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Sample records for prion protein sequence

  1. What Makes a Protein Sequence a Prion?

    PubMed Central

    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

  2. Sequence-dependent Prion Protein Misfolding and Neurotoxicity*

    PubMed Central

    Fernandez-Funez, Pedro; Zhang, Yan; Casas-Tinto, Sergio; Xiao, Xiangzhu; Zou, Wen-Quan; Rincon-Limas, Diego E.

    2010-01-01

    Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic scrapie conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated. PMID:20817727

  3. Amino acid sequence of the Amur tiger prion protein.

    PubMed

    Wu, Changde; Pang, Wanyong; Zhao, Deming

    2006-10-01

    Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank. PMID:16780982

  4. Porcine prion protein amyloid

    PubMed Central

    Hammarström, Per; Nyström, Sofie

    2015-01-01

    ABSTRACT 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. PMID:26218890

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

    PubMed

    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-?-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. Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China.

    PubMed

    Zhang, Zhuming; Wang, Renli; Xu, Lihua; Yuan, Fangzhong; Zhou, Xiangmei; Yang, Lifeng; Yin, Xiaomin; Xu, Binrui; Zhao, Deming

    2013-10-25

    Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of an abnormal isoform prion protein (PrP(Sc)) encoded by a single copy prion protein gene (PRNP). Prion disease has been reported in many herbivores but not in Equus and the species barrier might be playing a role in resistance of these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may help understand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia, China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequence in GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequence analysis showed that the Xiji donkey PRNP was consisted of an open reading frame of 768 nucleotides encoding 256 amino acids. Amino acid residues unique to donkey as compared with some Equus animals, mink, cow, sheep, human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence of Xiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species and close relation to the PRNP of sika deer, sheep, dog, camel, cow, mink, rabbit and hamster with 83.1-99.7% identity. The results provided the PRNP data for an additional Equus species, which should be useful to the study of the prion disease pathogenesis, resistance and cross species transmission. PMID:23954254

  7. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences. PMID:18397498

  8. Sequence analysis of the prion protein gene in Mongolian gazelles (Procapra gutturosa).

    PubMed

    Wang, Yiqin; Qin, Zhenkui; Bao, Yonggan; Qiao, Junwen; Yang, Lifeng; Zhao, Deming

    2009-10-01

    Prion diseases are a group of human and animal neurodegenerative conditions, which are caused by the deposition of an abnormal isoform prion protein (PrPSc) encoded by a single copy prion protein gene (Prnp). In sheep, genetic variations of Prnp were found to be associated with the incubation period, susceptibility, and species barrier to the scrapie disease. We investigated the sequence and polymorphisms of the prion protein gene of Mongolian gazelles (gPrnp). gPrnp gene sequence analysis of blood samples from 26 Mongolian gazelles showed high identity within species. The gPrnp gene was closely related to the Prnp genes of Thomsons gazelle, blackbuck, and cattle with 100, 100, and 98.5% identity, respectively, whereas the gPrnp gene with a deletion was closely related to the Prnp genes of wildebeest, Western roe deer, and sheep with 99.3, 99.3, and 98.9% identity, respectively. Polymorphisms of the open reading frame of Prnp as amino acid substitutions were detected at codons 119(N --> S), 143(S --> G) or 160(Y --> H), 172(V --> A), 182(N --> S) and 221(V --> A). There was also deletion of one octapeptide repeat at the N-terminal octapeptide repeat region. The polymorphisms of gPrnp will assist the study of prion disease pathogenesis, resistance, and cross species transmission. PMID:19579063

  9. Human prion protein sequence elements impede cross-species chronic wasting disease transmission

    PubMed Central

    Kurt, Timothy D.; Jiang, Lin; Fernández-Borges, Natalia; Bett, Cyrus; Liu, Jun; Yang, Tom; Spraker, Terry R.; Castilla, Joaquín; Eisenberg, David; Kong, Qingzhong; Sigurdson, Christina J.

    2015-01-01

    Chronic wasting disease (CWD) is a fatal prion disease of North American deer and elk and poses an unclear risk for transmission to humans. Human exposure to CWD occurs through hunting activities and consumption of venison from prion-infected animals. Although the amino acid residues of the prion protein (PrP) that prevent or permit human CWD infection are unknown, NMR-based structural studies suggest that the β2-α2 loop (residues 165–175) may impact species barriers. Here we sought to define PrP sequence determinants that affect CWD transmission to humans. We engineered transgenic mice that express human PrP with four amino acid substitutions that result in expression of PrP with a β2-α2 loop (residues 165–175) that exactly matches that of elk PrP. Compared with transgenic mice expressing unaltered human PrP, mice expressing the human-elk chimeric PrP were highly susceptible to elk and deer CWD prions but were concurrently less susceptible to human Creutzfeldt-Jakob disease prions. A systematic in vitro survey of amino acid differences between humans and cervids identified two additional residues that impacted CWD conversion of human PrP. This work identifies amino acids that constitute a substantial structural barrier for CWD transmission to humans and helps illuminate the molecular requirements for cross-species prion transmission. PMID:25705888

  10. Quantum dots and prion proteins

    PubMed Central

    Sobrova, Pavlina; Blazkova, Iva; Chomoucka, Jana; Drbohlavova, Jana; Vaculovicova, Marketa; Kopel, Pavel; Hubalek, Jaromir; Kizek, Rene; Adam, Vojtech

    2013-01-01

    A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrPSc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrPSc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels. PMID:24055838

  11. Treatment of Prion Disease with Heterologous Prion Proteins.

    PubMed

    Skinner, Pamela J; Kim, Hyeon O; Bryant, Damani; Kinzel, Nikilyn J; Reilly, Cavan; Priola, Suzette A; Ward, Anne E; Goodman, Patricia A; Olson, Katherine; Seelig, Davis M

    2015-01-01

    Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the host's own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans. PMID:26134409

  12. Treatment of Prion Disease with Heterologous Prion Proteins

    PubMed Central

    Skinner, Pamela J.; Kim, Hyeon O.; Bryant, Damani; Kinzel, Nikilyn J.; Reilly, Cavan; Priola, Suzette A.; Ward, Anne E.; Goodman, Patricia A.; Olson, Katherine; Seelig, Davis M.

    2015-01-01

    Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are fatal neurodegenerative diseases for which there is no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPsc or PrPres). Both in vitro (cell culture and cell free conversion assays) and in vivo (animal) studies have demonstrated the strong dependence of this conversion process on protein sequence homology between the initial prion inoculum and the hosts own cellular prion protein. The presence of non-homologous (heterologous) proteins is often inhibitory to this conversion process. We hypothesize that the presence of heterologous prion proteins from one species might therefore constitute an effective treatment for prion disease in another species. To test this hypothesis, we infected mice intracerebrally with murine adapted RML-Chandler scrapie and treated them with heterologous prion protein (purified bacterially expressed recombinant hamster prion protein) or vehicle alone. Treated animals demonstrated reduced disease associated pathology, decreased accumulation of protease-resistant disease-associated prion protein, with delayed onset of clinical symptoms and motor deficits. This was concomitant with significantly increased survival times relative to mock-treated animals. These results provide proof of principle that recombinant hamster prion proteins can effectively and safely inhibit prion disease in mice, and suggest that hamster or other non-human prion proteins may be a viable treatment for prion diseases in humans. PMID:26134409

  13. Generating recombinant C-terminal prion protein fragments of exact native sequence.

    PubMed

    Johanssen, V A; Barnham, K J; Masters, C L; Hill, A F; Collins, S J

    2012-02-01

    Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (∼10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations. PMID:22197912

  14. Prions and Prion-like Proteins

    PubMed Central

    Fraser, Paul E.

    2014-01-01

    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. PMID:24860092

  15. Prions and prion-like proteins.

    PubMed

    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. PMID:24860092

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

    PubMed Central

    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 pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-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 mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases. PMID:25206608

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

    PubMed

    Paul, Kacy R; Ross, Eric D

    2015-09-01

    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. PMID:26555096

  18. Amyloid Core Formed of Full-Length Recombinant Mouse Prion Protein Involves Sequence 127143 but Not Sequence 107126

    PubMed Central

    Chatterjee, Biswanath; Lee, Chung-Yu; Lin, Chen; Chen, Eric H.-L.; Huang, Chao-Li; Yang, Chien-Chih; Chen, Rita P.-Y.

    2013-01-01

    The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrPC) into its disease-causing isoform, PrPSc. This conversion is associated with a marked change in secondary structure from predominantly ?-helical to a high ?-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107143), mPrP(107126), and mPrP(127143). Our results showed that the amyloid fibrils formed from mPrP(107143) and mPrP(127143), but not those formed from mPrP(107126), were able to seed the amyloidogenesis of mPrP(23230), showing that the segment residing in sequence 127143 was used to form the amyloid core in the fibrillization of mPrP(23230). PMID:23844138

  19. Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle

    PubMed Central

    Choi, Sangho

    2012-01-01

    Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms. PMID:22705734

  20. Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus).

    PubMed

    Brandt, Adam L; Kelly, Amy C; Green, Michelle L; Shelton, Paul; Novakofski, Jan; Mateus-Pinilla, Nohra E

    2015-11-01

    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. Prion protein and aging

    PubMed Central

    Gasperini, Lisa; Legname, Giuseppe

    2014-01-01

    The cellular prion protein (PrPC) has been widely investigated ever since its conformational isoform, the prion (or PrPSc), was identified as the etiological agent of prion disorders. The high homology shared by the PrPC-encoding gene among mammals, its high turnover rate and expression in every tissue strongly suggest that PrPC may possess key physiological functions. Therefore, defining PrPC roles, properties and fate in the physiology of mammalian cells would be fundamental to understand its pathological involvement in prion diseases. Since the incidence of these neurodegenerative disorders is enhanced in aging, understanding PrPC functions in this life phase may be of crucial importance. Indeed, a large body of evidence suggests that PrPC plays a neuroprotective and antioxidant role. Moreover, it has been suggested that PrPC is involved in Alzheimer disease, another neurodegenerative pathology that develops predominantly in the aging population. In prion diseases, PrPC function is likely lost upon protein aggregation occurring in the course of the disease. Additionally, the aging process may alter PrPC biochemical properties, thus influencing its propensity to convert into PrPSc. Both phenomena may contribute to the disease development and progression. In Alzheimer disease, PrPC has a controversial role because its presence seems to mediate ?-amyloid toxicity, while its down-regulation correlates with neuronal death. The role of PrPC in aging has been investigated from different perspectives, often leading to contrasting results. The putative protein functions in aging have been studied in relation to memory, behavior and myelin maintenance. In aging mice, PrPC changes in subcellular localization and post-translational modifications have been explored in an attempt to relate them to different protein roles and propensity to convert into PrPSc. Here we provide an overview of the most relevant studies attempting to delineate PrPC functions and fate in aging. PMID:25364751

  2. Prions

    PubMed Central

    Prusiner, Stanley B.

    1998-01-01

    Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and CreutzfeldtJakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high ?-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases. PMID:9811807

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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 transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. IMPORTANCE Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection. PMID:25810548

  4. A bipolar personality of yeast prion proteins

    PubMed Central

    Kurahashi, Hiroshi; Oishi, Keita

    2011-01-01

    Prions are infectious, self-propagating protein conformations. [PSI+], [RNQ+] and [URE3] are well characterized prions in Saccharomyces cerevisiae and represent the aggregated states of the translation termination factor Sup35, a functionally unknown protein Rnq1 and a regulator of nitrogen metabolism Ure2, respectively. Overproduction of Sup35 induces the de novo appearance of the [PSI+] prion in [RNQ+] or [URE3] strain, but not in non-prion strain. However, [RNQ+] and [URE3] prions themselves, as well as overexpression of a mutant Rnq1 protein, Rnq1?100 and Lsm4, hamper the maintenance of [PSI+]. These findings point to a bipolar activity of [RNQ+], [URE3], Rnq1?100 and Lsm4, and probably other yeast prion proteins as well, for the fate of [PSI+] prion. Possible mechanisms underlying the apparent bipolar activity of yeast prions will be discussed. PMID:22156730

  5. Prions: The Chemistry of Infectious Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A prion is pathological protein that causes a set of rare fatal neurological diseases called transmissible spongiform encephalopathies (TSE). TSE diseases occur in humans, sheep, goats, deer, elk, mink, cows and other mammals. A prion and the normal cellular prion protein (PrPC) have the same primar...

  6. The Prion Protein Knockout Mouse

    PubMed Central

    Lindquist, Susan; Aguzzi, Adriano

    2007-01-01

    The key pathogenic event in prion disease involves misfolding and aggregation of the cellular prion protein (PrP). Beyond this fundamental observation, the mechanism by which PrP misfolding in neurons leads to injury and death remains enigmatic. Prion toxicity may come about by perverting the normal function of PrP. If so, understanding the normal function of PrP may help to elucidate the molecular mechansim of prion disease. Ablation of the Prnp gene, which encodes PrP, was instrumental for determining that the continuous production of PrP is essential for replicating prion infectivity. Since the structure of PrP has not provided any hints to its possible function, and there is no obvious phenotype in PrP KO mice, studies of PrP function have often relied on intuition and serendipity. Here, we enumerate the multitude of phenotypes described in PrP deficient mice, many of which manifest themselves only upon physiological challenge. We discuss the pleiotropic phenotypes of PrP deficient mice in relation to the possible normal function of PrP. The critical question remains open: which of these phenotypes are primary effects of PrP deletion and what do they tell us about the function of PrP? PMID:19164918

  7. [Significance of prion protein in transmission of prions and in pathogenesis of spongiform encephalopathies].

    PubMed

    Raeber, A J; Klein, M A; Frigg, R; Brandner, S; Blttler, T; Aguzzi, A

    1998-01-01

    Prion disease or transmissible spongiform encephalopathies are caused by novel pathogens termed prions. Unlike classical infectious agents such as viruses or bacteria, prions lack an independent genome and consist largely if not entirely of an abnormal form of the host-encoded prion protein. How prions multiply is not known. A wealth of experimental evidence supports an essential role for the host-encoded prion protein in susceptibility and pathogenesis of prion diseases and in the propagation and spread of prions. In addition, B lymphocytes have been found to play a crucial role in the neuroinvasiveness of prions. PMID:9611346

  8. Physiology of the prion protein.

    PubMed

    Linden, Rafael; Martins, Vilma R; Prado, Marco A M; Cammarota, Martn; Izquierdo, Ivn; Brentani, Ricardo R

    2008-04-01

    Prion diseases are transmissible spongiform encephalopathies (TSEs), attributed to conformational conversion of the cellular prion protein (PrP(C)) into an abnormal conformer that accumulates in the brain. Understanding the pathogenesis of TSEs requires the identification of functional properties of PrP(C). Here we examine the physiological functions of PrP(C) at the systemic, cellular, and molecular level. Current data show that both the expression and the engagement of PrP(C) with a variety of ligands modulate the following: 1) functions of the nervous and immune systems, including memory and inflammatory reactions; 2) cell proliferation, differentiation, and sensitivity to programmed cell death both in the nervous and immune systems, as well as in various cell lines; 3) the activity of numerous signal transduction pathways, including cAMP/protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt pathways, as well as soluble non-receptor tyrosine kinases; and 4) trafficking of PrP(C) both laterally among distinct plasma membrane domains, and along endocytic pathways, on top of continuous, rapid recycling. A unified view of these functional properties indicates that the prion protein is a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior. PMID:18391177

  9. Amyloid diseases of yeast: prions are proteins acting as genes.

    PubMed

    Wickner, Reed B; Edskes, Herman K; Bateman, David A; Kelly, Amy C; Gorkovskiy, Anton; Dayani, Yaron; Zhou, Albert

    2014-01-01

    The unusual genetic properties of the non-chromosomal genetic elements [URE3] and [PSI+] led to them being identified as prions (infectious proteins) of Ure2p and Sup35p respectively. Ure2p and Sup35p, and now several other proteins, can form amyloid, a linear ordered polymer of protein monomers, with a part of each molecule, the prion domain, forming the core of this ?-sheet structure. Amyloid filaments passed to a new cell seed the conversion of the normal form of the protein into the same amyloid form. The cell's phenotype is affected, usually from the deficiency of the normal form of the protein. Solid-state NMR studies indicate that the yeast prion amyloids are in-register parallel ?-sheet structures, in which each residue (e.g. Asn35) forms a row along the filament long axis. The favourable interactions possible for aligned identical hydrophilic and hydrophobic residues are believed to be the mechanism for propagation of amyloid conformation. Thus, just as DNA mediates inheritance by templating its own sequence, these proteins act as genes by templating their conformation. Distinct isolates of a given prion have different biological properties, presumably determined by differences between the amyloid structures. Many lines of evidence indicate that the Saccharomyces cerevisiae prions are pathological disease agents, although the example of the [Het-s] prion of Podospora anserina shows that a prion can have beneficial aspects. PMID:25131596

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

    PubMed Central

    Nystrm, Sofie; Hammarstrm, 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 A?1-40, A?1-42, 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. PMID:25960067

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

    PubMed

    Nystrm, Sofie; Hammarstrm, 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 A?1-40, A?1-42, 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. PMID:25960067

  12. Copper binding in the prion protein.

    PubMed

    Millhauser, Glenn L

    2004-02-01

    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 Cu(2+). The structural features of the Cu(2+) binding sites have now been characterized and are providing important clues about the normal function of the prion protein and perhaps how metals or loss of protein function play a role in disease. The link between prion protein and copper may provide insight into the general, and recently appreciated, role of metals in neurodegenerative disease. PMID:14967054

  13. Alzheimer's Disease and Prion Protein

    PubMed Central

    Zhou, Jiayi; Liu, Bingqian

    2013-01-01

    Summary Alzheimer's disease (AD) is a devastating neurodegenerative disease with progressive loss of memory and cognitive function, pathologically hallmarked by aggregates of the amyloid-beta (A?) peptide and hyperphosphorylated tau in the brain. Aggregation of A? under the form of amyloid fibrils has long been considered central to the pathogenesis of AD. However, recent evidence has indicated that soluble A? oligomers, rather than insoluble fibrils, are the main neurotoxic species in AD. The cellular prion protein (PrPC) has newly been identified as a cell surface receptor for A? oligomers. PrPC is a cell surface glycoprotein that plays a key role in the propagation of prions, proteinaceous infectious agents that replicate by imposing their abnormal conformation to PrPC molecules. In AD, PrPC acts to transduce the neurotoxic signals arising from A? oligomers, leading to synaptic failure and cognitive impairment. Interestingly, accumulating evidence has also shown that aggregated A? or tau possesses prion-like activity, a property that would allow them to spread throughout the brain. In this article, we review recent findings regarding the function of PrPC and its role in AD, and discuss potential therapeutic implications of PrPC-based approaches in the treatment of AD. PMID:25343100

  14. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    NASA Astrophysics Data System (ADS)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  15. Discovering putative prion sequences in complete proteomes using probabilistic representations of Q/N-rich domains

    PubMed Central

    2013-01-01

    Background Prion proteins conform a special class among amyloids due to their ability to transmit aggregative folds. Prions are known to act as infectious agents in neurodegenerative diseases in animals, or as key elements in transcription and translation processes in yeast. It has been suggested that prions contain specific sequential domains with distinctive amino acid composition and physicochemical properties that allow them to control the switch between soluble and β-sheet aggregated states. Those prion-forming domains are low complexity segments enriched in glutamine/asparagine and depleted in charged residues and prolines. Different predictive methods have been developed to discover novel prions by either assessing the compositional bias of these stretches or estimating the propensity of protein sequences to form amyloid aggregates. However, the available algorithms hitherto lack a thorough statistical calibration against large sequence databases, which makes them unable to accurately predict prions without retrieving a large number of false positives. Results Here we present a computational strategy to predict putative prion-forming proteins in complete proteomes using probabilistic representations of prionogenic glutamine/asparagine rich regions. After benchmarking our predictive model against large sets of non-prionic sequences, we were able to filter out known prions with high precision and accuracy, generating prediction sets with few false positives. The algorithm was used to scan all the proteomes annotated in public databases for the presence of putative prion proteins. We analyzed the presence of putative prion proteins in all taxa, from viruses and archaea to plants and higher eukaryotes, and found that most organisms encode evolutionarily unrelated proteins with susceptibility to behave as prions. Conclusions To our knowledge, this is the first wide-ranging study aiming to predict prion domains in complete proteomes. Approaches of this kind could be of great importance to identify potential targets for further experimental testing and to try to reach a deeper understanding of prions’ functional and regulatory mechanisms. PMID:23663289

  16. PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores

    PubMed Central

    Zambrano, Rafael; Conchillo-Sole, Oscar; Iglesias, Valentin; Illa, Ricard; Rousseau, Frederic; Schymkowitz, Joost; Sabate, Raimon; Daura, Xavier; Ventura, Salvador

    2015-01-01

    Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/. PMID:25977297

  17. PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores.

    PubMed

    Zambrano, Rafael; Conchillo-Sole, Oscar; Iglesias, Valentin; Illa, Ricard; Rousseau, Frederic; Schymkowitz, Joost; Sabate, Raimon; Daura, Xavier; Ventura, Salvador

    2015-07-01

    Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/. PMID:25977297

  18. The Unexposed Secrets of Prion Protein Oligomers.

    PubMed

    Wang, Gailing; Wang, Mingcheng; Li, Chuanfeng

    2015-08-01

    According to the "protein-only" hypothesis, the misfolding and conversion of host-derived cellular prion protein (PrP(C)) into pathogenically misfolded PrP are believed to be the key procedure in the pathogenesis of prion diseases. Intermediate, soluble oligomeric prion protein (PrP) aggregates were considered a critical process for prion diseases. Several independent studies on PrP oligomers gained insights into oligomers' formation, biophysical and biochemical characteristics, structure conversion, and neurotoxicity. PrP oligomers are rich in ?-sheet structure and slightly resistant to proteinase K digestion. PrP oligomers exhibited more neurotoxicity and induced neuronal apoptosis in vivo and/or in vitro. In this review, we summarized recent studies regarding PrP oligomers and the relationship between misfolded PrP aggregates and neuronal death in the course of prion diseases. PMID:25823438

  19. [Protein structure: Folding and prions].

    PubMed

    Rey-Gayo, Antonio; Calbo Torrecilla, Francisco

    2002-04-01

    Transmissible spongiform encephalopathies have become a subject of prime social concern in recent years because of its relation to "mad cow disease" and their potential for transmission to humans. Among the most important scientific aspects of these diseases are the peculiar characteristics of the agent involved in their transmission. In this article we briefly describe the outstanding features of prions, the most widely accepted hypothesis for these diseases. We focus on the molecular characteristics of this protein, coded in the genome of the affected host, and describe the conformational alterations in the protein's tertiary structure that have been blamed for its pathologic activity. Our aim is to summarize the state-of-the-art knowledge on prions, the hypotheses proposed to explain mechanisms of disease transmission without agents containing genetic material, and some specific peculiarities of this new infectious agent. The links between this knowledge and possible therapeutic strategies to overcome the disease justify, once again, close interaction among chemistry, molecular biology, and medicine. PMID:11996702

  20. Engineering the prion protein using chemical synthesis.

    PubMed

    Ball, H L; King, D S; Cohen, F E; Prusiner, S B; Baldwin, M A

    2001-11-01

    In recent years, the technology of solid-phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared several modified and wild-type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, alpha-helical cellular protein (PrPc) into a beta-sheet-rich pathogenic isoform (PrP(Sc)). The ability to form PrP(Sc) in transgenic mice is retained by a 106 residue 'mini-prion' (PrP106), with the deletions 23-88 and 141-176. Synthetic PrP106 (sPrP106) and a His-tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N-(2-chlorobenzyloxycarbonyloxy) succinimide. A single reversed-phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90-200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t-Boc chemistry. To mimic the glycosylphosphatidyl inositol (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surface, where the conversion of PrPc is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences. These groups enabled sPrP to be immobilized on either the cell surface or a streptavidin-coated ELISA plate, respectively, in an orientation analogous to that of membrane-bound, GPI-anchored PrPc. The chemical manipulation of such biologically relevant forms of PrP by the introduction of point mutations or groups that mimic post-translational modifications should enhance our understanding of the processes that cause prion diseases and may lead to the chemical synthesis of an infectious agent. PMID:11892845

  1. Spontaneous Variants of the [RNQ+] Prion in Yeast Demonstrate the Extensive Conformational Diversity Possible with Prion Proteins

    PubMed Central

    Huang, Vincent J.; Stein, Kevin C.; True, Heather L.

    2013-01-01

    Prion strains (or variants) are structurally distinct amyloid conformations arising from a single polypeptide sequence. The existence of prion strains has been well documented in mammalian prion diseases. In many cases, prion strains manifest as variation in disease progression and pathology, and in some cases, these prion strains also show distinct biochemical properties. Yet, the underlying basis of prion propagation and the extent of conformational possibilities available to amyloidogenic proteins remain largely undefined. Prion proteins in yeast that are also capable of maintaining multiple self-propagating structures have provided much insight into prion biology. Here, we explore the vast structural diversity of the yeast prion [RNQ+] in Saccharomyces cerevisiae. We screened for the formation of [RNQ+] in vivo, allowing us to calculate the rate of spontaneous formation as ~2.96x10-6, and successfully isolate several different [RNQ+] variants. Through a comprehensive set of biochemical and biological analyses, we show that these prion variants are indeed novel. No individual property or set of properties, including aggregate stability and size, was sufficient to explain the physical basis and range of prion variants and their resulting cellular phenotypes. Furthermore, all of the [RNQ+] variants that we isolated were able to facilitate the de novo formation of the yeast prion [PSI+], an epigenetic determinant of translation termination. This supports the hypothesis that [RNQ+] acts as a functional amyloid in regulating the formation of [PSI+] to produce phenotypic diversity within a yeast population and promote adaptation. Collectively, this work shows the broad spectrum of available amyloid conformations, and thereby expands the foundation for studying the complex factors that interact to regulate the propagation of distinct aggregate structures. PMID:24205387

  2. The Role of Crowded Physiological Environments in Prion and Prion-like Protein Aggregation

    PubMed Central

    Ma, Qian; Hu, Ji-Ying; Chen, Jie; Liang, Yi

    2013-01-01

    Prion diseases and prion- like protein misfolding diseases are related to the accumulation of abnormal aggregates of the normal host proteins including prion proteins and Tau protein. These proteins possess self-templating and transmissible characteristics. The crowded physiological environments where the aggregation of these amyloidogenic proteins takes place can be imitated in vitro by the addition of macromolecular crowding agents such as inert polysaccharides. In this review, we summarize the aggregation of prion proteins in crowded physiological environments and discuss the role of macromolecular crowding in prion protein aggregation. We also summarize the aggregation of prion- like proteins including human Tau protein, human ?-synuclein, and human copper, zinc superoxide dismutase under macromolecular crowding environments and discuss the role of macromolecular crowding in prion- like protein aggregation. The excluded-volume effects caused by macromolecular crowding could accelerate the aggregation of neurodegenerative disease-associated proteins while inhibiting the aggregation of the proteins that are not neurodegenerative disease-associated. PMID:24284393

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

    PubMed Central

    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

    2013-01-01

    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 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. PMID:24188521

  4. Prion search and cellular prion protein expression in stranded dolphins.

    PubMed

    Di Guardo, G; Cocumelli, C; Meoli, R; Barbaro, K; Terracciano, G; Di Francesco, C E; Mazzariol, S; Eleni, C

    2012-01-01

    The recent description of a prion disease (PD) case in a free-ranging bottlenose dolphin (Tursiops truncatus) prompted us to carry out an extensive search for the disease-associated isoform (PrPSc) of the cellular prion protein (PrPC) in the brain and in a range of lymphoid tissues from 23 striped dolphins (Stenella coeruleoalba), 5 bottlenose dolphins and 2 Risso s dolphins (Grampus griseus) found stranded between 2007 and 2012 along the Italian coastline. Three striped dolphins and one bottlenose dolphin showed microscopic lesions of encephalitis, with no evidence of spongiform brain lesions being detected in any of the 30 free-ranging cetaceans investigated herein. Nevertheless, we could still observe a prominent PrPC immunoreactivity in the brain as well as in lymphoid tissues from these dolphins. Although immunohistochemical and Western blot investigations yielded negative results for PrPSc deposition in all tissues from the dolphins under study, the reported occurrence of a spontaneous PD case in a wild dolphin is an intriguing issue and a matter of concern for both prion biology and intra/inter-species transmissibility, as well as for cetacean conservation medicine. PMID:23034277

  5. Cellular Prion Protein: From Physiology to Pathology

    PubMed Central

    Yusa, Sei-ichi; Oliveira-Martins, Jos B.; Sugita-Konishi, Yoshiko; Kikuchi, Yutaka

    2012-01-01

    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 ?-amyloid precursor protein (APP) in Alzheimer disease (AD), which can be physiologically processed by ?-, ?-, and ?-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. PMID:23202518

  6. Chronic wasting disease prions are not transmissible to transgenic mice overexpressing human prion protein

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sigurdson, Christina J.; Glatzel, Markus; O'Malley, Catherine; Powell, Caroline; Asante, Emmanuel A.; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Collinge, John

    2010-01-01

    Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids, including mule deer, white-tailed deer, Rocky Mountain elk and moose. CWD-infected cervids have been reported in 14 USA states, two Canadian provinces and in South Korea. The possibility of a zoonotic transmission of CWD prions via diet is of particular concern in North America where hunting of cervids is a popular sport. To investigate the potential public health risks posed by CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from CWD-infected mule deer transmits prion infection to transgenic mice overexpressing human prion protein with methionine or valine at polymorphic residue 129. These transgenic mice have been utilized in extensive transmission studies of human and animal prion disease and are susceptible to BSE and vCJD prions, allowing comparison with CWD. Here, we show that these mice proved entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier associated with this prion strain/host combination is greater than that observed with classical BSE prions. However, it is possible that CWD may be caused by multiple prion strains. Further studies will be required to evaluate the transmission properties of distinct cervid prion strains as they are characterized. PMID:20610667

  7. Biochemical insight into the prion protein family.

    PubMed

    Ciric, Danica; Rezaei, Human

    2015-01-01

    Prion protein family comprises proteins, which share not only similarity in their primary structure, but also similarity in their fold. These two groups of similarity presume a parceling in their respective biological function through the common biochemical properties. In this review, biochemical and structural similarities of PrP and two other proteins, Doppel and Shadoo, are evocated. Some evidence demonstrating respectively similarity between PrP N-terminal and C-terminal domain with respectively Shadoo and Doppel is presented. We extended primary structure similarity analysis to the other PrP subdomain as 166-176 polyNQ domain and compare it to proteins using aggregation as a support for structural information transference and structural epigenetic. Finally, we questioned if prion protein family have conserved the PrP structural bistability, which should be at the origin of Prion phenomenon and if Prion pathology is not, ultimately, an exaptation of the physiological propensity of PrP to undergo a structural switch and polymerize. PMID:25717473

  8. Biochemical insight into the prion protein family

    PubMed Central

    Ciric, Danica; Rezaei, Human

    2015-01-01

    Prion protein family comprises proteins, which share not only similarity in their primary structure, but also similarity in their fold. These two groups of similarity presume a parceling in their respective biological function through the common biochemical properties. In this review, biochemical and structural similarities of PrP and two other proteins, Doppel and Shadoo, are evocated. Some evidence demonstrating respectively similarity between PrP N-terminal and C-terminal domain with respectively Shadoo and Doppel is presented. We extended primary structure similarity analysis to the other PrP subdomain as 166-176 polyNQ domain and compare it to proteins using aggregation as a support for structural information transference and structural epigenetic. Finally, we questioned if prion protein family have conserved the PrP structural bistability, which should be at the origin of Prion phenomenon and if Prion pathology is not, ultimately, an exaptation of the physiological propensity of PrP to undergo a structural switch and polymerize. PMID:25717473

  9. Manganese Enhances Prion Protein Survival in Model Soils and Increases Prion Infectivity to Cells

    PubMed Central

    Davies, Paul; Brown, David R.

