for detection and characterisation of prion proteins (Stahl

  20. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  1. Immunization with recombinant prion protein leads to partial protection in a murine model of TSEs through a novel mechanism.

    Science.gov (United States)

    Xanthopoulos, Konstantinos; Lagoudaki, Rosa; Kontana, Anastasia; Kyratsous, Christos; Panagiotidis, Christos; Grigoriadis, Nikolaos; Yiangou, Minas; Sklaviadis, Theodoros

    2013-01-01

    Transmissible spongiform encephalopathies are neurodegenerative diseases, which despite fervent research remain incurable. Immunization approaches have shown great potential at providing protection, however tolerance effects hamper active immunization protocols. In this study we evaluated the antigenic potential of various forms of recombinant murine prion protein and estimated their protective efficacy in a mouse model of prion diseases. One of the forms tested provided a significant elongation of survival interval. The elongation was mediated via an acute depletion of mature follicular dendritic cells, which are associated with propagation of the prion infectious agent in the periphery and in part to the development of humoral immunity against prion protein. This unprecedented result could offer new strategies for protection against transmissible encephalopathies as well as other diseases associated with follicular dendritic cells. PMID:23554984

  2. Immunization with recombinant prion protein leads to partial protection in a murine model of TSEs through a novel mechanism.

    Directory of Open Access Journals (Sweden)

    Konstantinos Xanthopoulos

    Full Text Available Transmissible spongiform encephalopathies are neurodegenerative diseases, which despite fervent research remain incurable. Immunization approaches have shown great potential at providing protection, however tolerance effects hamper active immunization protocols. In this study we evaluated the antigenic potential of various forms of recombinant murine prion protein and estimated their protective efficacy in a mouse model of prion diseases. One of the forms tested provided a significant elongation of survival interval. The elongation was mediated via an acute depletion of mature follicular dendritic cells, which are associated with propagation of the prion infectious agent in the periphery and in part to the development of humoral immunity against prion protein. This unprecedented result could offer new strategies for protection against transmissible encephalopathies as well as other diseases associated with follicular dendritic cells.

  3. The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein.

    Science.gov (United States)

    Fonseca-Ornelas, Luis; Zweckstetter, Markus

    2016-08-01

    In a group of neurodegenerative diseases, collectively termed transmissible spongiform encephalopathies, the prion protein aggregates into β-sheet rich amyloid-like deposits. Because amyloid structure has been connected to different prion strains and cellular toxicity, it is important to obtain insight into the structural properties of prion fibrils. Using a combination of solution NMR spectroscopy, thioflavin-T fluorescence and electron microscopy we here show that within amyloid fibrils of a peptide containing residues 108-143 of the human prion protein [humPrP (108-143)]-the evolutionary most conserved part of the prion protein - residue H111 and S135 are in close spatial proximity and their interaction is critical for fibrillization. We further show that residues H111 and H140 share the same microenvironment in the unfolded, monomeric state of the peptide, but not in the fibrillar form. While protonation of H140 has little influence on fibrillization of humPrP (108-143), a positive charge at position 111 blocks the conformational change, which is necessary for amyloid formation of humPrP (108-143). Our study thus highlights the importance of protonation of histidine residues for protein aggregation and suggests point mutations to probe the structure of infectious prion particles. PMID:27184108

  4. The role of metals in protein conformational disorders - The case of prion protein and Aβ -peptide

    Science.gov (United States)

    De Santis, E.; Minicozzi, V.; Morante, S.; Rossi, G. C.; Stellato, F.

    2016-02-01

    Protein conformational disorders are members of a vast class of pathologies in which endogenous proteins or peptides undergo a misfolding process by switching from the physiological soluble configuration to a pathological fibrillar insoluble state. An important, but not yet fully elucidated, role in the process appears to be played by transition metal ions, mainly copper and zinc. X-ray absorption spectroscopy is one of the most suitable techniques for the structural characterization of biological molecules in complex with metal. Owing to its chemical selectivity and sensitivity to the local atomic geometry around the absorber, it can be successfully used to study the environment of metal ions in complex with proteins and peptides in physiological conditions. In this paper we present X-ray absorption spectroscopy studies of the metal ions coordination modes in systems where metals are complexed with specific amyloidogenic proteins and peptides. In particular, we show results concerning the Amyloid β peptide, that is involved in Alzheimer's disease, and the Prion protein, that is responsible for the Transmissible Spongiform Encephalopathy. Our findings suggest that the copper and zinc ions may play a crucial role in the aggregation and fibril formation process of these two biomolecules. Elucidating this kind of interaction could be a key preliminary step before any viable therapy can be conceived or designed.

  5. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    Energy Technology Data Exchange (ETDEWEB)

    Manno, D; Filippo, E; Fiore, R; Serra, A [Dipartimento di Scienza dei Materiali, Universita del Salento, Lecce (Italy); Urso, E; Rizzello, A; Maffia, M [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, Lecce (Italy)

    2010-04-23

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP{sup C}. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP{sup C} at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  6. Insights into alternative prion protein topologies induced under high hydrostatic pressure

    International Nuclear Information System (INIS)

    The critical step in the pathogenesis of transmissible spongiform encephalopathies (TSEs) appears to be a conformational transition of a normal prion protein (PrPC) into a misfolded isoform (PrPSc). To gain insight into the structural conversion of the prion protein we have exploited the use of high hydrostatic pressure combined with various spectroscopic techniques. In vitro transitions of the recombinant PrP to a scrapie-like form have never resulted in an infectious structure. It is our hypothesis that the acquisition of the disease-causing conformation depends on folding pathways which are difficult to attain. We attempt to favour, via specific reaction conditions at high pressure, alternative routes of misfolding leading to a stable infectious amyloidogenic conformer. Our results have demonstrated the potential of high pressure to reveal various prion structural changes, which are inaccessible by conventional methods. Especially, we have characterized a pressure-induced conformer in which the normal α-helical structure is changed into a highly aggregated β-sheet conformation showing markedly increased resistance to proteolysis (key markers of potential infectious agents). Our work may have important implications, not only for ultimately proving the protein-only hypothesis and for understanding the basic mechanism of the disease, but also for developing preventative and therapeutic measures

  7. Immunization with Recombinant Prion Protein Leads to Partial Protection in a Murine Model of TSEs through a Novel Mechanism

    OpenAIRE

    Konstantinos Xanthopoulos; Rosa Lagoudaki; Anastasia Kontana; Christos Kyratsous; Christos Panagiotidis; Nikolaos Grigoriadis; Minas Yiangou; Theodoros Sklaviadis

    2013-01-01

    Transmissible spongiform encephalopathies are neurodegenerative diseases, which despite fervent research remain incurable. Immunization approaches have shown great potential at providing protection, however tolerance effects hamper active immunization protocols. In this study we evaluated the antigenic potential of various forms of recombinant murine prion protein and estimated their protective efficacy in a mouse model of prion diseases. One of the forms tested provided a significant elongat...

  8. Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease.

    Science.gov (United States)

    Curcio, L; Sebastiani, C; Di Lorenzo, P; Lasagna, E; Biagetti, M

    2016-10-01

    Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection

  9. Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy

    Directory of Open Access Journals (Sweden)

    Maas Elke

    2005-10-01

    Full Text Available Abstract Background The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD in humans or bovine spongiform encephalopathy (BSE in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods. Results We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10-8 nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle. Conclusion We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an ante mortem diagnostic test for prion diseases.

  10. Hsp70/Hsp90 organising protein (hop): beyond interactions with chaperones and prion proteins.

    Science.gov (United States)

    Baindur-Hudson, Swati; Edkins, Adrienne L; Blatch, Gregory L

    2015-01-01

    The Hsp70/Hsp90 organising protein (Hop), also known as stress-inducible protein 1 (STI1), has received considerable attention for diverse cellular functions in both healthy and diseased states. There is extensive evidence that intracellular Hop is a co-chaperone of the major chaperones Hsp70 and Hsp90, playing an important role in the productive folding of Hsp90 client proteins. Consequently, Hop is implicated in a number of key signalling pathways, including aberrant pathways leading to cancer. However, Hop is also secreted and it is now well established that Hop also serves as a receptor for the prion protein, PrP(C). The intracellular and extracellular forms of Hop most likely represent two different isoforms, although the molecular determinants of these divergent functions are yet to be identified. There is also a growing body of research that reports the involvement of Hop in cellular activities that appear independent of either chaperones or PrP(C). While Hop has been shown to have various cellular functions, its biological function remains elusive. However, recent knockout studies in mammals suggest that Hop has an important role in embryonic development. This review provides a critical overview of the latest molecular, cellular and biological research on Hop, critically evaluating its function in healthy systems and how this function is adapted in diseases states.

  11. Effect of metal ions on de novo aggregation of full-length prion protein

    International Nuclear Information System (INIS)

    It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu2+, Mn2+, Ni2+, Co2+, and Zn2+) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn2+ at low micromolar concentrations that could be blocked by nanomolar concentration of Cu2+. These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo

  12. De novo prions

    OpenAIRE

    Legname, Giuseppe; Geschwind, Michael; Benetti, Federico

    2010-01-01

    Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases. They occur in three forms - sporadic, genetic, or acquired - and involve non-covalent post-translational modifications of the cellular prion protein (PrPC). Prions (PrPSc) are characterized by their infectious properties and intrinsic ability to act as a template, converting the normal, physiological PrPC into the pathological form, PrPSc. The ‘protein-only’ hypothesis, postulated by Stanley B Prusiner, impl...

  13. Neurotoxicity of prion peptides mimicking the central domain of the cellular prion protein.

    Directory of Open Access Journals (Sweden)

    Silvia Vilches

    Full Text Available The physiological functions of PrP(C remain enigmatic, but the central domain, comprising highly conserved regions of the protein may play an important role. Indeed, a large number of studies indicate that synthetic peptides containing residues 106-126 (CR located in the central domain (CD, 95-133 of PrP(C are neurotoxic. The central domain comprises two chemically distinct subdomains, the charge cluster (CC, 95-110 and a hydrophobic region (HR, 112-133. The aim of the present study was to establish the individual cytotoxicity of CC, HR and CD. Our results show that only the CD peptide is neurotoxic. Biochemical, Transmission Electron Microscopy and Atomic Force Microscopy experiments demonstrated that the CD peptide is able to activate caspase-3 and disrupt the cell membrane, leading to cell death.

  14. Prion and prion-like diseases in animals.

    Science.gov (United States)

    Aguilar-Calvo, Patricia; García, Consolación; Espinosa, Juan Carlos; Andreoletti, Olivier; Torres, Juan María

    2015-09-01

    Transmissible spongiform encephalopaties (TSEs) are fatal neurodegenerative diseases characterized by the aggregation and accumulation of the misfolded prion protein in the brain. Other proteins such as β-amyloid, tau or Serum Amyloid-A (SAA) seem to share with prions some aspects of their pathogenic mechanism; causing a variety of so called prion-like diseases in humans and/or animals such as Alzheimer's, Parkinson's, Huntington's, Type II diabetes mellitus or amyloidosis. The question remains whether these misfolding proteins have the ability to self-propagate and transmit in a similar manner to prions. In this review, we describe the prion and prion-like diseases affecting animals as well as the recent findings suggesting the prion-like transmissibility of certain non-prion proteins.

  15. Techniques to elucidate the conformation of prions

    OpenAIRE

    Daus, Martin L.

    2015-01-01

    Proteinaceous infectious particles (prions) are unique pathogens as they are devoid of any coding nucleic acid. Whilst it is assumed that prion disease is transmitted by a misfolded isoform of the cellular prion protein, the structural insight of prions is still vague and research for high resolution structural information of prions is still ongoing. In this review, techniques that may contribute to the clarification of the conformation of prions are presented and discussed.

  16. Techniques to elucidate the conformation of prions

    Institute of Scientific and Technical Information of China (English)

    Martin; L; Daus

    2015-01-01

    Proteinaceous infectious particles(prions) are unique pathogens as they are devoid of any coding nucleic acid.Whilst it is assumed that prion disease is transmitted by a misfolded isoform of the cellular prion protein, the structural insight of prions is still vague and research for high resolution structural information of prions is still ongoing. In this review, techniques that may contribute to the clarification of the conformation of prions are presented and discussed.

  17. A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

    Science.gov (United States)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2013-06-01

    A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

  18. A direct assessment of human prion adhered to steel wire using real-time quaking-induced conversion.

    Science.gov (United States)

    Mori, Tsuyoshi; Atarashi, Ryuichiro; Furukawa, Kana; Takatsuki, Hanae; Satoh, Katsuya; Sano, Kazunori; Nakagaki, Takehiro; Ishibashi, Daisuke; Ichimiya, Kazuko; Hamada, Masahisa; Nakayama, Takehisa; Nishida, Noriyuki

    2016-01-01

    Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies have directly evaluated human prions. A newly developed in vitro amplification system, designated real-time quaking-induced conversion (RT-QuIC), has allowed the activity of abnormal prion proteins to be assessed within a few days. RT-QuIC using human recombinant prion protein (PrP) showed high sensitivity for prions as the detection limit of our assay was estimated as 0.12 fg of active prions. We applied this method to detect human prion activity on stainless steel wire. When we put wires contaminated with human Creutzfeldt-Jakob disease brain tissue directly into the test tube, typical PrP-amyloid formation was observed within 48 hours, and we could detect the activity of prions at 50% seeding dose on the wire from 10(2.8) to 10(5.8) SD50. Using this method, we also confirmed that the seeding activities on the wire were removed following treatment with NaOH. As seeding activity closely correlated with the infectivity of prions using the bioassay, this wire-QuIC assay will be useful for the direct evaluation of decontamination methods for human prions. PMID:27112110

  19. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  20. Differential expression of calcium-related genes in gastric cancer cells transfected with cellular prion protein.

    Science.gov (United States)

    Liang, Jie; Luo, Guanhong; Ning, Xiaoxuan; Shi, Yongquan; Zhai, Huihong; Sun, Shiren; Jin, Haifeng; Liu, Zhenxiong; Zhang, Faming; Lu, Yuanyuan; Zhao, Yunping; Chen, Xiong; Zhang, Hongbo; Guo, Xuegang; Wu, Kaichun; Fan, Daiming

    2007-06-01

    The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies, which causes prion disorders partially due to Ca2+ dysregulation. In our previous work, we found that overexpressed PrPC in gastric cancer was involved in apoptosis, cell proliferation, and metastasis of gastric cancer. To better understand how PrPC acts in gastric cancer, a human microarray was performed to select differentially regulated genes that correlate with the biological function of PrPC. The microarray data were analyzed and revealed 3798 genes whose expression increased at least 2-fold in gastric cancer cells transfected with PrPC. These genes encode proteins involved in several aspects of cell biology, among which, we specially detected molecules related to calcium, especially the S100 calcium-binding proteins, and found that PrPC upregulates S100A1, S100A6, S100B, and S100P but downregulates CacyBP in gastric cancer cells. We also found that intracellular Ca2+ levels in cells transfected with PrPC increased, whereas these levels decreased in knockdowns of these cells. Taken together, PrPC might increase intracellular Ca2+, partially through calcium-binding proteins, or PrPC might upregulate the expression of S100 proteins, partially through stimulating the intracellular calcium level in gastric cancer. Though the underlying mechanisms need further exploration, this study provides a new insight into the role of PrPC in gastric cancer and enriches our knowledge of prion protein. PMID:17612632

  1. Mouse-hamster chimeric prion protein (PrP) devoid of N-terminal residues 23-88 restores susceptibility to 22L prions, but not to RML prions in PrP-knockout mice.

    Science.gov (United States)

    Uchiyama, Keiji; Miyata, Hironori; Yano, Masashi; Yamaguchi, Yoshitaka; Imamura, Morikazu; Muramatsu, Naomi; Das, Nandita Rani; Chida, Junji; Hara, Hideyuki; Sakaguchi, Suehiro

    2014-01-01

    Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.

  2. Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity

    Science.gov (United States)

    Yen, Chi-Fu; Harischandra, Dilshan S.; Kanthasamy, Anumantha; Sivasankar, Sanjeevi

    2016-01-01

    Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. Copper exposure has been linked to prion pathogenesis; however, its mechanistic basis is unknown. We resolve, with single-molecule precision, the molecular mechanism of Cu2+-induced misfolding of PrP under physiological conditions. We also demonstrate that misfolded PrPs serve as seeds for templated formation of aggregates, which mediate inflammation and degeneration of neuronal tissue. Using a single-molecule fluorescence assay, we demonstrate that Cu2+ induces PrP monomers to misfold before oligomer assembly; the disordered amino-terminal region mediates this structural change. Single-molecule force spectroscopy measurements show that the misfolded monomers have a 900-fold higher binding affinity compared to the native isoform, which promotes their oligomerization. Real-time quaking-induced conversion demonstrates that misfolded PrPs serve as seeds that template amyloid formation. Finally, organotypic slice cultures show that misfolded PrPs mediate inflammation and degeneration of neuronal tissue. Our study establishes a direct link, at the molecular level, between copper exposure and PrP neurotoxicity. PMID:27419232

  3. Do prion protein gene polymorphisms induce apoptosis in non-mammals?

    Indian Academy of Sciences (India)

    Tugce Birkan; Mesut Sahin; Zubeyde Oztel; Erdal Balcan

    2016-03-01

    Genetic variations such as single nucleotide polymorphisms (SNPs) in prion protein coding gene, Prnp, greatly affect susceptibility to prion diseases in mammals. Here, the coding region of Prnp was screened for polymorphisms in redeared turtle, Trachemys scripta. Four polymorphisms, L203V, N205I, V225A and M237V, were common in 15 out of 30 turtles; in one sample, three SNPs, L203V, N205I and M237V, and in the remaining 14 samples, only L203V and N205I polymorphisms, were investigated. Besides, C658T, C664T, C670A and C823A SNPs were silent mutations. To elucidate the relationship between the SNPs and apoptosis, TUNEL assays and active caspase-3 immunodetection techniques in brain sections of the polymorphic samples were performed. The results revealed that TUNEL-positive cells and active caspase-3-positive cells in the turtles with four polymorphisms were significantly increased compared with those of the turtles with two polymorphisms (P<0.01 and P<0.05, respectively). In conclusion, this study provides preliminary information about the possible relationship between SNPs within the Prnp locus and apoptosis in a non-mammalian species, Trachemys scripta, in which prion disease has never been reported.

  4. Intraepithelial and interstitial deposition of pathological prion protein in kidneys of scrapie-affected sheep.

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    Ciriaco Ligios

    Full Text Available Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE. The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrP(Sc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrP(Sc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla and occasional interstitial (papilla deposition of PrP(Sc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrP(Sc. PrP(Sc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrP(Sc in kidney varied from approximately 27% (subclinical to 73.6% (clinical in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrP(Sc. Here we demonstrate unexpectedly frequent deposition of high levels of PrP(Sc in ovine kidneys of various flocks. Renal deposition of PrP(Sc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep.

  5. Preclinical deposition of pathological prion protein in muscle of experimentally infected primates.

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    Susanne Krasemann

    Full Text Available Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and animals. A central step in disease progression is the accumulation of a misfolded form (PrP(Sc of the host encoded prion protein (PrP(C in neuronal and non-neuronal tissues. The involvement of peripheral tissues in preclinical states increases the risk of accidental transmission. On the other hand, detection of PrP(Sc in non-neuronal easy-accessible compartments such as muscle may offer a novel diagnostic tool. Primate models have proven invaluable to investigate prion diseases. We have studied the deposition of PrP(Sc in muscle and central nervous system of rhesus monkeys challenged with sporadic Creutzfeldt-Jakob disease (sCJD, variant CJD (vCJD and bovine spongiform encephalopathy (BSE in preclinical and clinical stage using biochemical and morphological methods. Here, we show the preclinical presence of PrP(Sc in muscle and central nervous system of rhesus monkeys experimentally infected with vCJD.

  6. The physical relationship between infectivity and prion protein aggregates is strain-dependent.

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    Philippe Tixador

    2010-04-01

    Full Text Available Prions are unconventional infectious agents thought to be primarily composed of PrP(Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C. They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP(Sc conformation could encode this 'strain' diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP(Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP(Sc aggregates from PrP(C. The distribution of PrP(Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP(Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12-30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP(Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

  7. Prion propagation in vitro: are we there yet?

    OpenAIRE

    Ryou, Chongsuk; Mays, Charles E.