    2009-01-01

    Prion diseases are considered to be transmissible. The existence of sporadic forms of prion diseases such as scrapie implies an environmental source for the infectious agent. This would suggest that under certain conditions the prion protein, the accepted agent of transmission, can survive in the environment. We have developed a novel technique to extract the prion protein from soil matrices. Previous studies have suggested that environmental manganese is a possible risk factor for prion diseases. We have shown that exposure to manganese is a soil matrix causes a dramatic increase in prion protein survival (?10 fold) over a two year period. We have also shown that manganese increases infectivity of mouse passaged scrapie to culture cells by 2 logs. These results clearly verify that manganese is a risk factor for both the survival of the infectious agent in the environment and its transmissibility. PMID:19844576

  10. Recombinant human prion protein fragment 90-231, a useful model to study prion neurotoxicity.

    PubMed

    Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Aceto, Antonio; Florio, Tullio

    2012-01-01

    Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of fatal neurodegenerative disorders of animals and humans. Human diseases include Creutzfeldt-Jakob (CJD) and Gerstmann-Straussler-Scheinker (GSSD) diseases, fatal familial insomnia, and Kuru. Human and animal TSEs share a common histopathology with a pathognomonic triad: spongiform vacuolation of the grey matter, neuronal death, glial proliferation, and, more inconstantly, amyloid deposition. According to the "protein only" hypothesis, TSEs are caused by a unique post-translational conversion of normal, host-encoded, protease-sensitive prion protein (PrP(sen) or PrP(C)) to an abnormal disease-associated isoform (PrP(res) or PrP(Sc)). To investigate the molecular mechanism of neurotoxicity induced by PrP(Sc) we developed a protocol to obtain millimolar amounts of soluble recombinant polypeptide encompassing the amino acid sequence 90-231 of human PrP (hPrP90-231). This protein corresponds to the protease-resistant prion protein fragment that originates after amino-terminal truncation. Importantly, hPrP90-231 has a flexible backbone that, similar to PrP(C), can undergo to structural rearrangement. This peptide, structurally resembling PrP(C), can be converted in a PrP(Sc)-like conformation, and thus represents a valuable model to study prion neurotoxicity. In this article we summarized our experimental evidence on the molecular and structural mechanisms responsible of hPrP90-231 neurotoxicity on neuroectodermal cell line SHSY5Y and the effects of some PrP pathogen mutations identified in familial TSE. PMID:22321015

  11. Monitoring prion protein stability by NMR.

    PubMed

    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

  12. Interaction Networks of Prion, Prionogenic and Prion-Like Proteins in Budding Yeast, and Their Role in Gene Regulation

    PubMed Central

    Harbi, Djamel; Harrison, Paul M.

    2014-01-01

    Prions are transmissible, propagating alternative states of proteins. Prions in budding yeast propagate heritable phenotypes and can function in large-scale gene regulation, or in some cases occur as diseases of yeast. Other ‘prionogenic’ proteins are likely prions that have been determined experimentally to form amyloid in vivo, and to have prion-like domains that are able to propagate heritable states. Furthermore, there are over 300 additional ‘prion-like’ yeast proteins that have similar amino-acid composition to prions (primarily a bias for asparagines and glutamines). Here, we examine the protein functional and interaction networks that involve prion, prionogenic and prion-like proteins. Set against a marked overall preference for N/Q-rich prion-like proteins not to interact with each other, we observe a significant tendency of prion/prionogenic proteins to interact with other, N/Q-rich prion-like proteins. This tendency is mostly due to a small number of networks involving the proteins NUP100p, LSM4p and PUB1p. In general, different data analyses of functional and interaction networks converge to indicate a strong linkage of prionogenic and prion-like proteins, to stress-granule assembly and related biological processes. These results further elucidate how prions may impact gene regulation, and reveal a broader horizon for the functional relevance of N/Q-rich prion-like domains. PMID:24972093

  13. Transmissible Proteins: Expanding the Prion Heresy

    PubMed Central

    Soto, Claudio

    2012-01-01

    The once-heretical concept that a misfolded protein is the infectious agent responsible for prion diseases is now widely accepted. Recent exciting research has led not only to the end of the skepticism that proteins can transmit disease but also to expanding the concept that transmissible proteins might be at the root of some of the most prevalent human illnesses. At the same time, the idea that biological information can be transmitted by propagation of protein (mis)folding raises the possibility that heritable protein agents may be operating as epigenetic factors in normal biological functions and participating in evolutionary adaptation. PMID:22632966

  14. Scrapie-infected cells, isolated prions, and recombinant prion protein: a comparative study.

    PubMed

    Kneipp, J; Miller, L M; Spassov, S; Sokolowski, F; Lasch, P; Beekes, M; Naumann, D

    Fourier -transform infrared microscopic spectra of scrapie-infected nervous tissue measured at high spatial resolution (approximately 6 microm) were compared with those obtained from the purified, partly proteinase K digested scrapie isoform of the prion protein isolated from nervous tissue of hamsters infected with the same scrapie strain (263K) to elucidate similarities/dissimilarities between prion structure investigated in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP(90-232) after in vitro conformational transition from the predominantly alpha-helical isoform to beta-sheet-rich structures. It is shown that prion protein structure can be investigated within tissue and that detectability of regions with elevated beta-sheet content as observed in microspectra of prion-infected tissue strongly depends on spatial resolution of the experiment. PMID:15137116

  15. Knocked-out and still walking: prion protein-deficient cattle are resistant to prion disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Transmissible spongiform encephalopathies (TSEs) or prion diseases are caused by the propagation of a misfolded form (PrP**d) of the normal cellular prion protein, PrP**c. Disruption of PrP**c expression in the mouse results in resistance to PrP-propagation and disease. However, the impa...

  16. Prions, prionoids and pathogenic proteins in Alzheimer disease

    PubMed Central

    Ashe, Karen H.; Aguzzi, Adriano

    2013-01-01

    Like patients with prion disease, Alzheimer patients suffer from a fatal, progressive form of dementia. There is growing evidence that amyloid-β (Aβ) aggregates may be transmissible similar to prions, at least under extreme experimental conditions. However, unlike mice infected with prion protein (PrP) prions, those inoculated with Aβ do not die. The transmission of Aβ and PrP thus differs conspicuously in the neurological effects they induce in their hosts, the difference being no less than a matter of life and death. Far from being a mere academic nuance, this distinction between Aβ and PrP begs the crucial questions of what, exactly, controls prion toxicity and how prion toxicity relates to prion infectivity. PMID:23208281

  17. Prions, prionoids and pathogenic proteins in Alzheimer disease.

    PubMed

    Ashe, Karen H; Aguzzi, Adriano

    2013-01-01

    Like patients with prion disease, Alzheimer patients suffer from a fatal, progressive form of dementia. There is growing evidence that amyloid-β (Aβ) aggregates may be transmissible similar to prions, at least under extreme experimental conditions. However, unlike mice infected with prion protein (PrP) prions, those inoculated with Aβ do not die. The transmission of Aβ and PrP thus differs conspicuously in the neurological effects they induce in their hosts, the difference being no less than a matter of life and death. Far from being a mere academic nuance, this distinction between Aβ and PrP begs the crucial questions of what, exactly, controls prion toxicity and how prion toxicity relates to prion infectivity. PMID:23208281

  18. Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases.

    PubMed

    Schmitz, Matthias; Lüllmann, Katharina; Zafar, Saima; Ebert, Elisabeth; Wohlhage, Marie; Oikonomou, Panteleimon; Schlomm, Markus; Mitrova, Eva; Beekes, Michael; Zerr, Inga

    2014-05-01

    The present study investigates whether posttranslational modifications of cellular prion protein (PrP(C)) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrP(Sc)) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrP(C) charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrP(C) were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrP(C) isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrP(Sc) type 2 (vs. PrP(Sc) type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrP(C) species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrP(C) in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors. PMID:24360565

  19. Evolutionary implications of metal binding features in different species' prion protein: an inorganic point of view.

    PubMed

    La Mendola, Diego; Rizzarelli, Enrico

    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 ?-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. PMID:24970230

  20. Structural determinants in prion protein folding and stability.

    PubMed

    Benetti, Federico; Biarns, Xevi; Attanasio, Francesco; Giachin, Gabriele; Rizzarelli, Enrico; Legname, Giuseppe

    2014-11-11

    Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein. Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrP(C) is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region in prion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species, and (iii) the role of the disulfide bridge in prion protein folding. PMID:25280897

  1. [Functions of prion protein PrPc].

    PubMed

    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

  2. Mechanisms of prion protein assembly into amyloid

    PubMed Central

    Sthr, Jan; Weinmann, Nicole; Wille, Holger; Kaimann, Tina; Nagel-Steger, Luitgard; Birkmann, Eva; Panza, Giannantonio; Prusiner, Stanley B.; Eigen, Manfred; Riesner, Detlev

    2008-01-01

    The conversion of the ?-helical, cellular isoform of the prion protein (PrPC) to the insoluble, ?-sheet-rich, infectious, disease-causing isoform (PrPSc) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, ?-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions. PMID:18268326

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

    USGS Publications Warehouse

    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.

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

    PubMed

    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. PMID:26061765

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26631378

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

    PubMed Central

    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

    2015-01-01

    Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP) 1. 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 state2. We have now 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. Here we demonstrate that transgenic mice expressing both variant and wild type human PrP are completely resistant to both kuru and classical CJD prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to BSE prions to which the Fore were not exposed. Remarkably however, 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. PMID:26061765

  7. Prion protein induced signaling cascades in monocytes

    SciTech Connect

    Krebs, Bjarne; Dorner-Ciossek, Cornelia; Vassallo, Neville; Herms, Jochen; Kretzschmar, Hans A. . E-mail: Hans.Kretzschmar@med.uni-muenchen.de

    2006-02-03

    Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP{sup C}), 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 PrP{sup C} fusion proteins synthesized with a human Fc-tag. PrP{sup C} 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 ERK{sub 1,2} 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 PrP{sup C} in monocytes and macrophages.

  8. Attachment of Pathogenic Prion Protein to Model Oxide Surfaces

    PubMed Central

    Jacobson, Kurt H.; Kuech, Thomas R.; Pedersen, Joel A.

    2014-01-01

    Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The N-terminal portion of the protein appeared to facilitate attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions preferentially associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides). PMID:23611152

  9. In silico analysis of prion protein mutants: a comparative study by molecular dynamics approach.

    PubMed

    Doss, C George Priya; Rajith, B; Rajasekaran, R; Srajan, Jain; Nagasundaram, N; Debajyoti, C

    2013-01-01

    Polymorphisms in the human prion proteins lead to amino acid substitutions by the conversion of PrPC to PrPSc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease and fatal familial insomnia. Cation-π interaction is a non-covalent binding force that plays a significant role in protein stability. Here, we employ a novel approach by combining various in silico tools along with molecular dynamics simulation to provide structural and functional insight into the effect of mutation on the stability and activity of mutant prion proteins. We have investigated impressions of prevalent mutations including 1E1S, 1E1P, 1E1U, 1E1P, 1FKC and 2K1D on the human prion proteins and compared them with wild type. Structural analyses of the models were performed with the aid of molecular dynamics simulation methods. According to our results, frequently occurred mutations were observed in conserved sequences of human prion proteins and the most fluctuation values appear in the 2K1D mutant model at around helix 4 with residues ranging from 190 to 194. Our observations in this study could help to further understand the structural stability of prion proteins. PMID:23723004

  10. Prevalent mutations of human prion protein: a molecular modeling and molecular dynamics study.

    PubMed

    Behmard, Esmaeil; Abdolmaleki, Parviz; Asadabadi, Ebrahim Barzegari; Jahandideh, Samad

    2011-10-01

    Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrPC to PrPSc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144-152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species. PMID:21875156

  11. [Modification of [PSI+] prion properties by the combination of amino acid changes within Sup35 protein N-domain].

    PubMed

    Bondarev, S A; Shirokolobova, E D; Trubitsyna, N P; Zhuravleva, G A

    2014-01-01

    [PSI+] prion is an amyloid isoform of a release factor Sup35p (eRF3). The structure of these protein aggregates remains unclear despite a long term history of prion amyloids investigations. The N-terminal domain of Sup35p (which is responsible for a propagation of prion) shapes superpleated beta-structure, according to modern concepts. Recently we constructed five double mutations within SUP35 sequence encoding the N-terminal prion-forming domain and investigated properties of mutant proteins. Mutations sup35-M1 (YQ46-47KK) and sup35-M2 (QQ61-62KK) lead to [PSI+] prion loss, while other mutant alleles (sup35-M3 QQ70-71KK; sup35-M4 QQ80-81KK; sup35-M5 QQ89-90KK) maintained prion. For the detail analysis of effects of mutant alleles on Sup35p aggregation we characterized propagation and properties of [PSI] prion in yeast strains bearing different mutant allele combinations. The data obtained have refined a supposed organization of beta-sheets forming by different regions of Sup35p prion-forming domain within amyloid. Also we obtained evidences that mutant sup35-M2 and sup35-M4 alleles change structure of prion aggregates. The prion destabilization by these mutations possibly is connected with decrease of heteroaggregate fragmentation by chaperones. PMID:25850301

  12. Prion disease: a deadly disease for protein misfolding.

    PubMed

    Chakraborty, Chiranjib; Nandi, Shyam; Jana, Snehasis

    2005-04-01

    An infectious particle, termed prion, composed largely and perhaps solely of a single protein, is the likely causative agent of prion disease. It produces lethal decline of cognitive and motor function. The responsible protein arrives at a pathogenic state by misfolding from a normal form that has ubiquitous tissue distribution. Prion diseases are often called spongiform encephalopathies. Probably most mammalian species develop these diseases. Specific examples in various animals are -Scrapie, Transmissible Mink Encephalopathy (TME ), Chronic Wasting Disease(CWD) and bovine spongiform encephalopathy (BSE). Humans are also susceptible to several prion diseases: Creutzfeld-Jacob Disease (CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), Fatal Familial Insomnia (FFI), Kuru and Alpers Syndrome. This paper reviews transmission of this diseases, protein involvement, nature of protein, the conversion process from PrP(c) to PrP(Sc), conversion of prion protein in vitro, the different proposed models for the conversion of PrP(c) to PrP(Sc), prion and other amyloid diseases, prion strains, structure of PrP(c) the particular process that may induce prion disease, and immunization against these diseases. PMID:15853695

  13. Prion protein as a mediator of synaptic transmission

    PubMed Central

    Steinert, Joern R

    2015-01-01

    Neurodegenerative disorders are characterized by synaptic and neuronal dysfunction which precedes general neuronal loss and subsequent cognitive or behavioral anomalies. Although the exact early cellular signaling mechanisms involved in neurodegenerative diseases are largely unknown, a view is emerging that compromised synaptic function may underlie the initial steps in disease progression. Much recent research has been aimed at understanding these early underlying processes leading to dysfunctional synaptic signaling, as this knowledge could identify putative sites of interventions, which could potentially slow progression and delay onset of disease. We have recently reported that synaptic function in a Drosophila melanogaster model can be modulated by the presence of native mouse prion protein and this modulation is negatively affected by a mutation within the protein which is associated with the Gerstmann-Strussler-Scheinker syndrome, a human form of prion disease. Indeed, wild-type prion protein facilitates synaptic release, whereas the mutated form induced diminished phenotypes. It is believed that together with the gain-of-function of neurotoxic misfolded prion signaling, the lack of prion protein contributes to the pathology in prion diseases. Therefore, our study investigated a potential endogenous role of prion protein in synaptic signaling, the lack of which could resemble a lack-of-function phenotype in prion disease. PMID:26478992

  14. [Unfolding chaperone as a prion protein relating molecule].

    PubMed

    Hachiya, Naomi S; Sakasegawa, Yuji; Kaneko, Kiyotoshi

    2003-11-01

    Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPc and PrPSc are identical, but their conformations are rather different; PrPc rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPc and PrPSc. To examine the protein unfolding activities against collectively folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone. Unfolding, from S. cerevisiae. Unfolding consists of oligomeric ring-like structure with the central cavity and has an ATP-dependent protein Unfoldingg activity with broad specificity in vitro, of which targets included PrP in beta-sheet form, alpha-synuclein, and A beta protein. We have also found that mouse neuroblastoma N2a cells contained the activity. Treatment of this factor with an ATP-hydrolyzing enzyme, apyrase, caused the decrease in its protein Unfoldingg activity. It was suggested that the purified protein probably formed homo-oligomer consisting of 4-5 subunits and its activity was ATP-dependent. PMID:15152473

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

    PubMed Central

    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 ?-helical content along with an increase of ?-strand structure. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two ?-helices by one and two residues, as well as an extension of two ?-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 which are prone to conversion, than in WT prions due to 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 of prion proteins. PMID:21338080

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

    PubMed

    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 ?-helical content along with an increase in ?-strand content. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two ?-helices by one and two residues, as well as an extension of two ?-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. Peptide sequences converting polyglutamine into a prion in yeast.

    PubMed

    Odani, Wataru; Urata, Kazuhiro; Okuda, Momoko; Okuma, Shunsuke; Koyama, Hiroko; Pack, Chan-Gi; Fujiwara, Kei; Nojima, Tatsuya; Kinjo, Masataka; Kawai-Noma, Shigeko; Taguchi, Hideki

    2015-02-01

    Amyloids are ordered protein aggregates composed of cross-? sheet structures. Amyloids include prions, defined as infectious proteins, which are responsible for mammalian transmissible spongiform encephalopathies, and fungal prions. Although the conventional view is that typical amyloids are associated with nontransmissible mammalian neurodegenerative diseases such as Alzheimer's disease, increasing evidence suggests that the boundary between transmissible and nontransmissible amyloids is ambiguous. To clarify the mechanism underlying the difference in transmissibility, we investigated the dynamics and the properties of polyglutamine (polyQ) amyloids in yeast cells, in which the polyQ aggregates are not transmissible but can be converted into transmissible amyloids. We found that polyQ had an increased tendency to form aggregates compared to the yeast prion Sup35. In addition, we screened dozens of peptides that converted the nontransmissible polyQ to transmissible aggregates when they flanked the polyQ stretch, and also investigated their cellular dynamics aiming to understand the mechanism of transmission. PMID:25406629

  18. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sharps, Bernadette; De Oliveira, Michael Wiggins; Schmidt, Christian; Richard-Londt, Angela; Lyall, Sarah; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Collinge, John

    2014-01-01

    Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent. PMID:25005024

  19. Epitope scanning indicates structural differences in brain-derived monomeric and aggregated mutant prion proteins related to genetic prion diseases.

    PubMed

    Tapella, Laura; Stravalaci, Matteo; Bastone, Antonio; Biasini, Emiliano; Gobbi, Marco; Chiesa, Roberto

    2013-09-15

    Genetic Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia and prion protein cerebral amyloid angiopathy are clinically and neuropathologically distinct neurodegenerative diseases linked to mutations in the PRNP gene encoding the cellular prion protein (PrPC). How sequence variants of PRNP encode the information to specify these disease phenotypes is not known. It is suggested that each mutation produces a misfolded variant of PrPC with specific neurotoxic properties. However, structural studies of recombinant PrP did not detect major differences between wild-type and mutant molecules, pointing to the importance of investigating mutant PrPs from mammalian brains. We used surface plasmon resonance and a slot-blot immunoassay to analyse the antibody-binding profiles of soluble and insoluble PrP molecules extracted from the brains of transgenic mice modelling different prion diseases. By measuring the reactivity of monoclonal antibodies against different PrP epitopes, we obtained evidence of conformational differences between wild-type and mutant PrPs, and among different mutants. We detected structural heterogeneity in both monomeric and aggregated PrP, supporting the hypothesis that the phenotype of genetic prion diseases is encoded by mutant PrP conformation and assembly state. PMID:23808898

  20. Effect of Divalent Metals on the Neuronal Proteasomal System, Prion Protein Ubiquitination and Aggregation

    PubMed Central

    Kanthasamy, A. G.; Choi, C.; Jin, H.; Harischandra, D.S.; Anantharam, V.; Kanthasamy, A.

    2012-01-01

    The role of normal cellular prion protein (PrP) remains to be fully elucidated; however, the protein is crucial for the infection and progression of prion diseases. Recent evidence indicates that PrP is a metalloprotein since the octapeptide repeat sequences in the protein have high affinity for various divalent cations and the binding sites appear to play a role in the pathogenesis of prion diseases. In our present study, we tested several divalent metals including manganese and cadmium and determined their effects on protein degradation and protein aggregation in mouse neuronal cells expressing PrP protein. Cadmium was more neurotoxic than manganese following 24 h exposure. Manganese did not show any significant effect on the inhibition of proteasomal activity or formation of high molecular weight ubiquitinated PrP proteins. Interestingly, treatment with cadmium profoundly inhibited proteasomal activity, which resulted in greatly increased formation of high molecular weight ubiquitinated PrP proteins. Immunohistochemical analysis also revealed a dramatic increase in formation of oligomers after cadmium treatment. Cadmium also increased the formation of ubiquitinated prion PrP, but it did not lead to the formation of proteinase-K resistant PrP. Collectively, our results show that a divalent metal, cadmium affects proteasomal function and prion protein aggregation, which promote neurotoxicity. PMID:22995398

  1. Prions and the Potential Transmissibility of Protein Misfolding Diseases*

    PubMed Central

    Kraus, Allison; Groveman, Bradley R.; Caughey, Byron

    2016-01-01

    Prions, or infectious proteins, represent a major frontier in the study of infectious agents. The prions responsible for mammalian transmissible spongiform encephalopathies (TSEs) are due primarily to infectious self-propagation of misfolded prion proteins. TSE prion structures remain ill-defined, other than being highly structured, self-propagating, and often fibrillar protein multimers with the capacity to seed, or template, the conversion of their normal monomeric precursors into a pathogenic form. Purified TSE prions usually take the form of amyloid fibrils, which are self-seeding ultrastructures common to many serious protein misfolding diseases such as Alzheimer’s, Parkinson’s, Huntington’s and Lou Gehrig’s (amytrophic lateral sclerosis). Indeed, recent reports have now provided evidence of prion-like propagation of several misfolded proteins from cell to cell, if not from tissue to tissue or individual to individual. These findings raise concerns that various protein misfolding diseases might have spreading, prion-like etiologies that contribute to pathogenesis or prevalence. PMID:23808331

  2. SSCP analysis and sequencing of the human prion protein gene (PRNP) detects two different 24 bp deletions in an atypical Alzheimer`s disease family

    SciTech Connect

    Perry, R.T.; Go, R.C.P.; Harrell, L.E.; Acton, R.T.

    1995-02-27

    Alzheimer`s disease (AD) is a progressive, degenerative neurological disorder of the central nervous system. AD is the fourth leading cause of death in elderly persons 65 years or older in Western industrialized societies. The etiology of AD is unknown, but clinical, pathological, epidemiological, and molecular investigations suggest it is etiologically heterogeneous. Mutations in the amyloid protein are rare and segregate with the disease in a few early-onset familial AD (FAD) families. Similarities between AD and the unconventional viral (UCV) diseases, and between the amyloid and prion proteins, implicate the human prion protein gene (PRNP) as another candidate gene. Single strand conformation polymorphism (SSCP) analysis was used to screen for mutations at this locus in 82 AD patients from 54 families (30 FAD), vs. 39 age-matched controls. A 24-bp deletion around codon 68 that codes for one of five Gly-Pro rich octarepeats was identified in two affected sibs and one offspring of one late-onset FAD family. Two other affected sibs, three unaffected sibs, and three offspring from this family, in addition to one sporadic AD patient and three age-matched controls, were heterozygous for another octarepeat deletion located around codon 82. Two of the four affected sibs had features of PD, including one who was autopsy-verified AD and PD. Although these deletions were found infrequently in other AD patients and controls, they appear to be a rare polymorphism that is segregating in this FAD family. It does not appear that mutations at the PRNP locus are frequently associated with AD in this population. 54 refs., 4 figs.

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

    PubMed

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

    PubMed Central

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

    2011-01-01

    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 control mice with anchored PrP, intracerebral or sciatic nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue, ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231 days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion. Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS neuroinvasion of prion infection. PMID:21123371

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

    PubMed

    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. PMID:25746597

  6. Insights into prion protein function from atomistic simulations.

    PubMed

    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

  7. The cellular prion protein mediates neurotoxic signalling of ?-sheet-rich conformers independent of prion replication

    PubMed Central

    Resenberger, Ulrike K; Harmeier, Anja; Woerner, Andreas C; Goodman, Jessica L; Mller, Veronika; Krishnan, Rajaraman; Vabulas, R Martin; Kretzschmar, Hans A; Lindquist, Susan; Hartl, F Ulrich; Multhaup, Gerd; Winklhofer, Konstanze F; Tatzelt, Jrg

    2011-01-01

    Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrPSc), a ?-sheet-rich isoform of the cellular PrP (PrPC), are dependent on neuronal expression of PrPC. In this study, we demonstrate that PrPC has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ?-sheet-rich (?) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ?-peptide, (iii) yeast prion proteins or (iv) designed ?-peptides. Toxic signalling via PrPC requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrPC with ?-conformers. Interestingly, a secreted version of N-PrP associated with ?-conformers and antagonized their toxic signalling via PrPC. Moreover, PrPC-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrPC can mediate toxic signalling of various ?-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases. PMID:21441896

  8. Persistence of Pathogenic Prion Protein during Simulated Wastewater Treatment Processes

    PubMed Central

    Hinckley, Glen T.; Johnson, Christopher J.; Jacobson, Kurt H.; Bartholomay, Christian; McMahon, Katherine D.; McKenzie, Debbie; Aiken, Judd M.; Pedersen, Joel A.

    2009-01-01

    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 (PrPTSE) 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., 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 10-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 of the agent would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. PMID:18754377

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

    USGS Publications Warehouse

    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.

  10. Prions are affected by evolution at two levels.

    PubMed

    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

  11. Prions on the move.

    PubMed

    Weissmann, Charles; Li, Jiali; Mahal, Sukhvir P; Browning, Shawn

    2011-11-01

    Prions consist mainly, if not entirely, of PrP(Sc), an aggregated conformer of the host protein PrP(C). Prions come in different strains, all based on the same PrP(C) sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected. PMID:21997298

  12. Sialylation of the prion protein glycans controls prion replication rate and glycoform ratio

    PubMed Central

    Katorcha, Elizaveta; Makarava, Natallia; Savtchenko, Regina; Baskakov, Ilia V.

    2015-01-01

    Prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded and aggregated form of a sialoglycoprotein called prion protein or PrPC. PrPC has two sialylated N-linked carbohydrates. In PrPSc, the glycans are directed outward, with the terminal sialic acid residues creating a negative charge on the surface of prion particles. The current study proposes a new hypothesis that electrostatic repulsion between sialic residues creates structural constraints that control prion replication and PrPSc glycoform ratio. In support of this hypothesis, here we show that diglycosylated PrPC molecules that have more sialic groups per molecule than monoglycosylated PrPC were preferentially excluded from conversion. However, when partially desialylated PrPC was used as a substrate, recruitment of three glycoforms into PrPSc was found to be proportional to their respective populations in the substrate. In addition, hypersialylated molecules were also excluded from conversion in the strains with the strongest structural constraints, a strategy that helped reduce electrostatic repulsion. Moreover, as predicted by the hypothesis, partial desialylation of PrPC significantly increased the replication rate. This study illustrates that sialylation of N-linked glycans creates a prion replication barrier that controls replication rate and glycoform ratios and has broad implications. PMID:26576925

  13. Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP**C) into an abnormal form of scrapie prion (PrP**Sc). The cellular mechanisms underlying the misfolding of PrP**C are not well understood. Since cellular prion proteins harbor divalent metal b...

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

    PubMed

    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

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

    USGS Publications Warehouse

    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.

  16. Strain-dependent profile of misfolded prion protein aggregates

    PubMed Central

    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 (PrPSc) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrPSc aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrPSc aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrPSc 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 PrPSc aggregates and the incubation periods for the strains studied. The relative presence of PrPSc 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 PrPSc aggregates in prion-induced neurodegeneration. PMID:26877167

  17. Disruption of Amyloid Prion Protein Aggregates by Cationic Pyridylphenylene Dendrimers.

    PubMed

    Sorokina, Svetlana A; Stroylova, Yulia Yu; Shifrina, Zinaida B; Muronetz, Vladimir I

    2016-02-01

    Disruption of amyloid protein aggregates is one of the potential therapies for treatment of neurodegenerative disorders such as prion diseases. Here, for the first time we report that pH-independent cationic pyridylphenylene dendrimers are able to disrupt amyloid protein aggregates at physiological pH as exemplified by inclusion bodies of ovine prion protein. The results show that exposure of inclusion bodies to the dendrimers leads to its partial disaggregation and release of the nanosize protein-dendrimer complexes. The complexes were characterized by SDS PAGE, DLS, and Western blotting methods. Thioflavin T fluorescence clearly demonstrated a decrease of amyloidogenic capability of the prion protein upon exposure to the dendrimers. The complexes formed are stable and do not show further aggregation. PMID:26445143

  18. Prion Diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prion diseases comprise a set of rare fatal neurological diseases found in humans and other mammals. A prion is a protein capable of converting a normal cellular protein (PrPC) into a prion and thereby propagating an infection. A prion and PrPC differ solely in their conformation. There are differen...

  19. Probing Structural Differences in Prion Protein Isoforms by Tyrosine Nitration

    PubMed Central

    Lennon, Christopher W.; Cox, Holly D.; Hennelly, Scott P.; Chelmo, Sam J.; McGuirl, Michele A.

    2008-01-01

    Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the ?-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in ?-sheet. PMID:17397138

  20. Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein.

    PubMed

    Wu, Emilia L; Qi, Yifei; Park, Soohyung; Mallajosyula, Sairam S; MacKerell, Alexander D; Klauda, Jeffery B; Im, Wonpil

    2015-11-17

    Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrPSc) converted from a normal host cellular prion protein (PrPC). Experimental studies suggest that PrPC is enriched with ?-helical structure, whereas PrPSc contains a high proportion of ?-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrPC leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface. PMID:26588568

  1. Prion Protein Accumulation in Lipid Rafts of Mouse Aging Brain

    PubMed Central

    Agostini, Federica; Dotti, Carlos G.; Prez-Caams, Azucena; Ledesma, Maria Dolores; Benetti, Federico; Legname, Giuseppe

    2013-01-01

    The cellular form of the prion protein (PrPC) 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 PrPC. In old mice, this change favors PrPC accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrPC translocation into detergent-resistant membranes (DRMs), we looked at PrPC 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 PrPC 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. PMID:24040215

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Purified prion proteins and scrapie infectivity copartition into liposomes.

    PubMed Central

    Gabizon, R; McKinley, M P; Prusiner, S B

    1987-01-01

    Considerable evidence indicates that the scrapie prion protein (PrP 27-30) is required for infectivity. Aggregates of PrP 27-30 form insoluble amyloid rods that resist dissociation by nondenaturing detergents. Mixtures of the detergent cholate and phospholipids were found to solubilize purified PrP 27-30 in the form of detergent-lipid-protein complexes. Removal of the cholate by dialysis resulted in the formation of closed liposomes. Both the detergent-lipid-protein complexes and the liposomes often but not always exhibited a 10-fold increase in scrapie infectivity compared to that observed with the rods. No evidence for a prion-associated nucleic acid could be found when the phospholipid vesicles containing PrP 27-30 were digested with nucleases and Zn2+ under conditions that allowed hydrolysis of exogenously added nucleic acids. No filamentous or rod-shaped particles were found amongst prion liposomes by electron microscopy in our search for a putative filamentous "scrapie virus." The partitioning of PrP 27-30 and scrapie infectivity into phospholipid vesicles contends that PrP 27-30 has a central role in scrapie pathogenesis, establishes that the prion amyloid rods are not essential for infectivity, and argues that prions are fundamentally different from viruses. Images PMID:3108886

  4. Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease

    PubMed Central

    White, Melanie D.; Farmer, Michael; Mirabile, Ilaria; Brandner, Sebastian; Collinge, John; Mallucci, Giovanna R.

    2008-01-01

    Prion diseases are fatal neurodegenerative conditions for which there is no effective treatment. Prion propagation involves the conversion of cellular prion protein, PrPC, to its conformational isomer, PrPSc, which accumulates in disease. Here, we show effective therapeutic knockdown of PrPC expression using RNAi in mice with established prion disease. A single administration of lentivirus expressing a shRNA targeting PrP into each hippocampus of mice with established prion disease significantly prolonged survival time. Treated animals lived 19% and 24% longer than mice given an empty lentivirus, or not treated, respectively. Lentivirally mediated RNAi of PrP also prevented the onset of behavioral deficits associated with early prion disease, reduced spongiform degeneration, and protected against neuronal loss. In contrast, mice receiving empty virus or no treatment developed early cognitive impairment and showed severe spongiosis and neuronal loss. The focal use of RNAi therapeutically in prion disease further supports strategies depleting PrPC, which we previously established to be a valid target for prion-based treatments. This approach can now be used to define the temporal, quantitative, and regional requirements for PrP knockdown for effective treatment of prion disease and to explore mechanisms involved in predegenerative neuronal dysfunction and its rescue. PMID:18632556

  5. Hot spot of structural ambivalence in prion protein revealed by secondary structure principal component analysis.

    PubMed

    Yamamoto, Norifumi

    2014-08-21

    The conformational conversion of proteins into an aggregation-prone form is a common feature of various neurodegenerative disorders including Alzheimer's, Huntington's, Parkinson's, and prion diseases. In the early stage of prion diseases, secondary structure conversion in prion protein (PrP) causing ?-sheet expansion facilitates the formation of a pathogenic isoform with a high content of ?-sheets and strong aggregation tendency to form amyloid fibrils. Herein, we propose a straightforward method to extract essential information regarding the secondary structure conversion of proteins from molecular simulations, named secondary structure principal component analysis (SSPCA). The definite existence of a PrP isoform with an increased ?-sheet structure was confirmed in a free-energy landscape constructed by mapping protein structural data into a reduced space according to the principal components determined by the SSPCA. We suggest a "spot" of structural ambivalence in PrP-the C-terminal part of helix 2-that lacks a strong intrinsic secondary structure, thus promoting a partial ?-helix-to-?-sheet conversion. This result is important to understand how the pathogenic conformational conversion of PrP is initiated in prion diseases. The SSPCA has great potential to solve various challenges in studying highly flexible molecular systems, such as intrinsically disordered proteins, structurally ambivalent peptides, and chameleon sequences. PMID:25101991

  6. Inherited Prion Disease A117V Is Not Simply a Proteinopathy but Produces Prions Transmissible to Transgenic Mice Expressing Homologous Prion Protein

    PubMed Central

    Asante, Emmanuel A.; Linehan, Jacqueline M.; Smidak, Michelle; Tomlinson, Andrew; Grimshaw, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Powell, Caroline; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Collinge, John

    2013-01-01

    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-Strussler-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 (PrPSc), 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 PrPSc was demonstrated in the brains of recipient transgenic mice. This PrPSc 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. PMID:24086135

  7. X-ray structural and molecular dynamical studies of the globular domains of cow, deer, elk and Syrian hamster prion proteins.