    2008-01-01

    Prion diseases are caused by proteinaceous pathogens termed prions. Although the details of the mechanism of prion propagation are not fully understood, conformational conversion of cellular prion protein (PrPC) to misfolded, disease-associated scrapie prion protein (PrPSc) is considered the essential biochemical event for prion replication. Currently, studying prion replication in vitro is difficult due to the lack of a system which fully recapitulates the in vivo phenomenon. Over the last 1...

  8. Accumulation of pathological prion protein PrPSc in the skin of animals with experimental and natural scrapie.

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    Achim Thomzig

    2007-05-01

    Full Text Available Prion infectivity and its molecular marker, the pathological prion protein PrP(Sc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrP(Sc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles. Here, we address the question of whether prions target the skin and show widespread PrP(Sc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrP(Sc was detected before the onset of symptoms, but the bulk of skin-associated PrP(Sc accumulated in the clinical phase. PrP(Sc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrP(Sc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrP(Sc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.

  9. Cells and prions: a license to replicate.

    Science.gov (United States)

    Nuvolone, Mario; Aguzzi, Adriano; Heikenwalder, Mathias

    2009-08-20

    Prion diseases are neurodegenerative, infectious disorders characterized by the aggregation of a misfolded isoform of the cellular prion protein (PrP(C)). The infectious agent - termed prion - is mainly composed of misfolded PrP(Sc). In addition to the central nervous system prions can colonize secondary lymphoid organs and inflammatory foci. Follicular dendritic cells are important extraneural sites of prion replication. However, recent data point to a broader range of cell types that can replicate prions. Here, we review the state of the art in regards to peripheral prion replication, neuroinvasion and the determinants of prion replication competence. PMID:19527722

  10. Disinfectants and Prions

    Science.gov (United States)

    Prions are novel pathogens that are believed to be composed solely of protein. They are capable of converting a normal cellular protein into the infectious isoform and thereby propagating an infection. Prion infections are characterized by a long asymptomatic incubation period followed by a relative...

  11. Dissection and design of yeast prions.

    OpenAIRE

    Osherovich, Lev Z; Cox, Brian S; Mick F Tuite; Weissman, Jonathan S

    2004-01-01

    Many proteins can misfold into beta-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, ...

  12. The comprehensive native interactome of a fully functional tagged prion protein.

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    Dorothea Rutishauser

    Full Text Available The enumeration of the interaction partners of the cellular prion protein, PrP(C, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrP(C. When expressed in transgenic mice, PrP(myc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrP(C. PrP(myc antagonized the toxicity of truncated PrP, restored prion infectibility of PrP(C-deficient mice, and was physically incorporated into PrP(Sc aggregates, indicating that it possessed all functional characteristics of genuine PrP(C. We then immunopurified myc epitope-containing protein complexes from PrP(myc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrP(C and may represent component of a multiprotein complex. Selected PrP(C interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.

  13. The complexity and implications of yeast prion domains

    OpenAIRE

    Du, Zhiqiang

    2011-01-01

    Prions are infectious proteins with altered conformations converted from otherwise normal host proteins. While there is only one known mammalian prion protein, PrP, a handful of prion proteins have been identified in the yeast Saccharomyces cerevisiae. Yeast prion proteins usually have a defined region called prion domain (PrD) essential for prion properties, which are typically rich in glutamine (Q) and asparagine (N). Despite sharing several common features, individual yeast PrDs are genera...

  14. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    Science.gov (United States)

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  15. Familial Creutzfeldt-Jakob disease associated with a point mutation at codon 210 of the prion protein gene

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    Huang Nancy

    2001-01-01

    Full Text Available Creutzfeldt-Jakob disease (CJD, the most known human prion disease, is usually sporadic but approximately 15% of the cases are familial. To date, seven CJD cases with codon 210 mutation (GTT to ATT have been reported in the literature. We describe a case of a 57 year-old woman who presented gait disturbances and rapidly progressive dementia, leading to death four months after onset. Electroencephalogram revealed periodic activity, diffusion-weighted magnetic resonance imaging showed hypersignal in basal ganglia, and test for 14-3-3 protein was strongly positive in the CSF. The complete prion protein gene coding region was sequenced after PCR amplification, showing a point mutation in codon 210. This is the first case of CJD with codon 210 mutation diagnosed in Brazil. We emphasize the role of genetic search for prion protein gene mutation, even in patients presenting clinical features resembling sporadic CJD.

  16. Molecular dynamics studies on the NMR structures of rabbit prion protein wild-type and mutants: surface electrostatic charge distributions

    CERN Document Server

    Zhang, Jiapu

    2014-01-01

    Prion is a misfolded protein found in mammals that causes infectious diseases of the nervous system in humans and animals. Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species such as sheep and goats, cattle, deer, elk and humans etc. Recent studies have shown that rabbits have a low susceptibility to be infected by prion diseases with respect to other animals including humans. The present study employs molecular dynamics (MD) means to unravel the mechanism of rabbit prion proteins (RaPrPC) based on the recently available rabbit NMR structures (of the wild-type and its two mutants of two surface residues). The electrostatic charge distributions on the protein surface are the focus when analysing the MD trajectories. It is found that we can conclude that surface electrostatic charge distributions indeed contribute to the structural stability of wild-type RaPrPC; this may be useful for the medicinal treatment of prion diseases.

  17. Limited transcriptional response of ovine microglia to prion accumulation

    Science.gov (United States)

    Sheep scrapie (Sc) is the classical transmissible spongiform encephalopathy (prion disease). The conversion of normal cellular prion protein (PrPC) to disease-associated prion protein (PrPSc) is the fundamental pathogenesis of prion diseases. Many of the molecular mechanisms contributing to prion ...

  18. The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep.

    Science.gov (United States)

    Mesquita, P; Garcia, V; Marques, M R; Santos Silva, F; Oliveira Sousa, M C; Carolino, I; Pimenta, J; Fontes, C M G A; Horta, A E M; Prates, J A M; Pereira, R M

    2016-02-01

    The objective of this study was to search for polymorphisms in the ovine prion-related protein (testis-specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene-coding region was analyzed by single-strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine-X-X-serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C-112G-129T-144A,17CT-112GC-129CT-144AG and 17T-112C-129C-144G), and the only three animals found homozygous at c.78A had the 17C-112G-129C-144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP). PMID:26538093

  19. Degradation of the disease-associated prion protein by a serine protease from lichens.

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    Christopher J Johnson

    Full Text Available The disease-associated prion protein (PrP(TSE, the probable etiological agent of the transmissible spongiform encephalopathies (TSEs, is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria have the ability to degrade prion protein (PrP from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  20. Degradation of the disease-associated prion protein by a serine protease from lichens.

    Science.gov (United States)

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J. C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  1. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

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    Jia Shao

    Full Text Available Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.

  2. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    Science.gov (United States)

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis. PMID:24714645

  3. CRISPR-Cas9-based knockout of the prion protein and its effect on the proteome.

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    Mohadeseh Mehrabian

    Full Text Available The molecular function of the cellular prion protein (PrPC and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer's disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼ 120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.

  4. [Gerstmann-Sträussler-Scheinker syndrome with a Pro102Leu mutation in the prion protein gene and atypical MRI findings, hyperthermia, tachycardia, and hyperhidrosis].

    Science.gov (United States)

    Imaiso, Y; Mitsuo, K

    1998-01-01

    A 64-year-old Japanese woman with Gerstmann-Sträussler-Scheinker syndrome (GSS) is reported. She was admitted to our hospital for progressive amnesia, twitching of the right upper limb, and difficulty in speaking and walking for 5 months. Physical examination revealed a fever, tachycardia, and hyperhidrosis without any evidence of inflammation or infection. Neurological examinations demonstrated dementia, frontal lobe signs, and spontaneous myoclonus. She developed akinetic mutism 4 months later. The levels of neuron-specific enolase and 14-3-3 protein were elevated in the cerebrospinal fluid, and serial EEG showed periodic synchronous discharges. DNA analysis of the prion protein gene revealed a Pro102Leu mutation and therefore she was diagnosed as GSS102. Head MRI showed abnormal high signal intensity by T2 weighted image in bilateral caudate nuclei, putamen, frontal lobes, and white matter around the posterior horn of lateral ventricles at admission, and extension to global cerebral cortex and diffuse deep white matter with marked atrophy of bilateral frontal and cerebellar cortices 4 months later. In 123I-IMP SPECT study, uptake of RI decreased slightly only in left frontal region at admission, but decreased markedly in bilateral frontal region 4 months later. Analysis of autonomic function (analysis of noradrenarine in plasma and urine, coefficient of variation of R-R intervals before and after giving atenolol, Aschner's eyeball pressure test, intracutaneous atropine and adrenaline injection test) revealed sympathetic hyperactivity but normal parasympathetic activity. This is a very rare case of GSS102 with atypical MRI findings and clinical features like Creutzfeldt-Jakob disease rather than GSS102, presenting hyperthermia, tachycardia, and hyperhidrosis caused presumably by sympathetic hyperactivity as well as fatal familial insomnia. Therefore it is suggested that some factors besides the codon mutation in the prion protein gene may influence clinical

  5. A Review on the Salt Bridge Between ASP177 and ARG163 of Wild-Type Rabbit Prion Protein

    CERN Document Server

    Zhang, Jiapu

    2014-01-01

    Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species such as sheep and goats, cattle, deer, elks, humans and mice etc., but rabbits have a low susceptibility to be infected by prion diseases with respect to other species. The stability of rabbit prion protein is due to its highly ordered beta2-alpha2 loop [PLoS One 5 (10) e13273 (2010); Journal of Biological Chemistry 285 (41) 31682-31693 (2010)] and a helix-capping motif within this loop [PLoS One 8 (5) e63047 (2013)]. The beta2-alpha2 loop has been a focus in prion studies. For this loop we found a salt bridge linkage ASP177-ARG163 (O-N) [Journal of Theoretical Biology 342 (7 February 2014) 70-82 (2014)]. Some scientists said on the 2FJ3.pdb NMR file of the rabbit prion protein, the distance of ASP177-ARG163 (O-N) gives the salt bridge of about 10 angstroms which is nearly null in terms of energy thus think our result is wrong. This opinion is clearly wrong simply due to the 3O7...

  6. The prion protein constitutively controls neuronal store-operated Ca2+ entry through Fyn kinase

    Directory of Open Access Journals (Sweden)

    Agnese eDe Mario

    2015-10-01

    Full Text Available The prion protein (PrPC is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-β (Aβ oligomers associated with Alzheimer’s disease (AD, and that PrPC-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn.Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons expressing, or not, PrPC allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons.We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1-42 oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanism for Aβ-induced neuronal Ca2+ dyshomeostasis.

  7. Physiological and environmental control of yeast prions

    OpenAIRE

    Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

    2013-01-01

    Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion ...

  8. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    International Nuclear Information System (INIS)

    Research highlights: → We develop a method to track a quantum dot-conjugated protein in yeast cells. → We incorporate the conjugated quantum dot proteins into yeast spheroplasts. → We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  9. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

    2011-02-25

    Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  10. The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6.

    Science.gov (United States)

    Küffer, Alexander; Lakkaraju, Asvin K K; Mogha, Amit; Petersen, Sarah C; Airich, Kristina; Doucerain, Cédric; Marpakwar, Rajlakshmi; Bakirci, Pamela; Senatore, Assunta; Monnard, Arnaud; Schiavi, Carmen; Nuvolone, Mario; Grosshans, Bianka; Hornemann, Simone; Bassilana, Frederic; Monk, Kelly R; Aguzzi, Adriano

    2016-08-25

    Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options. PMID:27501152

  11. Inherited prion disease with A117V mutation of the prion protein gene: a novel Hungarian family

    OpenAIRE

    Kovacs, G; Ertsey, C; Majtenyi, C; Jelencsik, I; Laszlo, L; Flicker, H; Strain, L.; Szirmai, I; Budka, H.

    2001-01-01

    Three members of a family with inherited prion disease are reported. One additional family member had a progressive neurological disease without details. Two developed symptoms of ataxia, dementia, myoclonus, rigidity, and hemiparesis, and one had a different phenotype with the combination of lower motor neuron deficit, parkinsonism, intellectual decline, and ataxia. In this last patient cell loss of the anterior horn motor neurons and chronic neurogenic muscle atrophy was evid...

  12. Nanoimaging for prion related diseases.

    Science.gov (United States)

    Krasnoslobodtsev, Alexey V; Portillo, Alexander M; Deckert-Gaudig, Tanja; Deckert, Volker; Lyubchenko, Yuri L

    2010-01-01

    Misfolding and aggregation of prion proteins is linked to a number of neurodegenerative disorders such as Creutzfeldt-Jacob disease (CJD) and its variants: Kuru, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. In prion diseases, infectious particles are proteins that propagate by transmitting a misfolded state of a protein, leading to the formation of aggregates and ultimately to neurodegeneration. Prion phenomenon is not restricted to humans. There are a number of prion-related diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle. All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal. Prion proteins were also found in some fungi where they are responsible for heritable traits. Prion proteins in fungi are easily accessible and provide a powerful model for understanding the general principles of prion phenomenon and molecular mechanisms of mammalian prion diseases. Presently, several fundamental questions related to prions remain unanswered. For example, it is not clear how prions cause the disease. Other unknowns include the nature and structure of infectious agent and how prions replicate. Generally, the phenomenon of misfolding of the prion protein into infectious conformations that have the ability to propagate their properties via aggregation is of significant interest. Despite the crucial importance of misfolding and aggregation, very little is currently known about the molecular mechanisms of these processes. While there is an apparent critical need to study molecular mechanisms underlying misfolding and aggregation, the detailed characterization of these single molecule processes is hindered by the limitation of conventional methods. Although some issues remain unresolved, much progress has been recently made primarily due to the application of nanoimaging tools. The use of nanoimaging methods shows

  13. Enzymatic degradation of prion protein by keratinase producing proteolytic micro-organisms

    OpenAIRE

    Okoroma, Emeka A.

    2012-01-01

    Prions are highly resistant to common proteases and conventional sterilisation processes. Consequently, prion infectivity is destroyed by methods such as incineration, alkaline and thermal hydrolysis. These harsh, destructive and potentially hazardous methods are unsuitable for processing specified risk materials (SRM) and animal by-products, and in the decontamination of medical and laboratory devices, and prion contaminated environments. Thus an environmentally friendly, enzymatic degradati...

  14. The chemistry of prions: small molecules, protein conformers and mass spectrometry

    Science.gov (United States)

    Background/Introduction. Prions propagate by converting a normal cellular isoform (PrPC) into the prion isoform (PrPSc) in a template-driven process. The lysines in PrPC are highly conserved and strongly influence prion propagation, based on studies using natural polymorphisms of PrPC and transg...

  15. Temporal resolution of misfolded prion protein transport, accumulation, glial activation, and neuronal death in the retinas of mice inoculated with scrapie

    Science.gov (United States)

    Currently, there is a lack of pathologic landmarks to describe the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between the transport of misfolded prion protein from the brain to the retina, the accumulation of PrPSc in the retina, the respon...

  16. Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9.

    Science.gov (United States)

    Kaczmarczyk, Lech; Mende, Ylva; Zevnik, Branko; Jackson, Walker S

    2016-01-01

    The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program. PMID:27128441

  17. Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9.

    Directory of Open Access Journals (Sweden)

    Lech Kaczmarczyk

    Full Text Available The mammalian prion protein (PrP, encoded by Prnp is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9, have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs. It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.

  18. β-sheet-like formation during the mechanical unfolding of prion protein

    International Nuclear Information System (INIS)

    Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220

  19. Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9

    Science.gov (United States)

    Kaczmarczyk, Lech; Mende, Ylva; Zevnik, Branko; Jackson, Walker S.

    2016-01-01

    The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program. PMID:27128441

  20. Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

    International Nuclear Information System (INIS)

    Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrPC) into a disease associated form (PrPSc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrPC or a β-sheet rich, protease resistant form similar to PrPSc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrPSc from PrPC. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.

  1. β-sheet-like formation during the mechanical unfolding of prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Weiwei; Cao, Penghui; Park, Harold S., E-mail: parkhs@bu.edu [Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215 (United States); Yoon, Gwonchan [Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215 (United States); Department of Mechanical Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Eom, Kilho [Biomechanics Laboratory, College of Sport Science, Sungkyunkwan University, Suwon 16419 (Korea, Republic of)

    2015-09-28

    Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP{sup C}, whose misfolded form PrP{sup Sc} can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

  2. β-sheet-like formation during the mechanical unfolding of prion protein

    Science.gov (United States)

    Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S.

    2015-09-01

    Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

  3. Codon 129 polymorphism of prion protein gene in is not a risk factor for Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Jerusa Smid

    2013-07-01

    Full Text Available Interaction of prion protein and amyloid-b oligomers has been demonstrated recently. Homozygosity at prion protein gene (PRNP codon 129 is associated with higher risk for Creutzfeldt-Jakob disease. This polymorphism has been addressed as a possible risk factor in Alzheimer disease (AD. Objective To describe the association between codon 129 polymorphisms and AD. Methods We investigated the association of codon 129 polymorphism of PRNP in 99 AD patients and 111 controls, and the association between this polymorphism and cognitive performance. Other polymorphisms of PRNP and additive effect of apolipoprotein E gene (ApoE were evaluated. Results Codon 129 genotype distribution in AD 45.5% methionine (MM, 42.2% methionine valine (MV, 12.1% valine (VV; and 39.6% MM, 50.5% MV, 9.9% VV among controls (p>0.05. There were no differences of cognitive performance concerning codon 129. Stratification according to ApoE genotype did not reveal difference between groups. Conclusion Codon 129 polymorphism is not a risk factor for AD in Brazilian patients.

  4. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    International Nuclear Information System (INIS)

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation

  5. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    Energy Technology Data Exchange (ETDEWEB)

    Haigh, Cathryn L., E-mail: chaigh@unimelb.edu.au [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); Drew, Simon C., E-mail: sdrew@unimelb.edu.au [Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010 (Australia)

    2015-06-05

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation.

  6. Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

    Directory of Open Access Journals (Sweden)

    Rodrigo Diaz-Espinoza

    Full Text Available Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP, is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(- or SO(4(-2. The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc. The aggregates also exhibited PrP(Sc-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

  7. Mass spectrometric detection of proteins in non-aqueous media : the case of prion proteins in biodiesel

    Energy Technology Data Exchange (ETDEWEB)

    Douma, M.D.; Kerr, G.M.; Brown, R.S.; Keller, B.O.; Oleschuk, R.D. [Queen' s Univ., Kingston, ON (Canada). Dept. of Chemistry

    2008-08-15

    This paper presented a filtration method for detecting protein traces in non-aqueous media. The extraction technique used a mixture of acetonitrile, non-ionic detergent and water along with filter disks with embedded C{sub 8}-modified silica particles to capture the proteins from non-aqueous samples. The extraction process was then followed by an elution of the protein from the filter disk and direct mass spectrometric detection and tryptic digestion with peptide mapping and MS/MS fragmentation of protein-specific peptides. The method was used to detect prion proteins in spiked biodiesel samples. A tryptic peptide with the sequence YGQGSPGGNR was used for unambiguous identification. Results of the study showed that the method is suitable for the large-scale testing of protein impurities in tallow-based biodiesel production processes. 33 refs., 6 figs.

  8. Computational Calculation Of The Ionization Energies Of The Human Prion Protein By The Coarse-grain Method

    Science.gov (United States)

    Lyu, Justin; Andrianarijaona, V. M.

    2016-05-01

    The causes of the misfolding of prion protein -i.e. the transformation of PrPC to PrPSc - have not been clearly elucidated. Many studies have focused on identifying possible chemical conditions, such as pH, temperature and chemical denaturation, that may trigger the pathological transformation of prion proteins (Weiwei Tao, Gwonchan Yoon, Penghui Cao, `` β-sheet-like formation during the mechanical unfolding of prion protein'', The Journal of Chemical Physics, 2015, 143, 125101). Here, we attempt to calculate the ionization energies of the prion protein, which will be able to shed light onto the possible causes of the misfolding. We plan on using the coarse-grain method which allows for a more feasible calculation time by means of approximation. We believe that by being able to approximate the ionization potential, particularly that of the regions known to form stable β-strands of the PrPSc form, the possible sources of denaturation, be it chemical or mechanical, may be narrowed down.

  9. PrionHome: a database of prions and other sequences relevant to prion phenomena.

    Directory of Open Access Journals (Sweden)

    Djamel Harbi

    Full Text Available Prions are units of propagation of an altered state of a protein or proteins; prions can propagate from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We report the bioinformatic construction of the PrionHome, a database of >2000 prion-related sequences. The data was collated from various public and private resources and filtered for redundancy. The data was then processed according to a transparent classification system of prionogenic sequences (i.e., sequences that can make prions, prionoids (i.e., proteins that propagate like prions between individual cells, and other prion-related phenomena. There are eight PrionHome classifications for sequences. The first four classifications are derived from experimental observations: prionogenic sequences, prionoids, other prion-related phenomena, and prion interactors. The second four classifications are derived from sequence analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences. Database entries list: supporting information for PrionHome classifications, prion-determinant areas (where relevant, and disordered and compositionally-biased regions. Also included are literature references for the PrionHome classifications, transcripts and genomic coordinates, and structural data (including comparative models made for the PrionHome from manually curated alignments. We provide database usage examples for both vertebrate and fungal prion contexts. Using the database data, we have performed a detailed analysis of the compositional biases in known budding-yeast prionogenic

  10. Prion Disease Induces Alzheimer Disease-Like Neuropathologic Changes

    Science.gov (United States)

    Tousseyn, Thomas; Bajsarowicz, Krystyna; Sánchez, Henry; Gheyara, Ania; Oehler, Abby; Geschwind, Michael; DeArmond, Bernadette; DeArmond, Stephen J.

    2016-01-01

    We examined the brains of 266 patients with prion diseases (PrionD) and found that 46 (17%) had Alzheimer disease (AD)-like changes. To explore potential mechanistic links between PrionD and AD, we exposed human brain aggregates (Hu BrnAggs) to brain homogenate from a patient with sporadic Creutzfeldt-Jakob disease (CJD) and found that the neurons in the Hu BrnAggs produced many β-amyloid (β42) inclusions, whereas uninfected, control-exposed Hu BrnAggs did not. Western blots of 20-pooled CJD-infected BrnAggs verified higher Aβ42 levels than controls. We next examined the CA1 region of the hippocampus from 14 patients with PrionD and found that 5 patients had low levels of scrapie-associated prion protein (PrPSc), many Aβ42 intraneuronal inclusions, low APOE-4, and no significant nerve cell loss. Seven patients had high levels of PrPSc, low Aβ42, high APOE-4 and 40% nerve cell loss, suggesting that APOE-4 and PrPSc together cause neuron loss in PrionD. There were also increased levels of hyperphosphorylated tau protein (Hτ) and Hτ-positive neuropil threads and neuron bodies in both PrionD and AD groups. The brains of 6 age-matched control patients without dementia did not contain Aβ42 deposits; however, there were rare Hτ-positive threads in 5 controls and 2 controls had a few Hτ-positive nerve cell bodies. We conclude that PrionD may trigger biochemical changes similar to AD and suggest that PrionD are diseases of PrPSc, Aβ42, APOE-4 and abnormal tau. PMID:26226132

  11. Pathological Propagation through Cell-to-Cell Transmission of Non-Prion Protein Aggregates in Neurodegenerative Disorders

    Science.gov (United States)

    Lee, Seung-Jae; Desplats, Paula; Sigurdson, Christina; Tsigelny, Igor; Masliah, Eliezer

    2016-01-01

    Neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, fronto-temporal dementia, Huntington's Disease and Creutzfeldt-Jakob Disease (CJD) are characterized by progressive accumulation of protein aggregates in selected brain regions. Protein misfolding and templated assembly into aggregates might result from an imbalance between protein synthesis, aggregation and clearance. While protein misfolding and aggregation occur in most neurodegenerative disorders, the concept of spreading and infectivity of aggregates in the CNS has been reserved to prion diseases such as CJD and bovine spongiform encephalopathy. Emerging evidence suggests that prion-like spreading may occur in other neurodegenerative disorders, taking place with secreted proteins, such as amyloid-β,) and cytosolic proteins, such as tau, huntingtin and α-synuclein. Underlying molecular mechanisms and therapeutic implications are discussed. PMID:21045796

  12. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    International Nuclear Information System (INIS)

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases

  13. Expression profiles of prion and doppel proteins and of their receptors in mouse splenocytes.

    Science.gov (United States)

    Cordier-Dirikoc, Sevda; Zsürger, Nicole; Cazareth, Julie; Ménard, Baptiste; Chabry, Joëlle

    2008-08-01

    Doppel (Dpl) shares common structural features with the prion protein (PrP) whose pathologic isoform is considered as the causative agent of prion diseases. Although their physiological functions in the immune system remain largely unknown, we demonstrated that substantial amounts of PrP and Dpl are expressed by spleen cells notably B lymphocytes, granulocytes and DC, but not T lymphocytes and NK. To characterize trans-interacting partners of PrP and Dpl on mouse splenocytes, fluorescent PrP and Dpl tetramers were produced and used as tracers. Both tetramers specifically bind to B lymphocytes, dendritic cells, macrophages and granulocytes and in a lesser extend to T lymphocytes. No binding was observed on NK, follicular dendritic cells and mesenchymal spleen cells. The activation of intracellular transduction signals (i.e. intracellular calcium concentration and activation of the MAP kinase pathway) suggested that PrP and Dpl tetramers bind to functional receptors on B cells. None of the previously described PrP partners account for the binding sites characterized here. Our study suggests a possible role for PrP and Dpl in the cell-cell interactions in the immune system. PMID:18604867

  14. Copper attachment to a non-octarepeat site in prion protein

    Science.gov (United States)

    Hodak, Miroslav; Bernholc, Jerry

    2010-03-01

    Prion protein, PrP, plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The PrP is known to efficiently bind copper ions and this ability has been linked to its function. PrP contains up to six binding sites, four of which are located in the so-called octarepeat region and are now well known. The binding sites outside this region are still largely undetermined, despite evidence of their relevance to prion diseases. Using a hybrid DFT/DFT, which combines Kohn-Sham DFT with orbital-free DFT to achieve accurate and efficient description of solvent effects in ab initio calculations, we have investigated copper attachment to the sequence GGGTH, which represents the copper binding site located at His96. We have considered both NNNN and NNNO types of copper coordination, as suggested by experiments. Our calculations have determined the geometry of copper attachment site and its energetics. Comparison to the already known binding sites provides insight into the process of copper uptake in PrP.

  15. Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

    Directory of Open Access Journals (Sweden)

    Béringue Vincent

    2010-07-01

    Full Text Available Abstract Background The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages. Results Only subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences. Conclusions These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

  16. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein.

    Science.gov (United States)

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E; Hong, Seo Jung; Chapman, Daniel C; Westaway, David; Williams, David B

    2016-03-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrP(C)) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrP(C) expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrP(C) expression. We show that FK506 treatment results in a profound reduction in PrP(C) expression due to a defect in the translocation of PrP(C) into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrP(C) signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrP(C). However, this occurred at a later stage, after translocation of PrP(C) into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrP(Sc) propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrP(C) biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  17. Complement protein C3 exacerbates prion disease in a mouse model of chronic wasting disease.

    Science.gov (United States)

    Michel, Brady; Ferguson, Adam; Johnson, Theodore; Bender, Heather; Meyerett-Reid, Crystal; Wyckoff, A Christy; Pulford, Bruce; Telling, Glenn C; Zabel, Mark D

    2013-12-01

    Accumulating evidence shows a critical role of the complement system in facilitating attachment of prions to both B cells and follicular dendritic cells and assisting in prion replication. Complement activation intensifies disease in prion-infected animals, and elimination of complement components inhibits prion accumulation, replication and pathogenesis. Chronic wasting disease (CWD) is a highly infectious prion disease of captive and free-ranging cervid populations that utilizes the complement system for efficient peripheral prion replication and most likely efficient horizontal transmission. Here we show that complete genetic or transient pharmacological depletion of C3 prolongs incubation times and significantly delays splenic accumulation in a CWD transgenic mouse model. Using a semi-quantitative prion amplification scoring system we show that C3 impacts disease progression in the early stages of disease by slowing the rate of prion accumulation and/or replication. The delayed kinetics in prion replication correlate with delayed disease kinetics in mice deficient in C3. Taken together, these data support a critical role of C3 in peripheral CWD prion pathogenesis. PMID:24038599

  18. Grass plants bind, retain, uptake and transport infectious prions

    OpenAIRE

    Sandra Pritzkow; Rodrigo Morales; Fabio Moda; Uffaf Khan; Glenn C. Telling; Edward Hoover; Claudio Soto

    2015-01-01

    Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrPSc) to plants. Small quantities of PrPSc contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-...

  19. A non Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast

    OpenAIRE

    Taneja, Vibha; Maddelein, Marie-Lise; Talarek, Nicolas; J. Saupe, Sven; Liebman, Susan W

    2007-01-01

    Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrPSc, causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine(Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins, lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions: transient overexpression of the HET-s fusion induces r...

  20. The many shades of prion strain adaptation.

    Science.gov (United States)

    Baskakov, Ilia V

    2014-01-01

    In several recent studies transmissible prion disease was induced in animals by inoculation with recombinant prion protein amyloid fibrils produced in vitro. Serial transmission of amyloid fibrils gave rise to a new class of prion strains of synthetic origin. Gradual transformation of disease phenotypes and PrP(Sc) properties was observed during serial transmission of synthetic prions, a process that resembled the phenomenon of prion strain adaptation. The current article discusses the remarkable parallels between phenomena of prion strain adaptation that accompanies cross-species transmission and the evolution of synthetic prions occurring within the same host. Two alternative mechanisms underlying prion strain adaptation and synthetic strain evolution are discussed. The current article highlights the complexity of the prion transmission barrier and strain adaptation and proposes that the phenomenon of prion adaptation is more common than previously thought.

  1. High-level expression and secondary structure analysis of the bovine mature prion protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By using the recombinant DNA technology, the gene of the bovine mature prion protein (bPrPCL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPCL is 23 630 u, the bPrPCL has a high α-helix content (36.1%) and low β-sheet content (11.9%).

  2. Induced prion protein controls immune-activated retroviruses in the mouse spleen.

    Directory of Open Access Journals (Sweden)

    Marius Lötscher

    Full Text Available The prion protein (PrP is crucially involved in transmissible spongiform encephalopathies (TSE, but neither its exact role in disease nor its physiological function are known. Here we show for mice, using histological, immunochemical and PCR-based methods, that stimulation of innate resistance was followed by appearance of numerous endogenous retroviruses and ensuing PrP up-regulation in germinal centers of the spleen. Subsequently, the activated retroviruses disappeared in a PrP-dependent manner. Our results reveal the regular involvement of endogenous retroviruses in murine immune responses and provide evidence for an essential function of PrP in the control of the retroviral activity. The interaction between PrP and ubiquitous endogenous retroviruses may allow new interpretations of TSE pathophysiology and explain the evolutionary conservation of PrP.

  3. Prions are affected by evolution at two levels.

    Science.gov (United States)

    Wickner, Reed B; Kelly, Amy C

    2016-03-01

    Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals. PMID:26713322

  4. Prions are affected by evolution at two levels.

    Science.gov (United States)

    Wickner, Reed B; Kelly, Amy C

    2016-03-01

    Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals.

  5. Distinct transmissibility features of TSE sources derived from ruminant prion diseases by the oral route in a transgenic mouse model (TgOvPrP4 overexpressing the ovine prion protein.

    Directory of Open Access Journals (Sweden)

    Jean-Noël Arsac

    Full Text Available Transmissible spongiform encephalopathies (TSEs are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP. Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE, transmissible mink encephalopathy (TME, kuru and variant Creutzfeldt-Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4 overexpressing the ovine prion protein (A136R154Q171 under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.

  6. Human prion diseases: surgical lessons learned from iatrogenic prion transmission.

    Science.gov (United States)

    Bonda, David J; Manjila, Sunil; Mehndiratta, Prachi; Khan, Fahd; Miller, Benjamin R; Onwuzulike, Kaine; Puoti, Gianfranco; Cohen, Mark L; Schonberger, Lawrence B; Cali, Ignazio

    2016-07-01

    The human prion diseases, or transmissible spongiform encephalopathies, have captivated our imaginations since their discovery in the Fore linguistic group in Papua New Guinea in the 1950s. The mysterious and poorly understood "infectious protein" has become somewhat of a household name in many regions across the globe. From bovine spongiform encephalopathy (BSE), commonly identified as mad cow disease, to endocannibalism, media outlets have capitalized on these devastatingly fatal neurological conditions. Interestingly, since their discovery, there have been more than 492 incidents of iatrogenic transmission of prion diseases, largely resulting from prion-contaminated growth hormone and dura mater grafts. Although fewer than 9 cases of probable iatrogenic neurosurgical cases of Creutzfeldt-Jakob disease (CJD) have been reported worldwide, the likelihood of some missed cases and the potential for prion transmission by neurosurgery create considerable concern. Laboratory studies indicate that standard decontamination and sterilization procedures may be insufficient to completely remove infectivity from prion-contaminated instruments. In this unfortunate event, the instruments may transmit the prion disease to others. Much caution therefore should be taken in the absence of strong evidence against the presence of a prion disease in a neurosurgical patient. While the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) have devised risk assessment and decontamination protocols for the prevention of iatrogenic transmission of the prion diseases, incidents of possible exposure to prions have unfortunately occurred in the United States. In this article, the authors outline the historical discoveries that led from kuru to the identification and isolation of the pathological prion proteins in addition to providing a brief description of human prion diseases and iatrogenic forms of CJD, a brief history of prion disease nosocomial transmission

  7. Comparison of 2 synthetically generated recombinant prions

    OpenAIRE

    Zhang, Yi; Wang, Fei; Wang, Xinhe; Zhang, Zhihong; Xu, Yuanyuan; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2014-01-01

    Prion is a protein-conformation-based infectious agent causing fatal neurodegenerative diseases in humans and animals. Our previous studies revealed that in the presence of cofactors, infectious prions can be synthetically generated in vitro with bacterially expressed recombinant prion protein (PrP). Once initiated, the recombinant prion is able to propagate indefinitely via serial protein misfolding cyclic amplification (sPMCA). In this study, we compared 2 separately initiated recombinant p...

  8. Biology and Genetics of Prions Causing Neurodegeneration

    OpenAIRE

    Prusiner, SB

    2013-01-01

    Prions are proteins that acquire alternative conformations that become self-propagating. Transformation of proteins into prions is generally accompanied by an increase in β-sheet structure and a propensity to aggregate into oligomers. Some prions are beneficial and perform cellular functions, whereas others cause neurodegeneration. In mammals, more than a dozen proteins that become prions have been identified, and a similar number has been found in fungi. In both mammals and fungi, variations...

  9. Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue

    Science.gov (United States)

    Mehrabian, Mohadeseh; Brethour, Dylan; Williams, Declan; Wang, Hansen; Arnould, Hélène; Schneider, Benoit; Schmitt-Ulms, Gerold

    2016-01-01

    A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP), best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types. PMID:27327609

  10. Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue.

    Science.gov (United States)

    Mehrabian, Mohadeseh; Brethour, Dylan; Williams, Declan; Wang, Hansen; Arnould, Hélène; Schneider, Benoit; Schmitt-Ulms, Gerold

    2016-01-01

    A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP), best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types.

  11. How does domain replacement affect fibril formation of the rabbit/human prion proteins.

    Directory of Open Access Journals (Sweden)

    Xu Yan

    Full Text Available It is known that in vivo human prion protein (PrP have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2 have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different

  12. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms.

    Directory of Open Access Journals (Sweden)

    Pedro Fernandez-Funez

    2009-06-01

    Full Text Available Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C converts into a misfolded isoform (PrP(Sc with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc-specific conformational epitopes. In contrast to PrP(Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover

  13. Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

    Science.gov (United States)

    Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

    2016-05-01

    The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

  14. Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

    Directory of Open Access Journals (Sweden)

    Katsufumi Tomobe

    2016-05-01

    Full Text Available The conformational change from the cellular prion protein (PrPc to scrapie prion protein (PrPsc is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

  15. Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126.

    Science.gov (United States)

    Kawahara, Masahiro; Koyama, Hironari; Nagata, Tetsuya; Sadakane, Yutaka

    2011-07-01

    Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases. PMID:21442127

  16. Membrane binding of prion protein N-terminal peptides characterised by neutron reflectometry

    International Nuclear Information System (INIS)

    The prion protein (PrP) is widely recognised to mis-fold into the causative agent of the transmissible spongiform encephalopathies, known as Creutzfeldt–Jakob disease (CJD) in humans, scrappie in sheep or Bovine spongiform encephalopathy in cows (BSE, “mad cow disease”). PrP has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the mis-folding events associated with prion pathogenesis, PrP can undergo various post-translational modifications, including internal cleavage events. Alpha and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2 respectively, which interact specifically with negatively charged phospholipids at low pH. Previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption [1]. This work aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, neutron reflectometry was used to define the structural details of the interactions in combination with quartz crystal microbalance interrogation and calcein release assays. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with previous studies, interactions were stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. Overall, the data shows that the N1 and N2 peptides interact with the anionic phospholipid headgroups of supported lipid bilayers, inducing lipid ordering in the absence of significant penetration into the acyl tails or permeation of the membrane.

  17. Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus).

    Science.gov (United States)

    Brandt, Adam L; Kelly, Amy C; Green, Michelle L; Shelton, Paul; Novakofski, Jan; Mateus-Pinilla, Nohra E

    2015-01-01

    The sequence of the prion protein gene (PRNP) affects susceptibility to spongiform encephalopathies, or prion diseases in many species. In white-tailed deer, both coding and non-coding single nucleotide polymorphisms have been identified in this gene that correlate to chronic wasting disease (CWD) susceptibility. Previous studies examined individual nucleotide or amino acid mutations; here we examine all nucleotide polymorphisms and their combined effects on CWD. A 626 bp region of PRNP was examined from 703 free-ranging white-tailed deer. Deer were sampled between 2002 and 2010 by hunter harvest or government culling in Illinois and Wisconsin. Fourteen variable nucleotide positions were identified (4 new and 10 previously reported). We identified 68 diplotypes comprised of 24 predicted haplotypes, with the most common diplotype occurring in 123 individuals. Diplotypes that were found exclusively among positive or negative animals were rare, each occurring in less than 1% of the deer studied. Only one haplotype (C, odds ratio 0.240) and 2 diplotypes (AC and BC, odds ratios of 0.161 and 0.108 respectively) has significant associations with CWD resistance. Each contains mutations (one synonymous nucleotide 555C/T and one nonsynonymous nucleotide 286G/A) at positions reported to be significantly associated with reduced CWD susceptibility. Results suggest that deer populations with higher frequencies of haplotype C or diplotypes AC and BC might have a reduced risk for CWD infection--while populations with lower frequencies may have higher risk for infection. Understanding the genetic basis of CWD has improved our ability to assess herd susceptibility and direct management efforts within CWD infected areas.

  18. Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease.

    Science.gov (United States)

    Johnson, Chad; Johnson, Jody; Vanderloo, Joshua P; Keane, Delwyn; Aiken, Judd M; McKenzie, Debbie

    2006-07-01

    The primary sequence of the prion protein affects susceptibility to transmissible spongiform encephalopathies, or prion diseases, in mice, sheep and humans. The Prnp gene sequence of free-ranging, Wisconsin white-tailed deer was determined and the Prnp genotypes of chronic wasting disease (CWD)-positive and CWD-negative deer were compared. Six amino acid changes were identified, two of which were located in pseudogenes. Two alleles, a Q-->K polymorphism at codon 226 and a single octapeptide repeat insertion into the pseudogene, have not been reported previously. The predominant alleles--wild-type (Q95, G96 and Q226) and a G96S polymorphism--comprised almost 98% of the Prnp alleles in the Wisconsin white-tailed deer population. Comparison of the allelic frequencies in the CWD-positive and CWD-negative deer suggested that G96S and a Q95H polymorphism were linked to a reduced susceptibility to CWD. The G96S allele did not, however, provide complete resistance, as a CWD-positive G96S/G96S deer was identified. The G96S allele was also linked to slower progression of the disease in CWD-positive deer based on the deposition of PrP(CWD) in the obex region of the medulla oblongata. Although the reduced susceptibility of deer with at least one copy of the Q95H or G96S allele is insufficient to serve as a genetic barrier, the presence of these alleles may modulate the impact of CWD on white-tailed deer populations.