    PubMed

    Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

    2015-10-01

    Misfolded prion proteins are the cause of neurodegenerative diseases that affect many mammalian species, including humans. Transmission of the prion diseases poses a considerable public-health risk as a specific prion disease such as bovine spongiform encephalopathy can be transferred to humans and other mammalian species upon contaminant exposure. The underlying mechanism of prion propagation and the species barriers that control cross species transmission has been investigated quite extensively. So far a number of prion strains have been characterized and those have been intimately linked to species-specific infectivity and other pathophysiological manifestations. These strains are encoded by a protein-only agent, and have a high degree of sequence identity across mammalian species. The molecular events that lead to strain differentiation remain elusive. In order to contribute to the understanding of strain differentiation, we have determined the crystal structures of the globular, folded domains of four prion proteins (cow, deer, elk and Syrian hamster) bound to the POM1 antibody fragment Fab. Although the overall structural folds of the mammalian prion proteins remains extremely similar, there are several local structural variations observed in the misfolding-initiator motifs. In additional molecular dynamics simulation studies on these several prion proteins reveal differences in the local fluctuations and imply that these differences have possible roles in the unfolding of the globular domains. These local variations in the structured domains perpetuate diverse patterns of prion misfolding and possibly facilitate the strain selection and adaptation. PMID:26320075

  8. Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells

    PubMed Central

    Bellingham, Shayne A.; Coleman, Bradley M.; Hill, Andrew F.

    2012-01-01

    Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrPC, and the abnormal infectious form, PrPSc, are found associated with exosomes, which are small 50–130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease. PMID:22965126

  9. Humic substances interfere with detection of pathogenic prion protein

    USGS Publications Warehouse

    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.

  10. PRIONS: PATHOLOGICAL PROTEINS AT THE INTERFACE OF OIL AND WATER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prions are infectious proteins that cause of a set of rare fatal neurological diseases referred to as transmissible spongiform encephalopathies (TSEs). TSE diseases occur in humans, sheep, goats, deer, elk, mink, cows and other mammals. This presentation will include an historical review of the scie...

  11. Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein.

    PubMed Central

    Telling, G C; Scott, M; Hsiao, K K; Foster, D; Yang, S L; Torchia, M; Sidle, K C; Collinge, J; DeArmond, S J; Prusiner, S B

    1994-01-01

    Transgenic (Tg) mice were constructed that express a chimeric prion protein (PrP) in which a segment of mouse (Mo) PrP was replaced with the corresponding human (Hu) PrP sequence. The chimeric PrP, designated MHu2MPrP, differs from MoPrP by 9 amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease approximately 200 days after inoculation with brain homogenates from three patients dying of Creutzfeldt-Jakob disease (CJD). Inoculation of Tg(MHu2M) mice with CJD prions produced MHu2MPrPSc (where PrPSc is the scrapie isoform of PrP); inoculation with Mo prions produced Mo-PrPSc. The patterns of MHu2MPrPSc and MoPrPSc accumulation in the brains of Tg(MHu2M) mice were different. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease > 500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to Hu prions indicate that additional species-specific factors are involved in prion replication. Diagnosis, prevention, and treatment of Hu prion diseases should be facilitated by Tg(MHu2M) mice. Images PMID:7937921

  12. Pharmacological chaperone for the structured domain of human prion protein

    PubMed Central

    Nicoll, Andrew J.; Trevitt, Clare R.; Tattum, M. Howard; Risse, Emmanuel; Quarterman, Emma; Ibarra, Amaurys Avila; Wright, Connor; Jackson, Graham S.; Sessions, Richard B.; Farrow, Mark; Waltho, Jonathan P.; Clarke, Anthony R.; Collinge, John

    2010-01-01

    In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrPC). Numerous ligands have been reported to bind to human PrPC (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 1?1 complex via the structured Cterminus of huPrP with a Kd of 4.52?M, which is in the range of its IC50 for curing prion-infected cells of 1.60.4?M and the concentration required to inhibit protein-misfolding cyclic amplification. Therefore, this molecule tests the hypothesis that stabilization of huPrPC, as a principle, could be used in the treatment of human prion disease. The identification of a binding site with a defined 3D structure opens up the possibility of designing small molecules that stabilize huPrP and prevent its conversion into the disease-associated form. PMID:20876144

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

    PubMed

    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. PMID:26110609

  14. Prion protein modulates cellular iron uptake: a novel function with implications for prion disease pathogenesis.

    PubMed

    Singh, Ajay; Mohan, Maradumane L; Isaac, Alfred Orina; Luo, Xiu; Petrak, Jiri; Vyoral, Daniel; Singh, Neena

    2009-01-01

    Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP(C)) from a mainly alpha-helical to a beta-sheet rich PrP-scrapie (PrP(Sc)) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP(Sc), nor the normal function of PrP(C) is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP(C) mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP(C) increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP(C) on ferritin iron content is enhanced by stimulating PrP(C) endocytosis, and reversed by cross-linking PrP(C) on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP(102L) decreases ferritin iron content significantly relative to PrP(C) expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP(C) nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP(C) in cellular iron uptake and transport to ferritin, and dysfunction of PrP(C) as a significant contributing factor of brain iron imbalance in prion disorders. PMID:19212444

  15. Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

    PubMed Central

    Isaac, Alfred Orina; Luo, Xiu; Petrak, Jiri; Vyoral, Daniel; Singh, Neena

    2009-01-01

    Converging evidence leaves little doubt that a change in the conformation of prion protein (PrPC) from a mainly ?-helical to a ?-sheet rich PrP-scrapie (PrPSc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrPSc, nor the normal function of PrPC is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrPC mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrPC increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrPC on ferritin iron content is enhanced by stimulating PrPC endocytosis, and reversed by cross-linking PrPC on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP102L decreases ferritin iron content significantly relative to PrPC expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrPC nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrPC in cellular iron uptake and transport to ferritin, and dysfunction of PrPC as a significant contributing factor of brain iron imbalance in prion disorders. PMID:19212444

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

    PubMed Central

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

    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-?) and interleukin 1 beta (IL-1?) on PrPc expression was analyzed in gastric cell lines. RESULTS: PrPc expression was increased in H pylori-infection compared with non-infected controls and decreased to normal after successful eradication. Gastrin, PGE2, and IL-1? dose-dependently upregulated PrPc in gastric cells, while TNF-? 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-1? synthesis. H pylori creates a milieu for enhanced propagation of prions in the gastrointestinal tract. PMID:16437693

  17. Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins

    PubMed Central

    Colacino, Stefano; Tiana, Guido; Colombo, Giorgio

    2006-01-01

    Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrPC) is converted into the infectious PrPSc through a conformational process during which it acquires a high ?-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research. Results In this paper, the dynamical and energetic properties of the two proteins in solution is comparatively analyzed by means of long time scale explicit solvent, all-atom molecular dynamics in different temperature conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach (Tiana et al, Prot. Sci. 2004) aimed at identifying the key residues for the stabilization and folding of the protein. Our analysis shows that Prion and Doppel have two different cores stabilizing the native state and that the relative contribution of the nucleus to the global stability of the protein for Doppel is sensitively higher than for PrP. Moreover, under misfolding conditions the Doppel core is conserved, while the energy stabilization network of PrP is disrupted. Conclusion These observations suggest that different sequences can share similar native topology with different stabilizing interactions and that the sequences of the Prion and Doppel proteins may have diverged under different evolutionary constraints resulting in different folding and stabilization mechanisms. PMID:16857062

  18. Increasing Prion Propensity by Hydrophobic Insertion

    PubMed Central

    Petri, Michelina; Flores, Noe; Rogge, Ryan A.; Cascarina, Sean M.; Ross, Eric D.

    2014-01-01

    Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein. These data, combined with bioinformatics analysis, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. This limit may explain the under-representation of non-aromatic hydrophobic residues in yeast prion domains. Prion activity requires not only that a protein be able to form prion fibers, but also that these fibers be cleaved to generate new independently-segregating aggregates to offset dilution by cell division. Recent studies suggest that aromatic residues, but not non-aromatic hydrophobic residues, support the fiber cleavage step. Therefore, we propose that while both aromatic and non-aromatic hydrophobic residues promote prion formation, aromatic residues are favored in yeast prion domains because they serve a dual function, promoting both prion formation and chaperone-dependent prion propagation. PMID:24586661

  19. Kinetics of Ozone Inactivation of Infectious Prion Protein

    PubMed Central

    Ding, Ning; Price, Luke M.; Braithwaite, Shannon L.; Balachandran, Aru; Mitchell, Gordon; Belosevic, Miodrag

    2013-01-01

    The kinetics of ozone inactivation of infectious prion protein (PrPSc, scrapie 263K) was investigated in ozone-demand-free phosphate-buffered saline (PBS). Diluted infectious brain homogenates (IBH) (0.01%) were exposed to a predetermined ozone dose (10.8 ± 2.0 mg/liter) at three pHs (pH 4.4, 6.0, and 8.0) and two temperatures (4°C and 20°C). The inactivation of PrPSc was quantified by determining the in vitro destruction of PrPSc templating properties using the protein misfolding cyclic amplification (PMCA) assay and bioassay, which were shown to correlate well. The inactivation kinetics were characterized by both Chick-Watson (CW) and efficiency factor Hom (EFH) models. It was found that the EFH model fit the experimental data more appropriately. The efficacy of ozone inactivation of PrPSc was both pH and temperature dependent. Based on the EFH model, CT (disinfectant concentration multiplied by contact time) values were determined for 2-log10, 3-log10, and 4-log10 inactivation at the conditions under which they were achieved. Our results indicated that ozone is effective for prion inactivation in ozone-demand-free water and may be applied for the inactivation of infectious prion in prion-contaminated water and wastewater. PMID:23416994

  20. The cellular prion protein and its role in Alzheimer disease

    PubMed Central

    Irujo, A; Cuadrado-Tejedor, M; Paternain, B; Moleres, FJ; Ferrer, V

    2009-01-01

    The cellular prion protein (PrPC) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrPSc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrPC is needed for the establishment and further evolution of prions. The present work compares the expression and localization of PrPC between healthy human brains and those suffering from Alzheimer disease (AD). In both situations we have observed a rostrocaudal decrease in the amount of PrPC within the CNS, both by immunoblotting and immunohistochemistry techniques. PrPC is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrPC in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands. With regards to amyloid plaques, those present in AD cases were positively labeled for PrPC, a result which is further supported by the presence of PrPC in the amyloid plaques of a transgenic line of mice mimicking AD. The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre. PMID:19556894

  1. Prion protein degradation by lichens of the genus Cladonia

    USGS Publications Warehouse

    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.

  2. Role of the prion protein family in the gonads

    PubMed Central

    Allais-Bonnet, Aurlie; Pailhoux, Eric

    2014-01-01

    The prion-gene family comprises four members named PRNP (PRPc), PRND (Doppel), PRNT (PRT), and SPRN (Shadoo). According to species, PRND is located 1652 kb downstream from the PRNP locus, whereas SPRN is located on another chromosome. The fourth prion-family gene, PRNT, belongs to the same genomic cluster as PRNP and PRND in humans and bovidae. PRNT and PRND possibly resulted from a duplication event of PRND and PRNP, respectively, that occurred early during eutherian species divergence. Although most of the studies concerning the prion-family has been done on PRPc and its involvement in transmissible neurodegenerative disorders, different works report some potential roles of these proteins in the reproductive function of both sexes. Among them, a clear role of PRND, that encodes for the Doppel protein, in male fertility has been demonstrated through gene targeting studies in mice. In other species, Doppel seems to play a role in testis and ovary development but its cellular localization is variable according to the gonadal developmental stage and to the mammalian species considered. For the other three genes, their roles in reproductive function appear ill-defined and/or controversial. The present review aimed to synthesize all the available data on these prion-family members and their relations with reproductive processes, mainly in the gonad of both sexes. PMID:25364761

  3. Clearance of yeast prions by misfolded multi-transmembrane proteins.

    PubMed

    Arai, Chie; Kurahashi, Hiroshi; Ishiwata, Masao; Oishi, Keita; Nakamura, Yoshikazu

    2013-06-01

    Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the stress response to protect cells against toxicity by the unfolded protein response (UPR), heat shock response (HSR), and ER-associated degradation pathways. Here, we found that over-production of C-terminally truncated multi-transmembrane (MTM) mutant proteins triggers HSR, but not UPR, and clearance of yeast prions [PSI(+)] and [URE3]. One of the mutant MTM proteins, Dip5?C-v82, produces a disabled amino-acid permease. Fluorescence microscopy analysis revealed abnormal accumulation of Dip5?C-v82 in the ER. Importantly, the mutant defective in the GET pathway, which functions for ER membrane insertion of tail-anchored proteins, failed to translocate Dip5?C-v82 to the ER and disabled Dip5?C-v82-mediated prion clearance. These findings suggest that the GET pathway plays a pivotal role in quality assurance of MTM proteins, and entraps misfolded MTM proteins into ER compartments, leading to loss-of-prion through a yet undefined mechanism. PMID:23384482

  4. Gingerol prevents prion protein-mediated neuronal toxicity by regulating HIF prolyl hydroxylase 2 and prion protein.

    PubMed

    Park, Yang-Gyu; Park, Sang-Youel

    2014-11-01

    Prion diseases are a family of progressive neurodegenerative disorders, which are fatal in the majority of cases and affect both humans and domestic animals. Prion protein (PrP)(106-126) retains the neurotoxic properties of the entire pathological PrPsc and it is generally used as a reasonable model to study the mechanisms responsible for prion diseases. In our previous studies, we demonstrated that hypoxia-inducible factor (HIF)-1? is involved in the gingerol-mediated protection of neuronal cells. HIF mediates cellular adaptations to low oxygen. Prolyl hydroxylase domain-containing protein2 (PHD2) is an oxygen sensor that hydroxylates the HIF-?-subunit, promoting its proteasomal degradation under normoxic conditions. Thus, in the present study we wished to determine whether gingerol inhibits the catalytic activity of PHD2 and prevents HIF-1? protein proteasomal degradation, thereby preventing the occurrence of PrP(106-126)-induced neuronal apoptosis. We used the pharmacological inhibition of PHD2 by dimethyloxalylglycine (DMOG) or deferoxamine (DFO) and the genetic inhibition of HIF-1? by HIF-1? small interfering RNA (siRNA) to block the effects of gingerol against PrP(106-126)-induced neurotoxicity. Our results demonstrated that gingerol prevented PrP(106?126)-induced neuronal apoptosis by upregulating HIF-1? and inhibiting the catalytic activity of PHD2 under normoxic conditions. Moreover, the protective effects of gingerol against PrP(106-126)-induced neuronal apoptosis were associated with the upregulation of the expression of cellular prion protein (PrPc). In conclusion, our results indicate that gingerol has therapeutic potential for use in the treatment or prevention of prion diseases, and its inhibitory effects on the catalytic activity of PHD2 may be of clinical benefit. PMID:25231392

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26482532

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Yeast prions assembly and propagation

    PubMed Central

    2011-01-01

    Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI+], [URE3] and [PIN+] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the non-prion domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others. PMID:22052349

  8. Differential Toxicity of Antibodies to the Prion Protein

    PubMed Central

    Hornemann, Simone; Herrmann, Uli S.; Arand, Michael; Hawke, Simon; Aguzzi, Adriano

    2016-01-01

    Antibodies against the prion protein PrPC can antagonize prion replication and neuroinvasion, and therefore hold promise as possible therapeutics against prion diseases. However, the safety profile of such antibodies is controversial. It was originally reported that the monoclonal antibody D13 exhibits strong target-related toxicity, yet a subsequent study contradicted these findings. We have reported that several antibodies against certain epitopes of PrPC, including antibody POM1, are profoundly neurotoxic, yet antibody ICSM18, with an epitope that overlaps with POM1, was reported to be innocuous when injected into mouse brains. In order to clarify this confusing situation, we assessed the neurotoxicity of antibodies D13 and ICSM18 with dose-escalation studies using diffusion-weighted magnetic resonance imaging and various histological techniques. We report that both D13 and ICSM18 induce rapid, dose-dependent, on-target neurotoxicity. We conclude that antibodies directed to this region may not be suitable as therapeutics. No such toxicity was found when antibodies against the flexible tail of PrPC were administered. Any attempt at immunotherapy or immunoprophylaxis of prion diseases should account for these potential untoward effects. PMID:26821311

  9. Stability and Cu(II) binding of prion protein variants related to inherited human prion diseases.

    PubMed

    Cereghetti, Grazia M; Schweiger, Arthur; Glockshuber, Rudi; Van Doorslaer, Sabine

    2003-03-01

    All inherited forms of human prion diseases are linked with mutations in the prion protein (PrP) gene. Here we have investigated the stability and Cu(II) binding properties of three recombinant variants of murine full-length PrP(23-231)-containing destabilizing point mutations that are associated with human Gerstmann-Strussler-Scheinker disease (F198S), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (D178N) by electron paramagnetic resonance and circular dichroism spectroscopy. Furthermore, we analyzed the variants H140S, H177S, and H187S of the isolated C-terminal domain of murine PrP, mPrP(121-231), to test a role of the histidine residues in Cu(II) binding. The F198S and E200K variants of PrP(23-231) differed in Cu(II) binding from the wild-type mPrP(23-231). However, circular dichroism spectroscopy indicated that the variants and the wild type did not undergo conformational changes in the presence of Cu(II). The D178N variant showed a high tendency to aggregate at pH 7.4 both with and without Cu(II). At lower pH values, it showed the same Cu(II) binding behavior as the wild type. The analysis allowed for a better location of the Cu(II) binding sites in the C-terminal part of the protein. Our present data indicate that hereditary forms of prion diseases cannot be rationalized on the basis of altered Cu(II) binding or mutation-induced protein destabilization alone. PMID:12609901

  10. Disease Transmission by Misfolded Prion-Protein Isoforms, Prion-Like Amyloids, Functional Amyloids and the Central Dogma.

    PubMed

    Daus, Martin L

    2016-01-01

    In 1982, the term "prions" (proteinaceous infectious particles) was coined to specify a new principle of infection. A misfolded isoform of a cellular protein has been described as the causative agent of a fatal neurodegenerative disease. At the beginning of prion research scientists assumed that the infectious agent causing transmissible spongiform encephalopathy (TSE) was a virus, but some unconventional properties of these pathogens were difficult to bring in line with the prevailing viral model. The discovery that prions (obviously devoid of any coding nucleic acid) can store and transmit information similarly to DNA was initially even denoted as being "heretical" but is nowadays mainly accepted by the scientific community. This review describes, from a historical point of view, how the "protein-only hypothesis" expands the Central Dogma. Definition of both, the prion principle and the Central Dogma, have been essential steps to understand information storage and transfer within and among cells and organisms. Furthermore, the current understanding of the infectivity of prion-proteins after misfolding is summarized succinctly. Finally, prion-like amyloids and functional amyloids, as found in yeast and bacteria, will be discussed. PMID:26742083

  11. Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection

    PubMed Central

    Patel, Milan J.; Sporn, Zachary A.; Hines, Justin K.

    2014-01-01

    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 been maintained in eukaryotic chaperone evolution. PMID:25058638

  12. The Role of Activity in Synaptic Degeneration in a Protein Misfolding Disease, Prion Disease

    PubMed Central

    Vannini, Eleonora; Siskova, Zuzana; Al-Malki, Hussain; Morgan, Ruth; O'Connor, Vincent; Perry, V. Hugh

    2012-01-01

    In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in nave mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration. PMID:22815961

  13. Sensitive detection of aggregated prion protein via proximity ligation.

    PubMed

    Hammond, Maria; Wik, Lotta; Deslys, Jean-Philippe; Comoy, Emmanuel; Linn, Tommy; Landegren, Ulf; Kamali-Moghaddam, Masood

    2014-01-01

    The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases. PMID:25482604

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Copper and the prion protein: methods, structures, function, and disease.

    PubMed

    Millhauser, Glenn L

    2007-01-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrP(C) to PrP(Sc). 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 PrP(C) 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 PrP(C), with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrP(C) function, and emerging connections between copper and prion disease. PMID:17076634

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

    NASA Astrophysics Data System (ADS)

    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.

  17. The neurodegeneration in Alzheimer disease and the prion protein.

    PubMed

    Forloni, Gianluigi; Sclip, Alessandra; Borsello, Tiziana; Balducci, Claudia

    2013-01-01

    The concept of "prion-like" has been proposed to explain the pathogenic mechanism of the principal neurodegenerative disorders associated with protein misfolding, including Alzheimer disease (AD). Other evidence relates prion protein with AD: the cellular prion protein (PrP(C)) binds β amyloid oligomers, allegedly responsible for the neurodegeneration in AD, mediating their toxic effects. We and others have confirmed the high-affinity binding between β amyloid oligomers and PrP(C), but we were not able to assess the functional consequences of this interaction using behavioral investigations and in vitro tests. This discrepancy rather than being resolved with the classic explanations, differencies in methodological aspects, has been reinforced by new data from different sources. Here we present data obtained with PrP antibody that not interfere with the neurotoxic activity of β amyloid oligomers. Since the potential role of the PrP(C) in the neuronal dysfunction induced by β amyloid oligomers is an important issue, find reasonable explanation of the inconsistent results is needed. Even more important however is the relevance of this interaction in the context of the disease, so as to develop valid therapeutic strategies. PMID:23324596

  18. Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast.

    PubMed

    Ness, Frdrique; Ferreira, Paulo; Cox, Brian S; Tuite, Mick F

    2002-08-01

    [PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates. PMID:12101251

  19. Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates*

    PubMed Central

    Margalith, Ilan; Suter, Carlo; Ballmer, Boris; Schwarz, Petra; Tiberi, Cinzia; Sonati, Tiziana; Falsig, Jeppe; Nystrm, Sofie; Hammarstrm, Per; slund, Andreas; Nilsson, K. Peter R.; Yam, Alice; Whitters, Eric; Hornemann, Simone; Aguzzi, Adriano

    2012-01-01

    Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits. PMID:22493452

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

    PubMed Central

    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 (PrPC) into PrPSc, a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrPSc 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 PrPC may provide an opportunity to overcome these problems. PrPC 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 PrPC, 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 PrPC-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrPC-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer’s Disease. These results demonstrate that molecules binding to PrPC may produce a dual effect of blocking prion replication and inhibiting PrPC-mediated toxicity. PMID:26976106

  1. Prion protein fragment (106-126) induces prothrombotic state by raising platelet intracellular calcium and microparticle release.

    PubMed

    Mallick, Ram L; Kumari, Sharda; Singh, Nitesh; Sonkar, Vijay K; Dash, Debabrata

    2015-04-01

    Prion diseases are neurodegenerative disorders where infectious prion proteins (PrP) accumulate in brain leading to aggregation of amyloid fibrils and neuronal cell death. The amino acid sequence 106-126 from prion proteins, PrP(106-126), is highly amyloidogenic and implicated in prion-induced pathologies. As PrP is known to be expressed in blood following leakage from brain tissue, we sought to investigate its biological effects on human platelets, which have been widely employed as 'peripheral' model for neurons. Our findings suggested that, PrP(106-126) (20?M) induced dramatic 30-fold rise in intracellular calcium (from 10530 to 3425525nM) in platelets, which was attributable to influx from extracellular fluid with comparatively less contribution from intracellular stores. Calcium mobilization was associated with 8-10-fold stimulation in the activity of thiol protease calpain that led to partial cleavage of cytoskeleton-associated protein talin and extensive shedding of microparticles from platelets, thus transforming platelets to 'activated' phenotype. Both proteolysis of talin and microparticle release were precluded by calpeptin, a specific inhibitor of calpain. As microparticles are endowed with phosphatidylserine-enriched surface and hence are pro-coagulant in nature, exposure to prion favored a thrombogenic state in the organism. PMID:25749016

  2. Alzheimer disease and the prion disorders amyloid beta-protein and prion protein amyloidoses.

    PubMed Central

    Price, D L; Borchelt, D R; Sisodia, S S

    1993-01-01

    Alzheimer disease and the prion disorders/spongiform encephalopathies share many common features. These chronic, progressive, sometimes familial diseases of the central nervous system are characterized by the presence of different types of amyloid deposits in the brain. This review provides a perspective on these two types of neurodegenerative disorders. PMID:8101988

  3. Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

    PubMed Central

    Coleman, Bradley M.; Harrison, Christopher F.; Guo, Belinda; Masters, Colin L.; Barnham, Kevin J.; Lawson, Victoria A.

    2014-01-01

    ABSTRACT Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrPC, into the disease-associated isoform, PrPSc. Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrPC share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrPSc are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrPSc, giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion. IMPORTANCE Prion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that the mutants make highly infectious prions that are more sensitive to protease treatment. This study highlights a certain region of the prion protein as being involved in this effect and demonstrates that prions are not always resistant to protease treatment. PMID:24352465

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

    PubMed

    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. PMID:26086786

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

    PubMed Central

    Raymond, Lynne D.; Hughson, Andrew G.; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

    2015-01-01

    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. PMID:26086786

  6. Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

    PubMed Central

    Raymond, Gregory J.; Race, Brent; Hollister, Jason R.; Offerdahl, Danielle K.; Moore, Roger A.; Kodali, Ravindra; Raymond, Lynne D.; Hughson, Andrew G.; Rosenke, Rebecca; Long, Dan; Dorward, David W.

    2012-01-01

    Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrPres]) of the cellular prion protein (PrPC). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrPC. Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrPC and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrPC. To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrPres-like protease-resistant banding profile. These fibrils induced the formation of PrPres deposits in transgenic mice coexpressing wt and GPI-anchorless PrPC (wt/GPI?) at a combined level comparable to that of PrPC expression in wt mice. Secondary passage into mice expressing wt, GPI?, or wt plus GPI? PrPC induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI? PrPC and, in one case, caused disease only in GPI? mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrPC. These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrPC GPI anchor can modulate the propagation of synthetic TSE strains. PMID:22915801

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

    PubMed

    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

  8. In Vitro and In Vivo Neurotoxicity of Prion Protein Oligomers

    PubMed Central

    Simoneau, Steve; Rezaei, Human; Sals, Nicole; Kaiser-Schulz, Gunnar; Lefebvre-Roque, Maxime; Vidal, Catherine; Fournier, Jean-Guy; Comte, Julien; Wopfner, Franziska; Grosclaude, Jeanne; Schtzl, Hermann; Lasmzas, Corinne Ida

    2007-01-01

    The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers. PMID:17784787

  9. Contrasting Effects of Two Lipid Cofactors of Prion Replication on the Conformation of the Prion Protein

    PubMed Central

    Srivastava, Saurabh; Baskakov, Ilia V.

    2015-01-01

    Recent studies introduced two experimental protocols for converting full-length recombinant prion protein (rPrP) purified from E.coli into the infectious prion state (PrPSc) with high infectivity titers. Both protocols employed protein misfolding cyclic amplification (PMCA) for generating PrPSc de novo, but used two different lipids, 1-palmitoyl-2-oleolyl-sn-glycero-3-phospho(1’-rac-glycerol) (POPG) or phosphatidylethanolamine (PE), as conversion cofactors. The current study compares the effect of POPG and PE on the physical properties of native, α-helical full-length mouse rPrP under the solvent conditions used for converting rPrP into PrPSc. Surprisingly, the effects of POPG and PE on rPrP physical properties, including its conformation, thermodynamic stability, aggregation state and interaction with a lipid, were found to be remarkably different. PE was shown to have minimal, if any, effects on rPrP thermodynamic stability, cooperativity of unfolding, immediate solvent environment or aggregation state. In fact, little evidence indicates that PE interacts with rPrP directly. In contrast, POPG was found to bind to and induce dramatic changes in rPrP structure, including a loss of α-helical conformation and formation of large lipid-protein aggregates that were resistant to partially denaturing conditions. These results suggest that the mechanisms by which lipids assist conversion of rPrP into PrPSc might be fundamentally different for POPG and PE. PMID:26090881

  10. High CJD infectivity remains after prion protein is destroyed

    PubMed Central

    Miyazawa, Kohtaro; Emmerling, Kaitlin; Manuelidis, Laura

    2011-01-01

    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, PrPsc) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique 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 2hr 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 2hr, yet decreased titer by >2.5logs; few residual protein bands remained. FU-CJD infected cells with 10x the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 logs). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of ?7.8 logs remained. Our FU-CJD brain results are in good accord with the only other report on maximal PrP digestion and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles. PMID:21793041

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

    PubMed

    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 <0.2%, yet a very high titer of 8 logs survived. Our FU-CJD brain results are in good accord with the only other 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. PMID:21793041

  12. Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Martin, Dustin P.; Nicholson, Eric M.; Richt, Jrgen A.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2010-01-01

    Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrPC) into an abnormal form of scrapie prion (PrPSc). The cellular mechanisms underlying the misfolding of PrPC are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrPC processing in in vitro models of prion disease. Exposure to manganese significantly increased PrPC levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrPC upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrPC degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrPC turnover rate was significantly altered with manganese treatment, indicating increased stability of PrPC with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrPC. Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratoryinfected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis. PMID:20176619

  13. Iatrogenic and sporadic Creutzfeldt-Jakob disease in 2 sisters without mutation in the prion protein gene.

    PubMed

    Frontzek, Karl; Moos, Rita; Schaper, Elke; Jann, Lukas; Herfs, Gregor; Zimmermann, Dieter R; Aguzzi, Adriano; Budka, Herbert

    2015-11-01

    Human genetic prion diseases have invariably been linked to alterations of the prion protein (PrP) gene PRNP. Two sisters died from probable Creutzfeldt-Jakob disease (CJD) in Switzerland within 14 y. At autopsy, both patients had typical spongiform change in their brains accompanied by punctuate deposits of PrP. Biochemical analyses demonstrated proteinase K-resistant PrP. Sequencing of PRNP showed 2 wild-type alleles in both siblings. Retrospectively, clinical data revealed a history of dural transplantation in the initially deceased sister, compatible with a diagnosis of iatrogenic CJD. Clinical and familial histories provided no evidence for potential horizontal transmission. This observation of 2 siblings suffering from CJD without mutations in the PRNP gene suggests potential involvement of non-PRNP genes in prion disease etiology. PMID:26634863

  14. Species barrier in prion diseases: a kinetic interpretation based on the conformational adaptation of the prion protein.

    PubMed Central

    Kellershohn, N; Laurent, M

    1998-01-01

    Prion diseases are thought to result from the conformational change of the normal cellular prion protein to a pathogenic protease-resistant isoform. However, brain extracts not containing the protease-resistant isoform of the prion protein can be infectious following interspecies transmission. The 'protein-only' hypothesis of pathogenesis is extended to provide possible explanations which could be interpreted in terms of a different infectious agent. It is proposed that normal cellular protein (PrPC) may be transformed into a form (PrP*) that is conformationally distinct from the host-specific abnormal isoform (PrPSc). In infection from a heterologous donor, the dimeric forms of heterologous PrPSc, which may catalyse the formation of host PrP* from PrPC, host PrP* and host PrPSc are all considered to be capable of catalysing, to some extent, the conversion of PrPC into PrPSc. However, depending on the species involved, PrP* may, or may not, be pathogenic, and may, or may not, be sensitive to proteolysis. It is shown, by numerical integration of the differential rate equations derived from this model, that a strain may be stabilized after two or three passages through a different species and that transmission might occur in the absence of detectable protease-resistant prion protein. The natural transmission of scrapie to cattle is discussed in relation to the model. PMID:9729459

  15. The genetics of prion diseases.

    PubMed

    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

  16. Parallel In-register Intermolecular ?-Sheet Architectures for Prion-seeded Prion Protein (PrP) Amyloids*

    PubMed Central

    Groveman, Bradley R.; Dolan, Michael A.; Taubner, Lara M.; Kraus, Allison; Wickner, Reed B.; Caughey, Byron

    2014-01-01

    Structures of the infectious form of prion protein (e.g. PrPSc or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrPSc retain most of the native ?-helices of the normal, noninfectious prion protein, cellular prion protein (PrPC), but evidence is accumulating that these helices are absent in PrPSc amyloid. Moreover, recombinant PrPC can form amyloid fibrils in vitro that have parallel in-register intermolecular ?-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily ?-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrPSc fibrils, can have stable parallel in-register ?-sheet cores. These simulations revealed that the C-terminal residues ?124227 more readily adopt stable tightly packed structures than the N-terminal residues ?90123 in the absence of cofactors. Variations in the placement of turns and loops that link the ?-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures. PMID:25028516

  17. The Hofmeister effect on amyloid formation using yeast prion protein

    PubMed Central

    Yeh, Victor; Broering, James M; Romanyuk, Andrey; Chen, Buxin; Chernoff, Yury O; Bommarius, Andreas S

    2010-01-01

    A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross-? aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N-proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to ?-sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid-forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B-viscosity coefficient of the Jones-Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series. PMID:19890987

  18. Prion diseases.

    PubMed

    Takada, Leonel T; Geschwind, Michael D

    2013-09-01

    Prion diseases are a group of diseases caused by abnormally conformed infectious proteins, called prions. They can be sporadic (Jakob-Creutzfeldt disease [JCD]), genetic (genetic JCD, Gerstmann-Sträussler-Scheinker, and familial fatal insomnia), or acquired (kuru, variant JCD, and iatrogenic JCD). The clinical features associated with each form of prion disease, the neuroimaging findings, cerebrospinal fluid markers, and neuropathological findings are reviewed. Sporadic JCD is the most common form of human prion disease, and will be discussed in detail. Genetic prion diseases are caused by mutations in the prion-related protein gene (PRNP), and they are classified based on the mutation, clinical phenotype, and neuropathological features. Acquired prion diseases fortunately are becoming rarer, as awareness of transmission risk has led to implementation of measures to prevent such occurrences, but continued surveillance is necessary to prevent future cases. Treatment and management issues are also discussed. PMID:24234356

  19. Disease-associated prion protein oligomers inhibit the 26S proteasome.

    PubMed

    Kristiansen, Mark; Deriziotis, Pelagia; Dimcheff, Derek E; Jackson, Graham S; Ovaa, Huib; Naumann, Heike; Clarke, Anthony R; van Leeuwen, Fijs W B; Menndez-Benito, Victoria; Dantuma, Nico P; Portis, John L; Collinge, John; Tabrizi, Sarah J

    2007-04-27

    The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein. PMID:17466621

  20. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

    SciTech Connect

    Nieznanski, Krzysztof . E-mail: k.nieznanski@nencki.gov.pl; Podlubnaya, Zoya A.; Nieznanska, Hanna

    2006-10-13

    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 {approx}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.

  1. Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

    PubMed Central

    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

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

    PubMed

    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. Molecular dynamics studies on the buffalo prion protein.

    PubMed

    Zhang, Jiapu; Wang, Feng; Chatterjee, Subhojyoti

    2016-04-01

    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 Biomolecular Structure and Dynamics 28(3), 355-361)), (ii) at D178, there is a HB Y169-D178 and a polar contact R164-D178 for BufPrP(C) instead of a polar contact Q168-D178 for bovine PrP(C) (C.J. Cheng, & V. Daggett. (2014). Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH. Biomolecules 4(1), 181-201), (iii) BufPrP(C) owns three 310 helices at 125-127, 152-156, and in the β2-α2 loop, respectively, and (iv) in the β2-α2 loop, there is a strong π-π stacking and a strong π-cation F175-Y169-R164.(N)NH2, has been discovered. PMID:26043781

  4. Conservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity*

    PubMed Central

    Harrison, Christopher F.; Lawson, Victoria A.; Coleman, Bradley M.; Kim, Yong-Sun; Masters, Colin L.; Cappai, Roberto; Barnham, Kevin J.; Hill, Andrew F.

    2010-01-01

    Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrPC) into an abnormal isoform (PrPSc) that has infectious properties. The hydrophobic domain of PrPC is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrPC. Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrPC drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrPC are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrPSc, suggesting these residues provide conformational flexibility. These data suggest that this region of PrPC is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics. PMID:20356832

  5. Prion Protein Protects against Renal Ischemia/Reperfusion Injury

    PubMed Central

    Siedlak, Sandra; Abouelsaad, Mai; Zeng, Liang; Zhou, Xuefeng; O'Toole, John; Das, Alvin S.; Kofskey, Diane; Warren, Miriam; Bian, Zehua; Cui, Yuqi; Tan, Tao; Kresak, Adam; Wyza, Robert E.; Petersen, Robert B.; Wang, Gong-Xian; Kong, Qingzhong; Wang, Xinglong; Sedor, John; Zhu, Xiongwei; Zhu, Hua; Zou, Wen-Quan

    2015-01-01

    The cellular prion protein (PrPC), a protein most noted for its link to prion diseases, has been found to play a protective role in ischemic brain injury. To investigate the role of PrPC in the kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type (WT) and PrPC knockout (KO) mice that were subjected to 30-min of renal ischemia followed by 1, 2, or 3 days of reperfusion. Renal dysfunction and structural damage was more severe in KO than in WT mice. While PrP was undetectable in KO kidneys, Western blotting revealed an increase in PrP in IR-injured WT kidneys compared to sham-treated kidneys. Compared to WT, KO kidneys exhibited increases in oxidative stress markers heme oxygenase-1, nitrotyrosine, and Nε-(carboxymethyl)lysine, and decreases in mitochondrial complexes I and III. Notably, phosphorylated extracellular signal-regulated kinase (pERK) staining was predominantly observed in tubular cells from KO mice following 2 days of reperfusion, a time at which significant differences in renal dysfunction, histological changes, oxidative stress, and mitochondrial complexes between WT and KO mice were observed. Our study provides the first evidence that PrPC may play a protective role in renal IR injury, likely through its effects on mitochondria and ERK signaling pathways. PMID:26327228

  6. Transport of the pathogenic prion protein through landfill materials.

    PubMed

    Jacobson, Kurt H; Lee, Seunghak; McKenzie, Debbie; Benson, Craig H; Pedersen, Joel A

    2009-03-15

    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 RecentTSE outbreaks in domesticated and wild animal populations have created the need for safe and effective disposal of large quantities of potentially infected materials. Here, we report results of a study to evaluate the potential for transport of PrP(TSE) derived from carcasses and associated wastes in municipal solid waste (MSW) landfills. Column experiments were conducted to evaluate PrP(TSE) transport in quartz sand, two fine-textured burial soils currently used in landfill practice, a green waste residual material (a potential burial material), and fresh and aged MSW. PrP(TSE) was retained by quartz sand and the fine-textured burial soils, with no detectable PrP(TSE) eluted over more than 40 pore volumes. In contrast, PrP(TSE) was more mobile in MSW and green waste residual. Transport parameters were estimated from the experimental data and used to model PrP(TSE) migration in a MSW landfill. To the extent that the PrP(TSE) used mimics that released from decomposing carcasses and the column experiments adequately simulate prion transport through burial soils, burial of CWD-infected materials at MSW landfills could provide secure containment of PrP(TSE) provided reasonable burial strategies (e.g., encasement in fine-grained soil) are used. PMID:19368208

  7. Prion formation, but not clearance, is supported by protein misfolding cyclic amplification

    PubMed Central

    Shikiya, Ronald A; Eckland, Thomas E; Young, Alan J; Bartz, Jason C

    2014-01-01

    Prion diseases are fatal transmissible neurodegenerative disorders that affect animals including humans. The kinetics of prion infectivity and PrPSc accumulation can differ between prion strains and within a single strain in different tissues. The net accumulation of PrPSc in animals is controlled by the relationship between the rate of PrPSc formation and clearance. Protein misfolding cyclic amplification (PMCA) is a powerful technique that faithfully recapitulates PrPSc formation and prion infectivity in a cell-free system. PMCA has been used as a surrogate for animal bioassay and can model species barriers, host range, strain co-factors and strain interference. In this study we investigated if degradation of PrPSc and/or prion infectivity occurs during PMCA. To accomplish this we performed PMCA under conditions that do not support PrPSc formation and did not observe either a reduction in PrPSc abundance or an extension of prion incubation period, compared to untreated control samples. These results indicate that prion clearance does not occur during PMCA. These data have significant implications for the interpretation of PMCA based experiments such as prion amplification rate, adaptation to new species and strain interference where production and clearance of prions can affect the outcome. PMID:25482601

  8. Computational analysis of candidate prion-like proteins in bacteria and their role.

    PubMed

    Iglesias, Valentin; de Groot, Natalia S; Ventura, Salvador

    2015-01-01

    Prion proteins were initially associated with diseases such as Creutzfeldt Jakob and transmissible spongiform encephalopathies. However, deeper research revealed them as versatile tools, exploited by the cells to execute fascinating functions, acting as epigenetic elements or building membrane free compartments in eukaryotes. One of the most intriguing properties of prion proteins is their ability to propagate a conformational assembly, even across species. In this context, it has been observed that bacterial amyloids can trigger the formation of protein aggregates by interacting with host proteins. As our life is closely linked to bacteria, either through a parasitic or symbiotic relationship, prion-like proteins produced by bacterial cells might play a role in this association. Bioinformatics is helping us to understand the factors that determine conformational conversion and infectivity in prion-like proteins. We have used PrionScan to detect prion domains in 839 different bacteria proteomes, detecting 2200 putative prions in these organisms. We studied this set of proteins in order to try to understand their functional role and structural properties. Our results suggest that these bacterial polypeptides are associated to peripheral rearrangement, macromolecular assembly, cell adaptability, and invasion. Overall, these data could reveal new threats and therapeutic targets associated to infectious diseases. PMID:26528269

  9. Computational analysis of candidate prion-like proteins in bacteria and their role

    PubMed Central

    Iglesias, Valentin; de Groot, Natalia S.; Ventura, Salvador

    2015-01-01

    Prion proteins were initially associated with diseases such as Creutzfeldt Jakob and transmissible spongiform encephalopathies. However, deeper research revealed them as versatile tools, exploited by the cells to execute fascinating functions, acting as epigenetic elements or building membrane free compartments in eukaryotes. One of the most intriguing properties of prion proteins is their ability to propagate a conformational assembly, even across species. In this context, it has been observed that bacterial amyloids can trigger the formation of protein aggregates by interacting with host proteins. As our life is closely linked to bacteria, either through a parasitic or symbiotic relationship, prion-like proteins produced by bacterial cells might play a role in this association. Bioinformatics is helping us to understand the factors that determine conformational conversion and infectivity in prion-like proteins. We have used PrionScan to detect prion domains in 839 different bacteria proteomes, detecting 2200 putative prions in these organisms. We studied this set of proteins in order to try to understand their functional role and structural properties. Our results suggest that these bacterial polypeptides are associated to peripheral rearrangement, macromolecular assembly, cell adaptability, and invasion. Overall, these data could reveal new threats and therapeutic targets associated to infectious diseases. PMID:26528269

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

    PubMed

    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

  11. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  12. Chaperone Proteins Select and Maintain [PIN+] Prion Conformations in Saccharomyces cerevisiae

    PubMed Central

    Lancaster, David L.; Dobson, C. Melissa; Rachubinski, Richard A.

    2013-01-01

    Prions are proteins that can adopt different infectious conformations known as strains or variants, each with a distinct, epigenetically inheritable phenotype. Mechanisms by which prion variants are determined remain unclear. Here we use the Saccharomyces cerevisiae prion Rnq1p/[PIN+] as a model to investigate the effects of chaperone proteins upon prion variant determination. We show that deletion of specific chaperone genes alters [PIN+] variant phenotypes, including [PSI+] induction efficiency, Rnq1p aggregate morphology/size and variant dominance. Mating assays demonstrate that gene deletion-induced phenotypic changes are stably inherited in a non-Mendelian manner even after restoration of the deleted gene, confirming that they are due to a bona fide change in the [PIN+] variant. Together, our results demonstrate a role for chaperones in regulating the prion variant complement of a cell. PMID:23148221

  13. Characterization of Conformation-dependent Prion Protein Epitopes*

    PubMed Central

    Kang, Hae-Eun; Weng, Chu Chun; Saijo, Eri; Saylor, Vicki; Bian, Jifeng; Kim, Sehun; Ramos, Laylaa; Angers, Rachel; Langenfeld, Katie; Khaychuk, Vadim; Calvi, Carla; Bartz, Jason; Hunter, Nora; Telling, Glenn C.

    2012-01-01

    Whereas prion replication involves structural rearrangement of cellular prion protein (PrPC), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(2730), a protease-resistant counterpart of the pathogenic scrapie form (PrPSc) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(2730) both renature to a common structure that reconstitutes the globular domain. PMID:22948149

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

    NASA Astrophysics Data System (ADS)

    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.

  15. CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome

    PubMed Central

    Mehrabian, Mohadeseh; Brethour, Dylan; MacIsaac, Sarah; Kim, Jin Kyu; Gunawardana, C . Geeth; Wang, Hansen; Schmitt-Ulms, Gerold

    2014-01-01

    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. PMID:25490046

  16. Structural Studies of Truncated Forms of the Prion Protein PrP

    PubMed Central

    Wan, William; Wille, Holger; Stöhr, Jan; Kendall, Amy; Bian, Wen; McDonald, Michele; Tiggelaar, Sarah; Watts, Joel C.; Prusiner, Stanley B.; Stubbs, Gerald

    2015-01-01

    Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrPSc. PMID:25809267

  17. Follicular Dendritic Cell-Specific Prion Protein (PrPc) Expression Alone Is Sufficient to Sustain Prion Infection in the Spleen

    PubMed Central

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

    2011-01-01

    Prion diseases are characterised by the accumulation of PrPSc, an abnormally folded isoform of the cellular prion protein (PrPC), in affected tissues. Following peripheral exposure high levels of prion-specific PrPSc accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrPC 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 PrPC expression was specifically switched on or off only on FDC. We show that PrPC-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrPC-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 PrPC expression on FDC. PMID:22144895

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

    PubMed Central

    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 expressing anchorless or anchored PrP. Results To create a localized initial point of infection, we microinjected scrapie in a 0.5 microliter volume in the striatum. In this experiment, PrPres and gliosis were first detected in both types of mice at 40 days post-inoculation near the needle track. In mice with anchored PrP, PrPres appeared to spread via neurons to distant connected brain areas by the clinical endpoint at 150 days post-inoculation. This PrPres was rarely associated with blood vessels. In contrast, in mice with anchorless PrP, PrPres spread did not follow neuronal circuitry, but instead followed a novel slower pattern utilizing the drainage system of the brain interstitial fluid (ISF) including perivascular areas adjacent to blood vessels, subependymal areas and spaces between axons in white matter tracts. Conclusions In transgenic mice expressing anchorless PrP small amyloid-seeding PrPres aggregates appeared to be transported in the ISF, thus spreading development of cerebral amyloid angiopathy (CAA) throughout the brain. Spread of amyloid seeding by ISF may also occur in multiple human brain diseases involving CAA. PMID:24447368

  19. Mouse Prion Protein Polymorphism Phe-108/Val-189 Affects the Kinetics of Fibril Formation and the Response to Seeding

    PubMed Central

    Cortez, Leonardo M.; Kumar, Jitendra; Renault, Ludovic; Young, Howard S.; Sim, Valerie L.

    2013-01-01

    Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrPC) into an amyloidogenic ?-sheet infectious form (PrPSc). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnpa, encoding the polymorphism Leu-108/Thr-189) or b (Prnpb, Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrPSc. The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrPC into PrPSc remain unclear. Here we show that recombinant PrPa and PrPb aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrPb exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrPb is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP. PMID:23283973

  20. Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1

    PubMed Central

    Kayatekin, Can; Matlack, Kent E. S.; Hesse, William R.; Guan, Yinghua; Chakrabortee, Sohini; Russ, Jenny; Wanker, Erich E.; Shah, Jagesh V.; Lindquist, Susan

    2014-01-01

    Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings. PMID:25092318

  1. Prions in Yeast

    PubMed Central

    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-? aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

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

    PubMed

    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

  3. A sequencing strategy for identifying variation throughout the prion gene of BSE-affected cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle prion gene (PRNP) polymorphisms have been associated with bovine spongiform encephalopathy (BSE) susceptibility. We developed a method for sequencing bovine PRNP through all exons, introns and part of the promoter (25.2 kb) that accounts for known variation. The method can be used to detect...

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

    PubMed

    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

  5. The story of stolen chaperones: how overexpression of Q/N proteins cures yeast prions.

    PubMed

    Derkatch, Irina L; Liebman, Susan W

    2013-01-01

    Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller "seeds." Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI(+)] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI(+)] aggregates to enlarge. This is incompatible with a previously proposed "capping" model where the overexpressed Q/N-rich protein poisons, or "caps," the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI(+)] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI(+)] aggregates in a way that blocks their shearing. PMID:23924684

  6. Application of protein misfolding cyclic amplification to detection of prions in anaerobic digestate.

    PubMed

    Gilroyed, Brandon H; Braithwaite, Shannon L; Price, Luke M; Reuter, Tim; Czub, Stefanie; Graham, Catherine; Balachandran, Arumuga; McAllister, Tim A; Belosevic, Miodrag; Neumann, Norman F

    2015-11-01

    The exceptional physio-chemical resistance of prions to established decontamination procedures poses a challenge to assessing the suitability of applied inactivation methods. Prion detection is limited by the sensitivity level of Western blotting or by the cost and time factors of bioassays. In addition, prion detection assays can be limited by either the unique or complex nature of matrices associated with environmental samples. To investigate anaerobic digestion (AD) as a practical and economical approach for potential conversion of specified risk materials (SRM) into value added products (i.e., renewable energy), challenges associated with detection of prions in a complex matrix need to be overcome to determine potential inactivation. Protein misfolding cyclic amplification (PMCA) assay, with subsequent Western blot visualization, was used to detect prions within the AD matrix. Anaerobic digestate initially inhibited the PMCA reaction and/or Western blot detection. However, at concentrations of ≤1% of anaerobic digestate, 263K scrapie prions could be amplified and semi-quantitatively detected. Infectious 263K prions were also proven to be bioavailable in the presence of high concentrations of digestate (10-90%). Development of the PMCA application to digestate provides extremely valuable insight into the potential degradation and/or fate of prions in complex biological matrices without requiring expensive and time-consuming bioassays. PMID:26272376

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

    PubMed

    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

  8. The prion protein is neuroprotective against retinal degeneration in vivo.

    PubMed

    Frigg, Rico; Wenzel, Andreas; Samardzija, Marijana; Oesch, Bruno; Wariwoda, Hedwig; Navarini, Alexander A; Seeliger, Mathias W; Tanimoto, Naoyuki; Rem, Charlotte; Grimm, Christian

    2006-12-01

    A common feature of neurodegenerative disorders is acute or progressive loss of neurons due to apoptosis. The pathological isoform of the prion protein is associated with retinal apoptosis and the cellular isoform (PrPc) has been shown to mediate protection from apoptosis in cell culture and in neonatal retinal explants. Using a model of light-induced photoreceptor apoptosis, we show in vivo that the levels of PrPc expression in the retina inversely correlate with the susceptibility of photoreceptors to light damage. Dissection of apoptotic signalling cascades suggests that PrPc acts neuroprotectively downstream of AP-1 induction. Our results reveal PrP as a neuroprotective/anti-apoptotic factor in vivo and suggest that PrPc may function as a guardian of neuronal integrity. PMID:16952355

  9. Interaction between Prion Protein and Aβ Amyloid Fibrils Revisited

    PubMed Central

    2014-01-01

    Recent studies indicate that the pathogenesis of Alzheimer disease may be related to the interaction between prion protein (PrP) and certain oligomeric species of Aβ peptide. However, the mechanism of this interaction remains unclear and controversial. Here we provide direct experimental evidence that, in addition to previously demonstrated binding to Aβ oligomers, PrP also interacts with mature Aβ fibrils. However, contrary to the recent claim that PrP causes fragmentation of Aβ fibrils into oligomeric species, no evidence for such a disassembly could be detected in the present study. In contrast, our data indicate that the addition of PrP to preformed Aβ fibrils results in a lateral association of individual fibrils into larger bundles. These findings have potentially important implications for understanding the mechanism by which PrP might impact Aβ toxicity as well as for the emerging efforts to use PrP-derived compounds as inhibitors of Aβ-induced neurodegeneration. PMID:24669873

  10. The Cellular Prion Protein: A Player in Immunological Quiescence

    PubMed Central

    Bakkebø, Maren K.; Mouillet-Richard, Sophie; Espenes, Arild; Goldmann, Wilfred; Tatzelt, Jörg; Tranulis, Michael A.

    2015-01-01

    Despite intensive studies since the 1990s, the physiological role of the cellular prion protein (PrPC) remains elusive. Here, we present a novel concept suggesting that PrPC contributes to immunological quiescence in addition to cell protection. PrPC is highly expressed in diverse organs that by multiple means are particularly protected from inflammation, such as the brain, eye, placenta, pregnant uterus, and testes, while at the same time it is expressed in most cells of the lymphoreticular system. In this paradigm, PrPC serves two principal roles: to modulate the inflammatory potential of immune cells and to protect vulnerable parenchymal cells against noxious insults generated through inflammation. Here, we review studies of PrPC physiology in view of this concept. PMID:26388873

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

    PubMed

    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

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

    PubMed

    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

  13. Capillary electromigration based techniques in diagnostics of prion protein caused diseases.

    PubMed

    Sobrova, Pavlina; Ryvolova, Marketa; Adam, Vojtech; Kizek, Rene

    2012-12-01

    Transmissible spongiform encephalopathies are a group of fatal neurodegenerative diseases with long incubation time. This group includes Creutzfeld-Jakob disease, kuru, scrapie, chronic wasting disease, and bovine spongiform encephalopathy. Sensitive and specific detection of abnormal prion protein as "a source agent" of the above-mentioned diseases in blood could provide a diagnostic test or a screening assay for animal and human prion protein diseases diagnostics. Therefore, diagnostic tests for prion protein diseases represent unique challenge requiring development of novel assays exploiting properties of prion protein complex. Presently, diagnostic methods such as protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, fluorescence correlation spectroscopy, and/or flow microbead immunoassay are used for abnormal prion protein (PrP(Sc) ) detection. On the other hand, using of CE for PrP(Sc) detection in body fluids is an attractive alternative; it has been already applied for the blood samples of infected sheep, elk, chimpanzee, as well as humans. In this review, assays for prion protein detection are summarized with special attention to capillary electromigration based techniques, such as CE, CIEF, and/or CGE. The potential of the miniaturized and integrated lab-on-chip devices is highlighted, emphasizing recent advances of this field in the proteomic analysis. PMID:23161211

  14. The unfolded state of the murine prion protein and properties of single-point mutants related to human prion diseases.

    PubMed

    Gerum, Christian; Schlepckow, Kai; Schwalbe, Harald

    2010-08-01

    The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process. PMID:20541558

  15. Mouse Prion Protein (PrP) Segment 100 to 104 Regulates Conversion of PrPC to PrPSc in Prion-Infected Neuroblastoma Cells

    PubMed Central

    Hara, Hideyuki; Okemoto-Nakamura, Yuko; Shinkai-Ouchi, Fumiko; Hanada, Kentaro; Yamakawa, Yoshio

    2012-01-01

    Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrPSc; PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrPC) to PrPSc and the subsequent conversion of PrPC to PrPSc. We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrPC and PrPSc. Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrPSc state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrPSc and interfered with the conversion of endogenous MoPrPC. The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrPSc. Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrPC reduced the accumulation of PrPSc after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrPC plays a key role in conversion after binding to MoPrPSc. PMID:22398286

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

    PubMed

    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-1?), 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-1? 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-1? 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-1? stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106-126)-induced neurotoxicity via the up-regulation of HIF-1? 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

  17. PrionScan: an online database of predicted prion domains in complete proteomes

    PubMed Central

    2014-01-01

    Background Prions are a particular type of amyloids related to a large variety of important processes in cells, but also responsible for serious diseases in mammals and humans. The number of experimentally characterized prions is still low and corresponds to a handful of examples in microorganisms and mammals. Prion aggregation is mediated by specific protein domains with a remarkable compositional bias towards glutamine/asparagine and against charged residues and prolines. These compositional features have been used to predict new prion proteins in the genomes of different organisms. Despite these efforts, there are only a few available data sources containing prion predictions at a genomic scale. Description Here we present PrionScan, a new database of predicted prion-like domains in complete proteomes. We have previously developed a predictive methodology to identify and score prionogenic stretches in protein sequences. In the present work, we exploit this approach to scan all the protein sequences in public databases and compile a repository containing relevant information of proteins bearing prion-like domains. The database is updated regularly alongside UniprotKB and in its present version contains approximately 28000 predictions in proteins from different functional categories in more than 3200 organisms from all the taxonomic subdivisions. PrionScan can be used in two different ways: database query and analysis of protein sequences submitted by the users. In the first mode, simple queries allow to retrieve a detailed description of the properties of a defined protein. Queries can also be combined to generate more complex and specific searching patterns. In the second mode, users can submit and analyze their own sequences. Conclusions It is expected that this database would provide relevant insights on prion functions and regulation from a genome-wide perspective, allowing researches performing cross-species prion biology studies. Our database might also be useful for guiding experimentalists in the identification of new candidates for further experimental characterization. PMID:24498877

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

    SciTech Connect

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

    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.

  19. Nitric oxide induces prion protein via MEK and p38 MAPK signaling.

    PubMed

    Wang, Vinchi; Chuang, Tzu-Chao; Hsu, Yaw-Don; Chou, Wei-Yuan; Kao, Ming-Ching

    2005-07-22

    The prion diseases or transmissible spongiform encephalopathy, such as human Creutzfeldt-Jakob disease (CJD) and so-called mad cow disease, are attributed to the causative agent, the scrapie variant of prion protein (PrP(Sc)) which causes fatal neurodegeneration. To investigate if stresses such as nitric oxide (NO) induced the cellular isoform of prion protein (PrP(C)), lipopolysaccharide, and sodium nitroprusside were used to treat N2a and NT2 cells, which resulted in elevated levels of the PRNP mRNA and prion protein. The signaling pathway for the NO-induced PrP(C) production involved guanylyl cyclase, MEK, and p38 MAPK as shown by the effect of specific pharmacological inhibitors ODQ, PD98059, and SB203580, respectively. Knowing the PrP induction by the biologically existing stimulus, this study provides useful information about the possible cellular mechanism and strategies for the treatment of CJD. PMID:15936714

  20. Prion Protein-Specific Antibodies-Development, Modes of Action and Therapeutics Application

    PubMed Central

    Rovis, Tihana Lenac; Legname, Giuseppe

    2014-01-01

    Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are lethal neurodegenerative disorders involving the misfolding of the host encoded cellular prion protein, PrPC. This physiological form of the protein is expressed throughout the body, and it reaches the highest levels in the central nervous system where the pathology occurs. The conversion into the pathogenic isoform denoted as prion or PrPSc is the key event in prion disorders. Prominent candidates for the treatment of prion diseases are antibodies and their derivatives. Anti-PrPC antibodies are able to clear PrPSc from cell culture of infected cells. Furthermore, application of anti-PrPC antibodies suppresses prion replication in experimental animal models. Major drawbacks of immunotherapy are immune tolerance, the risks of neurotoxic side effects, limited ability of compounds to cross the blood-brain barrier and their unfavorable pharmacokinetic. The focus of this review is to recapitulate the current understanding of the molecular mechanisms for antibody mediated anti-prion activity. Although relevant for designing immunotherapeutic tools, the characterization of key antibody parameters shaping the molecular mechanism of the PrPC to PrPSc conversion remains elusive. Moreover, this review illustrates the various attempts towards the development of anti-PrP antibody compounds and discusses therapeutic candidates that modulate PrP expression. PMID:25275428

  1. 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

    PubMed Central

    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

  2. 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.

    PubMed

    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

  3. Prion protein promotes kidney iron uptake via its ferrireductase activity.

    PubMed

    Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

    2015-02-27

    Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrP(C)) from its normal conformation to an aggregated, PrP-scrapie (PrP(Sc)) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrP(C) in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrP(C) is lacking. Kidney provides a relevant model for this evaluation because PrP(C) is expressed in the kidney, and ?370 ?g of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrP(C) promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of (59)Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP(-/-)) mouse kidney relative to PrP(+/+) controls. Selective in vivo radiolabeling of plasma NTBI with (59)Fe revealed similar results. Expression of exogenous PrP(C) in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of (59)Fe-NTBI and to a smaller extent (59)Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP(?51-89)) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrP(C) to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrP(C) promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

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

    PubMed Central

    Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    ABSTRACT 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 (PrPSc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrPSc 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 PrPSc. 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 PrPSc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both. PMID:26284507

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

    PubMed

    Edskes, H K; Wickner, R B

    2000-06-01

    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 for de novo prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras-cAMP pathway. We find that in mks1Delta strains, de novo generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2(Val19) increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation. PMID:10823922

  6. POLYMORPHIC DISTRIBUTION OF THE PRION PROTEIN (PRNP) GENE IN SCRAPIE-INFECTED SHEEP FLOCKS IN WHICH EMBRYO TRANSFER WAS USED TO CIRCUMVENT THE TRANSMISSIONS OF SCRAPIE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic sequence of the ovine prion protein (PrP) gene between codons 102 and 175, with emphasis on ovine PrP gene codons 136 and 171, was determined in scrapie-exposed Suffolk embryo donors and in offspring from those donors that had been transferred to scrapie-free recipient ewes. The most com...

  7. Detection of Prion Protein in Urine-Derived Injectable Fertility Products by a Targeted Proteomic Approach

    PubMed Central

    Van Dorsselaer, Alain; Carapito, Christine; Delalande, François; Schaeffer-Reiss, Christine; Thierse, Daniele; Diemer, Hélène; McNair, Douglas S.; Krewski, Daniel; Cashman, Neil R.

    2011-01-01

    Background Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD) is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD), a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG). Methodology/Principal Findings Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG) and highly-purified urinary human menopausal gonadotropin (hMG-HP) products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. Conclusions/Significance The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and young women exposed to prions can be expected to survive an incubation period associated with a minimal inoculum. The risks of urine-derived fertility products could now outweigh their benefits, particularly considering the availability of recombinant products. PMID:21448279

  8. Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein.

    PubMed

    Satheeshkumar, K S; Jayakumar, R

    2003-07-01

    Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127). PMID:12829502

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Systemic Delivery of siRNA Down Regulates Brain Prion Protein and Ameliorates Neuropathology in Prion Disorder

    PubMed Central

    Resina, Sarah; Brillaud, Elsa; Casanova, Danielle; Vincent, Charles; Hamela, Claire; Poupeau, Sophie; Laffont, Mathieu; Gabelle, Audrey; Delaby, Constance; Belondrade, Maxime; Arnaud, Jacques-Damien; Alvarez, Maria-Teresa; Maurel, Jean-Claude; Maurel, Patrick; Crozet, Carole

    2014-01-01

    One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrPC level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders. PMID:24551164

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

    SciTech Connect

    Magzoub, Mazin; Sandgren, Staffan; Lundberg, Pontus; Oglecka, Kamila; Lilja, Johanna; Wittrup, Anders; Goeran Eriksson, L.E.; Langel, Ulo; Belting, Mattias . E-mail: mattias.belting@med.lu.se; Graeslund, Astrid . E-mail: astrid@dbb.su.se

    2006-09-22

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation

    PubMed Central

    Winkler, Juliane; Tyedmers, Jens

    2012-01-01

    Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA+ hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70Hsp100 cooperation at the surface of protein aggregates and prion fibrils. PMID:22869599

  14. Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation.

    PubMed

    Winkler, Juliane; Tyedmers, Jens; Bukau, Bernd; Mogk, Axel

    2012-08-01

    Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70-Hsp100 cooperation at the surface of protein aggregates and prion fibrils. PMID:22869599

  15. Amyloid-? nanotubes are associated with prion protein-dependent synaptotoxicity

    PubMed Central

    Nicoll, Andrew J.; Panico, Silvia; Freir, Darragh B.; Wright, Daniel; Terry, Cassandra; Risse, Emmanuel; Herron, Caroline E.; OMalley, Tiernan; Wadsworth, Jonathan D. F.; Farrow, Mark A.; Walsh, Dominic M.; Saibil, Helen R.; Collinge, John

    2013-01-01

    Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-? protein (A?) have important roles in Alzheimers disease with toxicities mimicked by synthetic A?142. However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of A?142 after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient A? assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in A?-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of A? nanotubes or their interaction with PrP might have a role in treatment of Alzheimers disease. PMID:24022506

  16. Combined copper/zinc attachment to prion protein

    NASA Astrophysics Data System (ADS)

    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.