  19. Polymorphisms of the prion protein gene (PRNP) in a Korean population.

    Science.gov (United States)

    Jeong, Byung-Hoon; Nam, Jae-Hwan; Lee, Yun-Jung; Lee, Kyung-Hee; Jang, Myoung-Kuk; Carp, Richard I; Lee, Ho-Dong; Ju, Young-Ran; Ahn Jo, Sangmee; Park, Keun-Yong; Kim, Yong-Sun

    2004-01-01

    Human prion protein gene (PRNP) has been considered to be involved in the susceptibility of humans to prion diseases. Polymorphisms of methionine (Met)/valine (Val) at codon 129 and of glutamic acid (Glu)/lysine (Lys) at codon 219 are thought to play an important role in susceptibility to sporadic, iatrogenic and variant Creutzfeldt-Jakob disease (CJD). Although the genotype distribution of polymorphisms in PRNP open reading frame (ORF) has been reported in many European populations, among Asian groups, it has been reported only in the Japanese population. We examined the PRNP polymorphisms in 529 healthy Koreans. We observed that genotype frequencies at codon 129 was 94.33% Met/Met, 5.48% Met/Val, and 0.19% Val/Val with an allele frequency of 0.971:0.029 Met:Val, and that genotype frequencies at codon 219 was 92.06% Glu/Glu, 7.94% Glu/Lys, and 0% Lys/Lys with an allele frequency of 0.96:0.04 Glu:Lys. The frequencies of the Glu/Glu genotype ( chi(2)=10.075, P=0.0015) and of the Glu allele ( chi(2)=9.486, P=0.0021) at codon 219 were significantly higher in the Korean population than the Japanese population. In addition, the genotype frequency of heterozygotes (12.7%) at codons 129 or/and 219 was significantly lower in Koreans than in people from Great Britain ( chi(2)=89.52, P<0.0001). The deletion rate of one octarepeat (R2 deletion) was 0.38%, with 99.62% undeleted homozygotes and 0% deleted homozygote. To our knowledge, the R2 octarepeat deletion has never been found in people from countries other than Korea. The data of PRNP polymorphism at codon 219 suggest that Koreans may be more sensitive to sporadic CJD than the Japanese population.

  20. Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus).

    Science.gov (United States)

    Brandt, Adam L; Kelly, Amy C; Green, Michelle L; Shelton, Paul; Novakofski, Jan; Mateus-Pinilla, Nohra E

    2015-01-01

    The sequence of the prion protein gene (PRNP) affects susceptibility to spongiform encephalopathies, or prion diseases in many species. In white-tailed deer, both coding and non-coding single nucleotide polymorphisms have been identified in this gene that correlate to chronic wasting disease (CWD) susceptibility. Previous studies examined individual nucleotide or amino acid mutations; here we examine all nucleotide polymorphisms and their combined effects on CWD. A 626 bp region of PRNP was examined from 703 free-ranging white-tailed deer. Deer were sampled between 2002 and 2010 by hunter harvest or government culling in Illinois and Wisconsin. Fourteen variable nucleotide positions were identified (4 new and 10 previously reported). We identified 68 diplotypes comprised of 24 predicted haplotypes, with the most common diplotype occurring in 123 individuals. Diplotypes that were found exclusively among positive or negative animals were rare, each occurring in less than 1% of the deer studied. Only one haplotype (C, odds ratio 0.240) and 2 diplotypes (AC and BC, odds ratios of 0.161 and 0.108 respectively) has significant associations with CWD resistance. Each contains mutations (one synonymous nucleotide 555C/T and one nonsynonymous nucleotide 286G/A) at positions reported to be significantly associated with reduced CWD susceptibility. Results suggest that deer populations with higher frequencies of haplotype C or diplotypes AC and BC might have a reduced risk for CWD infection--while populations with lower frequencies may have higher risk for infection. Understanding the genetic basis of CWD has improved our ability to assess herd susceptibility and direct management efforts within CWD infected areas. PMID:26634768

  1. INSIGHTS ON SCRAPIE PRION PROTEIN (PrPSc) STRUCTURE OBTAINED BY LIMITED PROTEOLYSIS AND MASS SPECTROMETRY

    Science.gov (United States)

    Elucidation of the structure of PrPSc, essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research, and is hampered by the insolubility and polymeric character of PrPSc. Limited proteolysis is a useful tool to obtain insight on...

  2. Human Tonsil-Derived Follicular Dendritic-Like Cells are Refractory to Human Prion Infection in Vitro and Traffic Disease-Associated Prion Protein to Lysosomes

    OpenAIRE

    Krejciova, Zuzana; De Sousa, Paul; Manson, Jean; Ironside, James W.; Mark W Head

    2014-01-01

    The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of...

  3. Prions, prion-like prionoids, and neurodegenerative disorders.

    Science.gov (United States)

    Verma, Ashok

    2016-01-01

    Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by the aggregation and deposition of the misfolded prion protein in the brain. α-synuclein (α-syn)-associated multiple system atrophy has been recently shown to be caused by a bona fide α-syn prion strain. Several other misfolded native proteins such as β-amyloid, tau and TDP-43 share some aspects of prions although none of them is shown to be transmissible in nature or in experimental animals. However, these prion-like "prionoids" are causal to a variety of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The remarkable recent discovery of at least two new α-syn prion strains and their transmissibility in transgenic mice and in vitro cell models raises a distinct question as to whether some specific strain of other prionoids could have the capability of disease transmission in a manner similar to prions. In this overview, we briefly describe human and other mammalian prion diseases and comment on certain similarities between prion and prionoid and the possibility of prion-like transmissibility of some prionoid strains. PMID:27293325

  4. Prions, prion-like prionoids, and neurodegenerative disordersVacancy

    Directory of Open Access Journals (Sweden)

    Ashok Verma

    2016-01-01

    Full Text Available Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by the aggregation and deposition of the misfolded prion protein in the brain. α-synuclein (α-syn-associated multiple system atrophy has been recently shown to be caused by a bona fide α-syn prion strain. Several other misfolded native proteins such as β-amyloid, tau and TDP-43 share some aspects of prions although none of them is shown to be transmissible in nature or in experimental animals. However, these prion-like “prionoids” are causal to a variety of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The remarkable recent discovery of at least two new α-syn prion strains and their transmissibility in transgenic mice and in vitro cell models raises a distinct question as to whether some specific strain of other prionoids could have the capability of disease transmission in a manner similar to prions. In this overview, we briefly describe human and other mammalian prion diseases and comment on certain similarities between prion and prionoid and the possibility of prion-like transmissibility of some prionoid strains.

  5. Prions, prion-like prionoids, and neurodegenerative disorders.

    Science.gov (United States)

    Verma, Ashok

    2016-01-01

    Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by the aggregation and deposition of the misfolded prion protein in the brain. α-synuclein (α-syn)-associated multiple system atrophy has been recently shown to be caused by a bona fide α-syn prion strain. Several other misfolded native proteins such as β-amyloid, tau and TDP-43 share some aspects of prions although none of them is shown to be transmissible in nature or in experimental animals. However, these prion-like "prionoids" are causal to a variety of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The remarkable recent discovery of at least two new α-syn prion strains and their transmissibility in transgenic mice and in vitro cell models raises a distinct question as to whether some specific strain of other prionoids could have the capability of disease transmission in a manner similar to prions. In this overview, we briefly describe human and other mammalian prion diseases and comment on certain similarities between prion and prionoid and the possibility of prion-like transmissibility of some prionoid strains.

  6. Prion disease. The characteristics and diagnostic points in Japan

    International Nuclear Information System (INIS)

    Prion disease develops when normal prion proteins change into transmissible abnormal prion proteins and the converted proteins accumulate in the brain. The Japanese Creutzfeldt-Jakob Disease (CJD) Surveillance Committee has identified 1,320 patients with prion diseases in the 10 years since 1999 (classified into 3 types: sporadic, 77.2%; hereditary, 16.7%; and environmentally acquired, 6.1%). Compared with patients in other countries, a relatively larger number of Japanese patients characteristically have dura mater graft-associated CJD and hereditary prion diseases. All the environmentally acquired cases, except 1 case of variant CJD, were acquired from dura grafts. Although most patients were diagnosed with a classical subtype of sporadic CJD (sCJD), whose features include rapidly progressing dementia, myoclonus, hyperintensity in the cerebral cortex and basal ganglia in diffusion-weighted magnetic resonance imaging, and periodic synchronous discharge in electroencephalography, the number of cases with atypical symptoms, such as MM2 (0.8%), MV2 (0.2%), VV1 (0%), and VV2 (0.2%) subtypes of sCJD cases, was not negligible. Appropriate diagnosis should be made based on clinical features, neuroradiological findings, cerebrospinal fluid (CSF) findings (14-3-3 and total tau proteins), and genetic analysis of polymorphisms. Hereditary prion diseases are classified into 3 major phenotypes: familial CJD (fCJD); Gerstmann-Straeussler-Scheinker disease (GSS), which mainly presents as spinocerebellar ataxia; and fatal familial insomnia. Many mutations of the prion protein gene have been identified, but V1801 (fCJD), P102L (GSS), and E200K (fCJD) mutations were the most common among the fCJD cases in Japan. Without a family history, genetic testing is necessary to distinguish even seemingly ''sporadic'' CJD from fCJD. Accurate diagnosis is important for clarification of the pathological process, prevention of secondary infection, and also psychological support. (author)

  7. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    Science.gov (United States)

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-01-01

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country. PMID:27622622

  8. Peripheral prion disease pathogenesis is unaltered in the absence of sialoadhesin (Siglec-1/CD169).

    Science.gov (United States)

    Bradford, Barry M; Crocker, Paul R; Mabbott, Neil A

    2014-09-01

    Prions are a unique group of pathogens, which are considered to comprise solely of an abnormally folded isoform of the cellular prion protein. The accumulation and replication of prions within secondary lymphoid organs is important for their efficient spread from the periphery to the brain where they ultimately cause neurodegeneration and death. Mononuclear phagocytes (MNP) play key roles in prion disease pathogenesis. Some MNP appear to facilitate the propagation of prions to and within lymphoid tissues, whereas others may aid their clearance by phagocytosis and by destroying them. Our recent data show that an intact splenic marginal zone is important for the efficient delivery of prions into the B-cell follicles where they subsequently replicate upon follicular dendritic cells before infecting the nervous system. Sialoadhesin is an MNP-restricted cell adhesion molecule that binds sialylated glycoproteins. Sialoadhesin is constitutively expressed upon splenic marginal zone metallophilic and lymph node sub-capsular sinus macrophage populations, where it may function to bind sialylated glycoproteins, pathogens and exosomes in the blood and lymph via recognition of terminal sialic acid residues. As the prion glycoprotein is highly sialylated, we tested the hypothesis that sialoadhesin may influence prion disease pathogenesis. We show that after peripheral exposure, prion pathogenesis was unaltered in sialoadhesin-deficient mice; revealing that lymphoid sequestration of prions is not mediated via sialoadhesin. Hence, although an intact marginal zone is important for the efficient uptake and delivery of prions into the B-cell follicles of the spleen, this is not influenced by sialoadhesin expression by the MNP within it. PMID:24684244

  9. α-Helical to β-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein

    Science.gov (United States)

    Singh, Jesse; Whitford, Paul; Hayre, Natha; Cox, Daniel; Onuchic, José.

    2011-03-01

    We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (α H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation.

  10. Localization of A11-reactive oligomeric species in prion diseases

    DEFF Research Database (Denmark)

    Aidt, Frederik H; Hasholt, Lis F; Christiansen, Michael;

    2013-01-01

    To investigate in prion diseases the in-situ localization of prion protein oligomers sharing a common epitope with amyloid oligomers involved in a range of neurodegenerative diseases.......To investigate in prion diseases the in-situ localization of prion protein oligomers sharing a common epitope with amyloid oligomers involved in a range of neurodegenerative diseases....

  11. Mass Spectrometric Approaches to Detecting and Quantifying Prions

    Science.gov (United States)

    Prions are infectious proteins that replicate by converting a normal cellular protein (PrPC)into a prion. Although prions and PrPC are isoforms, they have dramatically different physicochemical properties. Prions are resistant to proteinase K (PK) degradation, while PrPC is completely degraded by PK...

  12. Characterization and polyanion-binding properties of purified recombinant prion protein.

    Science.gov (United States)

    Brimacombe, D B; Bennett, A D; Wusteman, F S; Gill, A C; Dann, J C; Bostock, C J

    1999-09-15

    Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein. PMID:10477271

  13. Investigation of the effect of glycosylation on human prion protein by molecular dynamics.

    Science.gov (United States)

    Zhong, Linghao; Xie, Jimin

    2009-04-01

    Prion protein conformational isomerization, PrP(C)-->PrP(Sc), has been attributed as the cause of TSE diseases such as mad-cow disease. The mechanism of such isomerization, however, is little known due the experimental difficulties in studying the scrapie form. Among factors that affect PrP isomerization, the role which glycosylation plays remains vague. The number of innumerous glycan species, together with their high flexibility, leads to ineffective structural characterization. In this research, we studied the effect of chitobiose glycosylation on human PrP, in both monomeric (huPrP(mono)) and dimeric (huPrP(dimer)) forms, by molecular dynamics (MD) simulations. Our results show that this glycosylation has minimal impact on the structure of huPrP(mono). However, it affects the secondary structure of dimeric protein. An additional beta-sheet strand is found while the glycosylation is absent in the huPrP(dimer). Comparatively, when the protein is glycosylated with chitobiose, such beta-sheet addition is not observed. PMID:19236103

  14. Functions of the cellular prion protein, the end of Moore's law, and Ockham's razor theory

    Science.gov (United States)

    del Río, José A.; Gavín, Rosalina

    2016-01-01

    ABSTRACT Since its discovery the cellular prion protein (encoded by the Prnp gene) has been associated with a large number of functions. The proposed functions rank from basic cellular processes such as cell cycle and survival to neural functions such as behavior and neuroprotection, following a pattern similar to that of Moore's law for electronics. In addition, particular interest is increasing in the participation of Prnp in neurodegeneration. However, in recent years a redefinition of these functions has begun, since examples of previously attributed functions were increasingly re-associated with other proteins. Most of these functions are linked to so-called “Prnp-flanking genes” that are close to the genomic locus of Prnp and which are present in the genome of some Prnp mouse models. In addition, their role in neuroprotection against convulsive insults has been confirmed in recent studies. Lastly, in recent years a large number of models indicating the participation of different domains of the protein in apoptosis have been uncovered. However, after more than 10 years of molecular dissection our view is that the simplest mechanistic model in PrPC-mediated cell death should be considered, as Ockham's razor theory suggested. PMID:26890218

  15. Crystallization and preliminary X-ray crystallographic analysis of yeast prion protein Ure2p with shortened N-terminal

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    An orthorhombic crystal form of a recombinant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffusion technique against a mother liquor containing imidazole. Crystals belong to the primitive orthorhombic lattice with the cell parameters a = 54.5 ?, b = 74.7 ?, c = 131.0 ?. The crystals diffract to beyond 3.0 ? resolution at a synchrotron beamline.

  16. Introducing a rigid loop structure from deer into mouse prion protein increases its propensity for misfolding in vitro.

    Directory of Open Access Journals (Sweden)

    Leah M Kyle

    Full Text Available Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrP(c into the disease-associated isoform (PrP(Sc that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids.

  17. Activation of human natural killer cells by the soluble form of cellular prion protein

    International Nuclear Information System (INIS)

    Cellular prion protein (PrPC) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrPC in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrPC protein on human natural killer (NK) cells. Recombinant soluble PrPC protein was generated by fusion of human PrPC with the Fc portion of human IgG1 (PrPC-Fc). PrPC-Fc binds to the surface of human NK cells, particularly to CD56dim NK cells. PrPC-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrPC-Fc facilitated the IL-15-induced proliferation of NK cells. PrPC-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrPC-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrPC-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrPC (PrPC-Fc) was generated by fusion of human PrPC with IgG1 Fc portion. • PrPC-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrPC-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrPC-Fc protein activates human NK cells via the ERK and JNK signaling pathways

  18. Activation of human natural killer cells by the soluble form of cellular prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  19. Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171.

    Directory of Open Access Journals (Sweden)

    James F Striebel

    2011-09-01

    Full Text Available Transmissible spongiform encephalopathies (TSE or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP to a misfolded, protease-resistant form (PrPres. Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA, did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.

  20. Impact of SDS surfactant on the interactions of Cu(2+) ions with the amyloidogenic region of human prion protein.

    Science.gov (United States)

    Hecel, Aleksandra; Migliorini, Caterina; Valensin, Daniela; Luczkowski, Marek; Kozlowski, Henryk

    2015-08-01

    Prion diseases, known as Transmissible Spongiform Encephalopathies (TSEs), are a group of fatal neuronal, and to some extent infectious disorders, associated with a pathogenic protein agent called prion protein (PrP). The human prion protein (hPrP) fragment encompassing the 91-127 region, also known as the amyloidogenic domain, comprises two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for Cu(2+) binding. In this work, we investigated Cu(2+) interaction with hPrP91-127 in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), which induces a partial α-helix folding of the peptide. Our data indicate that the Cu(2+) coordination ability of the amyloidogenic fragment in the presence of SDS micelles is significantly different to that observed in aqueous solution. This is mainly due to the fact that SDS micelles strongly stabilize the formation of the α-helical structure of the peptide backbone, which is well conserved also upon Cu(2+) binding, contrary to the random coil conformation mainly assumed by hPrP91-127 in aqueous solutions. Potentiometric and spectroscopic studies clearly indicate that in the case of SDS containing solutions, Cu(2+) ions coordinate simultaneously to both imidazoles, while in the case of water solutions, metal ion coordination involves only a single His side chain, which individually acts as an independent Cu(2+) anchoring site.

  1. Doppel: more rival than double to prion.

    Science.gov (United States)

    Qin, K; O'Donnell, M; Zhao, R Y

    2006-08-11

    Conversion of normal cellular prion protein to the diseased form plays an essential role in transmissible spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jakob disease. However, the normal physiological function of prion protein remains elusive. Doppel, a German synonym of double, was initially identified as a prion-like protein due to its structural and biochemical similarities. However, emerging evidence suggests that function of prion protein is more antagonistic to Doppel than synergistic. In this review, basic biochemical and structural similarities of prion protein and Doppel are introduced; evidence demonstrating antagonistic interaction of prion protein with Doppel is presented; and a potential novel activity of Doppel and prion protein in spermatogenesis, which could stimulate new avenues for research, is discussed. PMID:16781817

  2. Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins.

    Science.gov (United States)

    Petrotchenko, Evgeniy V; Serpa, Jason J; Hardie, Darryl B; Berjanskii, Mark; Suriyamongkol, Bow P; Wishart, David S; Borchers, Christoph H

    2012-07-01

    Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including

  3. The Relationship of Prions and Translation

    OpenAIRE

    Wickner, Reed B.; Edskes, Herman K.; Shewmaker, Frank P.; Kryndushkin, Dmitry; Nemecek, Julie; McGlinchey, Ryan; Bateman, David

    2010-01-01

    Prions are infectious proteins, without the need for an accompanying nucleic acid. Nonetheless, there are connections of prions with translation and RNA, which we explore here. Most prions are based on self-propagating amyloids. The yeast [PSI+] prion is an amyloid of Sup35p, a subunit of the translation termination factor. The normal function of the Sup35p prion domain is in shortening the 3' polyA of mRNAs and thus in mRNA turnover. The [ISP+] prion is so named because it produces anti-supp...