  17. Superoxide dismutase activity of Cu-bound prion protein

    NASA Astrophysics Data System (ADS)

    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.

  18. Circadian regulation of prion protein messenger RNA in the rat forebrain: a widespread and synchronous rhythm.

    PubMed

    Cagampang, F R; Whatley, S A; Mitchell, A L; Powell, J F; Campbell, I C; Coen, C W

    1999-01-01

    Although the expression of the normal prion protein in the host is critical to the development of transmissible spongiform encephalopathies, the physiological role of this protein and the processes regulating its expression remain obscure. We now report that the messenger RNA for the prion protein is regulated in the rat brain in a marked circadian manner not only in the suprachiasmatic nuclei, the principal site for the generation of mammalian circadian rhythms, but also in other forebrain regions. The data show a remarkable consistency in the concurrence of a single peak of prion protein messenger RNA at each of the sites early in the animal's phase of increased locomotor activity; behavioural arousal does not, however, appear to affect this expression. We believe this to be the first study demonstrating that the expression of prion protein messenger RNA can change over a relatively short period in vivo. The results are discussed with reference to the range of recently discovered "clock-related" transcripts which also have widespread tissue expression; these include the messenger RNAs for D-box binding protein and thyroid embryonic factor, transcription factors which bind to the prion protein promoter. PMID:10391428

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

    PubMed

    Yang, Zi; Hong, Joo Y; Derkatch, Irina L; Liebman, Susan W

    2013-01-01

    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(+)]. PMID:23358669

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

    PubMed

    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. PMID:26721882

  1. Scrapie-like prion protein accumulates in aggresomes of cyclosporinA-treated cells

    PubMed Central

    Cohen, Ehud; Taraboulos, Albert

    2003-01-01

    Prion diseases are infectious, sporadic and inherited fatal neurodegenerations that are propagated by an abnormal refolding of the cellular prion protein PrPC. Which chaperones assist the normal folding of PrPC is unknown. The linkage of familial Gerstmann StrusslerScheinker (GSS) syndrome with proline substitutions in PrP raised the prospect that peptidylprolyl cistrans isomerases (PPIases) may play a role in normal PrP metabolism. Here we used cyclo sporinA (CsA), an immunosuppressant, to inhibit the cyclophilin family of PPIases in cultured cells. CsA-treated cells accumulated proteasome-resistant, prion-like PrP species, which deposited in long-lived aggresomes. PrP aggresomes also formed with disease-linked proline mutants when proteasomes were inhibited. These results suggest mechanisms whereby abnormally folded cytosolic PrP may in some cases participate in the development of spontaneous and inherited prion diseases. PMID:12554642

  2. Ectopic expression of prion protein (PrP) in T lymphocytes or hepatocytes of PrP knockout mice is insufficient to sustain prion replication

    PubMed Central

    Raeber, Alex J.; Sailer, Andreas; Hegyi, Ivan; Klein, Michael A.; Rlicke, Thomas; Fischer, Marek; Brandner, Sebastian; Aguzzi, Adriano; Weissmann, Charles

    1999-01-01

    The cellular form of the Prion protein (PrPC) is necessary for prion replication in mice. To determine whether it is also sufficient, we expressed PrP under the control of various cell- or tissue-specific regulatory elements in PrP knockout mice. The interferon regulatory factor-1 promoter/E? enhancer led to high PrP levels in the spleen and low PrP levels in the brain. Following i.p. scrapie inoculation, high prion titers were found in the spleen but not in the brain at 2 weeks and 6 months, showing that the lymphoreticular system by itself is competent to replicate prions. PrP expression directed by the Lck promoter resulted in high PrP levels on T lymphocytes only but, surprisingly, did not allow prion replication in the thymus, spleen, or brain following i.p. inoculation. A third transgenic line, which expressed PrP in the liver under the control of the albumin promoter/enhanceralbeit at low levelsalso failed to replicate prions. These results show that expression of PrP alone is not sufficient to sustain prion replication and suggest that additional components are needed. PMID:10097150

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

    PubMed

    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 vitro prion 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. PMID:25867521

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

    PubMed Central

    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

  5. Prions and Protein Assemblies that Convey Biological Information in Health and Disease.

    PubMed

    Sanders, David W; Kaufman, Sarah K; Holmes, Brandon B; Diamond, Marc I

    2016-02-01

    Prions derived from the prion protein (PrP) were first characterized as infectious agents that transmit pathology between individuals. However, the majority of cases of neurodegeneration caused by PrP prions occur sporadically. Proteins that self-assemble as cross-beta sheet amyloids are a defining pathological feature of infectious prion disorders and all major age-associated neurodegenerative diseases. In fact, multiple non-infectious proteins exhibit properties of template-driven self-assembly that are strikingly similar to PrP. Evidence suggests that like PrP, many proteins form aggregates that propagate between cells and convert cognate monomer into ordered assemblies. We now recognize that numerous proteins assemble into macromolecular complexes as part of normal physiology, some of which are self-amplifying. This review highlights similarities among infectious and non-infectious neurodegenerative diseases associated with prions, emphasizing the normal and pathogenic roles of higher-order protein assemblies. We propose that studies of the structural and cellular biology of pathological versus physiological aggregates will be mutually informative. PMID:26844828

  6. A Novel Human Disease with Abnormal Prion Protein Sensitive to Protease

    PubMed Central

    Gambetti, Pierluigi; Dong, Zhiqian; Yuan, Jue; Xiao, Xiangzhu; Zheng, Mengjie; Alshekhlee, Amer; Castellani, Rudy; Cohen, Mark; Barria, Marcelo A; Gonzalez-Romero, D; Belay, Ermias D.; Schonberger, Lawrence B.; Marder, Karen; Harris, Carrie; Burke, James R.; Montine, Thomas; Wisniewski, Thomas; Dickson, Dennis W.; Soto, Claudio; Hulette, Christine M.; Mastrianni, James A.; Kong, Qingzhong; Zou, Wen-Quan

    2009-01-01

    Objective To report a novel prion disease characterized by distinct histopathological and immunostaining features, and associated with an abnormal isoform of the prion protein (PrP) that, contrary to the common prion diseases, is predominantly sensitive to protease digestion. Methods Eleven subjects were investigated at the National Prion Disease Pathology Surveillance Center (NPDPSC) for clinical, histopathological, immunohistochemical, genotypical and PrP characteristics. Results Patients presented with behavioral and psychiatric manifestations on average at 62 years while mean disease duration was 20 months. The type of spongiform degeneration, the PrP immunostaining pattern and the presence of micro-plaques distinguished these cases from those with known prion diseases. Typical protease-resistant PrP was undetectable in the cerebral neocortex with standard diagnostic procedures. Following enrichment, abnormal PrP was detected at concentrations 16 times lower than common prion diseases; it included nearly four times less protease-resistant PrP, which formed a distinct electrophoretic profile. The subjects examined comprised about 3% of sporadic cases evaluated by the NPDPSC. Although several subjects had family history of dementia, no mutations were found in the PrP gene open reading frame (ORF). Interpretation The distinct histopathological, PrP immunohistochemical and physical-chemical features along with the homogeneous genotype indicate that this is a previously unidentified type of disease involving the prion protein, which we designated protease-sensitive prionopathy or PSPr. PSPr is not very rare among prion diseases, and might be even more prevalent than our data indicate since PSPr cases are likely to be also classified within the group of non-Alzheimer dementias. PMID:18571782

  7. Prion protein lacks robust cytoprotective activity in cultured cells

    PubMed Central

    Christensen, Heather M; Harris, David A

    2008-01-01

    Background The physiological function of the cellular prion protein (PrPC) remains unknown. However, PrPC has been reported to possess a cytoprotective activity that prevents death of neurons and other cells after a toxic stimulus. To explore this effect further, we attempted to reproduce several of the assays in which a protective activity of PrP had been previously demonstrated in mammalian cells. Results In the first set of experiments, we found that PrP over-expression had a minimal effect on the death of MCF-7 breast carcinoma cells treated with TNF-? and Prn-p0/0 immortalized hippocampal neurons (HpL3-4 cells) subjected to serum deprivation. In the second set of assays, we observed only a small difference in viability between cerebellar granule neurons cultured from PrP-null and control mice in response to activation of endogenous or exogenous Bax. Conclusion Taken together, our results suggest either that cytoprotection is not a physiologically relevant activity of PrPC, or that PrPC-dependent protective pathways operative in vivo are not adequately modeled by these cell culture systems. We suggest that cell systems capable of mimicking the neurotoxic effects produced in transgenic mice by N-terminally deleted forms of PrP or Doppel may represent more useful tools for analyzing the cytoprotective function of PrPC. PMID:18718018

  8. Genomic structure of the human prion protein gene.

    PubMed

    Puckett, C; Concannon, P; Casey, C; Hood, L

    1991-08-01

    Creutzfeld-Jacob disease and Gerstmann-Strussler syndrome are rare degenerative disorders of the nervous system which have been genetically linked to the prion protein (PrP) gene. The PrP gene encodes a host glycoprotein of unknown function and is located on the short arm of chromosome 20, a region with few known genes or anonymous markers. The complete structure of the PrP gene in man has not been determined despite considerable interest in its relationship to these unusual disorders. We have determined that the human PrP gene has the same simple genomic structure seen in the hamster gene and consists of two exons and a single intron. In contrast to the hamster PrP gene the human gene appears to have a single major transcriptional start site. The region immediately 5' of the transcriptional start site of the human PrP gene demonstrates the GC-rich features commonly seen in housekeeping genes. Curiously, the genomic clone we have isolated contains a 24-bp deletion that removes one of five octameric peptide repeats predicted to form a B-pleated sheet in this region of the PrP. We have also identified 5' of the PrP gene an RFLP which has a high degree of heterozygosity and which should serve as a useful marker for the pter-12 region of human chromosome 20. PMID:1678248

  9. The prion protein family: a view from the placenta

    PubMed Central

    Makzhami, Samira; Passet, Bruno; Halliez, Sophie; Castille, Johan; Moazami-Goudarzi, Katayoun; Duchesne, Amandine; Vilotte, Marthe; Laude, Hubert; Mouillet-Richard, Sophie; Bringue, Vincent; Vaiman, Daniel; Vilotte, Jean-Luc

    2014-01-01

    Based on its developmental pattern of expression, early studies suggested the implication of the mammalian Prion protein PrP, a glycosylphosphatidylinositol-anchored ubiquitously expressed and evolutionary conserved glycoprotein encoded by the Prnp gene, in early embryogenesis. However, gene invalidation in several species did not result in obvious developmental abnormalities and it was only recently that it was associated in mice with intra-uterine growth retardation and placental dysfunction. A proposed explanation for this lack of easily detectable developmental-related phenotype is the existence in the genome of one or more gene (s) able to compensate for the absence of PrP. Indeed, two other members of the Prnp gene family have been recently described, Doppel and Shadoo, and the consequences of their invalidation alongside that of PrP tested in mice. No embryonic defect was observed in mice depleted for Doppel and PrP. Interestingly, the co-invalidation of PrP and Shadoo in two independent studies led to apparently conflicting observations, with no apparent consequences in one report and the observation of a developmental defect of the ectoplacental cone that leads to early embryonic lethality in the other. This short review aims at summarizing these recent, apparently conflicting data highlighting the related biological questions and associated implications in terms of animal and human health. PMID:25364742

  10. The Toxicity of the PrP106-126 Prion Peptide on Cultured Photoreceptors Correlates with the Prion Protein Distribution in the Mammalian and Human Retina

    PubMed Central

    Gong, Jie; Jellali, Abdeljelil; Forster, Valrie; Mutterer, Jrme; Dubus, Elisabeth; Altrock, Wilko D.; Sahel, Jos A.; Rendon, Alvaro; Picaud, Serge

    2007-01-01

    In patients affected by Creutzfeldt-Jakob disease and in animals affected by transmissible spongiform encephalopathies, retinal functions are altered, and major spongiform changes are observed in the outer plexiform layer where photoreceptors have their synaptic terminals. In the present study, the prion protein PrPc was found to form aggregates in rod photoreceptor terminals from both rat and human retina, whereas no labeling was observed in cone photoreceptors. Discrete staining was also detected in the inner plexiform layer where the prion protein was located at human amacrine cell synapses. In mixed porcine retinal cell cultures, the PrP106-126 prion peptide triggered a 61% rod photoreceptor cell loss by apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling, whereas cone photoreceptors were not affected. Amacrine cells were also reduced by 47% in contrast to ganglion cells. Although this cell loss was associated with a 5.5-fold increase in microglial cells, the strict correlation between the PrPc prion protein expression and the peptide toxicity suggested that this toxicity did not rely on the release of a toxic compound by glial cells. These results provide new insights into the retinal pathophysiology of prion diseases and illustrate advantages of adult retinal cell cultures to investigate prion pathogenic mechanisms. PMID:17392170

  11. Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie

    PubMed Central

    Wolf, Hanna; Hossinger, Andr; Fehlinger, Andrea; Bttner, Sven; Sim, Valerie; McKenzie, Debbie; Vorberg, Ina M.

    2015-01-01

    Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system. PMID:26581229

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

    PubMed Central

    Black, Stefanie A. G.; Stys, Peter K.; Zamponi, Gerald W.; Tsutsui, Shigeki

    2014-01-01

    Although it is well established that misfolding of the cellular prion protein (PrPC) into the β-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 (Aβ) peptides, suggesting a role for PrPC in Alzheimer's disease (AD). Our recent findings suggest that Aβ 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 AD and other neurodegenerative disorders involving dysfunction of PrPC. PMID:25364752

  13. Potential roles for prions and protein-only inheritance in cancer

    PubMed Central

    Antony, H; Wiegmans, AP; Wei, MQ; Chernoff, YO; Khanna, KK; Munn, AL

    2011-01-01

    Inherited mutations are known to cause familial cancers. However, the cause of sporadic cancers, which likely represent the majority of cancers, is yet to be elucidated. Sporadic cancers contain somatic mutations (including oncogenic mutations), however, the origin of these mutations is unclear. An intriguing possibility is that a stable alteration occurs in somatic cells prior to oncogenic mutations and promotes the subsequent accumulation of oncogenic mutations. This review explores the possible role of prions and protein-only inheritance in cancer. Genetic studies using lower eukaryotes, primarily yeast, have identified a large number of proteins as prions that confer dominant phenotypes with cytoplasmic (non-Mendelian) inheritance. Many of these have mammalian functional homologs. The human prion protein (PrP) is known to cause neurodegenerative diseases and has now been found to be up-regulated in multiple cancers. PrP expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Epigenetic changes in gene expression and hyper-activation of MAP kinase (MAPK) signalling, processes that in lower eukaryotes are affected by prions, play important roles in oncogenesis in humans. Prion phenomena in yeast appear to be influenced by stresses and there is considerable evidence for association of some amyloids with biologically positive functions. This suggests that if protein-only somatic inheritance exists in mammalian cells, it might contribute to cancer phenotypes. Here we highlight evidence in the literature for an involvement of prion or prion-like mechanisms in cancer and how they may in the future be viewed as diagnostic markers and potential therapeutic targets. PMID:22138778

  14. Molecular Features of the Copper Binding Sites in the Octarepeat Domain of the Prion Protein

    PubMed Central

    Burns, Colin S.; Aronoff-Spencer, Eliah; Dunham, Christine M.; Lario, Paula; Avdievich, Nikolai I.; Antholine, William E.; Olmstead, Marilyn M.; Vrielink, Alice; Gerfen, Gary J.; Peisach, Jack; Scott, William G.; Millhauser, Glenn L.

    2010-01-01

    Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 6091. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 1376013771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(2328, 5791) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified GlyCu linkage is unstable below pH ?6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form. PMID:11900542

  15. Prion protein NMR structures of elk and of mouse/elk hybrids.

    PubMed

    Gossert, Alvar D; Bonjour, Sophie; Lysek, Dominikus A; Fiorito, Francesco; Wüthrich, Kurt

    2005-01-18

    The NMR structure of the recombinant elk prion protein (ePrP), which represents the cellular isoform (ePrPC) in the healthy organism, is described here. As anticipated from the highly conserved amino acid sequence, ePrPC has the same global fold as other mammalian prion proteins (PrPs), with a flexibly disordered "tail" of residues 23-124 and a globular domain 125-226 with three alpha-helices and a short antiparallel beta-sheet. However, ePrPC shows a striking local structure variation when compared with most other mammalian PrPs, in particular human, bovine, and mouse PrPC. A loop of residues 166-175, which links the beta-sheet with the alpha2-helix and is part of a hypothetical "protein X" epitope, is outstandingly well defined, whereas this loop is disordered in the other species. Based on NMR structure determinations of two mouse PrP variants, mPrP[N174T] and mPrP[S170N,N174T], this study shows that the structured loop in ePrPC relates to these two local amino acid exchanges, so that mPrP[S170N,N174T] exactly mimics ePrPC. These results are evaluated in the context of recent reports on chronic wasting disease (CWD) in captive and free-ranging deer and elk in the U.S. and Canada, and an animal model is proposed for support of future research on CWD. PMID:15647363

  16. STI571 protects neuronal cells from neurotoxic prion protein fragment-induced apoptosis.

    PubMed

    Pan, Yaoqian; Sun, Liyong; Wang, Jihong; Fu, Wenzhuo; Fu, Yongyao; Wang, Jin; Tong, Yigang; Pan, Bo

    2015-06-01

    Prion diseases are neurodegenerative disorders caused by the accumulation of misfolded prion proteins [scrapie form of PrP (PrP(Sc))]. PrP(Sc) accumulation in the brain causes neurotoxicity by inducing mitochondrial-apoptotic pathways. Neurodegeneration can be prevented by imatinib mesylate (Gleevec or STI571) that regulates c-Abl tyrosine kinases, which elicit protective effects in neurodegenerative disease models. However, the protective effect of STI571 against prion disease remains unknown. In the present study, the effect of STI571 on prion peptide-induced neuronal death was investigated. Results showed that STI571 rescued neurons from PrP106-126-induced neurotoxicity by preventing mitochondrial dysfunction. STI571-inhibited c-Abl tyrosine kinases prevented PrP106-126-induced reduction in mitochondrial potential, Bax translocation to the mitochondria and cytochrome c release. The protective effect of STI571 against mitochondrial dysfunction was related to the activation of BIM expression. This study is the first to demonstrate the protective effect of STI571 against prion-mediated neurotoxicity. Our results suggested that imatinib mesylate treatment may be a novel therapeutic strategy to treat prion-mediated neurotoxicity. PMID:25681617

  17. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

    PubMed

    Haas, Laura T; Salazar, Santiago V; Kostylev, Mikhail A; Um, Ji Won; Kaufman, Adam C; Strittmatter, Stephen M

    2016-02-01

    Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-? oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-? oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

  18. Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent.

    PubMed

    Bera, Alakesh; Nandi, Pradip K

    2014-12-01

    Nucleic acid can catalyze the conversion of ?-helical cellular prion protein to ?-sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered ?-helical structure is considered to be a necessary step for the structural conversion to its ?-sheet rich isoform, we have studied the unfolding of the ?-helical globular 121-231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein. PMID:25271002

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

    PubMed

    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

  20. Neuroimmunoendocrine Regulation of the Prion Protein in Neutrophils*

    PubMed Central

    Mariante, Rafael M.; Nóbrega, Alberto; Martins, Rodrigo A. P.; Areal, Rômulo B.; Bellio, Maria; Linden, Rafael

    2012-01-01

    The prion protein (PrPC) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrPC may be implicated in other degenerative conditions often associated with inflammation. PrPC is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrPC in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrPC in mouse neutrophils. Up-regulation of PrPC was dependent on the serum content of TGF-β and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-β, either alone or in combination, directly up-regulated PrPC in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrPC. Moreover, GC also mediated up-regulation of PrPC in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrPC presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrPC in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems. PMID:22910907

  1. Molecular dynamics simulations of wild-type and point mutation human prion protein at normal and elevated temperature.

    PubMed

    el-Bastawissy, E; Knaggs, M H; Gilbert, I H

    2001-01-01

    This paper describes molecular dynamics simulations of prion protein at 300 and 500 K. This was undertaken to gain insight into the factors involved in the stability of prion protein. Simulations were done using the Particle Mesh Ewald (PME) method using a homology model of the C-terminal fragment of human prion protein and the NMR structure of the human prion protein. The simulations at both 300 and 500 K were stable. Simulations were also undertaken with a mutant known to be associated with prion disease: Asp178Asn. The Asp178Asn simulation trajectory was observed to be much less stable than for the wild-type protein trajectory. Significant breakdown in secondary structure was observed for Asp178Asn at 500 K. PMID:11775001

  2. SIRP? polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells

    PubMed Central

    Nuvolone, Mario; Kana, Veronika; Hutter, Gregor; Sakata, Daiji; Mortin-Toth, Steven M.; Russo, Giancarlo

    2013-01-01

    Prnp?/? mice lack the prion protein PrPC and are resistant to prion infections, but variable phenotypes have been reported in Prnp?/? mice and the physiological function of PrPC remains poorly understood. Here we examined a cell-autonomous phenotype, inhibition of macrophage phagocytosis of apoptotic cells, previously reported in Prnp?/? mice. Using formal genetic, genomic, and immunological analyses, we found that the regulation of phagocytosis previously ascribed to PrPC is instead controlled by a linked locus encoding the signal regulatory protein ? (Sirpa). These findings indicate that control of phagocytosis was previously misattributed to the prion protein and illustrate the requirement for stringent approaches to eliminate confounding effects of flanking genes in studies modeling human disease in gene-targeted mice. The plethora of seemingly unrelated functions attributed to PrPC suggests that additional phenotypes reported in Prnp?/? mice may actually relate to Sirpa or other genetic confounders. PMID:24145514

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

    PubMed

    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 the same pH environment. In addition, we found an interesting result that the salt bridges such as ASP201-ARG155, ASP177-ARG163 contribute greatly to the structural stability of RaPrP. PMID:24184221

  4. Lack of TAR-DNA binding protein-43 (TDP-43) pathology in human prion diseases

    PubMed Central

    Isaacs, A M; Powell, C; Webb, T E; Linehan, J M; Collinge, J; Brandner, S

    2008-01-01

    Aims TAR-DNA binding protein-43 (TDP-43) is the major ubiquitinated protein in the aggregates in frontotemporal dementia with ubiquitin-positive, tau-negative inclusions and motor neurone disease. Abnormal TDP-43 immunoreactivity has also been described in Alzheimer's disease, Lewy body diseases and Guam parkinsonism–dementia complex. We therefore aimed to determine whether there is TDP-43 pathology in human prion diseases, which are characterised by variable deposition of prion protein (PrP) aggregates in the brain as amyloid plaques or more diffuse deposits. Material and methods TDP-43, ubiquitin and PrP were analysed by immunohistochemistry and double-labelling immunofluorescence, in sporadic, acquired and inherited forms of human prion disease. Results Most PrP plaques contained ubiquitin, while synaptic PrP deposits were not associated with ubiquitin. No abnormal TDP-43 inclusions were identified in any type of prion disease case, and TDP-43 did not co-localize with ubiquitin-positive PrP plaques or with diffuse PrP aggregates. Conclusions These data do not support a role for TDP-43 in prion disease pathogenesis and argue that TDP-43 inclusions define a distinct group of neurodegenerative disorders. PMID:18657254

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

    PubMed

    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

  6. Stress-dependent Proteolytic Processing of the Actin Assembly Protein Lsb1 Modulates a Yeast Prion*

    PubMed Central

    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.

    2014-01-01

    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

  7. Investigating the spreading and toxicity of prion-like proteins using the metazoan model organism C. elegans.

    PubMed

    Nussbaum-Krammer, Carmen I; Neto, Mrio F; Brielmann, Rene M; Pedersen, Jesper S; Morimoto, Richard I

    2015-01-01

    Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans. PMID:25591151

  8. Accumulation of prion protein in muscle fibers of experimental chloroquine myopathy: in vivo model for deposition of prion protein in non-neuronal tissues.

    PubMed

    Furukawa, Hisako; Doh-ura, Katsumi; Sasaki, Kensuke; Iwaki, Toru

    2004-07-01

    Prion protein (PrP) is known to accumulate in some non-neuronal tissues under conditions unrelated to prion diseases. The biochemical and biological nature of such accumulated PrP molecules, however, has not been fully evaluated. In this study, we established experimental myopathy in hamsters by long-term administration of chloroquine, and we examined the nature of the PrP molecules that accumulated. PrP accumulation was immunohistochemically demonstrated in autophagic vacuoles in degenerated muscle fibers, and this was accompanied by the accumulation of other molecules related to the neuropathogenesis of prion diseases such as clathrin, cathepsin B, heparan sulfate, and apolipoprotein J. Accumulated PrP molecules were partially insoluble in detergent solution and were slightly less sensitive to proteinase K digestion than normal cellular PrP. Muscle homogenates containing these PrP molecules did not cause disease in inoculated hamsters. The findings indicate that the PrP molecules that accumulated in muscle fibers have distinct biochemical and biological properties. Therefore, experimental chloroquine myopathy is a novel and useful model to investigate the mechanism of deposition of PrP in non-neuronal tissues and might provide new insights in the pathogenesis of prion diseases. PMID:15122307

  9. Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

    PubMed Central

    Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J.; Iyer, K. Swaminathan; Ho, Diwei; Newcombe, Estella A.; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S.

    2015-01-01

    Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration. PMID:26283796

  10. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    PubMed Central

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-01-01

    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. PMID:26490404

  11. Complex folding and misfolding effects of deer-specific amino acid substitutions in the ?2-?2 loop of murine prion protein

    NASA Astrophysics Data System (ADS)

    Agarwal, Sonya; Dring, 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 ?2?2 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 ?2?2 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 ?2?2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  12. Cellular Prion Protein Promotes Brucella Infection into Macrophages

    PubMed Central

    Watarai, Masahisa; Kim, Suk; Erdenebaatar, Janchivdorj; Makino, Sou-ichi; Horiuchi, Motohiro; Shirahata, Toshikazu; Sakaguchi, Suehiro; Katamine, Shigeru

    2003-01-01

    The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complexassociated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection. PMID:12847134

  13. Deficiency of prion protein induces impaired autophagic flux in neurons

    PubMed Central

    Shin, Hae-Young; Park, Jeong-Ho; Carp, Richard I.; Choi, Eun-Kyoung; Kim, Yong-Sun

    2014-01-01

    Normal cellular prion protein (PrPC) is highly expressed in the central nervous system. The Zrich I Prnp-deficient mouse strain did not show an abnormal phenotype in initial studies, however, in later studies, deficits in exploratory behavior and short- and long-term memory have been revealed. In the present study, numerous autophagic vacuoles were found in neurons from Zrich I Prnp-deficient mice. The autophagic accumulation in the soma of cortical neurons in Zrich I Prnp-deficient mice was observed as early as 3 months of age, and in the hippocampal neurons at 6 months of age. Specifically, there is accumulation of electron dense pigments associated with autophagy in the neurons of Zrich I Prnp-deficient mice. Furthermore, autophagic accumulations were observed as early as 3 months of age in the CA3 region of hippocampal and cerebral cortical neuropils. The autophagic vacuoles increased with age in the hippocampus of Zrich I Prnp-deficient mice at a faster rate and to a greater extent than in normal C57BL/6J mice, whereas the cortex exhibited high levels that were maintained from 3 months old in Zrich I Prnp-deficient mice. The pigmented autophagic accumulation is due to the incompletely digested material from autophagic vacuoles. Furthermore, a deficiency in PrPC may disrupt the autophagic flux by inhibiting autophagosome-lysosomal fusion. Overall, our results provide insight into the protective role of PrPC in neurons, which may play a role in normal behavior and other brain functions. PMID:25202268

  14. Hematological shift in goat kids naturally devoid of prion protein

    PubMed Central

    Reiten, Malin R.; Bakkeb, Maren K.; Brun-Hansen, Hege; Lewandowska-Sabat, Anna M.; Olsaker, Ingrid; Tranulis, Michael A.; Espenes, Arild; Boysen, Preben

    2015-01-01

    The physiological role of the cellular prion protein (PrPC) is incompletely understood. The expression of PrPC in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrPC knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrPC. Here we report hematological and immunological analyses of homozygous goat kids lacking PrPC (PRNPTer/Ter) compared to heterozygous (PRNP+/Ter) and normal (PRNP+/+) kids. Levels of cell surface PrPC and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNPTer/Ter, intermediate in PRNP+/Ter and high in PRNP+/+ kids. The PRNPTer/Ter animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrPC has a role in bone marrow physiology and warrant further studies of PrPC in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrPC-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrPC. PMID:26217662

  15. Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains

    PubMed Central

    Duque Velásquez, Camilo; Kim, Chiye; Herbst, Allen; Daude, Nathalie; Garza, Maria Carmen; Wille, Holger; Aiken, Judd

    2015-01-01

    ABSTRACT Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrPC). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrPC modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95+) with distinct biological properties. Transmission of first-passage tg60CWD-H95+ isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrPC primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrPC molecules results in the generation of novel CWD strains. IMPORTANCE CWD prions are contagious among wild and captive cervids in North America and in South Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrPC) to the emergence of a novel CWD strain (H95+). We show that, upon infection, deer expressing H95-PrPC molecules accumulated a mixture of CWD strains that selectively propagated depending on the PRNP genotype of the host in which they were passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95+ CWD prions. The potential for the generation of novel strains raises the possibility of an expanded host range for CWD. PMID:26423950

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

    SciTech Connect

    Ji, Hong-Fang; Zhang, Hong-Yu . E-mail: zhanghy@sdut.edu.cn

    2007-08-03

    To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP{sup C} and CkPrP{sup C}), 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 HuPrP{sup C} is comparable with that of CkPrP{sup C}, which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP{sup C}.

  17. [Protective Activity of Prion Protein Fragments after Immunization of Annimals with Experimentally Induced Alzheimer's Disease].

    PubMed

    Volpina, O M; Volkova, T D; Medvinskaya, N I; Kamynina, A V; Zaporozhskaya, Y V; Aleksandrova, I J; Koroev, D O; Samokhin, A N; Nesterova, I V; Deygin, V I; Bobkova, N V

    2015-01-01

    The prion protein is considered as one of the membrane targets of neurotoxic beta-amyloid during Alzheimer's disease development. We have chosen and synthesized 17-33, 23-33, 95-110 and 101-115 prion fragments involved in beta-amyloid binding. The effect of immunization with the peptides on the features of Alzheimer's disease was investigated in animals with an experimentally induced form of the disease. It was shown that immunization either with peptide 17-33 or with protein conjugates of peptides 23-33 and 101-115 increases the level of brain beta-amyloid and improves morfofunctional state of the brain. PMID:26165121

  18. A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity.

    PubMed Central

    Harris, D A; Falls, D L; Johnson, F A; Fischbach, G D

    1991-01-01

    The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor-inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well. Images PMID:1715573

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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,...

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

    PubMed

    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. PMID:18443224

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

    PubMed

    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

  2. Baicalein prevents human prion protein-induced neuronal cell death by regulating JNK activation.

    PubMed

    Moon, Ji-Hong; Park, Sang-Youel

    2015-02-01

    Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal isoform of the protease-insensitive isoform (PrPSc) of prion protein. Human prion protein fragment 106?126 [PrP (106?126)] contains most of the pathological characteristics associated with PrPSc. Although a number of compounds have been identified to inhibit PrP accumulation or dissolve fibrils and aggregates in vitro, there is currenlty no treatment available for these progressive neurodegenerative diseases. Baicalein, the dried root of Scutellaria baicalensis (S. baicalensis) Georgi (known as Huang-qin in traditional Chinese medicine) has been reported to exert neuroprotective effects on neurodegenerative diseases. In the present study, we investigated the effects of baicalein on the development of prion diseases using SH-SY5Y and SK-N-SH cells in vitro. We found that baicalein protected the cells against PrP?induced neuronal cell death by inhibiting the production of reactive oxygen species (ROS) and mitochondrial dysfunction using ROS detection assay and MTP assay. We demonstrated that baicalein treatment regulated the phosphorylation of c-Jun N-terminal kinase (JNK) by using western blot analysis and Annexin V assay. Our data suggest that baicalein has potential for use as a therapeutic drug for the treatment of various neurodegenerative diseases, including prion diseases. PMID:25435015

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

    USGS Publications Warehouse

    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.

  4. Mechanisms of triggering H1 helix in prion proteins unfolding revealed by molecular dynamic simulation

    NASA Astrophysics Data System (ADS)

    Tseng, Chih-Yuan; Lee, H. C.

    2006-03-01

    In template-assistance model, normal Prion protein (PrP^C), the pathogen to cause several prion diseases such as Creutzfeldt-Jakob (CJD) in human, Bovine Spongiform Encephalopathy (BSE) in cow, and scrapie in sheep, converts to infectious prion (PrP^Sc) through a transient interaction with PrP^Sc. Furthermore, conventional studies showed S1-H1-S2 region in PrP^C to be the template of S1-S2 β-sheet in PrP^Sc, and Prion protein's conformational conversion may involve an unfolding of H1 and refolding into β-sheet. Here we prepare several mouse prion peptides that contain S1-H1-S2 region with specific different structures, which are corresponding to specific interactions, to investigate possible mechanisms to trigger H1 α-helix unfolding process via molecular dynamic simulation. Three properties, conformational transition, salt-bridge in H1, and hydrophobic solvent accessible surface (SAS) are analyzed. From these studies, we found the interaction that triggers H1 unfolding to be the one that causes dihedral angle at residue Asn^143 changes. Whereas interactions that cause S1 segment's conformational changes play a minor in this process. These studies offers an additional evidence for template-assistance model.

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

    USGS Publications Warehouse

    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.

  6. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease

    PubMed Central

    King, Oliver D.; Gitler, Aaron D.; Shorter, James

    2012-01-01

    Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable prion domain enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimers disease and Huntingtons disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion-like domains of RNA-binding proteins could underlie the classical non-cell-autonomous emanation of neurodegenerative pathology from originating epicenters to neighboring portions of the nervous system. PMID:22445064

  7. Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to ?-Sheet Rich Oligomers and Fibrils

    PubMed Central

    Ladner-Keay, Carol L.; Griffith, Bethany J.; Wishart, David S.

    2014-01-01

    The formation of ?-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into ?-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to ?-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of ?-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to ?-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable ?-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC). PMID:24892647

  8. Selective vulnerability to neurodegenerative disease: the curious case of Prion Protein

    PubMed Central

    Jackson, Walker S.

    2014-01-01

    The mechanisms underlying the selective targeting of specific brain regions by different neurodegenerative diseases is one of the most intriguing mysteries in medicine. For example, it is known that Alzheimers disease primarily affects parts of the brain that play a role in memory, whereas Parkinsons disease predominantly affects parts of the brain that are involved in body movement. However, the reasons that other brain regions remain unaffected in these diseases are unknown. A better understanding of the phenomenon of selective vulnerability is required for the development of targeted therapeutic approaches that specifically protect affected neurons, thereby altering the disease course and preventing its progression. Prion diseases are a fascinating group of neurodegenerative diseases because they exhibit a wide phenotypic spectrum caused by different sequence perturbations in a single protein. The possible ways that mutations affecting this protein can cause several distinct neurodegenerative diseases are explored in this Review to highlight the complexity underlying selective vulnerability. The premise of this article is that selective vulnerability is determined by the interaction of specific protein conformers and region-specific microenvironments harboring unique combinations of subcellular components such as metals, chaperones and protein translation machinery. Given the abundance of potential contributory factors in the neurodegenerative process, a better understanding of how these factors interact will provide invaluable insight into disease mechanisms to guide therapeutic discovery. PMID:24396151

  9. MANGANESE UPREGULATES CELLULAR PRION PROTEINS AND INHIBITS THE RATE OF PROTEINASE-K DEPENDENT LIMITED PROTEOLYSIS IN NEURONAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The key 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 cations such as copper to th...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Clinical features in prion protein-deficient and wild-type cattle inoculated with transmissible mink encephalopathy (TME)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Transmissible spongiform encephalopathies (TSEs) or prion diseases are caused by the propagation of a misfolded form (PrP**d) of the normal cellular prion protein, PrP**c. Recently, we have reported the generation and characterization of PrP**C-deficient cattle (PrP-/-) produced by a seq...

  13. Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

    PubMed

    Moore, Roger A; Herzog, Christian; Errett, John; Kocisko, David A; Arnold, Kevin M; Hayes, Stanley F; Priola, Suzette A

    2006-03-01

    A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD. PMID:16452616

  14. Peptidylarginine deiminase and protein citrullination in prion diseases: strong evidence of neurodegeneration.

    PubMed

    Jang, Byungki; Ishigami, Akihito; Maruyama, Naoki; Carp, Richard I; Kim, Yong-Sun; Choi, Eun-Kyoung

    2013-01-01

    The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration. PMID:23022892

  15. Requirements for Mutant and Wild-Type Prion Protein Misfolding In Vitro

    PubMed Central

    Noble, Geoffrey P.; Walsh, Daniel J.; Miller, Michael B.; Jackson, Walker S.; Supattapone, Surachai

    2015-01-01

    Misfolding of the prion protein (PrP) plays a central role in the pathogenesis of infectious, sporadic, and inherited prion diseases. Here we use a chemically defined prion propagation system to study misfolding of the pathogenic PrP mutant D177N in vitro. This mutation causes PrP to misfold spontaneously in the absence of cofactor molecules in a process dependent on time, temperature, pH, and intermittent sonication. Spontaneously misfolded mutant PrP is able to template its unique conformation onto wild-type PrP substrate in a process that requires a phospholipid activity distinct from that required for the propagation of infectious prions. Similar results were obtained with a second pathogenic PrP mutant, E199K, but not with the polymorphic substitution M128V. Moreover, wild-type PrP inhibits mutant PrP misfolding in a dose-dependent manner, and cofactor molecules can antagonize this effect. These studies suggest that interactions between mutant PrP, wild-type PrP, and other cellular factors may control the rate of PrP misfolding in inherited prion diseases. PMID:25584902

  16. Variant Creutzfeldt-Jakob Disease With Extremely Low Lymphoreticular Deposition of Prion Protein

    PubMed Central

    Mead, Simon; Wadsworth, Jonathan D. F.; Porter, Marie-Claire; Linehan, Jacqueline M.; Pietkiewicz, Wojciech; Jackson, Graham S.; Brandner, Sebastian; Collinge, John

    2014-01-01

    IMPORTANCE Human transmission of bovine spongiform encephalopathy causes the fatal neurodegenerative condition variant Creutzfeldt-Jakob disease (vCJD) and, based on recent human prevalence studies, significant subclinical prion infection of the UK population. To date, all clinical cases have been fatal, totaling 228 mostly young adults residing in the United Kingdom. OBSERVATIONS Here we describe the investigation and case history of a patient recently diagnosed as having vCJD in the United Kingdom. Although his presentation, imaging findings, cerebrospinal fluid investigation results, and clinical progression were typical of other cases, tonsillar biopsy and subsequent examination of multiple tissues at autopsy showed minimal deposition of disease-associated prion protein in peripheral lymphoreticular tissue. The result of a blood test for vCJD, the Direct Detection Assay for vCJD, was negative. CONCLUSIONS AND RELEVANCE These findings suggest that some patients with vCJD have very low peripheral prion colonization and therefore may not have detectable prion deposition in diagnostic tonsillar biopsy or markers of prion infection in blood. These results have implications for accurate interpretation of diagnostic tests and prevalence studies based on lymphoreticular tissue or blood. PMID:24445428

  17. The CPEB3 Protein Is a Functional Prion that Interacts with the Actin Cytoskeleton.

    PubMed

    Stephan, Joseph S; Fioriti, Luana; Lamba, Nayan; Colnaghi, Luca; Karl, Kevin; Derkatch, Irina L; Kandel, Eric R

    2015-06-23

    The mouse cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a translational regulator implicated in long-term memory maintenance. Invertebrate orthologs of CPEB3 in Aplysia and Drosophila are functional prions that are physiologically active in the aggregated state. To determine if this principle applies to the mammalian CPEB3, we expressed it in yeast and found that it forms heritable aggregates that are the hallmark of known prions. In addition, we confirm in the mouse the importance of CPEB3's prion formation for CPEB3 function. Interestingly, deletion analysis of the CPEB3 prion domain uncovered a tripartite organization: two aggregation-promoting domains surround a regulatory module that affects interaction with the actin cytoskeleton. In all, our data provide direct evidence that CPEB3 is a functional prion in the mammalian brain and underline the potential importance of an actin/CPEB3 feedback loop for the synaptic plasticity underlying the persistence of long-term memory. PMID:26074072

  18. Inactivation of prion proteins via covalent grafting with methoxypoly(ethylene glycol).

    PubMed

    Scott, Mark D

    2006-01-01

    Transmissible spongiform encephalopathies (TSE) such as bovine spongiform encephalitis (BSE), Creutzfeld-Jakob disease (CJD) as well as other proteinaceous infectious particles (prions) mediated diseases have emerged as a significant concern in transfusion medicine. This concern is derived from both the disease causing potential of prion contaminated blood products but also due to tremendous impact of the active deferral of current and potential blood donors due to their extended stays in BSE prevalent countries (e.g., the United Kingdom). To date, there are no effective means by which infectious prion proteins can be inactivated in cellular and acellular blood products. Based on current work on the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to proteins, viruses, and anuclear, and nucleated cells, it is hypothesized that the conversion of the normal PrP protein to its mutant conformation can be prevented by the covalent grafting of mPEG to the mutant protein. Inactivation of infective protein particles (prions) in both cellular blood products as well as cell free solutions (e.g., clotting factors) could be of medical/commercial value. It is hypothesized that consequent to the covalent modification of donor-derived prions with mPEG the requisite nucleation of the normal and mutant PrP proteins is inhibited due to the increased solubility of the modified mutant PrP and that the conformational conversion arising from the mutant PrP is prevented due to obscuration of protein charge by the heavily hydrated and neutral mPEG polymers, as well as by direct steric hindrance of the interaction due to the highly mobile polymer graft. PMID:16242248

  19. Cellular prion protein (PrPC) and its role in stress responses

    PubMed Central

    Zeng, Liang; Zou, Wenquan; Wang, Gongxian

    2015-01-01

    Investigation of the physiological function of cellular prion protein (PrPC) has been developed by the generation of transgenic mice, however, the pathological mechanisms related to PrPC in prion diseases such as transmissible spongiform encephalopathies (TSEs) are still abstruse. Regardless of some differences, most studies describe the neuroprotective role of PrPC in environmental stresses. In this review, we will update the current knowledge on the responses of PrPC to various stresses, especially those correlated with cell signaling and neural degeneration, including ischemia, oxidative stress, inflammation and autophagy. PMID:26221369

  20. Heterogeneity of the Abnormal Prion Protein (PrPSc) of the Chandler Scrapie Strain

    PubMed Central

    Kasai, Kazuo; Iwamaru, Yoshifumi; Masujin, Kentaro; Imamura, Morikazu; Mohri, Shirou; Yokoyama, Takashi

    2013-01-01

    The pathological prion protein, PrPSc, displays various sizes of aggregates. In this study, we investigated the conformation, aggregation stability and proteinase K (PK)-sensitivity of small and large PrPSc aggregates of mouse-adapted prion strains. We showed that small PrPSc aggregates, previously thought to be PK-sensitive, are resistant to PK digestion. Furthermore, we showed that small PrPSc aggregates of the Chandler scrapie strain have greater resistance to PK digestion and aggregation-denaturation than large PrPSc aggregates of this strain. We conclude that this strain consists of heterogeneous PrPSc. PMID:25436883

  1. New Insights into Metal Interactions with the Prion Protein

    PubMed Central

    McDonald, Alex; Pushie, M. Jake; Millhauser, Glenn L.; George, Graham N.

    2013-01-01

    Copper coordination to the prion protein (PrP) has garnered considerable interest for almost 20 years, due in part to the possibility that this interaction may be part of the normal function of PrP. The most characterized form of copper binding to PrP has been Cu2+ interaction with the conserved tandem repeats in the N-terminal domain of PrP, termed the octarepeats, with many studies focusing on single and multiple repeats of PHGGGWGQ. Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used in several previous instances to characterize the solution structure of Cu2+ binding into the peptide backbone in the HGGG portion of the octarepeats. All previous EXAFS studies, however, have benefitted from crystallographic structure information for [CuII (Ac-HGGGW-NH2)–2H], but have not conclusively demonstrated that the complex EXAFS spectrum represents the same coordination environment for Cu2+ bound to the peptide backbone. Density functional structure calculations as well as full multiple scattering EXAFS curve fitting analysis are brought to bear on the predominant coordination mode for Cu2+ with the Ac-PHGGGWGQ-NH2 peptide at physiological pH, under high Cu2+ occupancy conditions. In addition to the structure calculations, which provide a thermodynamic link to structural information, methods are also presented for extensive deconvolution of the EXAFS spectrum. We demonstrate how the EXAFS data can be analyzed to extract the maximum structural information and arrive at a structural model that is significantly improved over previous EXAFS characterizations. The EXAFS spectrum for the chemically reduced form of copper binding to the Ac-PHGGGWGQ-NH2 peptide is presented, which is best modeled as a linear 2-coordinate species with a single His imidazole ligand and a water molecule. The extent of in situ photoreduction of the copper center during standard data collection is also presented and EXAFS curve fitting of the photoreduced species reveals an intermediate structure that is similar to the Cu2+ form with reduced coordination number. PMID:24102071

  2. Familial mutations and the thermodynamic stability of the recombinant human prion protein.

    PubMed

    Swietnicki, W; Petersen, R B; Gambetti, P; Surewicz, W K

    1998-11-20

    Hereditary forms of human prion disease are linked to specific mutations in the PRNP gene. It has been postulated that these mutations may facilitate the pathogenic process by reducing the stability of the prion protein (PrP). To test this hypothesis, we characterized the recombinant variants of human PrP(90-231) containing point mutations corresponding to Gerstmann-Straussler-Scheinker disease (P102L), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (M129/D178N). The first two of these mutants could be recovered form from the periplasmic space of Escherichia coli in a soluble form, whereas the D178N variant aggregated into inclusion bodies. The secondary structure of the two soluble variants was essentially identical to that of the wild-type protein. The thermodynamic stability of these mutants was assessed by unfolding in guanidine hydrochloride and thermal denaturation. The stability properties of the P102L variant were indistinguishable from those of wild-type PrP, whereas the E200K mutation resulted in a very small destabilization of the protein. These data, together with the predictive analysis of other familial mutations, indicate that some hereditary forms of prion disease cannot be rationalized using the concept of mutation-induced thermodynamic destabilization of the cellular prion protein. PMID:9813003

  3. IMMUNOHISTOCHEMICAL DETECTION AND DISTRIBUTION OF PRION PROTEIN IN A GOAT WITH NATURAL SCRAPIE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Formalin-fixed, paraffin-embedded tissue sections from a 3-year-old female Angora goat suffering from clinical scrapie were immunostained 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 iso...

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

    ERIC Educational Resources Information Center

    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

  5. Specific binding modes of Cu(I) and Ag(I) with neurotoxic domain of the human prion protein.

    PubMed

    Valensin, Daniela; Padula, Emilia Maria; Hecel, Aleksandra; Luczkowski, Marek; Kozlowski, Henryk

    2016-02-01

    Prion diseases are neurodegenerative disorders associated with a conformational change of the normal cellular isoform of the prion protein (PrP(C)) to an abnormal scrapie isoform (PrP(Sc)). human prion protein (hPrP(C)) is able to bind up to six Cu(II) ions. Four of them are distributed in the octarepeat domain, containing four tandem-repetitions of the sequence PHGGGWGQ. Immediately outside the octarepeat domain, in so called PrP amyloidogenic region, two additional and independent Cu(II) binding sites, encompassing His96 and His111 residues, respectively, are present. Considering the potential involvement of PrP in cellular redox homeostasis, investigations on Cu(I)-PrP interaction might be also biologically relevant. Interestingly, the amyloidogenic fragment of PrP contains a -M(X)nM- motif, known to act as Cu(I) binding site in different proteins. In order to shed more light on this issue, copper(I) and silver(I) interactions with model peptides derived from that region were analyzed. The results of our studies reveal that both metal ions are anchored to two thioether sulfurs of Met109 and Met112, respectively. Subsequent metal interaction and coordination to His96 and His111 imidazoles are primarily found for Cu(I) at physiological pH. Metal binding was also investigated in the presence of negatively charged micelles formed by the anionic surfactant, sodium dodecyl sulfate (SDS). Our results strongly support that metal binding mode strongly depends on the protein backbone structure. In particular we show that ?-helix structuring of the amyloid PrP domain influences both the metal coordination sphere and the binding affinity. PMID:26606290

  6. Generating new prions by targeted mutation or segment duplication.

    PubMed

    Paul, Kacy R; Hendrich, Connor G; Waechter, Aubrey; Harman, Madison R; Ross, Eric D

    2015-07-14

    Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity. PMID:26100899

  7. Generating new prions by targeted mutation or segment duplication

    PubMed Central

    Paul, Kacy R.; Hendrich, Connor G.; Waechter, Aubrey; Harman, Madison R.; Ross, Eric D.

    2015-01-01

    Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity. PMID:26100899

  8. The cellular prion protein: biochemistry, topology, and physiologic functions.

    PubMed

    Griffoni, Cristiana; Toni, Mattia; Spisni, Enzo; Bianco, Maria; Santi, Spartaco; Riccio, Massimo; Tomasi, Vittorio

    2003-01-01

    Studies on the transmission from man to animals of Creutzfeld-Jacob disease (CJD) led Prusiner to identify a proteinaceous infectious particle lacking nucleic acid, which was called prion. The identification of the infectious prion (PrPsc) then led to the discovery of the normal cellular counterpart (PrPc). One of the still enigmatic aspects regarding prion diseases is actually how, where, and when the transformation PrPc/PrPsc is occurring, this being due to the result of a large extent to the fact that so far most studies have been dedicated to the formation and transmission of PrPsc, whereas the understanding of physiologic roles of PrPc are in their infancy. In this review, we hope to identify the most reliable hypotheses for future experiments on PrPc. This is relevant not only for the understanding of PrPc functions but also to unravel the enigmatic nature of PrPc/PrPsc conversion. PMID:12794269

  9. Loss of prion protein leads to age-dependent behavioral abnormalities and changes in cytoskeletal protein expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellular prion protein (PrPC) is a multifunctional protein, whose exact physiological role remains elusive. Since previous studies indicated a neuroprotective function of PrPC, we investigated whether Prnp knockout mice(Prnp0/0)display age-dependent behavioral abnormalities. Matched sets of Prnp0/0 ...

  10. A bipolar functionality of Q/N-rich proteins: Lsm4 amyloid causes clearance of yeast prions

    PubMed Central

    Oishi, Keita; Kurahashi, Hiroshi; Pack, Chan-Gi; Sako, Yasushi; Nakamura, Yoshikazu

    2013-01-01

    Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI+]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI+], [URE3], and [RNQ+]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI+] elimination, the [PSI+] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi?]-like mother cells. We also found that the antiprion activity is a general property of [PSI+]-inducible factors. These data provoked a novel unified model that explains both prion induction and elimination by a single scheme. PMID:23512891

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

    SciTech Connect

    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 at ultra-low fields is realized by incorporating the high sensitivities of a dc superconducting quantum interference device (SQUID) with the high polarizations attainable through optica11y pumping {sup 129}Xe gas.

  12. Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

    PubMed Central

    Lehmann, S; Harris, D A

    1996-01-01

    Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643624

  13. N-terminal Domain of Prion Protein Directs Its Oligomeric Association*

    PubMed Central

    Trevitt, Clare R.; Hosszu, Laszlo L. P.; Batchelor, Mark; Panico, Silvia; Terry, Cassandra; Nicoll, Andrew J.; Risse, Emmanuel; Taylor, William A.; Sandberg, Malin K.; Al-Doujaily, Huda; Linehan, Jacqueline M.; Saibil, Helen R.; Scott, David J.; Collinge, John; Waltho, Jonathan P.; Clarke, Anthony R.

    2014-01-01

    The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar β-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined β-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23–90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91–231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein. PMID:25074940

  14. Assessing Proteinase K Resistance of Fish Prion Proteins in a Scrapie-Infected Mouse Neuroblastoma Cell Line

    PubMed Central

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

    2014-01-01

    The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrPC, into an aberrant and partially proteinase K resistant isoform, PrPSc. 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. PMID:25402173

  15. A sequencing strategy for identifying variation throughout the prion gene of BSE-affected cattle

    PubMed Central

    Clawson, Michael L; Heaton, Michael P; Keele, John W; Smith, Timothy PL; Harhay, Gregory P; Richt, Juergen A; Laegreid, William W

    2008-01-01

    Background Classical and atypical bovine spongiform encephalopathies (BSEs) are cattle prion diseases. Distinct bovine prion gene (PRNP) alleles have been associated with classical and atypical BSE susceptibility. However, the full extent of PRNP allele association with BSE susceptibility is not known. A systematic sequence-based genotyping method that detects variation throughout PRNP would be useful for: 1) detecting rare PRNP alleles that may be present in BSE-affected animals and 2) testing PRNP alleles for an association with either classical or atypical BSE susceptibility. Findings We improved a Sanger-based sequencing strategy for detecting bovine PRNP variation through all exons, introns, and part of the promoter (25.2 kb). Our current method can detect 389 known and other potentially unknown PRNP polymorphisms that may be present in BSE-affected cattle. We determined PRNP genotypes for the first U.S. BSE case and her sire. Previously unknown PRNP polymorphisms were not detected in either animal and all PRNP genotypes support the sire-daughter relationship. Conclusion The methodologies described here characterize variation throughout PRNP. Consequently, rare PRNP alleles that may be present in BSE-affected cattle can be detected. PMID:18710485

  16. Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle?

    PubMed Central

    Jeffrey, Martin; Perez, Belinda Baquero; Martin, Stuart; Terry, Linda; González, Lorenzo

    2008-01-01

    Background The epidemic form of Bovine Spongiform Encephalopathy (BSE) is generally considered to have been caused by a single prion strain but at least two strain variants of cattle prion disorders have recently been recognized. An additional neurodegenerative condition, idiopathic brainstem neuronal chromatolysis and hippocampal sclerosis (IBNC), a rare neurological disease of adult cattle, was also recognised in a sub-set of cattle submitted under the BSE Orders in which lesions of BSE were absent. Between the years of 1988 and 1991 IBNC occurred in Scotland with an incidence of 7 cases per 100,000 beef suckler cows over the age of 6 years. Results When the brains of 15 IBNC cases were each tested by immunohistochemistry, all showed abnormal labelling for prion protein (PrP). Immunohistological labelling for PrP was also present in the retina of a single case available for examination. The pattern of PrP labelling in brain is distinct from that seen in other ruminant prion diseases and is absent from brains with other inflammatory conditions and from normal control brains. Brains of IBNC cattle do not reveal abnormal PrP isoforms when tested by the commercial BioRad or Idexx test kits and do not reveal PrPres when tested by Western blotting using stringent proteinase digestion methods. However, some weakly protease resistant isoforms of PrP may be detected when tissues are examined using mild proteinase digestion techniques. Conclusion The study shows that a distinctive neurological disorder of cattle, which has some clinical similarities to BSE, is associated with abnormal PrP labelling in brain but the pathology and biochemistry of IBNC are distinct from BSE. The study is important either because it raises the possibility of a significant increase in the scope of prion disease or because it demonstrates that widespread and consistent PrP alterations may not be confined to prion diseases. Further studies, including transmission experiments, are needed to establish whether IBNC is a condition in which prion protein is abnormally regulated or it is yet a further example of an infectious cattle prion disease. PMID:18826563

  17. [Prion disease].

    PubMed

    Mizusawa, Hidehiro

    2010-11-01

    Human prion diseases are classified into 3 categories according to etiologies: idiopathic of unknown cause, acquired of infectious origin, and genetic by PRNP mutation. The surveillance committee have analyzed 2,494 cases and identified 1,402 as prion diseases. Most of them are idiopathic, namely sporadic CJD (77%) with less genetic and acquired prion diseases (17% and 5%, respectively). The number of patients identified by the surveillance committee in these years is about 120 which are less than the number of annual death of prion disease. The difference might be due to partly the fact our surveillance need the consent from patients' family and is not complete. The mean age at onset of prion disease is late 60s while the range is fairly wide. Brain MRIs and increase of CSF 14-3-3 and tau protein levels are very characteristic. Classical sporadic CJD could show completely normal T1WI with patchy high signals in the cerebral cortex and basal ganglia on DWI. In Japan, classical sporadic CJD (MM1) is most popular but there are some rare atypical subtypes. Among them, MM2-thalamic CJD is hardest to diagnose because it shows no high intensity signals on DWI, in addition to frequent absence of CSF and EEG characteristics. In this case, CBF decrease in the thalamus on SPECT is very helpful. Genetic prion diseases in Japan are quite distinct from those in Europe. V180I and M232R mutations are unique to Japan and show sporadic CJD phenotype. Dura graft-associated CJD (dCJD) are composed of 67% of classical sporadic CJD phenotype and 33% of atypical phenotype showing slower progression with amyloid plaques. Trace-back experiments suggested the PrP(sc) of the atypical dCJD was likely to be modified from infection of abnormal VV2 protein. Although there are some atypical forms of prion diseases as mentioned above, almost all prion cases could be diagnosed with EEG, MRI, genetic test, CSF test and SPECT. We also have some incidents in which brain surgery was done before the diagnosis of prion disease and many other patients were operated using the same operating instruments before their sterilization against prion disease had been done. The explanation of possibility of prion disease infection to the patients and their follow-up was started by the incident committee. It is very important for all the nations to cooperate with each other in order to overcome this intractable disease. PMID:21921445

  18. Recombinant scrapie-like prion protein of 106 amino acids is?soluble

    PubMed Central

    Muramoto, Tamaki; Scott, Michael; Cohen, Fred?E.; Prusiner, Stanley?B.

    1996-01-01

    The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies. PMID:8986833

  19. Translation of the Prion Protein mRNA Is Robust in Astrocytes but Does Not Amplify during Reactive Astrocytosis in the Mouse Brain

    PubMed Central

    Jackson, Walker S.; Krost, Clemens; Borkowski, Andrew W.; Kaczmarczyk, Lech

    2014-01-01

    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. PMID:24752288

  20. Lack of Prion Accumulation in Lymphoid Tissues of Scrapie-affected Sheep with the AA136, QR171 Prion Protein Genotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Sheep scrapie is a transmissible spongiform encephalopathy which can be transmitted horizontally through the shedding of an infectious conformer (PrP**Sc) of the normal cellular prion protein (PrP**c). Genetics profoundly influence the susceptibility of sheep to scrapie. PrP**c amino-aci...

  1. From cell protection to death: may Ca2+ signals explain the chameleonic attributes of the mammalian prion protein?

    PubMed

    Sorgato, M Catia; Bertoli, Alessandro

    2009-02-01

    It is now accepted that a conformational change of the cellular prion protein (PrP(C)) generates the prion, the infectious agent responsible for lethal neurodegenerative disorders, named transmissible spongiform encephalopathies, or prion diseases. The mechanisms of prion-associated neurodegeneration are still obscure, as is the cell role of PrP(C), although increasing evidence attributes to PrP(C) important functions in cell survival. Such a behavioral dichotomy thus enables the prion protein to switch from a benign role under normal conditions, to the execution of neurons during disease. By reviewing data from models of prion disease and PrP(C)-null paradigms, which suggest a relation between the prion protein and Ca(2+) homeostasis, here we discuss the possibility that Ca(2+) is the factor behind the enigma of the pathophysiology of PrP(C). Ca(2+) features in almost all processes of cell signaling, and may thus tell us much about a protein that pivots between health and disease. PMID:19101513

  2. Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants

    PubMed Central

    Wickner, Reed B.; Bezsonov, Evgeny; Bateman, David A.

    2014-01-01

    [URE3] is an amyloid prion of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. Overproduction of Btn2p, involved in late endosome to Golgi protein transport, or its paralog Cur1p, cures [URE3]. Btn2p, in curing, is colocalized with Ure2p in a single locus, suggesting sequestration of Ure2p amyloid filaments. We find that most [URE3] variants generated in a btn2 cur1 double mutant are cured by restoring normal levels of Btn2p and Cur1p, with both proteins needed for efficient curing. The [URE3] variants cured by normal levels of Btn2p and Cur1p all have low seed number, again suggesting a seed sequestration mechanism. Hsp42 overproduction also cures [URE3], and Hsp42p aids Btn2 overproduction curing. Cur1p is needed for Hsp42 overproduction curing of [URE3], but neither Btn2p nor Cur1p is needed for overproduction curing by the other. Although hsp42? strains stably propagate [URE3-1], hsp26? destabilizes this prion. Thus, Btn2p and Cur1p are antiprion system components at their normal levels, acting with Hsp42. Btn2p is related in sequence to human Hook proteins, involved in aggresome formation and other transport activities. PMID:24938787

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

    USGS Publications Warehouse

    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 animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.

  4. Implications of prion polymorphisms

    PubMed Central

    Cortez, Leonardo M; Sim, Valerie L

    2013-01-01

    The sequence of a hosts prion protein (PrP) can affect that hosts susceptibility to prion disease and is the primary basis for the species barrier to transmission. Yet within many species, polymorphisms of the prion protein gene (Prnp) exist, each of which can further affect susceptibility or influence incubation period, pathology and phenotype. As strains are defined by these features (incubation period, pathology, phenotype), polymorphisms may also lead to the preferential propagation or generation of certain strains. In our recent study of the mouse Prnpa and Prnpb polymorphisms (which produced the proteins PrPa and PrPb, respectively), we found differences in aggregation tendency, strain adaptability and conformational variability. Comparing our in vitro data with that of in vivo studies, we found that differing incubation periods between Prnpa and Prnpb mice can primarily be explained on the basis of faster or more efficient aggregation of PrPa. In addition, and more importantly, we found that the faithful propagation of strains in Prnpb mice can be explained by the ability of PrPb to adopt a wider range of conformations. This adaptability allows PrPb to successfully propagate the structural features of a seed. In contrast, Prnpa mice revert PrPb strains into PrPa -type strains, and overall they have a narrower distribution of incubation periods. This can be explained by PrPa having fewer preferred conformations. We propose that Prnp polymorphisms are one route by which certain prion strains may preferentially propagate. This has significant implications for prion disease, chronic wasting disease (CWD) in particular, as it is spreading through North America. Deer which are susceptible to CWD also carry polymorphisms which influence their susceptibility. If these polymorphisms also preferentially allow strain diversification and propagation, this may accelerate the crossing of species barriers and propagation of the disease up the food chain. PMID:23807178

  5. Perturbation of the Secondary Structure of the Scrapie Prion Protein Under Conditions that Alter Infectivity

    NASA Astrophysics Data System (ADS)

    Gasset, Maria; Baldwin, Michael A.; Fletterick, Robert J.; Prusiner, Stanley B.

    1993-01-01

    Limited proteolysis of the scrapie prion protein (PrPSc) generates PrP 27-30, which polymerizes into amyloid. By attenuated total reflection-Fourier transform infrared spectroscopy, PrP 27-30 polymers contained 54% ?-sheet, 25% ?-helix, 10% turns, and 11% random coil; dispersion into detergent-lipid-protein-complexes preserved infectivity and secondary structure. Almost 60% of the ?-sheet was low-frequency infrared-absorbing, reflecting intermolecular aggregation. Decreased low-frequency ?-sheet and increased turn content were found after SDS/PAGE, which disassembled the amyloid polymers, denatured PrP 27-30, and diminished scrapie infectivity. Acid-induced transitions were reversible, whereas alkali produced an irreversible transition centered at pH 10 under conditions that diminished infectivity. Whether PrPSc synthesis involves a transition in the secondary structure of one or more domains of the cellular prion protein from ?-helical, random coil, or turn into ?-sheet remains to be established.

  6. Prion-like properties of Tau protein: the importance of extracellular Tau as a therapeutic target.

    PubMed

    Holmes, Brandon B; Diamond, Marc I

    2014-07-18

    Work over the past 4 years indicates that multiple proteins associated with neurodegenerative diseases, especially Tau and α-synuclein, can propagate aggregates between cells in a prion-like manner. This means that once an aggregate is formed it can escape the cell of origin, contact a connected cell, enter the cell, and induce further aggregation via templated conformational change. The prion model predicts a key role for extracellular protein aggregates in mediating progression of disease. This suggests new therapeutic approaches based on blocking neuronal uptake of protein aggregates and promoting their clearance. This will likely include therapeutic antibodies or small molecules, both of which can be developed and optimized in vitro prior to preclinical studies. PMID:24860099

  7. Subcellular distribution of the prion protein in sickness and in health.

    PubMed

    Godsave, Susan F; Peters, Peter J; Wille, Holger

    2015-09-01

    The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus. PMID:25683509

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

    PubMed Central

    Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Takatsuki, Hanae; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    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. PMID:26368533

  9. Efficient Uptake and Dissemination of Scrapie Prion Protein by Astrocytes and Fibroblasts from Adult Hamster Brain

    PubMed Central

    Hollister, Jason R.; Lee, Kil Sun; Dorward, David W.; Baron, Gerald S.