  4. Follicular dendritic cells related to nerve fibres and cellular prion protein expression in ileal and jejunal Peyer’s patches of cows and calves

    OpenAIRE

    Defaweux, Valérie; Antoine, Nadine; Dorban, G.; C Demonceau; Piret, Joëlle; Heinen, Ernst

    2005-01-01

    Prion pathogenesis following oral exposure is thought to involve gut-associated lymphoid tissue, which includes Peyer’s patches (PP). Before neuroinvasion, early accumulation of infectious prion protein (PrPsc) takes place on follicular dendritic cells (FDC) which are resident cells in germinal centres. The strain, the infection pathway and the lesions in the central nervous system are similar between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt Jakob diseases. But in BSE, t...

  5. Prion and Fish Prion Proteins:Current Research Status%与朊毒体相关的鱼类朊蛋白研究概况

    Institute of Scientific and Technical Information of China (English)

    兰邹然; 王志亮; 张学成

    2006-01-01

    疯牛病(mad cow disease),即牛传染性海绵状脑病(bovine transmissible spongiform encephalopathy,BSE)的俗称,是一种慢性消耗性、致死性、中枢神经系统退行性疾病.疯牛病被认为与朊毒体(Prion)有关,朊毒体是由正常朊蛋白(Prion protein,或者prpC)发生构象改变后形成的异常蛋白(PrPSc).疯牛病的发生引起了世界各国政府和科学界的高度重视,PrP的起源及其功能研究已成为研究热点.鱼类PrP相关蛋白的研究正在展开中,由于鱼类PrP相关蛋白与朊蛋白的结构相似,鱼类感染TSE类似病存在理论上的风险.本文全面地综述了疯牛病的概况、朊毒体的特性、朊毒体与哺乳动物朊蛋白、鱼类PrP相关蛋白(PrP1、PrP2和PrP3)及鱼类其他PrP相关蛋白的研究情况,为国内水生动物PrP相关蛋白研究提供参考.

  6. Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

    International Nuclear Information System (INIS)

    The complex of MoPrP(120–232) and Fab POM1 has been crystallized (space group C2, unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°). Diffraction data to 2.30 Å resolution have been collected using synchrotron radiation. Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrPc to the pathogenic isoform PrPsc. Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120–232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°

  7. Uncovering molecular structural mechanisms of signaling mediated by the prion protein

    International Nuclear Information System (INIS)

    The glycosyl phosphatidylinositol (GPI) - anchored prion protein (PrPc), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrPc with the secretable cochaperone hop/STI 1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of Pr Pc and hop STI 1 entails structural rearrangements relevant for signaling. Circular dichroism and fluorescence spectroscopy showed that PrPc:hop/STI 1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the Pr Pc143-153 beta-helix. Novel SAXS models revealed a significant C-terminal compaction of hop/STI 1 when bound to PrPc. Differing from a recent dimeric model of human hop/STI 1, both size exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI 1. Changes in the Pr Pc143-153 beta-helix may engage the transmembrane signaling protein laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of Pr Pc, and further ligands may be engaged by the tertiary structural changes of hop/STI 1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by Pr Pc:hop/STI 1 interaction, consistent with the hypothesis that Pr Pc scaffolds multiprotein signaling complexes at the cell surface. (author)

  8. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M; Sim, Valerie L; Woodside, Michael T

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  9. Cloning and Expression Analysis of a Prion Protein Encoding Gene in Guppy (Poecilia reticulata)

    Institute of Scientific and Technical Information of China (English)

    WU Suihan; WEI Qiwei; YANG Guanpin; WANG Dengqiang; ZOU Guiwei; CHEN Daqing

    2008-01-01

    The full length eDNA of a prion protein (PrP) encoding gene of guppy (Poecilia reticulata) and the corresponding ge-nomic DNA were cloned.The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a pro-tein of 515 amino acids,which held all typical structural characteristics of the functional PrP.The cloned genomic DNA fragmentcorresponding to the eDNA was 3720 bp in length,consisting of 2 introns and 2 exons.The 5' untranslated region of eDNA origi-nated from the 2 exons,while the ORF originated from the second exon.Although the gene was transcribed in diverse tissues in-cluding brain,eye,liver,intestine,muscle and tail,its transcript was most abundant in the brain.In addition,the transcription of thegene was enhanced by 5 salinity,implying that it was associated with the response of guppy to saline stress.

  10. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-06-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

  11. Affinity Association Between Polynucleotide, Glycoprotein, or Sulfated Polysaccharides and Disease-Associated Prion Protein

    Directory of Open Access Journals (Sweden)

    Kazuo Tsukui

    2009-01-01

    Full Text Available Proteinase-K resistant prion protein (PrPres has the property to aggregate in TSE-injured animal tissues. We have developed a test method to discriminate scrapie-infected and mock-infected hamsters by detecting the PrPres in plasma. It seemed that aggregation of the PrPres with some heterogeneous molecule(s enabled successful detection by this method. In order to investigate which molecule became the partner in the PrPres aggregates; we examined some molecules that could presumably have this ability. As a result, we found synthetic Poly-A RNA, especially in its denatured form, to be the most effective entity although glycoprotein, sulfated polysaccharide showed less effectiveness. DNA in the denatured form also has a high affinity, although in the presence of protein the effectiveness unsuccessful. On the basis of this result, it is possible that the PrPres aggregate in scrapie-infected hamster plasma is composed of PrPres and RNA.

  12. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-01-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation. PMID:27346148

  13. Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates.

    Directory of Open Access Journals (Sweden)

    Enric Vidal

    2015-08-01

    Full Text Available Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab. Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD, experimental murine strains (ME7 and RML and experimentally obtained ruminant (sheepBSE and rabbit (de novo NZW strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrPd deposition profiles and proteinase-K (PK resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrPd originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.

  14. Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue.

    Directory of Open Access Journals (Sweden)

    Mohadeseh Mehrabian

    Full Text Available A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP, best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types.

  15. Immunologically induced, complement-dependent up-regulation of the prion protein in the mouse spleen: follicular dendritic cells versus capsule and trabeculae.

    Science.gov (United States)

    Lötscher, Marius; Recher, Mike; Hunziker, Lukas; Klein, Michael A

    2003-06-15

    The expression of the prion protein (PrP) in the follicular dendritic cell network of germinal centers in the spleen is critical for the splenic propagation of the causative agent of prion diseases. However, a physiological role of the prion protein in the periphery remains elusive. To investigate the role and function of PrP expression in the lymphoid system we treated naive mice i.v. with preformed immune complexes or vesicular stomatitis virus. Immunohistochemistry and Western blot analysis of the spleen revealed that 8 days after immunization, immune complexes and vesicular stomatitis virus had both induced a strong increase of PrP expression in the follicular dendritic cell network. Remarkably, this up-regulation did not occur in mice that lack an early factor of the complement cascade, C1q, a component which has been shown previously to facilitate early prion pathogenesis. In addition to the variable PrP level in the germinal centers, we detected steady and abundant PrP expression in the splenic capsule and trabeculae, which are structural elements that have not been associated before with PrP localization. The abundant trabeculo-capsular PrP expression was also evident in spleens of Rag-1-deficient mice, which have been shown before to be incapable of prion expansion. We conclude that trabeculocapsular PrP is not sufficient for splenic prion propagation. Furthermore, our observations may provide important clues for a physiological function of the prion protein and allow a new view on the role of complement and PrP in peripheral prion pathogenesis. PMID:12794132

  16. Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice.

    Science.gov (United States)

    Miyazawa, Kohtaro; Kanaya, Takashi; Tanaka, Sachi; Takakura, Ikuro; Watanabe, Kouichi; Ohwada, Shyuichi; Kitazawa, Haruki; Rose, Michael T; Sakaguchi, Suehiro; Katamine, Shigeru; Yamaguchi, Takahiro; Aso, Hisashi

    2007-03-01

    The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP(Sc)). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP(c)) to PrP(Sc). Therefore, PrP(c) expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP(c) in the gut is still a matter of controversy. We therefore investigated the localization of PrP(c) in the bovine and murine small intestine. In cattle, most PrP(c) positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP(c) was expressed in serotonin producing cells. In bovine Peyer's patches, PrP(c) was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP(c) was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP(c) was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer's patches. In this study, we demonstrate that there are a number of differences in the localization of PrP(c) between the murine and bovine small intestines. PMID:17165097

  17. Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

    International Nuclear Information System (INIS)

    The crystallization of a specific nanobody against mouse PrPC and preliminary diffraction analysis of a crystal that diffracted to 1.23 Å resolution are presented. Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrPC into the pathogenic isoform PrPSc. Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb-PrP-01) was raised against mouse PrPC. A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 30.04, b = 37.15, c = 83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrPC together with the known PrPC structure may help in understanding the PrPC/PrPSc transition mechanism

  18. Expression patterns of prion protein gene in differential genotypes sheep: quantification using molecular beacon real-time RT-PCR.

    Science.gov (United States)

    Wang, Chuan; Wu, Run; Li, Fa-Di; Liu, Lei; Zhang, Xiao-Li; Zhao, Chun-Lin; Diao, Xiao-Long; Guan, Hong-Wei

    2011-06-01

    Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its role in organisms and revealing mechanism of susceptibility and resistance to scrapie. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail in digestive tract which is important during scrapie spread through oral route. Herein, we report on measurement of sheep PrP mRNA using absolute quantitative real-time RT-PCR. Total RNA was isolated from five different regions of the central nervous system (CNS), four regions of lymphoid system, eleven regions of digestive tract, and two reproductive organ tissues of eight sheep of two different genotypes (ARR/ARQ and ARH/ARQ) and PrP mRNA was quantified by real-time RT-PCR using molecular beacon. The results showed that highest levels of PrP mRNA were expressed in thalamus and cerebrum (P mRNA expression in sheep for further studies of pathogenesis of prion diseases.

  19. The genetics of prion diseases.

    Science.gov (United States)

    Mastrianni, James A

    2010-04-01

    Prion diseases are a rare group of fatal neurodegenerative disorders of humans and animals that manifest primarily as progressive dementia and ataxia. Unique to these diseases is the prion, a misfolded isoform of the prion protein that can transmit disease from cell to cell or host to host by associating with, and transforming, normal prion protein into the misfolded isoform (the pathogenic scrapie-inducing form). Although the majority of cases occur on a sporadic basis, and rarely result from exposure to prions, such as mad cow disease, 10-15% are attributable to the presence of an autosomal dominant mutation of the prion protein gene (PRNP). Single base pair changes, or the insertion of one or more multiples of a 24 base pair repeat segment, make up the known sequence alterations of PRNP associated with genetic prion disease. The common polymorphic codon 129 of PRNP also plays an important and complex role in risk and phenotype of sporadic and genetic prion disease. This review will focus on the clinical and histopathologic features of the genetic prion diseases. Selected mutations will be highlighted as a way to illustrate general phenotype-genotype correlations. PMID:20216075

  20. A survey and a molecular dynamics study on the (central) hydrophobic region of prion proteins

    OpenAIRE

    Zhang, Jiapu; Wang, Feng

    2014-01-01

    Prion diseases are invariably fatal neurodegenerative diseases that affect humans and animals. Unlike most other amyloid forming neurodegenerative diseases, these can be highly infectious. Prion diseases occur in a variety of species. They include the fatal human neurodegenerative diseases Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, the bovine spongiform encephalopathy (BSE or 'mad-cow' disease) in cattle, the chronic wa...

  1. Protease-Sensitive Synthetic Prions

    OpenAIRE

    Colby, David W; Rachel Wain; Baskakov, Ilia V.; Giuseppe Legname; Palmer, Christina G.; Nguyen, Hoang-Oanh B.; Azucena Lemus; Cohen, Fred E.; Stephen J DeArmond; Prusiner, Stanley B.

    2010-01-01

    Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, ...

  2. Genes contributing to prion pathogenesis

    DEFF Research Database (Denmark)

    Tamgüney, Gültekin; Giles, Kurt; Glidden, David V;

    2008-01-01

    incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes...... show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1...

  3. Quantitative phosphoproteomic analysis of prion-infected neuronal cells

    Directory of Open Access Journals (Sweden)

    Löwer Johannes

    2010-09-01

    Full Text Available Abstract Prion diseases or transmissible spongiform encephalopathies (TSEs are fatal diseases associated with the conversion of the cellular prion protein (PrPC to the abnormal prion protein (PrPSc. Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.

  4. Plasma soluble prion protein, a potential biomarker for sport-related concussions: a pilot study.

    Science.gov (United States)

    Pham, Nam; Akonasu, Hungbo; Shishkin, Rhonda; Taghibiglou, Changiz

    2015-01-01

    Sport-related mild traumatic brain injury (mTBI) or concussion is a significant health concern to athletes with potential long-term consequences. The diagnosis of sport concussion and return to sport decision making is one of the greatest challenges facing health care clinicians working in sports. Blood biomarkers have recently demonstrated their potential in assisting the detection of brain injury particularly, in those cases with no obvious physical injury. We have recently discovered plasma soluble cellular prion protein (PrP(C)) as a potential reliable biomarker for blast induced TBI (bTBI) in a rodent animal model. In order to explore the application of this novel TBI biomarker to sport-related concussion, we conducted a pilot study at the University of Saskatchewan (U of S) by recruiting athlete and non-athlete 18 to 30 year-old students. Using a modified quantitative ELISA method, we first established normal values for the plasma soluble PrP(C) in male and female students. The measured plasma soluble PrP(C) in confirmed concussion cases demonstrated a significant elevation of this analyte in post-concussion samples. Data collected from our pilot study indicates that the plasma soluble PrP(C) is a potential biomarker for sport-related concussion, which may be further developed into a clinical diagnostic tool to assist clinicians in the assessment of sport concussion and return-to-play decision making.

  5. Plasma soluble prion protein, a potential biomarker for sport-related concussions: a pilot study.

    Directory of Open Access Journals (Sweden)

    Nam Pham

    Full Text Available Sport-related mild traumatic brain injury (mTBI or concussion is a significant health concern to athletes with potential long-term consequences. The diagnosis of sport concussion and return to sport decision making is one of the greatest challenges facing health care clinicians working in sports. Blood biomarkers have recently demonstrated their potential in assisting the detection of brain injury particularly, in those cases with no obvious physical injury. We have recently discovered plasma soluble cellular prion protein (PrP(C as a potential reliable biomarker for blast induced TBI (bTBI in a rodent animal model. In order to explore the application of this novel TBI biomarker to sport-related concussion, we conducted a pilot study at the University of Saskatchewan (U of S by recruiting athlete and non-athlete 18 to 30 year-old students. Using a modified quantitative ELISA method, we first established normal values for the plasma soluble PrP(C in male and female students. The measured plasma soluble PrP(C in confirmed concussion cases demonstrated a significant elevation of this analyte in post-concussion samples. Data collected from our pilot study indicates that the plasma soluble PrP(C is a potential biomarker for sport-related concussion, which may be further developed into a clinical diagnostic tool to assist clinicians in the assessment of sport concussion and return-to-play decision making.

  6. Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH.

    Science.gov (United States)

    Cheng, Chin Jung; Daggett, Valerie

    2014-01-01

    Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is α-helix rich; and PrPSc is the β-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative β-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative β-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative β-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative β-strand formation, thereby improving our understanding of how PrPC misfolds into the β-sheet rich PrPSc and how pH factors into the process. PMID:24970211

  7. Early embryonic gene expression profiling of zebrafish prion protein (Prp2 morphants.

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    Rasoul Nourizadeh-Lillabadi

    Full Text Available BACKGROUND: The Prion protein (PRNP/Prp plays a crucial role in transmissible spongiform encephalopathies (TSEs like Creutzfeldt-Jakob disease (CJD, scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. METHODOLOGY/PRINCIPAL FINDINGS: The zebrafish (Danio rerio genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. CONCLUSIONS/SIGNIFICANCE: The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development.

  8. Molecular Dynamics Simulations Capture the Misfolding of the Bovine Prion Protein at Acidic pH

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    Chin Jung Cheng

    2014-02-01

    Full Text Available Bovine spongiform encephalopathy (BSE, or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP, which adopts two conformers; PrPC is the native innocuous form, which is α-helix rich; and PrPSc is the β-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative β-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative β-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative β-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative β-strand formation, thereby improving our understanding of how PrPC misfolds into the β-sheet rich PrPSc and how pH factors into the process.

  9. Copper attachment to prion protein at a non-octarepeat site

    Science.gov (United States)

    Hodak, Miroslav; Bernholc, Jerry

    2011-03-01

    Prion protein (PrP) plays a causative role in a group of neurodegenerative diseases, which include ``mad cow disease'' or its human form variant Creutzfeld-Jacob disease. Normal function of PrP remains unknown, but it is now well established that PrP can efficiently bind copper ions and this ability has been linked to its function. The primary binding sites are located in the so-called octarepeat region located between residues 60-91. While these are by now well characterized, the sites located outside these region remain mostly undetermined. In this work, we investigate the properties of Cu binding site located at His 111 using recently developed hybrid Kohn-Sham/orbital-free density functional simulations. Experimental data indicate that copper is coordinated by either four nitrogens or three nitrogens and one oxygen. We investigate both possibilities, comparing their energetics and attachment geometries. Similarities and differences with other binding sites and implications for PrP function will also be discussed.

  10. Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

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    José B Oliveira-Martins

    Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

  11. Conformational diversity in prion protein variants influences intermolecular [beta]-sheet formation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seungjoo; Antony, Lizamma; Hartmann, Rune; Knaus, Karen J.; Surewicz, Krystyna; Surewicz, Witold K.; Yee, Vivien C. (Case Western); (Cleveland Clinic)

    2010-04-19

    A conformational transition of normal cellular prion protein (PrP{sup C}) to its pathogenic form (PrP{sup Sc}) is believed to be a central event in the transmission of the devastating neurological diseases known as spongiform encephalopathies. The common methionine/valine polymorphism at residue 129 in the PrP influences disease susceptibility and phenotype. We report here seven crystal structures of human PrP variants: three of wild-type (WT) PrP containing V129, and four of the familial variants D178N and F198S, containing either M129 or V129. Comparison of these structures with each other and with previously published WT PrP structures containing M129 revealed that only WT PrPs were found to crystallize as domain-swapped dimers or closed monomers; the four mutant PrPs crystallized as non-swapped dimers. Three of the four mutant PrPs aligned to form intermolecular {beta}-sheets. Several regions of structural variability were identified, and analysis of their conformations provides an explanation for the structural features, which can influence the formation and conformation of intermolecular {beta}-sheets involving the M/V129 polymorphic residue.

  12. Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

    Science.gov (United States)

    Bate, Clive; Nolan, William; McHale-Owen, Harriet; Williams, Alun

    2016-08-12

    Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal. PMID:27325697

  13. Activation and function of murine primary microglia in the absence of the prion protein.

    Science.gov (United States)

    Pinheiro, Lívia P; Linden, Rafael; Mariante, Rafael M

    2015-09-15

    The prion protein (PrP(C)) is predominantly expressed in the nervous and immune systems and is involved in relevant cell signaling. Microglia participate in neuroimmune interactions, and their regulatory mechanisms are critical for both health and disease. Despite recent reports with a microglial cell line, little is known about the relevance of PrP(C) in brain microglia. We investigated the role of PrP(C) in mouse primary microglia, and found no differences between wild type and Prnp-null cells in cell morphology or the expression of a microglial marker. Translocation of NF-κB to the nucleus also did not differ, nor did cytokine production. The levels of iNOS were also similar and, finally, microglia of either genotype showed no differences in either rates of phagocytosis or migration, even following activation. Thus, functional roles of PrP(C) in primary microglial cells are - if present - much more subtle than in transformed microglial cell lines.

  14. The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

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    Ramón A. Lorca

    2011-01-01

    Full Text Available Although the physiological function of the cellular prion protein (PrPC remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+.

  15. An overview of animal prion diseases

    OpenAIRE

    Imran Muhammad; Mahmood Saqib

    2011-01-01

    Abstract Prion diseases are transmissible neurodegenerative conditions affecting human and a wide range of animal species. The pathogenesis of prion diseases is associated with the accumulation of aggregates of misfolded conformers of host-encoded cellular prion protein (PrPC). Animal prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, transmissible mink encephalopathy, feline spongiform encephalopathy, exotic ungulate spongiform encep...

  16. Mouse Models for Studying the Formation and Propagation of Prions*

    OpenAIRE

    Watts, JC; Prusiner, SB

    2014-01-01

    Prions are self-propagating protein conformers that cause a variety of neurodegenerative disorders in humans and animals. Mouse models have played key roles in deciphering the biology of prions and in assessing candidate therapeutics. The development of transgenic mice that form prions spontaneously in the brain has advanced our understanding of sporadic and genetic prion diseases. Furthermore, the realization that many proteins can become prions has necessitated the development of mouse mode...

  17. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

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    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  18. Mutation and polymorphism of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease (CJD)

    Energy Technology Data Exchange (ETDEWEB)

    Gabizon, R.; Rosenmann, H.; Meiner, Z.; Kahana, I. (Hadassah Univ., Jerusalem (Israel)); Kahana, E. (Barzilai Medical Center, Ashkelon (Israel)); Shugart, Y.; Ott, J. (Columbia Univ., New York, NY (United States)); Prusiner, S.B. (Univ. of California, San Francisco, CA (United States))

    1993-10-01

    The inherited prion diseases are neurodegenerative disorders which are not only genetic but also transmissible. More than a dozen mutations in the prion protein gene that result in nonconservative amino acid substitutions segregate with the inherited prion diseases including familial Creutzfeldt-Jakob disease (CJD). In Israel, the incidence of CJD is about 1 case/10[sup 4] Libyan Jews. A Lys[sub 200] substitution segregates with CJD and is reported here to be genetically linked to CJD with a lod score of >4.8. Some healthy elderly Lys[sub 200] carriers > age 65 years were identified, suggesting the possibility of incomplete penetrance. In contrast, no linkage was found between the development of familial CJD and a polymorphism encoding either Met[sub 129] or Val[sub 129]. All Libyan Jewish CJD patients with the Lys[sub 200] mutation encode a Met[sub 129] on the mutant allele. Homozygosity for Met[sub 129] did not correlate with age at disease onset or the duration of illness. The frequency of the Met[sub 129] allele was higher in the affected pedigrees than in a control population of Libyan Jews. The frequency of the Met[sub 129] and Val[sub 129] alleles in the control Libyan population was similar to that found in the general Caucasian population. The identification of three Libyan Jews homozygous for the Lys[sub 200] mutation suggests frequent intrafamilial marriages, a custom documented by genealogical investigations. 26 refs., 3 figs., 6 tabs.

  19. Species-Dependent Differences in Cofactor Utilization for Formation of the Protease-Resistant Prion Protein in Vitro†

    OpenAIRE

    Deleault, Nathan R.; Kascsak, Richard; Geoghegan, James C.; Supattapone, Surachai

    2010-01-01

    The cofactor preferences for in vitro propagation of the protease-resistant isoforms of the prion protein (PrPSc) from various rodent species were investigated using the serial protein misfolding cyclic amplification (sPMCA) technique. Whereas RNA molecules facilitate hamster PrPSc propagation, RNA and several other polyanions do not promote the propagation of mouse and vole PrPSc molecules. Pretreatment of crude Prnp0/0 (PrP knockout) brain homogenate with RNase A or micrococcal nuclease inh...

  20. A comparison of classical and H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism in wild type and EK211 cattle following intracranial inoculation

    Science.gov (United States)

    In 2006, a case of H-type bovine spongiform encephalopathy (BSE-H) was diagnosed in a cow that was associated with a heritable polymorphism in the bovine prion protein gene (PRNP) resulting in a lysine for glutamine amino acid substitution at codon 211 (called E211K) of the prion protein. Although t...

  1. Uncovering molecular structural mechanisms of signaling mediated by the prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Sebastian A.; Linden, Rafael [Universidade Federal do Rio de Janeiro (IBCCF/UFRl), RJ (Brazil). Inst. de Biofisica Carlos Chagas Filho; Cordeiro, Yraima; Rocha e Lima, Luis M.T. da [Universidade Federal do Rio de Janeiro (FF/UFRl), RJ (Brazil). Fac. de Farmacia; Lopes, Marilene H. [Instituto Ludwig de Pesquisa de Cancer, Sao Paulo, SP (Brazil); Silva, Jerson L.; Foguel, Debora [Universidade Federal do Rio de Janeiro (IBqM/UFRl), RJ (Brazil). Inst. de Bioquimica Medica

    2009-07-01

    The glycosyl phosphatidylinositol (GPI) - anchored prion protein (PrP{sup c}), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrP{sup c} with the secretable cochaperone hop/STI 1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of Pr P{sup c} and hop STI 1 entails structural rearrangements relevant for signaling. Circular dichroism and fluorescence spectroscopy showed that PrP{sup c}:hop/STI 1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the Pr P{sup c}{sub 143-153} beta-helix. Novel SAXS models revealed a significant C-terminal compaction of hop/STI 1 when bound to PrP{sup c}. Differing from a recent dimeric model of human hop/STI 1, both size exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI 1. Changes in the Pr P{sup c}{sub 143-153} beta-helix may engage the transmembrane signaling protein laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of Pr P{sup c}, and further ligands may be engaged by the tertiary structural changes of hop/STI 1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by Pr P{sup c}:hop/STI 1 interaction, consistent with the hypothesis that Pr P{sup c} scaffolds multiprotein signaling complexes at the cell surface. (author)

  2. Secretory pathway retention of mutant prion protein induces p38-MAPK activation and lethal disease in mice.

    Science.gov (United States)

    Puig, Berta; Altmeppen, Hermann C; Ulbrich, Sarah; Linsenmeier, Luise; Krasemann, Susanne; Chakroun, Karima; Acevedo-Morantes, Claudia Y; Wille, Holger; Tatzelt, Jörg; Glatzel, Markus

    2016-01-01

    Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrP(C)) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrP(Sc) leading to prion diseases. We show that a deletion in the C-terminal domain of PrP(C) (PrPΔ214-229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrP(C). Transgenic (Tg(PrPΔ214-229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214-229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice. PMID:27117504

  3. Copper Binding in the Prion Protein†

    OpenAIRE

    Millhauser, Glenn L.

    2004-01-01

    A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt–Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for ...

  4. Analysis of Protein Levels of 24 Cytokines in Scrapie Agent-Infected Brain and Glial Cell Cultures from Mice Differing in Prion Protein Expression Levels ▿

    OpenAIRE

    Tribouillard-Tanvier, Déborah; Striebel, James F; Peterson, Karin E.; Chesebro, Bruce

    2009-01-01

    Activation of microglia and astroglia is seen in many neurodegenerative diseases including prion diseases. Activated glial cells produce cytokines as a protective response against certain pathogens and as part of the host inflammatory response to brain damage. In addition, cytokines might also exacerbate tissue damage initiated by other processes. In the present work using multiplex assays to analyze protein levels of 24 cytokines in scrapie agent-infected C57BL/10 mouse brains, we observed e...

  5. FATE AND TRANSPORT OF PRIONS FROM CHRONIC WASTING DISEASE (CWD) WASTE IN MUNICIPAL SOLID WASTE LANDFILLS

    Science.gov (United States)

    CWD is a fatal neurologic disease of deer and elk caused by an infectious abnormal protein called a prion. Infected free-ranging or captive deer and elk have been found in several states including Wisconsin, Illinois and Minnesota in Region 5. The management of CWD may call for...

  6. A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

    Science.gov (United States)

    Classical scrapie is a form of transmissible spongiform encephalopathies (TSE) affecting domestic goats and sheep and disease is characterized by the accumulation of abnormal conformational isoform (PrP-Sc) of normal cellular prion protein (PrP-C) in the central nervous system and, in most cases, ly...

  7. Defining the conformational features of anchorless, poorly neuroinvasive prions.

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    Cyrus Bett

    Full Text Available Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion.

  8. Effect of electrostatics on aggregation of prion protein Sup35 peptide

    International Nuclear Information System (INIS)

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ∼5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers. (paper)

  9. Effect of electrostatics on aggregation of prion protein Sup35 peptide

    Science.gov (United States)

    Portillo, Alexander M.; Krasnoslobodtsev, Alexey V.; Lyubchenko, Yuri L.

    2012-04-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ˜5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers.

  10. The Role of Prion Protein Expression in Predicting Gastric Cancer Prognosis

    Science.gov (United States)

    Tang, Zhaoqing; Ma, Ji; Zhang, Wei; Gong, Changguo; He, Jing; Wang, Ying; Yu, Guohua; Yuan, Chonggang; Wang, Xuefei; Sun, Yihong; Ma, Jiyan; Liu, Fenglin; Zhao, Yulan

    2016-01-01

    Previous reports indicated that prion protein (PrP) is involved in gastric cancer (GC) development and progression, but its role in GC prognosis has been poorly characterized. A total of 480 GC patients were recruited in this retrospective study. PrP expression in cancerous and non-cancerous gastric tissues was detected by using the tissue microarray and immunohistochemical staining techniques. Our results showed that the PrP expression in GC was significantly less frequent than that in the non-cancerous gastric tissue (44.4% vs 66.4%, P < 0.001). Cox regression analysis revealed that PrP expression was associated with TNM stage, survival status and survival time. GC patients with higher TNM stages (stages II, III and IV) had significantly lower PrP expression levels in tumors than those with lower TNM stages (stages 0 and I). Kaplan-Meier survival curves revealed that negative PrP expression was associated with poor overall survival (log-rank test: P < 0.001). The mean survival time for patients with negative PrP expression was significant lower than those with positive PrP expression (43.0±28.5m vs. 53.9±31.1m, P<0.001). In multivariate Cox hazard regression, PrP expression was an independent prognostic factor for GC survival, with a HR (hazard ratio) of 0.687 (95%CI:0.520-0.907, P=0.008). Our results revealed that negative PrP expression could independently predict worse outcome in GC and thereby could be used to guide the clinical practice. PMID:27313789

  11. Solution structure and dynamics of the I214V mutant of the rabbit prion protein.

    Directory of Open Access Journals (Sweden)

    Yi Wen

    Full Text Available BACKGROUND: The conformational conversion of the host-derived cellular prion protein (PrP(C into the disease-associated scrapie isoform (PrP(Sc is responsible for the pathogenesis of transmissible spongiform encephalopathies (TSEs. Various single-point mutations in PrP(Cs could cause structural changes and thereby distinctly influence the conformational conversion. Elucidation of the differences between the wild-type rabbit PrP(C (RaPrP(C and various mutants would be of great help to understand the ability of RaPrP(C to be resistant to TSE agents. METHODOLOGY/PRINCIPAL FINDINGS: We determined the solution structure of the I214V mutant of RaPrP(C(91-228 and detected the backbone dynamics of its structured C-terminal domain (121-228. The I214V mutant displays a visible shift of surface charge distribution that may have a potential effect on the binding specificity and affinity with other chaperones. The number of hydrogen bonds declines dramatically. Urea-induced transition experiments reveal an obvious decrease in the conformational stability. Furthermore, the NMR dynamics analysis discloses a significant increase in the backbone flexibility on the pico- to nanosecond time scale, indicative of lower energy barrier for structural rearrangement. CONCLUSIONS/SIGNIFICANCE: Our results suggest that both the surface charge distribution and the intrinsic backbone flexibility greatly contribute to species barriers for the transmission of TSEs, and thereby provide valuable hints for understanding the inability of the conformational conversion for RaPrP(C.

  12. Dynamic changes and surveillance function of prion protein expression in gastric cancer drug resistance

    Institute of Scientific and Technical Information of China (English)

    Ji-Heng Wang; Jing-Ping Du; Ying-Hai Zhang; Xiao-Jun Zhao; Ru-Ying Fan; Zhi-Hong Wang; Zi-Tao Wu; Ying Han

    2011-01-01

    AIM: To explore the dynamic changes of prion protein (PrPc) in the process of gastric cancer drug resistance and the role of PrPc expression in the prognosis of gastric cancer patients receiving chemotherapy. METHODS: A series of gastric cancer cell lines resistant to different concentrations of adriamycin was established,and the expression of PrPc, Bcl-2 and Bax was detected in these cells. Apoptosis was determined using Annexin V staining. Western blotting and immunohistochemistry were performed to detect the expression of PrPc in patients receiving chemotherapy and to explore the role of PrPc expression in predicting the chemosensitivity and the outcome of gastric cancer patients receiving chemotherapy. Follow-up was performed for 2 years. RESULTS: PrPc expression was increased with the increase in drug resistance. Bcl-2, together with PrPc, increased the level of anti-apoptosis of cancer cells. Increased PrPc expression predicted the enhanced level of anti-apoptosis and resistance to anticancer drugs. PrPc expression could be used as a marker for predicting the efficacy of chemotherapy and the prognosis of gastric cancer. Increased PrPc expression predicted both poor chemosensitivity and a low 2-year survival rate. Contrarily, low PrPc expression predicted favorable chemosensitivity and a relatively high 2-year survival rate.CONCLUSION: PrPc expression is associated with histological types and differentiation of gastric cancer cells; The PrPc expression level might be a valuable marker in predicting the efficacy of chemotherapy and the prognosis of gastric cancer patients receiving chemotherapy.

  13. Prion protein gene analysis in three kindreds with fatal familial insomnia (FFI): codon 178 mutation and codon 129 polymorphism.

    OpenAIRE

    Medori, R; Tritschler, H J

    1993-01-01

    Fatal familial insomnia (FFI) is a disease linked to a GAC(Asp)-->AAC(Asn) mutation in codon 178 of the prion protein (PrP) gene. FFI is characterized clinically by untreatable progressive insomnia, dysautonomia, and motor dysfunctions and is characterized pathologically by selective thalamic atrophy. We confirmed the 178Asn mutation in the PrP gene of a third FFI family of French ancestry. Three family members who are under 40 years of age and who inherited the mutation showed only reduced p...

  14. Defining the Conformational Features of Anchorless, Poorly Neuroinvasive Prions

    OpenAIRE

    Cyrus Bett; Kurt, Tim D.; Melanie Lucero; Margarita Trejo; Rozemuller, Annemieke J.; Qingzhong Kong; K. Peter R. Nilsson; Eliezer Masliah; Oldstone, Michael B.; Sigurdson, Christina J.

    2013-01-01

    Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the p...

  15. Detecting prions and discriminating among prion strains by discerning the differences in absence.

    Science.gov (United States)

    Prions are molecular pathogens, able to convert a normal cellular prion protein (PrPC) into a prion (PrPSc). The only demonstrated difference between PrPC and PrPSc is conformational. This means that the information necessary for this conversion is contained solely in the conformation of PrPSc. It ...

  16. Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease.

    Directory of Open Access Journals (Sweden)

    Natallia Makarava

    2011-12-01

    Full Text Available The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrP(Sc, which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP(Sc template. Here we report that authentic PrP(Sc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP amyloid fibrils, which are structurally different from PrP(Sc and lack any detectable PrP(Sc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres before authentic PrP(Sc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP(Sc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as "deformed templating" postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrP(Sc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.

  17. Protease-sensitive synthetic prions.

    Directory of Open Access Journals (Sweden)

    David W Colby

    2010-01-01

    Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

  18. The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

    Science.gov (United States)

    Huang, Danzhi; Caflisch, Amedeo

    2015-01-01

    Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

  19. An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Du, Zhiqiang; Valtierra, Stephanie; Li, Liming

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI(+)]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI(+)] and [PIN(+)] ([RNQ(+)]) (Genetics, Vol. 197, 685-700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI(+)] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ(+)] or [SWI(+)]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI(+)] prion stabilizes. Our finding provides strong evidence supporting the "cross-seeding" model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.

  20. A new perspective on beta-sheet structures using vibrational Raman optical activity: From poly(L-lysine) to the prion protein

    DEFF Research Database (Denmark)

    McColl, L.H.; Blanch, E.W.; Gill, A.C.;

    2003-01-01

    (L-lySine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP94-233 that is rich...... patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study....

  1. Statistical Mechanics of Prion Diseases

    International Nuclear Information System (INIS)

    We present a two-dimensional, lattice based, protein-level statistical mechanical model for prion diseases (e.g., mad cow disease) with concomitant prion protein misfolding and aggregation. Our studies lead us to the hypothesis that the observed broad incubation time distribution in epidemiological data reflect fluctuation dominated growth seeded by a few nanometer scale aggregates, while much narrower incubation time distributions for innoculated lab animals arise from statistical self-averaging. We model ''species barriers'' to prion infection and assess a related treatment protocol

  2. Statistical Mechanics of Prion Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Slepoy, A.; Singh, R. R. P.; Pazmandi, F.; Kulkarni, R. V.; Cox, D. L.

    2001-07-30

    We present a two-dimensional, lattice based, protein-level statistical mechanical model for prion diseases (e.g., mad cow disease) with concomitant prion protein misfolding and aggregation. Our studies lead us to the hypothesis that the observed broad incubation time distribution in epidemiological data reflect fluctuation dominated growth seeded by a few nanometer scale aggregates, while much narrower incubation time distributions for innoculated lab animals arise from statistical self-averaging. We model ''species barriers'' to prion infection and assess a related treatment protocol.

  3. Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie

    OpenAIRE

    Matthias Schmitz; Maria Cramm; Franc Llorens; Niccolò Candelise; Dominik Müller-Cramm; Daniela Varges; Schulz-Schaeffer, Walter J.; Saima Zafar; Inga Zerr

    2016-01-01

    In vitro amplification assays, such as real-time quaking-induced conversion (RT-QuIC) are used to detect aggregation activity of misfolded prion protein (PrP) in brain, cerebrospinal fluid (CSF) and urine samples from patients with a prion disease. We believe that the method also has a much broader application spectrum. In the present study, we applied RT-QuIC as a pre-screening test for substances that potentially inhibit the aggregation process of the cellular PrP (PrPC) to proteinase (PK)-...

  4. Panencephalopathic Creutzfeldt-Jakob disease with distinct pattern of prion protein deposition in a patient with D178N mutation and homozygosity for valine at codon 129 of the prion protein Gene.

    Science.gov (United States)

    Marcon, Gabriella; Indaco, Antonio; Di Fede, Giuseppe; Suardi, Silvia; Finato, Nicoletta; Moretti, Valentino; Micoli, Sandro; Fociani, Paolo; Zerbi, Pietro; Pincherle, Alessandro; Redaelli, Veronica; Tagliavini, Fabrizio; Giaccone, Giorgio

    2014-03-01

    Prion diseases include sporadic, acquired and genetic forms linked to mutations of the prion protein (PrP) gene (PRNP). In subjects carrying the D178N PRNP mutation, distinct phenotypes can be observed, depending on the methionine/valine codon 129 polymorphism. We present here a 53-year-old woman with D178N mutation in the PRNP gene and homozygosity for valine at codon 129. The disease started at age 47 with memory deficits, progressive cognitive impairment and ataxia. The clinical picture slowly worsened to a state of akinetic mutism in about 2 years and the disease course was 6 years. The neuropathologic examination demonstrated severe diffuse cerebral atrophy with neuronal loss, spongiosis and marked myelin loss and tissue rarefaction in the hemispheric white matter, configuring panencephalopathic Creutzfeldt-Jakob disease. PrP deposition was present in the cerebral cortex, basal ganglia and cerebellum with diffuse synaptic-type pattern of immunoreactivity and clusters of countless, small PrP deposits, particularly evident in the lower cortical layers, in the striatum and in the molecular layer of the cerebellum. Western blot analysis showed the presence of type 1 PrP(Sc) (Parchi classification). These findings underline the clear-cut distinction between the neuropathological features of Creutzfeldt-Jakob disease associated with D178N PRNP mutation and those of fatal familial insomnia.