    2015-01-01

    Prion infections target neurons and lead to neuronal loss. However, the role of non-neuronal cells in the initiation and spread of infection throughout the brain remains unclear despite the fact these cells can also propagate prion infectivity. To evaluate how different brain cells process scrapie prion protein (PrPres) during acute infection, we exposed neuron-enriched and non-neuronal cell cultures from adult hamster brain to fluorescently-labeled purified PrPres and followed the cultures by live cell confocal imaging over time. Non-neuronal cells present in both types of cultures, specifically astrocytes and fibroblasts, internalized PrPres more efficiently than neurons. PrPres was trafficked to late endosomal/lysosomal compartments and rapidly transported throughout the cell bodies and processes of all cell types, including contacts between astrocytes and neurons. These observations suggest that astrocytes and meningeal fibroblasts play an as yet unappreciated role in prion infections via efficient uptake and dissemination of PrPres. PMID:25635871

  10. Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation.

    PubMed

    Kraus, Allison; Anson, Kelsie J; Raymond, Lynne D; Martens, Craig; Groveman, Bradley R; Dorward, David W; Caughey, Byron

    2015-08-28

    Human prion diseases can have acquired, sporadic, or genetic origins, each of which results in the conversion of prion protein (PrP) to transmissible, pathological forms. The genetic prion disease Gerstmann-Straussler-Scheinker syndrome can arise from point mutations of prolines 102 or 105. However, the structural effects of these two prolines, and mutations thereof, on PrP misfolding are not well understood. Here, we provide evidence that individual mutations of Pro-102 or Pro-105 to noncyclic aliphatic residues such as the Gerstmann-Straussler-Scheinker-linked leucines can promote the in vitro formation of PrP amyloid with extended protease-resistant cores reminiscent of infectious prions. This effect was enhanced by additional charge-neutralizing mutations of four nearby lysine residues comprising the so-called central lysine cluster. Substitution of these proline and lysine residues accelerated PrP conversion such that spontaneous amyloid formation was no longer slower than scrapie-seeded amyloid formation. Thus, Pro-102 and Pro-105, as well as the lysines in the central lysine cluster, impede amyloid formation by PrP, implicating these residues as key structural modulators in the conversion of PrP to disease-associated types of amyloid. PMID:26175152

  11. Proteomics analyses for the global proteins in the brain tissues of different human prion diseases.

    PubMed

    Shi, Qi; Chen, Li-Na; Zhang, Bao-Yun; Xiao, Kang; Zhou, Wei; Chen, Cao; Zhang, Xiao-Mei; Tian, Chan; Gao, Chen; Wang, Jing; Han, Jun; Dong, Xiao-Ping

    2015-04-01

    Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases. PMID:25616867

  12. Proteomics Analyses for the Global Proteins in the Brain Tissues of Different Human Prion Diseases*

    PubMed Central

    Shi, Qi; Chen, Li-Na; Zhang, Bao-Yun; Xiao, Kang; Zhou, Wei; Chen, Cao; Zhang, Xiao-Mei; Tian, Chan; Gao, Chen; Wang, Jing; Han, Jun; Dong, Xiao-Ping

    2015-01-01

    Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases. PMID:25616867

  13. Yeast Prions: Structure, Biology, and Prion-Handling Systems

    PubMed Central

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

    2015-01-01

    SUMMARY 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. PMID:25631286

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

    PubMed Central

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

    2015-01-01

    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. PMID:26531259

  15. Conformational stability of mammalian prion protein amyloid fibrils is dictated by a packing polymorphism within the core region.

    PubMed

    Cobb, Nathan J; Apostol, Marcin I; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K

    2014-01-31

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP(Sc). One key operational parameter used to define differences between strains has been conformational stability of PrP(Sc) as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in ?-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP(Sc), especially because large strain-specific differences in PrP(Sc) stability are often observed despite a similar size of the PrP(Sc) core region. PMID:24338015

  16. Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region*

    PubMed Central

    Cobb, Nathan J.; Apostol, Marcin I.; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K.

    2014-01-01

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrPSc. One key operational parameter used to define differences between strains has been conformational stability of PrPSc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrPSc, especially because large strain-specific differences in PrPSc stability are often observed despite a similar size of the PrPSc core region. PMID:24338015

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

    NASA Astrophysics Data System (ADS)

    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.

  18. A rapid sequence-based method for comprehensive polymorphism identification within a 25.2-kb region of the bovine prion gene in BSE-affected cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine spongiform encephalopathy (BSE) is a fatal neurological disorder characterized by abnormal deposits of a protease-resistant isoform of the prion protein. Typical and atypical BSEs have been identified in cattle and the relationships of prion gene (PRNP) variation with susceptibility to these...

  19. Spongiform encephalopathy in siblings with no evidence of protease-resistant prion protein or a mutation in the prion protein gene.

    PubMed

    Varges, Daniela; Schulz-Schaeffer, Walter J; Wemheuer, Wiebke M; Damman, Insa; Schmitz, Matthias; Cramm, Maria; Kallenberg, Kai; Shirneshan, Katayoon; Elkenani, Manar; Markwort, Susanne; Faist, Michael; Kohlhase, Jrgen; Windl, Otto; Zerr, Inga

    2013-07-01

    We discuss relevant aspects in two siblings with a neurodegenerative process of unclear aetiology who developed progressive dementia with global aphasia and hyperoral behaviour at the ages of 39 and 46 years and who died 6 and 5 years after disease onset. The cases were reported to the National Reference Center for TSE Surveillance in Gttingen, Germany. Detailed clinical examinations, CSF, blood samples, and copies of the important diagnostic tests (magnetic resonance imaging, electroencephalogram, laboratory tests) were obtained. Further neuropathological and genetic analyses were performed. Cerebral magnetic resonance imaging of both siblings showed prominent changes in signal intensity, especially in the left medial temporal cortex, but also the hippocampal formation. Neuropathological examination revealed spongiform changes, neuronal loss, and astrocytic gliosis, which are typical in Creutzfeldt-Jakob disease. However, no prion protein deposits were detectable by immunohistochemical analysis, Western blot, or PET blot, though abundant tau protein deposits were observed. A mutation in the coding region of the prion protein genes of both siblings was excluded. A detailed search of the literature revealed no other cases with a similar clinical and neuropathological appearance. While the disease aetiology remains unclear, the findings point to a neurodegenerative process and most likely a genetic disease. PMID:23546304

  20. Protein Transmission, Seeding and Degradation: Key Steps for ?-Synuclein Prion-Like Propagation

    PubMed Central

    Ximerakis, Methodios; Vekrellis, Kostas

    2014-01-01

    Converging lines of evidence suggest that cell-to-cell transmission and the self-propagation of pathogenic amyloidogenic proteins play a central role in the initiation and the progression of several neurodegenerative disorders. This "prion-like" hypothesis has been recently reported for ?-synuclein, a presynaptic protein implicated in the pathogenesis of Parkinson's disease (PD) and related disorders. This review summarizes recent findings on ?-synuclein prion-like propagation, focusing on its transmission, seeding and degradation and discusses some key questions that remain to be explored. Understanding how ?-synuclein exits cells and propagates from one brain region to another will lead to the development of new therapeutic strategies for the treatment of PD, aiming at slowing or stopping the disease progression. PMID:25548532

  1. Review: membrane-associated misfolded protein propagation in natural transmissible spongiform encephalopathies (TSEs), synthetic prion diseases and Alzheimer's disease.

    PubMed

    Jeffrey, M

    2013-04-01

    Protein misfolding has long been recognized as a primary cause of systemic amyloidosis and, increasingly, template-mediated misfolding of native host proteins is now also considered to be central pathogenetic events in some neurodegenerative diseases. Alzheimer's disease, naturally occurring transmissible spongiform encephalopathies (TSEs) and experimental disorders caused by misfolded prion protein (PrP) generated in vitro all share an imbalance of protein synthesis, aggregation and clearance that leads to protein aggregation, prompting some to suggest that Alzheimer's disease is caused by a prion-like mechanism. In TSEs, the host-coded, glycosyl-phosphoinositol (GPI) membrane-anchored prion protein (PrP(c) ) is misfolded into disease-associated, putatively infectious aggregates known as prions. In Alzheimer's disease the membrane-spanning Alzheimer's precursor protein (APP) is progressively cleaved within the plasmalemma to form Aβ peptide fragments that can form pathogenic extracellular aggregates while microtubule-associated tau proteins may also aggregate within neurones. Oligomeric Aβ peptides and full-length misfolded PrP show a common potential to convert native protein and aggregate on plasma membranes before subsequent release to form amyloid fibrils in the extracellular space. However, the nature, membrane topography and processing of the precursor and propagated proteins in prion and Alzheimer's disease all differ, and each group of diseases has distinctive spectra of additional pathological changes and clinical signs suggesting that fundamentally different disease mechanisms are involved. PMID:23171056

  2. Swaps in protein sequences.

    PubMed

    Fliess, Amit; Motro, Benny; Unger, Ron

    2002-08-01

    An important question in protein evolution is to what extent proteins may have undergone swaps (switches of domain or fragment order) during evolution. Such events might have occurred in several forms: Swaps of short fragments, swaps of structural and functional motifs, or recombination of domains in multidomain proteins. This question is important for the theoretical understanding of the evolution of proteins, and has practical implications for using swaps as a design tool in protein engineering. In order to analyze the question systematically, we conducted a large scale survey of possible swaps and permutations among all pairs of protein from the Swissport database. A swap is defined as a specific kind of sequence mutation between two proteins in which two fragments that appear in both sequences have different relative order in the two sequences. For example, aXbYc and dYeXf are defined as a swap, where X and Y represent sequence fragments that switched their order. Identifying such swaps is difficult using standard sequence comparison packages. One of the main problems in the analysis stems from the fact that many sequences contain repeats, which may be identified as false-positive swaps. We have used two different approaches to detect pairs of proteins with swaps. The first approach is based on the predefined list of domains in Pfam. We identified all the proteins that share at least two domains and analyzed their relative order, looking for pairs in which the order of these domains was switched. We designed an algorithm to distinguish between real swaps and duplications. In the second approach, we used Blast to detect pairs of proteins that share several fragments. Then, we used an automatic procedure to select pairs that are likely to contain swaps. Those pairs were analyzed visually, using a graphical tool, to eliminate duplications. Combining these approaches, about 140 different cases of swaps in the Swissprot database were found (after eliminating multiple pairs within the same family). Some of the cases have been described in the literature, but many are novel examples. Although each new example identified may be interesting to analyze, our main conclusion is that cases of swaps are rare in protein evolution. This observation is at odds with the common view that proteins are very modular to the point that modules (e.g., domains) can be shuffled between proteins with minimal constraints. Our study suggests that sequential constraints, i.e., the relative order between domains, are highly conserved. PMID:12112704

  3. Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy

    PubMed Central

    Kachel, Norman; Kremer, Werner; Zahn, Ralph; Kalbitzer, Hans Robert

    2006-01-01

    Background Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins as well as the dynamics and structure of folding intermediates. Results Conformational intermediates of the human prion protein huPrPC were characterized by a combination of hydrostatic pressure (up to 200 MPa) with two-dimensional NMR spectroscopy. All pressure effects showed to be reversible and there is virtually no difference in the overall pressure response between the folded core of the N-terminal truncated huPrPC(121230) and the full-length huPrPC(23230). The only significant differences in the pressure response of full-length and truncated PrP suggest that E168, H187, T192, E207, E211 and Y226 are involved in a transient interaction with the unfolded N-terminus. High-pressure NMR spectroscopy indicates that the folded core of the human prion protein occurs in two structural states N1and N2 in solution associated with rather small differences in free enthalpies (3.0 kJ/mol). At atmospheric pressure approximately 29% of the protein are already in the pressure favored conformation N2. There is a second process representing two possible folding intermediates I1 and I2 with corresponding average free enthalpies of 10.8 and 18.6 kJ/mol. They could represent preaggregation states of the protein that coexist at ambient pressure with a very small population of approximately 1.2% and less than 0.1%. Further the pressure response of the N-terminus indicates that four different regions are in a fast equilibrium with non-random structural states whose populations are shifted by pressure. Conclusion We identified pressure stabilized folding intermediates of the human prion protein. The regions reflecting most strongly the transition to the intermediate states are the ?1/?1-loop and the solvent exposed side of ?3. The most pressure-sensitive region (representing mainly intermediate I1) is the loop between ?-strand 1 and ?-helix 1 (residue 139141), indicating that this region might be the first entry point for the infectious conformer to convert the cellular protein. PMID:16846506

  4. Modulation of Proteinase K-resistant Prion Protein in Cells and Infectious Brain Homogenate by Redox Iron: Implications for Prion Replication and Disease Pathogenesis

    PubMed Central

    Basu, Subhabrata; Mohan, Maradumane L.; Luo, Xiu; Kundu, Bishwajit; Kong, Qingzhong

    2007-01-01

    The principal infectious and pathogenic agent in all prion disorders is a ?-sheetrich isoform of the cellular prion protein (PrPC) termed PrP-scrapie (PrPSc). Once initiated, PrPSc is self-replicating and toxic to neuronal cells, but the underlying mechanisms remain unclear. In this report, we demonstrate that PrPC binds iron and transforms to a PrPSc-like form (*PrPSc) when human neuroblastoma cells are exposed to an inorganic source of redox iron. The *PrPSc thus generated is itself redox active, and it induces the transformation of additional PrPC, simulating *PrPSc propagation in the absence of brain-derived PrPSc. Moreover, limited depletion of iron from prion disease-affected human and mouse brain homogenates and scrapie-infected mouse neuroblastoma cells results in 4- to 10-fold reduction in proteinase K (PK)-resistant PrPSc, implicating redox iron in the generation, propagation, and stability of PK-resistant PrPSc. Furthermore, we demonstrate increased redox-active ferrous iron levels in prion disease-affected brains, suggesting that accumulation of PrPSc is modulated by the combined effect of imbalance in brain iron homeostasis and the redox-active nature of PrPSc. These data provide information on the mechanism of replication and toxicity by PrPSc, and they evoke predictable and therapeutically amenable ways of modulating PrPSc load. PMID:17567949

  5. Functional Role of Tia1/Pub1 and Sup35 Prion Domains: Directing Protein Synthesis Machinery to the Tubulin Cytoskeleton

    PubMed Central

    Li, Xiang; Rayman, Joseph B.; Kandel, Eric R; Derkatch, Irina L.

    2014-01-01

    SUMMARY Tia1/Pub1 is a stress granule component carrying a Q/N-rich prion domain. We provide direct evidence that Tia1 forms a prion in yeast. Moreover, Tia1/Pub1 acts co-operatively with release factor Sup35/eRF3 to establish a two-protein self-propagating state. This two-protein prion driven by the Q/N-rich prion domains of Sup35 and Tia1/Pub1 can be visualized as distinctive line structures along tubulin cytoskeleton. Furthermore, we find that tubulin-associated complex containing Pub1 and Sup35 oligomers normally exists in yeast, and its assembly depends on prion domains of Pub1 and Sup35. This Sup35/Pub1 complex, which also contains TUB1 mRNA and components of translation machinery, is important for the integrity of the tubulin cytoskeleton: PUB1 disruption and Sup35 depletion from the complex lead to cytoskeletal defects. We propose that the complex is implicated in protein synthesis at the site of microtubule assembly. Thus our study identifies the role for prion domains in the assembly of multi-protein complexes. PMID:24981173

  6. Novel N-terminal domain mutation in prion protein detected in 2 patients diagnosed with frontotemporal lobar degeneration syndrome.

    PubMed

    Bernardi, Livia; Cupidi, Chiara; Frangipane, Francesca; Anfossi, Maria; Gallo, Maura; Conidi, Maria Elena; Vasso, Franca; Colao, Rosanna; Puccio, Gianfranco; Curcio, Sabrina A M; Mirabelli, Maria; Clodomiro, Alessandra; Di Lorenzo, Raffaele; Smirne, Nicoletta; Maletta, Raffaele; Bruni, Amalia C

    2014-11-01

    Prion protein gene mutations have been associated with clinical pictures mimicking neurodegenerative diseases different from inherited prion diseases (IPD). We report a novel missense P39L mutation in the N-terminal domain of prion protein in 2 patients affected by frontotemporal lobar degeneration syndrome, negative for mutations in genes causative of dementia. Neither the first carrier, a 67-year-old male in which the onset was a progressive non-fluent aphasia, nor the second carrier, a 78-year-old male affected by frontotemporal dementia and parkinsonism, showed any clinical or instrumental findings suggestive of IPD. Genetic screening of healthy controls and in silico analysis provide support for the potential pathogenicity of this variant. Patient phenotypes, unclassifiable as prion disease, may depend on the location of the mutation in the N-terminal domain, outside the amyloid core of pathologic prion protein, although further functional studies are required to determine whether and how this mutation exerts its pathogenic effect. However, genetic screening of prion protein gene becomes relevant in familial degenerative dementia, particularly in geographical areas with high IPD prevalence. PMID:25022973

  7. The role of the cellular prion protein in the immune system

    PubMed Central

    Isaacs, J D; Jackson, G S; Altmann, D M

    2006-01-01

    Prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrPC) is expressed widely in the immune system, in haematopoietic stem cells and mature lymphoid and myeloid compartments in addition to cells of the central nervous system. It is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocyte. Furthermore, antibody cross-linking of surface PrP modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signalling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Although PrP–/– mice have been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self-renewal of haematopoietic stem cells. Here, we consider the evidence for a distinctive role for PrPC in the immune system and what the effects of anti-prion therapeutics may be on immune function. PMID:16968391

  8. Mice deficient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus.

    PubMed Central

    Lledo, P M; Tremblay, P; DeArmond, S J; Prusiner, S B; Nicoll, R A

    1996-01-01

    We recorded in the CA1 region from hippocampal slices of prion protein (PrP) gene knockout mice to investigate whether the loss of the normal form of prion protein (PrPC) affects neuronal excitability as well as synaptic transmission in the central nervous system. No deficit in synaptic inhibition was found using field potential recordings because (i) responses induced by stimulation in stratum radiatum consisted of a single population spike in PrP gene knockout mice similar to that recorded from control mice and (ii) the plot of field excitatory postsynaptic potential slope versus the population spike amplitude showed no difference between the two groups of mice. Intracellular recordings also failed to detect any difference in cell excitability and the reversal potential for inhibitory postsynaptic potentials. Analysis of the kinetics of inhibitory postsynaptic current revealed no modification. Finally, we examined whether synaptic plasticity was altered and found no difference in long-term potentiation between control and PrP gene knockout mice. On the basis of our findings, we propose that the loss of the normal form of prion protein does not alter the physiology of the CA1 region of the hippocampus. PMID:8637886

  9. Mice Deficient for Prion Protein Exhibit Normal Neuronal Excitability and Synaptic Transmission in the Hippocampus

    NASA Astrophysics Data System (ADS)

    Lledo, Pierre-Marie; Tremblay, Patrick; Dearmond, Stephen J.; Prusiner, Stanley B.; Nicoll, Roger A.

    1996-03-01

    We recorded in the CA1 region from hippocampal slices of prion protein (PrP) gene knockout mice to investigate whether the loss of the normal form of prion protein (PrPC) affects neuronal excitability as well as synaptic transmission in the central nervous system. No deficit in synaptic inhibition was found using field potential recordings because (i) responses induced by stimulation in stratum radiatum consisted of a single population spike in PrP gene knockout mice similar to that recorded from control mice and (ii) the plot of field excitatory postsynaptic potential slope versus the population spike amplitude showed no difference between the two groups of mice. Intracellular recordings also failed to detect any difference in cell excitability and the reversal potential for inhibitory postsynaptic potentials. Analysis of the kinetics of inhibitory postsynaptic current revealed no modification. Finally, we examined whether synaptic plasticity was altered and found no difference in long-term potentiation between control and PrP gene knockout mice. On the basis of our findings, we propose that the loss of the normal form of prion protein does not alter the physiology of the CA1 region of the hippocampus.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Life cycle of cytosolic prions.

    PubMed

    Hofmann, Julia; Vorberg, Ina

    2013-01-01

    Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions. PMID:24021964

  12. Life cycle of cytosolic prions

    PubMed Central

    Hofmann, Julia; Vorberg, Ina

    2013-01-01

    Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions. PMID:24021964

  13. The prion protein gene polymorphisms associated with bovine spongiform encephalopathy susceptibility differ significantly between cattle and buffalo.

    PubMed

    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. PMID:26319996

  14. BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein

    PubMed Central

    Asante, Emmanuel A.; Linehan, Jacqueline M.; Desbruslais, Melanie; Joiner, Susan; Gowland, Ian; Wood, Andrew L.; Welch, Julie; Hill, Andrew F.; Lloyd, Sarah E.; Wadsworth, Jonathan D.F.; Collinge, John

    2002-01-01

    Variant CreutzfeldtJakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrPSc type2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure. PMID:12456643

  15. Prions in skeletal muscle.

    PubMed

    Bosque, Patrick J; Ryou, Chongsuk; Telling, Glenn; Peretz, David; Legname, Giuseppe; DeArmond, Stephen J; Prusiner, Stanley B

    2002-03-19

    Considerable evidence argues that consumption of beef products from cattle infected with bovine spongiform encephalopathy (BSE) prions causes new variant Creutzfeldt-Jakob disease. In an effort to prevent new variant Creutzfeldt-Jakob disease, certain "specified offals," including neural and lymphatic tissues, thought to contain high titers of prions have been excluded from foods destined for human consumption [Phillips, N. A., Bridgeman, J. & Ferguson-Smith, M. (2000) in The BSE Inquiry (Stationery Office, London), Vol. 6, pp. 413-451]. Here we report that mouse skeletal muscle can propagate prions and accumulate substantial titers of these pathogens. We found both high prion titers and the disease-causing isoform of the prion protein (PrP(Sc)) in the skeletal muscle of wild-type mice inoculated with either the Me7 or Rocky Mountain Laboratory strain of murine prions. Particular muscles accumulated distinct levels of PrP(Sc), with the highest levels observed in muscle from the hind limb. To determine whether prions are produced or merely accumulate intramuscularly, we established transgenic mice expressing either mouse or Syrian hamster PrP exclusively in muscle. Inoculating these mice intramuscularly with prions resulted in the formation of high titers of nascent prions in muscle. In contrast, inoculating mice in which PrP expression was targeted to hepatocytes resulted in low prion titers. Our data demonstrate that factors in addition to the amount of PrP expressed determine the tropism of prions for certain tissues. That some muscles are intrinsically capable of accumulating substantial titers of prions is of particular concern. Because significant dietary exposure to prions might occur through the consumption of meat, even if it is largely free of neural and lymphatic tissue, a comprehensive effort to map the distribution of prions in the muscle of infected livestock is needed. Furthermore, muscle may provide a readily biopsied tissue from which to diagnose prion disease in asymptomatic animals and even humans. PMID:11904434

  16. The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients.

    PubMed

    Moore, Roger A; Head, Mark W; Ironside, James W; Ritchie, Diane L; Zanusso, Gianluigi; Pyo Choi, Young; Priola, Suzette A

    2016-02-01

    Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrPSc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrPC) into infectious PrPSc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrPSc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrPC allotype to PrPSc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrPSc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrPC containing an M or V at residue 129 having a similar propensity to misfold into PrPSc thus causing sCJD. By contrast, PrPSc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrPSc containing V at residue 129. In both types of CJD, the PrPSc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrPSc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD. PMID:26840342

  17. The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients

    PubMed Central

    Moore, Roger A.; Head, Mark W.; Ironside, James W.; Ritchie, Diane L.; Zanusso, Gianluigi; Pyo Choi, Young; Priola, Suzette A.

    2016-01-01

    Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrPSc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrPC) into infectious PrPSc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrPSc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrPC allotype to PrPSc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrPSc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrPC containing an M or V at residue 129 having a similar propensity to misfold into PrPSc thus causing sCJD. By contrast, PrPSc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrPSc containing V at residue 129. In both types of CJD, the PrPSc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrPSc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD. PMID:26840342

  18. Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

    NASA Astrophysics Data System (ADS)

    Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

    2012-09-01

    Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a ?-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

  19. Functional role of Tia1/Pub1 and Sup35 prion domains: directing protein synthesis machinery to the tubulin cytoskeleton.

    PubMed

    Li, Xiang; Rayman, Joseph B; Kandel, Eric R; Derkatch, Irina L

    2014-07-17

    Tia1/Pub1 is a stress granule component carrying a Q/N-rich prion domain. We provide direct evidence that Tia1 forms a prion in yeast. Moreover, Tia1/Pub1 acts cooperatively with release factor Sup35/eRF3 to establish a two-protein self-propagating state. This two-protein prion driven by the Q/N-rich prion domains of Sup35 and Tia1/Pub1 can be visualized as distinctive line structures along tubulin cytoskeleton. Furthermore, we find that tubulin-associated complex containing Pub1 and Sup35 oligomers normally exists in yeast, and its assembly depends on prion domains of Pub1 and Sup35. This Sup35/Pub1 complex, which also contains TUB1 mRNA and components of translation machinery, is important for the integrity of the tubulin cytoskeleton: PUB1 disruption and Sup35 depletion from the complex lead to cytoskeletal defects. We propose that the complex is implicated in protein synthesis at the site of microtubule assembly. Thus our study identifies the role for prion domains in the assembly of multiprotein complexes. PMID:24981173

  20. Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

    PubMed Central

    Kamatari, Yuji O; Ohta, Shinri; Inoshima, Yasuo; Oda, Masayuki; Maruno, Takahiro; Kobayashi, Yuji; Ishiguro, Naotaka

    2014-01-01

    We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174247 of the chicken prion protein (ChPrPC). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrPC, the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrPC and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrPC (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent KD values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 10?8 and 1.6 10?8 M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways. PMID:24863561

  1. Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication*

    PubMed Central

    Salamat, Khalid; Moudjou, Mohammed; Chapuis, Jrme; Herzog, Laetitia; Jaumain, Emilie; Bringue, Vincent; Rezaei, Human; Pastore, Annalisa; Laude, Hubert; Dron, Michel

    2012-01-01

    The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrPSc. We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrPSc and elucidate the conformational changes underlying prions generation. PMID:22511770

  2. Ablation of prion protein immunoreactivity by heating in saturated calcium hydroxide

    PubMed Central

    Greenlee, Justin J; Nicholson, Eric M; Hamir, Amir N; Noyes, Gary P; Holtzapple, Mark T; Kehrli, Marcus E

    2008-01-01

    Background Prions, the infectious agents that cause transmissible spongiform encephalopathies (TSEs), are relatively resistant to destruction by physical, enzymatic, and chemical treatments. Hydrolysis in boiling saturated calcium hydroxide (limewater) utilizes inexpensive chemicals to digest protein components of offal. The purpose of this work was to determine if incubating brain material from scrapie-infected sheep in near-boiling saturated calcium hydroxide solution (Ca(OH)2) would abolish immunoreactivity of the infectious prion (PrPSc) as determined by western blot. Findings After incubating for as few as 10 minutes in saturated calcium hydroxide at 99C, immunoreactivity of protease resistant bands by western blot analysis is completely lost. Conclusion Boiling in limewater may offer an alternative for disposal of carcasses and enable alternative uses for rendered products from potentially infected carcasses. PMID:18957103

  3. Reversible monomer-oligomer transition in human prion protein

    PubMed Central

    Sasaki, Ken; Gaikwad, Jyoti; Hashiguchi, Shuhei; Kubota, Toshiya; Sugimura, Kazuhisa; Kremer, Werner; Kalbitzer, Hans Robert

    2008-01-01

    The structure and the dissociation reaction of oligomers PrPoligo from reduced human prion huPrPC(23231) have been studied by 1H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The 1H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105?210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrPC*, a rare metastable form of PrPC stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrPoligo is in dynamic equilibrium with the monomeric forms via PrPC*, namely huPrPC ? huPrPC* ? huPrPoligo. PMID:19158507

  4. Techniques to elucidate the conformation of prions

    PubMed Central

    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. PMID:26322176

  5. The normal cellular prion protein is strongly expressed by myeloid dendritic cells.

    PubMed

    Burthem, J; Urban, B; Pain, A; Roberts, D J

    2001-12-15

    Abnormal isoforms of the prion protein (PrP(Sc)) that cause prion diseases are propagated and spread within the body by "carrier" cell(s). Cells of the immune system have been strongly implicated in this process. In particular, PrP(Sc) is known to accumulate on follicular dendritic cells (FDCs) in individuals affected by variant Creutzfeld-Jakob disease. However, FDCs do not migrate widely and the natural history of prion disorders suggests other cells may be required for the transport of PrP(Sc) from the site of ingestion to lymphoid organs and the central nervous system. Substantial evidence suggests that the spread of PrP(Sc) requires bone marrow-derived cells that express normal cellular prion protein (PrP(C)). This study examined the expression of PrP(C) on bone marrow-derived cells that interact with lymphoid follicles. High levels of PrP(C) are present on myeloid dendritic cells (DCs) that surround the splenic white pulp. These myeloid DCs are ontologically and functionally distinct from the FDCs. Consistent with these observations, expression of PrP(C) was strongly induced during the generation of mature myeloid DCs in vitro. In these cells PrP(C) colocalized with major histocompatibility complex class II molecules at the level of light microscopy. Furthermore, given the close anatomic and functional connection of myeloid DCs with lymphoid follicles, these results raise the possibility that myeloid DCs may play a role in the propagation of PrP(Sc) in humans. PMID:11739179

  6. Repeats are one of the main characteristics of RNA-binding proteins with prion-like domains.

    PubMed

    Galzitskaya, Oxana V

    2015-08-01

    It is not surprising that a large number of diseases related to amyloid fibril depositions are formed in various organs. Therefore, it is necessary to understand the transformation of native proteins into amyloid fibrils in order to clarify which key elements of this process determine the pathway of protein misfolding. Significant attention has been directed recently to investigating the mechanism of formation of cross-? structures that have the properties of liquids but can also exist in gel-like forms, thus facilitating the retention of both RNAs and RNA-binding proteins. Proteins that form stress granules are believed to do this rapidly, and they are expected to contain a prion-like domain that can facilitate this process. By analyzing the known yeast prion proteins and 29 RNA-binding proteins with prion-like domains, we demonstrate here that the existence of repeats is one of the general characteristics of prion-like domains. The presence of repeats should help to determine the border of prion domains as in the case of Rnq1: five found repeats shift the border of the prion domain from the 153-rd to at least the 133-th residue. One can suggest that such repeats assist in the rapid initiation of the process of assembly and formation of cross-? structures and such domains most likely should be disordered. These repeats should contain aromatic amino acid residues for the formation of a hydrogel because its amino acid context modulates the strength of interaction. The key factors determined here can be used to control the process of aggregation to prevent the development of pathologies and diseases caused by prion-like domains. PMID:26022110

  7. Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein

    PubMed Central

    Yedidia, Yifat; Horonchik, Lior; Tzaban, Salit; Yanai, Anat; Taraboulos, Albert

    2001-01-01

    Prion diseases propagate by converting a normal glycoprotein of the host, PrPC, into a pathogenic prion conformation. Several misfolding mutants of PrPC are degraded through the ER-associated degradation (ERAD)proteasome pathway. In their infectious form, prion diseases such as bovine spongiform encephalopathy involve PrPC of wild-type sequence. In contrast to mutant PrP, wild-type PrPC was hitherto thought to be stable in the ER and thus immune to ERAD. Using proteasome inhibitors, we now show that ?10% of nascent PrPC molecules are diverted into the ERAD pathway. Cells incubated with N-acetyl-leucinal-leucinal-norleucinal (ALLN), lactacystin or MG132 accumulated both detergent-soluble and insoluble PrP species. The insoluble fraction included an unglycosylated 26 kDa PrP species with a protease-resistant core, and a Mr ladder that contained ubiquitylated PrP. Our results show for the first time that wild-type PrPC molecules are subjected to ERAD, in the course of which they are dislocated into the cytosol and ubiquitylated. The presence of wild-type PrP molecules in the cytosol may have potential pathogenic implications. PMID:11574470

  8. Doped diamond-like carbon coatings for surgical instruments reduce protein and prion-amyloid biofouling and improve subsequent cleaning.