  5. Yeast and Fungal Prions: Amyloid-Handling Systems, Amyloid Structure, and Prion Biology.

    Science.gov (United States)

    Wickner, R B; Edskes, H K; Gorkovskiy, A; Bezsonov, E E; Stroobant, E E

    2016-01-01

    Yeast prions (infectious proteins) were discovered by their outré genetic properties and have become important models for an array of human prion and amyloid diseases. A single prion protein can become any of many distinct amyloid forms (called prion variants or strains), each of which is self-propagating, but with different biological properties (eg, lethal vs mild). The folded in-register parallel β sheet architecture of the yeast prion amyloids naturally suggests a mechanism by which prion variant information can be faithfully transmitted for many generations. The yeast prions rely on cellular chaperones for their propagation, but can be cured by various chaperone imbalances. The Btn2/Cur1 system normally cures most variants of the [URE3] prion that arise. Although most variants of the [PSI+] and [URE3] prions are toxic or lethal, some are mild in their effects. Even the most mild forms of these prions are rare in the wild, indicating that they too are detrimental to yeast. The beneficial [Het-s] prion of Podospora anserina poses an important contrast in its structure, biology, and evolution to the yeast prions characterized thus far.

  6. A novel copper(II) coordination at His186 in full-length murine prion protein

    International Nuclear Information System (INIS)

    To explore Cu(II) ion coordination by His186 in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrPC.

  7. A novel copper(II) coordination at His186 in full-length murine prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Yasuko [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Hiraoka, Wakako [Laboratory of Biophysics, School of Science and Technology, Meiji University, Kawasaki 214-8571 (Japan); Igarashi, Manabu; Ito, Kimihito [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Shimoyama, Yuhei [Soft-Matter Physics Laboratory, Graduate School of Emergent Science, Muroran Institute of Technology, Muroran 050-8585 (Japan); Horiuchi, Motohiro [Laboratory of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Inagaki, Fuyuhiko [Laboratory of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 (Japan); Inanami, Osamu, E-mail: inanami@vetmed.hokudai.ac.jp [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan)

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  8. Low frequency of the scrapile resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed

    NARCIS (Netherlands)

    Acutis, P.L.; Sbaiz, L.; Verburg, F.J.; Riina, M.V.; Ru, G.; Moda, G.; Caramelli, M.; Bossers, A.

    2004-01-01

    Frequencies of polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene were studied in 1207 pure-bred and cross-bred Italian Biellese rams, a small ovine breed of about 65 000 head in Italy. Aside from the five most common alleles (VRQ, ARQ, ARR, AHQ and ARH), the rare ARK allele wa

  9. 3D local structure around copper site of rabbit prion-related protein: Quantitative determination by XANES spectroscopy combined with multiple-scattering calculations

    International Nuclear Information System (INIS)

    Prion-related protein (PrP), a cell-surface copper-binding glycoprotein, is considered to be responsible for a number of transmissible spongiform encephalopathies (TSEs). The structural conversion of PrP from the normal cellular isoform (PrPC) to the post-translationally modified form (PrPSc) is thought to be relevant to Cu2+ binding to histidine residues. Rabbits are one of the few mammalian species that appear to be resistant to TSEs, because of the structural characteristics of the rabbit prion protein (RaPrPC) itself. Here we determined the three-dimensional local structure around the C-terminal high-affinity copper-binding sites using X-ray absorption near-edge structure combined with ab initio calculations in the framework of the multiple-scattering (MS) theory. Result shows that two amino acid resides, Gln97 and Met108, and two histidine residues, His95 and His110, are involved in binding this copper(II) ion. It might help us understand the roles of copper in prion conformation conversions, and the molecular mechanisms of prion-involved diseases. - Highlights: ► The first structure of the metal ion binding site in RaPrP fifth copper-binding site. ► Quantitative determination by XANES spectroscopy combined with ab initio calculations. ► Provide a proof of the roles of copper in prion conformation conversions. ► Provide a proof of the molecular mechanisms of prion-involved diseases

  10. Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host

    OpenAIRE

    Warner Richard; Heasman Lindsay; Dexter Glenda E; Ryder Stephen J; Moore S Jo

    2009-01-01

    Abstract Background In order to study the sites of uptake and mechanisms of dissemination of scrapie prions in the natural host under controlled conditions, lambs aged 14 days and homozygous for the VRQ allele of the PrP gene were infected by the oral route. Infection occurred in all lambs with a remarkably short and highly consistent incubation period of approximately 6 months. Challenge of lambs at approximately eight months of age resulted in disease in all animals, but with more variable ...

  11. Stability of the β-structure in prion protein: A molecular dynamics study based on polarized force field

    Science.gov (United States)

    Xu, Zhijun; Lazim, Raudah; Mei, Ye; Zhang, Dawei

    2012-06-01

    Conformational changes of the antiparallel β-sheet in normal cellular prion protein (PrPC) of rat, bovine, and human are investigated by molecular dynamics simulations in both neutral and acidic environment. Using a recently developed simulation method based on an on-the-fly polarized protein-specific charge (PPC) update scheme during the simulation process, we evaluate and compare the cross-species performances of the β-sheet during the early stage transition from the PrPC to its mutant configuration. Through this study, we observe the growth of the β-sheet structure in all species studied with the extent of elongation in β-sheet being different across the three species.

  12. Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

    Directory of Open Access Journals (Sweden)

    Christine Pampeno

    Full Text Available The laminin receptor (LamR is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C and PrP(Sc. Indeed, LamR is a receptor for PrP(C. Whether LamR interacts with PrP(Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

  13. [Molecular bases of prion diseases].

    Science.gov (United States)

    Pokrovskiĭ, V I; Kiselev, O I

    1998-01-01

    The paper briefly analyzes the origin of priones and their association with the cellular gene and homologous protein of diseases in man and animals. There is evidence for a direct relationship of the agents that cause spongious encephalitis in the cattle and a new type of Creutzfeldt-Jacob disease in man. The molecular organization of priones and the conformational cellular protein changes underlying the infectious activation of the cell homologue of priones. Emphasis is first laid on the capacity of the cell homologue of priones and their infectiously active derivative to bind to DNA or RNA. In the context of concepts of the priones yeasts an attempt was made to explain the reproduction through the altered control of translation of mRNA that encodes the cellular homologue of priones, which accounts for the duration of the incubation period of the disease. The infections caused by priones are referred to as the so-called slow infections. But in the context of the proposed hypothesis, an infective process in the tissues did not really have some typical signs of infection and resembles accumulation diseases more without the replicative burst typical of infectious processes. The paper gives data on the vital cycle of priones in infected animals and changes in the accumulation of an infective agent. This assesses the currently available diagnostic methods and gives preference to the methods which will be based on the use of monoclonal antibodies that specifically recognize the conformationally altered form of an infectious prione or on the identification of primary oligomeric forms which manifest the onset of amyloidization of the damaged tissues. The main conclusion of the paper is that protein prionization is a common biological phenomenon and the diseases caused by these processes will increase in number in the near future, which makes it necessary to develop diagnostic methods and universal treatments of diseases, such as bacterial infections by using antibiotics.

  14. Continuous intraventricular infusion of pentosan polysulfate: clinical trial against prion diseases.

    Science.gov (United States)

    Tsuboi, Yoshio; Doh-Ura, Katsumi; Yamada, Tatsuo

    2009-10-01

    Prion diseases are progressive neurological disorders due to abnormal prion protein (PrP(Sc)) deposition in the central nervous system. At present, there is no effective treatment available for any form of prion disease. Pentosan polysulfate (PPS) has been shown to prolong significantly the incubation period in mice with PrP(Sc) infection when administered to the cerebral ventricles in preclinical trials. In human studies conducted in European countries and Japan, intraventricular PPS was administered to patients with different forms of prion disease and was well tolerated. We report 11 patients with prion disease treated with intraventricular PPS at Fukuoka University from 2004. Cases included three familial CJD (two with V180I mutation, one GSS with P102L mutation), two iatrogenic CJD, and six sporadic CJD cases. At present, average survival period after treatment was 24.2 months (range, 4-49). Seven cases died of sepsis and pneumonia. Subdural effusion with various degrees was seen on CT scan in most cases. Except for these, adverse effects did not occur in the treatment period. Although our preliminary study of the new treatment with PPS by continuous intraventricular infusion showed no apparent improvement of clinical features in patients with prion disease, the possibility of extended survival in some patients receiving long-term PPS was suggested.

  15. Structure-Based Drug Discovery for Prion Disease Using a Novel Binding Simulation

    Directory of Open Access Journals (Sweden)

    Daisuke Ishibashi

    2016-07-01

    Full Text Available The accumulation of abnormal prion protein (PrPSc converted from the normal cellular isoform of PrP (PrPC is assumed to induce pathogenesis in prion diseases. Therefore, drug discovery studies for these diseases have focused on the protein conversion process. We used a structure-based drug discovery algorithm (termed Nagasaki University Docking Engine: NUDE that ran on an intensive supercomputer with a graphic-processing unit to identify several compounds with anti-prion effects. Among the candidates showing a high-binding score, the compounds exhibited direct interaction with recombinant PrP in vitro, and drastically reduced PrPSc and protein-aggresomes in the prion-infected cells. The fragment molecular orbital calculation showed that the van der Waals interaction played a key role in PrPC binding as the intermolecular interaction mode. Furthermore, PrPSc accumulation and microgliosis were significantly reduced in the brains of treated mice, suggesting that the drug candidates provided protection from prion disease, although further in vivo tests are needed to confirm these findings. This NUDE-based structure-based drug discovery for normal protein structures is likely useful for the development of drugs to treat other conformational disorders, such as Alzheimer's disease.

  16. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies.

    Science.gov (United States)

    Krasnoslobodtsev, Alexey V; Deckert-Gaudig, Tanja; Zhang, Yuliang; Deckert, Volker; Lyubchenko, Yuri L

    2016-06-01

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. PMID:27060278

  17. Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease‡

    Science.gov (United States)

    Raymond, Gregory J.; Olsen, Emily A.; Lee, Kil Sun; Raymond, Lynne D.; Bryant, P. Kruger; Baron, Gerald S.; Caughey, Winslow S.; Kocisko, David A.; McHolland, Linda E.; Favara, Cynthia; Langeveld, Jan P. M.; van Zijderveld, Fred G.; Mayer, Richard T.; Miller, Michael W.; Williams, Elizabeth S.; Caughey, Byron

    2006-01-01

    Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo. PMID:16378962

  18. Newly identified prions in budding yeast, and their possible functions

    OpenAIRE

    Crow, Emily T.; Li, Liming

    2011-01-01

    Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we sum...

  19. Prions and Prion-Like Pathogens in Neurodegenerative Disorders

    OpenAIRE

    Caterina Peggion; Maria Catia Sorgato; Alessandro Bertoli

    2014-01-01

    Prions are unique elements in biology, being able to transmit biological information from one organism to another in the absence of nucleic acids. They have been identified as self-replicating proteinaceous agents responsible for the onset of rare and fatal neurodegenerative disorders—known as transmissible spongiform encephalopathies, or prion diseases—which affect humans and other animal species. More recently, it has been proposed that other proteins associated with common neurodegenerati...

  20. 3D local structure around copper site of rabbit prion-related protein: Quantitative determination by XANES spectroscopy combined with multiple-scattering calculations

    Science.gov (United States)

    Cui, P. X.; Lian, F. L.; Wang, Y.; Wen, Yi; Chu, W. S.; Zhao, H. F.; Zhang, S.; Li, J.; Lin, D. H.; Wu, Z. Y.

    2014-02-01

    Prion-related protein (PrP), a cell-surface copper-binding glycoprotein, is considered to be responsible for a number of transmissible spongiform encephalopathies (TSEs). The structural conversion of PrP from the normal cellular isoform (PrPC) to the post-translationally modified form (PrPSc) is thought to be relevant to Cu2+ binding to histidine residues. Rabbits are one of the few mammalian species that appear to be resistant to TSEs, because of the structural characteristics of the rabbit prion protein (RaPrPC) itself. Here we determined the three-dimensional local structure around the C-terminal high-affinity copper-binding sites using X-ray absorption near-edge structure combined with ab initio calculations in the framework of the multiple-scattering (MS) theory. Result shows that two amino acid resides, Gln97 and Met108, and two histidine residues, His95 and His110, are involved in binding this copper(II) ion. It might help us understand the roles of copper in prion conformation conversions, and the molecular mechanisms of prion-involved diseases.

  1. Translation of the prion protein mRNA is robust in astrocytes but does not amplify during reactive astrocytosis in the mouse brain.

    Directory of Open Access Journals (Sweden)

    Walker S Jackson

    Full Text Available Prion diseases induce neurodegeneration in specific brain areas for undetermined reasons. A thorough understanding of the localization of the disease-causing molecule, the prion protein (PrP, could inform on this issue but previous studies have generated conflicting conclusions. One of the more intriguing disagreements is whether PrP is synthesized by astrocytes. We developed a knock-in reporter mouse line in which the coding sequence of the PrP expressing gene (Prnp, was replaced with that for green fluorescent protein (GFP. Native GFP fluorescence intensity varied between and within brain regions. GFP was present in astrocytes but did not increase during reactive gliosis induced by scrapie prion infection. Therefore, reactive gliosis associated with prion diseases does not cause an acceleration of local PrP production. In addition to aiding in Prnp gene activity studies, this reporter mouse line will likely prove useful for analysis of chimeric animals produced by stem cell and tissue transplantation experiments.

  2. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    Science.gov (United States)

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-10-01

    The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  3. Accelerated high fidelity prion amplification within and across prion species barriers.

    Directory of Open Access Journals (Sweden)

    Kristi M Green

    Full Text Available Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP, as a substrate for in vitro generation of chronic wasting disease (CWD prions by protein misfolding cyclic amplification (PMCA. Characterization of this infectivity in Tg(CerPrP mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prion transmission, but also provide a facile means of generating and characterizing novel prion strains.

  4. Serial MRI in early Creutzfeldt-Jacob disease with a point mutation of prion protein at codon 180

    International Nuclear Information System (INIS)

    We report a 66-year-old woman with histologically diagnosed Creutzfeld-Jacob disease (CJD), followed with MRI from an early clinical stage. MRI demonstrated expansion of the high cortical signal on T2-weighted images, which differs from previous MRI reports of CJD. This patient followed an atypical clinical course: 16 months had passed before she developed akinetic mutism, and periodic sharp waves had not been detected on EEG after 2 years in spite of her akinetic mutism. Brain biopsy showed primary spongiform changes in the grey matter, and a point mutation of the prion protein gene at codon 180 was discovered using polymerase chain reaction direct sequencing and Tth 111 I cutting. This is the first case with the point mutation of the codon 180 variant with an atypical clinical course and characteristic MRI findings. (orig.)

  5. The presence of disease-associated prion protein in skeletal muscle of cattle infected with classical bovine spongiform encephalopathy.

    Science.gov (United States)

    Okada, Hiroyuki; Miyazawa, Kohtaro; Fukuda, Shigeo; Iwamaru, Yoshifumi; Imamura, Morikazu; Masujin, Kentaro; Matsuura, Yuichi; Fujii, Takashi; Fujii, Kei; Kageyama, Soichi; Yoshioka, Miyako; Murayama, Yuichi; Yokoyama, Takashi

    2014-01-01

    The aim of this study was to investigate the presence of disease-associated prion protein (PrP(Sc)) in the skeletal muscle of cattle infected with classical bovine spongiform encephalopathy (C-BSE). The study was carried out systematically in 12 different muscle samples from 43 (3 field and 40 experimental) cases of C-BSE; however, muscle spindles were not available in many of these cases. Therefore, analysis became restricted to a total of 31 muscles in 23 cattle. Even after this restriction, low levels of PrP(Sc) were detected in the muscle spindles of the masseter, intercostal, triceps brachii, psoas major, quadriceps femoris and semitendinosus muscles from 3 field and 6 experimental clinical-stage cases. The present data indicate that small amounts of PrP(Sc) are detectable by immunohistochemistry in the skeletal muscles of animals terminally affected with C-BSE.

  6. Lack of influence of prion protein gene expression on kainate-induced seizures in mice: studies using congenic, coisogenic and transgenic strains

    OpenAIRE

    Striebel, James F.; Race, Brent; Pathmajeyan, Melissa; Rangel, Alejandra; Chesebro, Bruce

    2013-01-01

    Prion protein (PrP) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein expressed by many cells, including those of the mammalian nervous system. At present the physiologic functions of PrP remain unclear. Deletion of Prnp, the gene encoding PrP in mice, has been shown to alter normal synaptic and electrophysiologic activities, indicating a potential role in seizure susceptibility. However, published efforts to link PrP with seizures, using both in vivo and in vitro models, ...

  7. Immune cell types involved in early uptake and transport of recombinant mouse prion protein in Peyer's patches of calves.

    Science.gov (United States)

    Lwin, Sein; Inoshima, Yasuo; Atoji, Yasuro; Ueno, Hiroshi; Ishiguro, Naotaka

    2009-12-01

    We have previously reported the early uptake and transport of foreign particles into Peyer's patches (PPs) of newborn and 2-month-old calves and shown that the peak uptake of particles occurs 6 h after inoculation, in addition to site- and size-related effects on particle uptake. We now report the distribution of immune cells within PPs of the distal ileum in newborn and 2-month-old calves inoculated with carbon black. The types of immune cells involved in the early uptake and transport of recombinant mouse prion protein (rMPrP) within PPs of newborn calf were investigated by using monoclonal antibodies CD11c, CD14, CD68, CD172a, and CD21. CD11c(+), CD14(+), CD68(+), CD172a(+), and CD21(+) immune cells were widely distributed in four tissue compartments (villi, dome, interfollicular region, and follicles) of PPs in the distal ileum of newborn and 2-month-old calves, whereas CD11c(+), CD14(+), CD172a(+), and CD21(+) immune cells were more prominently distributed in the dome areas of newborn calves than in 2-month-old calves. Moreover, CD11c(+) and CD14(+) dendritic cells, CD172a(+) and CD68(+) macrophages, and CD21(+) follicular dendritic cells containing rMPrP were primarily observed in the dome and inner follicular regions. The deposition of rMPrP within CD11c(+), CD14(+), CD172a(+), and CD68(+) cells, but not CD21(+) cells, was detected in villous regions. rMPrP-positive immune cells within the interfollicular regions included only CD11c(+) and CD172(+) cells. Although the particles used in this investigation do not include the infectious prion protein, PrP(Sc), our experimental setup provides a useful model for studying immune cells involved in the early uptake and transport of PrP(Sc). PMID:19834742

  8. The prion protein gene polymorphisms associated with bovine spongiform encephalopathy susceptibility differ significantly between cattle and buffalo.

    Science.gov (United States)

    Zhao, Hui; Du, Yanli; Chen, Shunmei; Qing, Lili; Wang, Xiaoyan; Huang, Jingfei; Wu, Dongdong; Zhang, Yaping

    2015-12-01

    Prion protein, encoded by the prion protein gene (PRNP), plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Several polymorphisms within the PRNP are known to be associated with influencing bovine spongiform encephalopathy (BSE) susceptibility in cattle, namely two insertion/deletion (indel) polymorphisms (a 23-bp indel in the putative promoter and a 12-bp indel in intron 1), the number of octapeptide repeats (octarepeats) present in coding sequence (CDS) and amino acid polymorphisms. The domestic buffaloes, Bubalus bubalis, are a ruminant involved in various aspects of agriculture. It is of interest to ask whether the PRNP polymorphisms differ between cattle and buffalo. In this study, we analyzed the previously reported polymorphisms associated with BSE susceptibility in Chinese buffalo breeds, and compared these polymorphisms in cattle with BSE, healthy cattle and buffalo by pooling data from the literature. Our analysis revealed three significant findings in buffalo: 1) extraordinarily low deletion allele frequencies of the 23- and 12-bp indel polymorphisms; 2) significantly low allelic frequencies of six octarepeats in CDS and 3) the presence of S4R, A16V, P54S, G108S, V123M, S154N and F257L substitutions in buffalo CDSs. Sequence alignments comparing the buffalo coding sequence to other species were analyzed using the McDonald-Kreitman test to reveal five groups (Bison bonasus, Bos indicus, Bos gaurus, Boselaphus tragocamelus, Syncerus caffer caffer) with significantly divergent non-synonymous substitutions from buffalo, suggesting potential divergence of buffalo PRNP and others. To the best of our knowledge this is the first study of PRNP polymorphisms associated with BSE susceptibility in Chinese buffalo. Our findings have provided evidence that buffaloes have a unique genetic background in the PRNP gene in comparison with cattle.

  9. Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

    Science.gov (United States)

    Mueller, Daniel A; Heinig, Lars; Ramljak, Sanja; Krueger, Astrid; Schulte, Reiner; Wrede, Arne; Stuke, Andreas W

    2010-12-01

    Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies. PMID:21087094

  10. The Expanding Universe of Prion Diseases

    OpenAIRE

    Watts, Joel C.; Aru Balachandran; David Westaway

    2006-01-01

    Prions cause fatal and transmissible neurodegenerative disease. These etiological infectious agents are formed in greater part from a misfolded cell-surface protein called PrP(C). Several mammalian species are affected by the diseases, and in the case of "mad cow disease" (BSE) the agent has a tropism for humans, with negative consequences for agribusiness and public health. Unfortunately, the known universe of prion diseases is expanding. At least four novel prion diseases--including human d...

  11. Effects of polymorphisms in ovine and caprine prion protein alleles on cell-free conversion

    Directory of Open Access Journals (Sweden)

    Eiden Martin

    2011-02-01

    Full Text Available Abstract In sheep polymorphisms of the prion gene (PRNP at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE infections. In goats a number of other gene polymorphisms were found which are suspected to trigger similar effects. However, no strong correlation between polymorphisms and TSE susceptibility in goats has yet been obtained from epidemiological studies and only a low number of experimental challenge data are available at present. We have therefore studied the potential impact of these polymorphisms in vitro by cell-free conversion assays using mouse scrapie strain Me7. Mouse scrapie brain derived PrPSc served as seeds and eleven recombinant single mutation variants of sheep and goat PrPC as conversion targets. With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals. Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.

  12. Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

    Science.gov (United States)

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-11-28

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

  13. Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

    Directory of Open Access Journals (Sweden)

    Rodolfo Villarreal-Calderon

    2013-11-01

    Full Text Available Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4 vs. high (n:26 air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005. Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

  14. Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

    Science.gov (United States)

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-01-01

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

  15. Prions: an evolutionary perspective.

    Science.gov (United States)

    Ogayar, A; Sánchez-Pérez, M

    1998-09-01

    Studies in both prion-due diseases in mammals and some non-Mendelian hereditary processes in yeasts have demonstrated that certain proteins are able to transmit structural information and self-replication. This induces the corresponding conformational changes in other proteins with identical or similar sequences. This ability of proteins may have been very useful during prebiotic chemical evolution, prior to the establishment of the genetic code. During this stage, proteins (proteinoids) must have molded and selected their structural folding units through direct interaction with the environment. The proteinoids that acquired the ability to propagate their conformations (which we refer to as conformons) would have acted as reservoirs and transmitters of a given structural information and hence could have acted as selectors for conformational changes. Despite the great advantage that arose from the establishment of the genetic code, the ability to propagate conformational changes did not necessarily disappear. Depending on the degree of involvement of this capacity in biological evolution, we propose two not mutually exclusive hypotheses: (i) extant prions could be an atavism of ancestral conformons, which would have co-evolved with cells, and (ii) the evolution of conformons would have produced cellular proteins, able to transmit structural information, and, in some cases, participating in certain processes of regulation and epigenesis. Therefore, prions could also be seen as conformons of a conventional infectious agent (or one that co-evolved with it independently) that, after a longer or shorter adaptive period, would have interacted with conformons from the host cells. PMID:10943358

  16. An overview of animal prion diseases.

    Science.gov (United States)

    Imran, Muhammad; Mahmood, Saqib

    2011-01-01

    Prion diseases are transmissible neurodegenerative conditions affecting human and a wide range of animal species. The pathogenesis of prion diseases is associated with the accumulation of aggregates of misfolded conformers of host-encoded cellular prion protein (PrPC). Animal prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, transmissible mink encephalopathy, feline spongiform encephalopathy, exotic ungulate spongiform encephalopathy, chronic wasting disease of cervids and spongiform encephalopathy of primates. Although some cases of sporadic atypical scrapie and BSE have also been reported, animal prion diseases have basically occurred via the acquisition of infection from contaminated feed or via the exposure to contaminated environment. Scrapie and chronic wasting disease are naturally sustaining epidemics. The transmission of BSE to human has caused more than 200 cases of variant Cruetzfeldt-Jacob disease and has raised serious public health concerns. The present review discusses the epidemiology, clinical neuropathology, transmissibility and genetics of animal prion diseases. PMID:22044871

  17. Loss of anti-Bax function in Gerstmann-Straussler-Scheinker syndrome-associated prion protein mutants.

    Directory of Open Access Journals (Sweden)

    Julie Jodoin

    Full Text Available Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD-associated prion protein (PrP mutants that are unable to generate cytosolic PrP (CyPrP. To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS-associated PrP mutants. These PrP mutants contained their respective methionine ((M or valine ((V at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V, D202N(V, P102L(V and Q217R(V retained, whereas the P102L(M, P105L(V, Y145stop(M and Q212P(M PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

  18. Biophysical and morphological studies on the dual interaction of non-octarepeat prion protein peptides with copper and nucleic acids.

    Science.gov (United States)

    Chaves, Juliana A P; Sanchez-López, Carolina; Gomes, Mariana P B; Sisnande, Tháyna; Macedo, Bruno; de Oliveira, Vanessa End; Braga, Carolina A C; Rangel, Luciana P; Silva, Jerson L; Quintanar, Liliana; Cordeiro, Yraima

    2014-08-01

    Conversion of prion protein (PrP) to an altered conformer, the scrapie PrP (PrP(Sc)), is a critical step in the development of transmissible spongiform encephalopathies. Both Cu(II) and nucleic acid molecules have been implicated in this conversion. Full-length PrP can bind up to six copper ions; four Cu(II) binding sites are located in the octarepeat domain (residues 60-91), and His-96 and His-111 coordinate two additional copper ions. Experimental evidence shows that PrP binds different molecules, resulting in diverse cellular signaling events. However, there is little information about the interaction of macromolecular ligands with Cu(II)-bound PrP. Both RNA and DNA sequences can bind PrP, and this interaction results in reciprocal conformational changes. Here, we investigated the interaction of Cu(II) and nucleic acids with amyloidogenic non-octarepeat PrP peptide models (comprising human PrP residues 106-126 and hamster PrP residues 109-149) that retain His-111 as the copper-anchoring residue. The effect of Cu(II) and DNA or RNA sequences in the aggregation, conformation, and toxicity of PrP domains was investigated at low and neutral pH. Circular dichroism and EPR spectroscopy data indicate that interaction of the PrP peptides with Cu(II) and DNA occurs at pH 7. This dual interaction induces conformational changes in the peptides, modulating their aggregation, and affecting the morphology of the aggregated species, resulting in different cytotoxic effects. These results provide new insights into the role of Cu(II) and nucleic acid sequences in the structural conversion and aggregation of PrP, which are both critical events related to prion pathogenesis.

  19. The landscape of the prion protein's structural response to mutation revealed by principal component analysis of multiple NMR ensembles.

    Directory of Open Access Journals (Sweden)

    Deena M A Gendoo

    Full Text Available Prion Proteins (PrP are among a small number of proteins for which large numbers of NMR ensembles have been resolved for sequence mutants and diverse species. Here, we perform a comprehensive principle components analysis (PCA on the tertiary structures of PrP globular proteins to discern PrP subdomains that exhibit conformational change in response to point mutations and clade-specific evolutionary sequence mutation trends. This is to our knowledge the first such large-scale analysis of multiple NMR ensembles of protein structures, and the first study of its kind for PrPs. We conducted PCA on human (n = 11, mouse (n = 14, and wildtype (n = 21 sets of PrP globular structures, from which we identified five conformationally variable subdomains within PrP. PCA shows that different non-local patterns and rankings of variable subdomains arise for different pathogenic mutants. These subdomains may thus be key areas for initiating PrP conversion during disease. Furthermore, we have observed the conformational clustering of divergent TSE-non-susceptible species pairs; these non-phylogenetic clusterings indicate structural solutions towards TSE resistance that do not necessarily coincide with evolutionary divergence. We discuss the novelty of our approach and the importance of PrP subdomains in structural conversion during disease.

  20. Detecting and quantifying prions: Mass spectrometry-based approaches

    Science.gov (United States)

    Prions are novel pathogens that cause a set of rare fatal neurological diseases know as transmissible spongiform encephalopathies. Examples of these diseases include Creutzfeldt-Jakob disease, scrapie and chronic wasting disease. Prions are able to recruit a normal cellular prion protein and convert...

  1. Prion disease tempo determined by host-dependent substrate reduction

    NARCIS (Netherlands)

    Mays, C.E.; Kim, C.; Haldiman, T.; Merwe, v.d. J.; Lau, A.; Yang, J.; Grams, J.; Bari, Di M.A.; Nonno, R.; Telling, G.C.; Kong, Q.; Langeveld, J.P.M.; McKenzie, D.; Westaway, D.; Safar, J.G.

    2014-01-01

    The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrPSc), which is derived from its cellular precursor (PrPC), as well as downregulation of the PrP-like Shadoo

  2. Mass Spectrometry of Prions: Approaches to Conformational Distinction

    Science.gov (United States)

    Prions are the agents that cause a set of fatal neurological diseases that include Creutzfeldt-Jakob disease. Prions are composed solely of protein. Unlike viral, bacterial, or fungal pathogens, the information necessary to propagate the infection is contained in the conformation of the prion isofor...

  3. De novo generation of prion strains.

    Science.gov (United States)

    Colby, David W; Prusiner, Stanley B

    2011-11-01

    Prions are self-replicating proteins that can cause neurodegenerative disorders such as bovine spongiform encephalopathy (also known as mad cow disease). Aberrant conformations of prion proteins accumulate in the central nervous system, causing spongiform changes in the brain and eventually death. Since the inception of the prion hypothesis - which states that misfolded proteins are the infectious agents that cause these diseases - researchers have sought to generate infectious proteins from defined components in the laboratory with varying degrees of success. Here, we discuss several recent studies that have produced an array of novel prion strains in vitro that exhibit increasingly high titres of infectivity. These advances promise unprecedented insight into the structure of prions and the mechanisms by which they originate and propagate. PMID:21947062

  4. Molecular Pathology of Human Prion Diseases

    Directory of Open Access Journals (Sweden)

    2009-03-01

    Full Text Available Prion diseases are fatal neurodegenerative conditions in humans and animals. In this review, we summarize the molecular background of phenotypic variability, relation of prion protein (PrP to other proteins associated with neurodegenerative diseases, and pathogenesis of neuronal vulnerability. PrP exists in different forms that may be present in both diseased and non-diseased brain, however, abundant disease-associated PrP together with tissue pathology characterizes prion diseases and associates with transmissibility. Prion diseases have different etiological background with distinct pathogenesis and phenotype. Mutations of the prion protein gene are associated with genetic forms. The codon 129 polymorphism in combination with the Western blot pattern of PrP after proteinase K digestion serves as a basis for molecular subtyping of sporadic Creutzfeldt-Jakob disease. Tissue damage may result from several parallel, interacting or subsequent pathways that involve cellular systems associated with synapses, protein processing, oxidative stress, autophagy, and apoptosis.

  5. Accelerating Yeast Prion Biology using Droplet Microfluidics

    Science.gov (United States)

    Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

    2012-02-01

    Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

  6. Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein.

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    Yuzuru Taguchi

    Full Text Available Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP(Sc of the host-encoded prion protein (PrP(c. A profound conformational change of PrP(c underlies formation of PrP(Sc and prion propagation involves conversion of PrP(c substrate by direct interaction with PrP(Sc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI effects of conversion-incompetent, internally-deleted PrP (ΔPrP on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2 which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP(C into PrP(Sc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale

  7. Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

    Science.gov (United States)

    Jacobs, J G; Bossers, A; Rezaei, H; van Keulen, L J M; McCutcheon, S; Sklaviadis, T; Lantier, I; Berthon, P; Lantier, F; van Zijderveld, F G; Langeveld, J P M

    2011-12-01

    Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

  8. Multiorgan detection and characterization of protease-resistant prion protein in a case of variant CJD examined in the United States.

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    Silvio Notari

    Full Text Available BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects. METHODOLOGY/PRINCIPAL FINDINGS: Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrP(res in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrP(res detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months of the disease. The PrP(res found in the brain had the typical characteristics of the PrP(res present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrP(res was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.

  9. Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

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    Xinhe Wang

    2015-07-01

    Full Text Available The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p. inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

  10. Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

    Science.gov (United States)

    Wang, Xinhe; McGovern, Gillian; Zhang, Yi; Wang, Fei; Zha, Liang; Jeffrey, Martin; Ma, Jiyan

    2015-07-01

    The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

  11. Scrapie prion liposomes and rods exhibit target sizes of 55,000 Da

    International Nuclear Information System (INIS)

    Scrapie is a degenerative neurologic disease in sheep and goats which can be experimentally transmitted to laboratory rodents. Considerable evidence suggests that the scrapie agent is composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc). Inactivation of scrapie prions by ionizing radiation exhibited single-hit kinetics and gave a target size of 55,000 +/- 9000 mol wt. The inactivation profile was independent of the form of the prion. Scrapie agent infectivity in brain homogenates, microsomal fractions, detergent-extracted microsomes, purified amyloid rods, and liposomes exhibited the same inactivation profile. Our data are consistent with the hypothesis that the infectious particle causing scrapie contains approximately 2 PrPSc molecules

  12. Coexistence of two forms of disease-associated prion protein in extracerebral tissues of cattle infected with H-type bovine spongiform encephalopathy.

    Science.gov (United States)

    Okada, Hiroyuki; Miyazawa, Kohtaro; Masujin, Kentaro; Yokoyama, Takashi

    2016-08-01

    H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in aged cattle. H-BSE is characterized by the presence of two proteinase K-resistant forms of disease-associated prion protein (PrP(Sc)), identified as PrP(Sc) #1 and PrP(Sc) #2, in the brain. To investigate the coexistence of different PrP(Sc) forms in the extracerebral tissues of cattle experimentally infected with H-BSE, immunohistochemical and molecular analyses were performed by using N-terminal-, core-region- and C-terminal-specific anti-prion protein antibodies. Our results demonstrated that two distinct forms of PrP(Sc) coexisted in the various extracerebral tissues. PMID:27010466

  13. Reduced response of splenocytes after mitogen-stimulation in the prion protein (PrP) gene-deficient mouse: PrPLP/Doppel production and cerebral degeneration

    International Nuclear Information System (INIS)

    Splenocytes of wild-type (Prnp +/+) and prion protein gene-deficient (Prnp -/-) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA) + ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrPC) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp +/+ splenocytes. Rikn Prnp -/- splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp -/- splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrPC and PrPLP/Doppel

  14. Bacoside-A, an anti-amyloid natural substance, inhibits membrane disruption by the amyloidogenic determinant of prion protein through accelerating fibril formation.

    Science.gov (United States)

    Malishev, Ravit; Nandi, Sukhendu; Kolusheva, Sofiya; Shaham-Niv, Shira; Gazit, Ehud; Jelinek, Raz

    2016-09-01

    Bacosides, class of compounds extracted from the Bacopa monniera plant, exhibit interesting therapeutic properties, particularly enhancing cognitive functions and putative anti-amyloid activity. We show that bacoside-A exerted significant effects upon fibrillation and membrane interactions of the amyloidogenic fragment of the prion protein [PrP(106-126)]. Specifically, when co-incubated with PrP(106-126), bacoside-A accelerated fibril formation in the presence of lipid bilayers and in parallel inhibited bilayer interactions of the peptide aggregates formed in solution. These interesting phenomena were studied by spectroscopic and microscopic techniques, which suggest that bacoside A-promoted fibrillation reduced the concentration of membrane-active pre-fibrillar species of the prion fragment. This study suggests that induction of fibril formation and corresponding inhibition of membrane interactions are likely the underlying factors for ameliorating amyloid protein toxicity by bacoside-A.

  15. Prion Protein Gene Variability in Spanish Goats. Inference through Susceptibility to Classical Scrapie Strains and Pathogenic Distribution of Peripheral PrPsc

    Science.gov (United States)

    Acín, Cristina; Martín-Burriel, Inmaculada; Monleón, Eva; Lyahyai, Jaber; Pitarch, José Luis; Serrano, Carmen; Monzón, Marta; Zaragoza, Pilar; Badiola, Juan José

    2013-01-01

    Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrPsc) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrPsc distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain. PMID:23580248

  16. PrP(Sc-specific antibodies with the ability to immunodetect prion oligomers.

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    Mourad Tayebi

    Full Text Available The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0 cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.

  17. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

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    Ewan West

    2015-06-01

    Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

  18. Doppel-induced cytotoxicity in human neuronal SH-SY5Y cells is antagonized by the prion protein

    Institute of Scientific and Technical Information of China (English)

    Ping Li; Kun Xu; Chan Tian; Jun Han; Xiaoping Dong; Chenfang Dong; Yanjun Lei; Bing Shan; Xinli Xiao; Huiying Jiang; Xin Wang; Chen Gao; Qi Shi

    2009-01-01

    Doppel(Dpl)is a prion(PrP)-like protein due to the structural and biochemical similarities;however,the natural functions of Dpl and PrP remain unclear.In this study,a 531-bp human PRND gene sequence encoding Dpl protein was amplified from human peripheral blood leucocytes.Full-length and various truncated human Dpi and PrP proteins were expressed and purified from Escherichia coll.Supplement of the full-length Dpl onto human neuroblastoma cell SH-SY5Y induced remarkable cytotoxicity,and the region responsible for its cytotoxicity was mapped at the middle segment of Dpl [amino acids(aa)81-122].Interestingly,Dpl-induced cytotoxicity was antagonized by the presence of fulllength wild-type PrP.Analysis on fragments of PrP mutants showed that the N-terminal fragment(aa 23-90)of PrP was responsible for the protective activity.A truncated PrP(PrPA32-121)with similar secondary structure as Dpl induced Dpl-like cytotoxicity on SHSY5Y cells.Furthermore,binding of copper ion could enhance the antagonizing effect of PrP on Dpl-induced cytotoxicity.Apoptosis assays revealed that cytotoxicity induced by Dpl occurred through an apoptotic mechanism.These results suggested that the function of Dpi is antagonistic to PrP rather than synergistic.

  19. Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.

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    Jeppe Falsig

    Full Text Available Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.

  20. Retinal function and morphology are altered in cattle infected with the prion disease transmissible mink encephalopathy.

    Science.gov (United States)

    Smith, J D; Greenlee, J J; Hamir, A N; Richt, J A; Greenlee, M H West

    2009-09-01

    Transmissible spongiform encephalopathies (TSEs) are a group of diseases that result in progressive and invariably fatal neurologic disease in both animals and humans. TSEs are characterized by the accumulation of an abnormal protease-resistant form of the prion protein in the central nervous system. Transmission of infectious TSEs is believed to occur via ingestion of prion protein-contaminated material. This material is also involved in the transmission of bovine spongiform encephalopathy ("mad cow disease") to humans, which resulted in the variant form of Creutzfeldt-Jakob disease. Abnormal prion protein has been reported in the retina of TSE-affected cattle, but despite these observations, the specific effect of abnormal prion protein on retinal morphology and function has not been assessed. The objective of this study was to identify and characterize potential functional and morphologic abnormalities in the retinas of cattle infected with a bovine-adapted isolate of transmissible mink encephalopathy. We used electroretinography and immunohistochemistry to examine retinas from 10 noninoculated and 5 transmissible mink encephalopathy-inoculated adult Holstein steers. Here we show altered retinal function, as evidenced by prolonged implicit time of the electroretinogram b-wave, in transmissible mink encephalopathy-infected cattle before the onset of clinical illness. We also demonstrate disruption of rod bipolar cell synaptic terminals, indicated by decreased immunoreactivity for the alpha isoform of protein kinase C and vesicular glutamate transporter 1, and activation of Müller glia, as evidenced by increased glial fibrillary acidic protein and glutamine synthetase expression, in the retinas of these cattle at the time of euthanasia due to clinical deterioration. This is the first study to identify both functional and morphologic alterations in the retinas of TSE-infected cattle. Our results support future efforts to focus on the retina for the development of