    PubMed

    Secker, T J; Herv, R; Zhao, Q; Borisenko, K B; Abel, E W; Keevil, C W

    2012-01-01

    Doped diamond-like carbon (DLC) coatings offer potential antifouling surfaces against microbial and protein attachment. In particular, stainless steel surgical instruments are subject to tissue protein and resilient prion protein attachment, making decontamination methods used in sterile service departments ineffective, potentially increasing the risk of iatrogenic Creutzfeldt-Jakob disease during surgical procedures. This study examined the adsorption of proteins and prion-associated amyloid to doped DLC surfaces and the efficacy of commercial cleaning chemistries applied to these spiked surfaces, compared to titanium nitride coating and stainless steel. Surfaces inoculated with ME7-infected brain homogenate were visualised using SYPRO Ruby/Thioflavin T staining and modified epi-fluorescence microscopy before and after cleaning. Reduced protein and prion amyloid contamination was observed on the modified surfaces and subsequent decontamination efficacy improved. This highlights the potential for a new generation of coatings for surgical instruments to reduce the risk of iatrogenic CJD infection. PMID:22694725

  9. [Physiopathology of prion diseases].

    PubMed

    Mandujano, Alejandra; Montes, Sara; Guzmn, Ada; Espinosa, Blanca; Rembao, Daniel; Martnez-Cairo, Salvador; Zenteno, Edgar; Guevara, Jorge

    2006-01-01

    Prion diseases are a group of degenerative disorders characterized by being progressive, fast growing, and fatal, they affect humans and animals. Due to their physiopathogeny, these disorders can be sporadic, genetic, or infectious. Prions are cellular proteins that lack nucleic acids; they are not viruses or microorganisms. Prions induce neuronal death, brain spongiosis, which are a hallmark of these diseases, as well as amyloid prion protein plaque aggregates. Although the causes that favor pathogenic prion proteins remain uncertain, it is possible that conformational changes of the prion protein allow them to create copies of themselves to form aggregates and induce neuronal death. Other theories suggest that quantitative and qualitative changes in the glycosylation pattern induce the pathological prion form. The latter allows to explain some of their interactions and to understand better the conformational changes and the physico-chemical properties of the prion protein. We review some of the first biological functions (as a transporter of Cu2+ ions) that have been described to this molecule. The present review focuses on different aspects of prion diseases aimed at understanding better their physiopathogenic characteristics. PMID:17128820

  10. Physiopathologic implications of the structural and functional domains of the prion protein.

    PubMed

    Sorgato, M Catia; Bertoli, Alessandro

    2006-01-01

    Prion diseases are invariably fatal neurodegenerative disorders affecting man and various animal species. A large body of evidence supports the notion that the causative agent of these diseases is the prion, which, devoid of nucleic acids, is composed largely, if not entirely, of a conformationally abnormal isoform (PrP(Sc) of the cellular prion protein (PrPc). PrPc is a highly conserved and ubiquitously expressed sialoglycoprotein, the normal function of which is, however, still ill defined. Several modules have been recognised in PrPc structure. Their extensive analysis by different experimental approaches, including transgenic animal models, has allowed to assigning to several modules a putative role in PrPc physiology. Concurrently, it has underscored the possibility that alteration of specific domains may determine the switching from a beneficial role of PrPc into one that becomes detrimental to neurons, and/or promote the conversion of PrPc into the pathogenic PrP(Sc) conformer. PMID:17274528

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

    PubMed

    Birkan, Tugce; Sahin, Mesut; Oztel, Zubeyde; Balcan, Erdal

    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 less than 0.01 and P less than 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. PMID:26949092

  12. Preclinical Deposition of Pathological Prion Protein in Muscle of Experimentally Infected Primates

    PubMed Central

    Krasemann, Susanne; Neumann, Melanie; Geissen, Markus; Bodemer, Walter; Kaup, Franz-Josef; Schulz-Schaeffer, Walter; Morel, Nathalie

    2010-01-01

    Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and animals. A central step in disease progression is the accumulation of a misfolded form (PrPSc) of the host encoded prion protein (PrPC) 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 PrPSc 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 PrPSc 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 PrPSc in muscle and central nervous system of rhesus monkeys experimentally infected with vCJD. PMID:21085647

  13. Functional characterization of the human prion protein promoter in neuronal and endothelial cells.

    PubMed

    Funke-Kaiser, H; Theis, S; Behrouzi, T; Thomas, A; Scheuch, K; Zollmann, F S; Paterka, M; Paul, M; Orzechowski, H D

    2001-09-01

    Human prion diseases such as Creutzfeld-Jakob disease and kuru are of major medical and biological importance because of their fatal course, epidemic potential, and unique pathophysiology. Endogenous expression of the normal cellular prion protein (PrP(C)) is necessary for infection and prion replication. However, knowledge of human PrP(C) gene regulation is rudimentary. We therefore cloned1543 bp of the 5' untranslated and promoter region of the PrP gene. Using transient transfection assays, the full-length promoter and serial deletion mutants subcloned in a luciferase reporter vector were analyzed in neuronal (KELLY) and endothelial (EA.hy926) cell lines, which both express PrP(C) as shown by RT/PCR. Analysis of promoter constructs in KELLY cells indicated two activating regions at -131/-284 and -1303/-1543, relative to the 3'-terminal end of exon 1, and also two repressing elements at -254/-567 and -567/-909 in neuronal cells. In EA.hy926 cells, activating elements were identified at -131/-284 and -284/-567, and one repressing region was localized at -567/-909. In addition, transcriptional start sites were determined by 5'-RACE reaction and RNase protection assay, revealing one major transcriptional start site located at -47 (in KELLY cells), -53 (in human thalamus) and at about -55 (in EA.hy926 cells). PMID:11692166

  14. Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells.

    PubMed

    Magalhes, Ana Cristina; Baron, Gerald S; Lee, Kil Sun; Steele-Mortimer, Olivia; Dorward, David; Prado, Marco A M; Caughey, Byron

    2005-05-25

    Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's Abeta1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells. PMID:15917460

  15. Conformational instability of human prion protein upon residue modification: a molecular dynamics simulation study

    PubMed Central

    Bamdad, Kourosh; Naderi-manesh, Hossein; Baumgaertner, Artur

    2014-01-01

    Technical strategies like amino acid substitution and residue modification have been widely used to characterize the importance of key amino acids and the role that each residue plays in the structural and functional properties of protein molecules. However, there is no systematic approach to assess the impact of the substituted/modified amino acids on the conformational dynamics of proteins. In this investigation to clarify the effects of residue modifications on the structural dynamics of human prion protein (PrP), a comparative molecular dynamics simulation study on the native and the amino acid-substituted analog at position 208 of PrP has been performed. It is believed that Arginine to Histidine mutation at position 208 is responsible for the structural transition of the native form of human prion protein to the pathogenic isoform causing Creutzfeldt-Jakob disease (CJD). So, three 10 ns molecular dynamics simulations on three model constructs have been performed. Simulation results indicated considerable differences of conformational fluctuations for Alanine substituted construct (PrPALA) and the analog form (PrPSB) comprising the neutralized state of the Arginine residue at position 208 of the human prion protein. According to our data, substitution of the Arginine residue by the uncharged state of this residue induces some reversible structural alterations in the intrinsically flexible loop area including residues 167171 of PrP. Thus, deprotonation of Arg208 is a weak perturbation to the structural fluctuations of the protein backbone and the resulting construct behaves almost identical as its native form. Otherwise, Alanine substitution at position 208 imposed an irreversible impact on the secondary and tertiary structure of the protein, which leads to conformational instabilities in the remote hot region comprising residues 190195 of the Cterminal part of helix 2. Based on the results, it could be deduced that the observed conformational transitions upon Arg208 to His point mutation, which is the main reason for CJD, may be mainly related to the structural instabilities due to the induced-conformational changes that caused alterations in local/spatial arrangements of the force distributions in the backbone of the human prion protein. PMID:26417255

  16. Stability of the ?-structure in prion protein: A molecular dynamics study based on polarized force field

    NASA Astrophysics Data System (ADS)

    Xu, Zhijun; Lazim, Raudah; Mei, Ye; Zhang, Dawei

    2012-06-01

    Conformational changes of the antiparallel ?-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 ?-sheet during the early stage transition from the PrPC to its mutant configuration. Through this study, we observe the growth of the ?-sheet structure in all species studied with the extent of elongation in ?-sheet being different across the three species.

  17. Amyloid-β Receptors: The Good, the Bad, and the Prion Protein*

    PubMed Central

    Jarosz-Griffiths, Heledd H.; Noble, Elizabeth; Rushworth, Jo V.; Hooper, Nigel M.

    2016-01-01

    Several different receptor proteins have been identified that bind monomeric, oligomeric, or fibrillar forms of amyloid-β (Aβ). “Good” receptors internalize Aβ or promote its transcytosis out of the brain, whereas “bad” receptors bind oligomeric forms of Aβ that are largely responsible for the synapticloss, memory impairments, and neurotoxicity that underlie Alzheimer disease. The prion protein both removes Aβ from the brain and transduces the toxic actions of Aβ. The clustering of distinct receptors in cell surface signaling platforms likely underlies the actions of distinct oligomeric species of Aβ. These Aβ receptor-signaling platforms provide opportunities for therapeutic intervention in Alzheimer disease. PMID:26719327

  18. Amyloid-β Receptors: The Good, the Bad, and the Prion Protein.

    PubMed

    Jarosz-Griffiths, Heledd H; Noble, Elizabeth; Rushworth, Jo V; Hooper, Nigel M

    2016-02-12

    Several different receptor proteins have been identified that bind monomeric, oligomeric, or fibrillar forms of amyloid-β (Aβ). "Good" receptors internalize Aβ or promote its transcytosis out of the brain, whereas "bad" receptors bind oligomeric forms of Aβ that are largely responsible for the synapticloss, memory impairments, and neurotoxicity that underlie Alzheimer disease. The prion protein both removes Aβ from the brain and transduces the toxic actions of Aβ. The clustering of distinct receptors in cell surface signaling platforms likely underlies the actions of distinct oligomeric species of Aβ. These Aβ receptor-signaling platforms provide opportunities for therapeutic intervention in Alzheimer disease. PMID:26719327

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

    PubMed

    Tixador, Philippe; Herzog, Latitia; Reine, Fabienne; Jaumain, Emilie; Chapuis, Jrme; Le Dur, Annick; Laude, Hubert; Bringue, Vincent

    2010-04-01

    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. PMID:20419156

  20. Expression of cellular prion protein (PrP(c)) in schizophrenia, bipolar disorder, and depression.

    PubMed

    Weis, Serge; Haybaeck, Johannes; Dulay, Jeannette R; Llenos, Ida C

    2008-05-01

    Cellular prion protein (PrP(c)) is a copper-binding, membrane-attached GPI-anchored glycoprotein characterized by a high degree of amino acid sequence conservation among mammals. PrP(c) expression has been demonstrated in neurons, microglia, lymphocytes, and keratinocytes. Recently, the concept that PrP(c) may be involved in the defense against oxidative stress was advanced. In the present study, we used immunohistochemistry for PrP(c) to investigate 60 brains from the Stanley Neuropathology Consortium (15 controls, 15 patients with schizophrenia, 15 with bipolar disorder, and 15 with major depression). Rating scores as well as the numerical density of PrP(c)-positive and -negative neurons and glial cells were determined in the cingulate gyrus. All four groups showed a very high interindividual variation. PrP(c)-positive glial cells were significantly reduced in the white matter of patients with schizophrenia, bipolar disorder, and major depression. A similar result was obtained for the white matter in bipolar patients using rating scales. From the confounding variables, use of medication (i.e. antipsychotics, antidepressants, and mood stabilizers) had a significant effect on the expression of PrP(c) by neurons and glial cells. PrP(c)-immunoreactivities were significantly reduced for white matter glial cells in all examined groups. However, the results are not indicative for the occurrence of oxidative stress in the brains of schizophrenic and bipolar patients. Since the effect of antipsychotic and antidepressant medication as well as of mood stabilizers on the expression of PrP(c) was significant, it needs further clarification in experimental models. PMID:18188498

  1. Can copper binding to the prion protein generate a misfolded form of the protein?

    PubMed

    Pushie, M Jake; Rauk, Arvi; Jirik, Frank R; Vogel, Hans J

    2009-02-01

    The native prion protein (PrP) has a two domain structure, with a globular folded alpha-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu(2+) via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in beta-sheet content. Cu(2+) bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu(+) can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu(2+) binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized beta-sheet structure, allowing us to suggest a potential mechanism for the initiation of beta-sheet misfolding in the C-terminal domain by Cu(2+). PMID:19140013

  2. Disinfectants and Prions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Hsp104 drives "protein-only" positive selection of Sup35 prion strains encoding strong [PSI(+)].

    PubMed

    DeSantis, Morgan E; Shorter, James

    2012-11-21

    Structurally distinct, self-templating prion "strains" can encode distinct phenotypes and amplify at different rates depending upon the environment. Indeed, prion strain ensembles can evolve in response to environmental challenges, which makes them highly challenging drug targets. It is not understood how the proteostasis network amplifies one prion strain at the expense of another. Here, we demonstrate that Hsp104 remodels the distinct intermolecular contacts of different synthetic Sup35 prion strains in a way that selectively amplifies prions encoding strong [PSI(+)] and simultaneously eliminates prions encoding weak [PSI(+)]. Hsp104 has reduced ability to fragment prions encoding weak [PSI(+)], but readily converts them to nontemplating forms. By contrast, Hsp104 readily fragments prions encoding strong [PSI(+)], but has reduced ability to eliminate their infectivity. Thus, we illuminate direct mechanisms underpinning how the proteostasis network can drive prion strain selection. PMID:23177195

  4. Interaction between a recombinant prion protein and organo-mineral complexes as evidenced by CPMAS 13C-NMR

    NASA Astrophysics Data System (ADS)

    Russo, F.; Scotti, R.; Gianfreda, L.; Conte, P.; Rao, M. A.

    2009-04-01

    Prion proteins (PrP) are the main responsible for Transmissible Spongiform Encephalopathies (TSE). The TSE etiological agent is a misfolded form of the normal cellular prion protein. The amyloidal aggregates accumulated in the brain of infected animals and mainly composed of PrPSc exhibit resistance to protease attack and many conventional inactivating procedures. The prion protein diseases cause an environmental issue because the environment and in particular the soil compartment can be contaminated and then become a potential reservoir and diffuser of TSEs infectivity as a consequence of (i) accidental dispersion from storage plants of meat and bone meal, (ii) incorporation of contaminated material in fertilizers, (iii) possible natural contamination of pasture soils by grazing herds, and (v) burial of carcasses. The environmental problem can be even more relevant because very low amounts of PrPSc are able to propagate the disease. Several studies evidenced that infectious prion protein remains active in soils for years. Contaminated soils result, thus, a possible critical route of TSE transmission in wild animals. Soil can also protect prion protein toward degradation processes due to the presence of humic substances and inorganic components such as clays. Mineral and organic colloids and the more common association between clay minerals and humic substances can contribute to the adsorption/entrapment of molecules and macromolecules. The polymerization of organic monomeric humic precursors occurring in soil in the presence of oxidative enzymes or manganese and iron oxides, is considered one of the most important processes contributing to the formation of humic substances. The process is very fast and produces a population of polymeric products of different molecular structures, sizes, shapes and complexity. Other molecules and possibly biomacromolecules such as proteins may be involved. The aim of the present work was to study by CPMAS 13C-NMR the interactions between a non pathogenic ovine recombinant prion protein and a model soil system represented by a manganese oxide in the form of birnessite (δ-MnO2), coated with a polymerized catechol. To better understand the effect of the polymerization process, PrP was added to the birnessite-cathecol system either before or after the polymerization processes. The NMR spectra of the prion protein interacting directly with birnessite revealed disappearance of the signals due to the paramagnetic nature of manganese oxide or abiotic degradation. Conversely, the signal pattern of the protein re-appeared as it is mixed to the soil-like system either during or after the catechol polymerization process. Results suggested that the possible interactions of the prion protein on soil systems can be mediated by natural organic matter. However, deeper studies on more complex real soil systems are needed to definitely confirm such hypothesis.

  5. Familial prion protein mutants inhibit Hrd1-mediated retrotranslocation of misfolded proteins by depleting misfolded protein sensor BiP.

    PubMed

    Peters, Sarah L; Déry, Marc-André; LeBlanc, Andrea C

    2016-03-01

    Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases. PMID:26740554

  6. Limited transcriptional response of ovine microglia to prion accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  7. Prions, From Structure to Epigenetics and Neuronal Functions

    NASA Astrophysics Data System (ADS)

    Lindquist, Susan

    2012-02-01

    Prions are a unique type of protein that can misfold and convert other proteins to the same shape. The well-characterized yeast prion [PSI+] is formed from an inactive amyloid fiber conformation of the translation-termination factor, Sup35. This altered conformation is passed from mother cells to daughters, acting as a template to perpetuate the prion state and providing a mechanism of protein-based inheritance. We employed a variety of methods to determine the structure of Sup35 amyloid fibrils. First, using fluorescent tags and cross-linking we identified specific segments of the protein monomer that form intermolecular contacts in a ``Head-to-Head,'' ``Tail-to-Tail'' fashion while a central region forms intramolecular contacts. Then, using peptide arrays we mapped the region responsible for the prion transmission barrier between two different yeast species. We have also used optical tweezers to reveal that the non-covalent intermolecular contacts between monomers are unusually strong, and maintain fibril integrity even under forces that partially unfold individual monomers and extend fibril length. Based on the handful of known yeast prion proteins we predicted sequences that could be responsible for prion-like amyloid folding. Our screen identified 19 new candidate prions, whose protein-folding properties and diverse cellular functions we have characterized using a combination of genetic and biochemical techniques. Prion-driven phenotypic diversity increases under stress, and can be amplified by the dynamic maturation of prion-initiating states. These qualities allow prions to act as ``bet-hedging'' devices that facilitate the adaptation of yeast to stressful environments, and might speed the evolution of new traits. Together with Kandel and Si, we have also found that a regulatory protein that plays an important role in synaptic plasticity behaves as a prion in yeast. Cytoplasmic polyAdenylation element binding protein, CPEB, maintains synapses by promoting the local translation of mRNAs. We postulate that the self-perpetuating folding of the prion domain acts as a molecular memory. Thus yeast prions have provided evidence for the surprising possibility that amyloid protein folds can serve as the basis for memory and inheritance.

  8. Modulation of proteinase K-resistant prion protein in cells and infectious brain homogenate by redox iron: implications for prion replication and disease pathogenesis.

    PubMed

    Basu, Subhabrata; Mohan, Maradumane L; Luo, Xiu; Kundu, Bishwajit; Kong, Qingzhong; Singh, Neena

    2007-09-01

    The principal infectious and pathogenic agent in all prion disorders is a beta-sheet-rich isoform of the cellular prion protein (PrP(C)) termed PrP-scrapie (PrP(Sc)). Once initiated, PrP(Sc) is self-replicating and toxic to neuronal cells, but the underlying mechanisms remain unclear. In this report, we demonstrate that PrP(C) binds iron and transforms to a PrP(Sc)-like form (*PrP(Sc)) when human neuroblastoma cells are exposed to an inorganic source of redox iron. The *PrP(Sc) thus generated is itself redox active, and it induces the transformation of additional PrP(C), simulating *PrP(Sc) propagation in the absence of brain-derived PrP(Sc). Moreover, limited depletion of iron from prion disease-affected human and mouse brain homogenates and scrapie-infected mouse neuroblastoma cells results in 4- to 10-fold reduction in proteinase K (PK)-resistant PrP(Sc), implicating redox iron in the generation, propagation, and stability of PK-resistant PrP(Sc). Furthermore, we demonstrate increased redox-active ferrous iron levels in prion disease-affected brains, suggesting that accumulation of PrP(Sc) is modulated by the combined effect of imbalance in brain iron homeostasis and the redox-active nature of PrP(Sc). These data provide information on the mechanism of replication and toxicity by PrP(Sc), and they evoke predictable and therapeutically amenable ways of modulating PrP(Sc) load. PMID:17567949

  9. Degradation of the Disease-Associated Prion Protein by a Serine Protease from Lichens

    PubMed Central

    Johnson, Christopher J.; Bennett, James P.; Biro, Steven M.; Duque-Velasquez, Juan Camilo; Rodriguez, Cynthia M.; Bessen, Richard A.; Rocke, Tonie 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. PMID:21589935

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

    USGS Publications Warehouse

    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 (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.

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

    USGS Publications Warehouse

    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.

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

    USGS Publications Warehouse

    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.

  13. Loss of amino-terminal acetylation suppresses a prion phenotype by modulating global protein folding.

    PubMed

    Holmes, William M; Mannakee, Brian K; Gutenkunst, Ryan N; Serio, Tricia R

    2014-01-01

    Amino-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. Although loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI(+)], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype. PMID:25023910

  14. Loss of N-terminal Acetylation Suppresses A Prion Phenotype By Modulating Global Protein Folding

    PubMed Central

    Holmes, William M.; Mannakee, Brian K.; Gutenkunst, Ryan N.; Serio, Tricia R.

    2014-01-01

    N-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. While loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI+], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype. PMID:25023910

  15. Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cellular prion protein (PrPC) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrPC expression is either up-regulated or depleted, its exact p...

  16. Effect of charged residues in the N-domain of Sup35 protein on prion [PSI+] stability and propagation.

    PubMed

    Bondarev, Stanislav A; Shchepachev, Vadim V; Kajava, Andrey V; Zhouravleva, Galina A

    2013-10-01

    Recent studies have shown that Sup35p prion fibrils probably have a parallel in-register ?-structure. However, the part(s) of the N-domain critical for fibril formation and maintenance of the [PSI(+)] phenotype remains unclear. Here we designed a set of five SUP35 mutant alleles (sup35(KK)) with lysine substitutions in each of five N-domain repeats, and investigated their effect on infectivity and ability of corresponding proteins to aggregate and coaggregate with wild type Sup35p in the [PSI(+)] strain. Alleles sup35-M1 (Y46K/Q47K) and sup35-M2 (Q61K/Q62K) led to prion loss, whereas sup35-M3 (Q70K/Q71K), sup35-M4 (Q80K/Q81K), and sup35-M5 (Q89K/Q90K) were able to maintain the [PSI(+)] prion. This suggests that the critical part of the parallel in-register ?-structure for the studied [PSI(+)] prion variant lies in the first 63-69 residues. Our study also reveals an unexpected interplay between the wild type Sup35p and proteins expressed from the sup35(KK) alleles during prionization. Both Sup35-M1p and Sup35-M2p coaggregated with Sup35p, but only sup35-M2 led to prion loss in a dominant manner. We suggest that in the fibrils, Sup35p can bind to Sup35-M1p in the same conformation, whereas Sup35-M2p only allowed the Sup35p conformation that leads to the non-heritable fold. Mutations sup35-M4 and sup35-M5 influence the structure of the prion forming region to a lesser extent, and can lead to the formation of new prion variants. PMID:23965990

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

    PubMed Central

    De Mario, Agnese; Castellani, Angela; Peggion, Caterina; Massimino, Maria Lina; Lim, Dmitry; Hill, Andrew F.; Sorgato, M. Catia; Bertoli, Alessandro

    2015-01-01

    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 (CGN) 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 mechanizm for Aβ-induced neuronal Ca2+ dyshomeostasis. PMID:26578881

  18. Prion protein "gamma-cleavage": characterizing a novel endoproteolytic processing event.

    PubMed

    Lewis, Victoria; Johanssen, Vanessa A; Crouch, Peter J; Klug, Genevieve M; Hooper, Nigel M; Collins, Steven J

    2016-02-01

    The cellular prion protein (PrP(C)) is a ubiquitously expressed protein of currently unresolved but potentially diverse function. Of putative relevance to normal biological activity, PrP(C) is recognized to undergo both α- and β-endoproteolysis, producing the cleavage fragment pairs N1/C1 and N2/C2, respectively. Experimental evidence suggests the likelihood that these processing events serve differing cellular needs. Through the engineering of a C-terminal c-myc tag onto murine PrP(C), as well as the selective use of a far-C-terminal anti-PrP antibody, we have identified a new PrP(C) fragment, nominally 'C3', and elaborating existing nomenclature, 'γ-cleavage' as the responsible proteolysis. Our studies indicate that this novel γ-cleavage event can occur during transit through the secretory pathway after exiting the endoplasmic reticulum, and after PrP(C) has reached the cell surface, by a matrix metalloprotease. We found that C3 is GPI-anchored like other C-terminal and full length PrP(C) species, though it does not localize primarily at the cell surface, and is preferentially cleaved from an unglycosylated substrate. Importantly, we observed that C3 exists in diverse cell types as well as mouse and human brain tissue, and of possible pathogenic significance, γ-cleavage may increase in human prion diseases. Given the likely relevance of PrP(C) processing to both its normal function, and susceptibility to prion disease, the potential importance of this previously underappreciated and overlooked cleavage event warrants further consideration. PMID:26298290

  19. The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation.

    PubMed

    Torrent, Joan; Lange, Reinhard; Rezaei, Human

    2015-08-14

    Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct ?-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains. PMID:26126829

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

    SciTech Connect

    Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi; Terajima, Hideki; Yajima, Junichiro; Nishizaka, Takayuki; Kinjo, Masataka; Taguchi, Hideki

    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.

  1. Nanoimaging for prion related diseases.

    PubMed

    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 great promise for understanding the molecular mechanisms of prion phenomenon, possibly leading toward early diagnosis and effective treatment of these devastating diseases. This review article summarizes recent reports which advanced our understanding of the prion phenomenon through the use of nanoimaging methods. PMID:20724837

  2. Identification and structural analysis of C-terminally truncated collapsin response mediator protein-2 in a murine model of prion diseases

    PubMed Central

    2010-01-01

    Background Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrPSc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood. Results In a proteomic analysis of soluble proteins in the brains of mice challenged intracerebrally with scrapie prion (Obihiro I strain), we found that the amount of the full-length form of collapsin response mediator protein-2 (CRMP-2; 61 kDa) decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-?C) lacked the sequence from serine518 to the C-terminus, including the C-terminal phosphorylation sites important for the regulation of axonal growth and axon-dendrite specification in developing neurons. The invariable size of the mRNA transcript in Northern blot analysis suggested that the truncation was due to post-translational proteolysis. By overexpression of CRMP-2-?C in primary cultured neurons, we observed the augmentation of the development of neurite branch tips to the same levels as for CRMP-2T514A/T555A, a non-phosphorylated mimic of the full-length protein. This suggests that the increased level of CRMP-2-?C in the brain modulates the integrity of neurons, and may be involved in the pathogenesis of the neuronal abnormalities observed in the late stages of the disease. Conclusions We identified the presence of CRMP-2-?C in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-?C in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders. PMID:20961402

  3. Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion.

    PubMed

    Silva, Jerson L; Vieira, Tuane C R G; Gomes, Mariana P B; Rangel, Luciana P; Scapin, Sandra M N; Cordeiro, Yraima

    2011-03-01

    The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, ?-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a ?-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies. PMID:21145399

  4. High affinity binding between copper and full-length prion protein identified by two different techniques.

    PubMed

    Thompsett, Andrew R; Abdelraheim, Salama R; Daniels, Maki; Brown, David R

    2005-12-30

    The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity. PMID:16258172

  5. The [PSI+] Prion Exists as a Dynamic Cloud of Variants

    PubMed Central

    Bateman, David A.; Wickner, Reed B.

    2013-01-01

    [PSI+] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion strains or variants are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, ?19, and E9, with limited transmissibility of [PSI+] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI+] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI+], all indistinguishably weak [PSI+], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI+]. Notably, a strong [PSI+] can have any of several different transmission efficiency patterns, showing that strong versus weak is insufficient to indicate prion variant uniformity. PMID:23382698

  6. The [PSI+] prion exists as a dynamic cloud of variants.

    PubMed

    Bateman, David A; Wickner, Reed B

    2013-01-01

    [PSI(+)] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion "strains" or "variants" are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, ?19, and E9, with limited transmissibility of [PSI(+)] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI(+)] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI(+)], all indistinguishably "weak" [PSI(+)], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI(+)]. Notably, a "strong [PSI(+)]" can have any of several different transmission efficiency patterns, showing that "strong" versus "weak" is insufficient to indicate prion variant uniformity. PMID:23382698

  7. PrP Expression Level and Sensitivity to Prion Infection

    PubMed Central

    Douet, Jean-Yves; Lacroux, Caroline; Corbire, Fabien; Litaise, Claire; Simmons, Hugh; Lugan, Sverine; Costes, Pierrette; Cassard, Herv; Weisbecker, Jean-Louis; Schelcher, Franois

    2014-01-01

    Mice overexpressing the prion protein (PrP) sequence from various host species are widely used for measuring infectious titers in prion disease. However, the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP results in similar estimates of the infectious titer. PMID:24574409

  8. ?-sheet-like formation during the mechanical unfolding of prion protein

    NASA Astrophysics Data System (ADS)

    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 ?-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 ?-helical structure to the ?-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.

  9. GFP-TAGGED MUTANT PRION PROTEIN FORMS INTRA-AXONAL AGGREGATES IN TRANSGENIC MICE

    PubMed Central

    Medrano, Andrea Z.; Barmada, Sami J.; Biasini, Emiliano; Harris, David A.

    2008-01-01

    A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis, PrP aggregation, and accumulation of a partially protease-resistant form of the mutant PrP. Strikingly, PG14-EGFP forms numerous fluorescent aggregates in the neuropil and white matter of multiple brain regions. These aggregates are particularly prominent along axonal tracts in both brain and peripheral nerve, and similar intracellular deposits are visible along the processes of cultured neurons. Our results reveal intra-axonal aggregates of a mutant PrP, which could contribute to the pathogenesis of familial prion disease by disrupting axonal transport. PMID:18514536

  10. The N-terminal, polybasic region is critical for prion protein neuroprotective activity.

    PubMed

    Turnbaugh, Jessie A; Westergard, Laura; Unterberger, Ursula; Biasini, Emiliano; Harris, David A

    2011-01-01

    Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (?32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (?23-31, ?23-111, and ?23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although ?23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases. PMID:21980526

  11. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers

    PubMed Central

    Younan, Nadine D.; Sarell, Claire J.; Davies, Paul; Brown, David R.; Viles, John H.

    2013-01-01

    There is now strong evidence to show that the presence of the cellular prion protein (PrPC) mediates amyloid-β (Aβ) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrPC and Aβ and discover that substoichiometric amounts of PrPC, as little as 1/20, relative to Aβ will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrPC. Electron microscopy indicates PrPC is able to trap Aβ in an oligomeric form. Unlike fibers, this oligomeric Aβ contains antiparallel β sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrPC, notably residues 95–113, binds to Aβ oligomers, but only once Aβ misfolds. The ability of PrPC to trap and concentrate Aβ in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrPC might confer Aβ toxicity in AD, as oligomers are thought to be the toxic form of Aβ. Identification of a specific recognition site on PrPC that traps Aβ in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.—Younan, N. D., Sarell, C. J., Davies, P., Brown, D. R., Viles, J. H. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers. PMID:23335053

  12. Evolutionary conserved Tyr169 stabilizes the ?2-?2 loop of the prion protein.

    PubMed

    Huang, Danzhi; Caflisch, Amedeo

    2015-03-01

    Experimental evidence indicates that the primary structure of the ?2-?2 loop region (residues 165-175) in mammalian prion proteins (PrP) influences the conversion from the cellular species (PrP(C)) to the ?-sheet-rich aggregate. Here, we captured the transition of the ?2-?2 loop from 310-helical turn to ? turn by unbiased molecular dynamics simulations of the single-point mutant Y169G. Multiple conformations along the spontaneous transition of the mutant were then used as starting point for sampling of the free-energy surface of the wild type and other single-point mutants. Using two different methods for the determination of free energy profiles, we found that the barrier for the 310-helical turn to ? turn transition of the wild type is higher by about 2.5 kcal/mol than for the Y169G mutant, which is due to favorable stacking of the aromatic rings of Y169 and F175, and a stable hydrogen bond between the side chains of Y169 and D178. The transition of the ?2-?2 loop to ? turn increases the solvent-exposure of the hydrophobic stretch 169-YSNQNNF-175. The simulations indicate that the strictly conserved Y169 in mammalian prion proteins stabilizes the 310-helical turn in the ?2-?2 loop, thus hindering the conversion to an aggregation-prone conformation. PMID:25671636

  13. MEK1 transduces the prion protein N2 fragment antioxidant effects.

    PubMed

    Haigh, C L; McGlade, A R; Collins, S J

    2015-04-01

    The prion protein (PrP(C)) when mis-folded is causally linked with a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies or prion diseases. PrP(C) normal function is still incompletely defined with such investigations complicated by PrP(C) post-translational modifications, such as internal cleavage, which feasibly could change, activate, or deactivate the function of this protein. Oxidative stress induces ?-cleavage and the N-terminal product of this cleavage event, N2, demonstrates a cellular protective response against oxidative stress. The mechanisms by which N2 mediates cellular antioxidant protection were investigated within an in vitro cell model. N2 protection was regulated by copper binding to the octarepeat domain, directing the route of internalisation, which stimulated MEK1 signalling. Precise membrane interactions of N2, determined by copper saturation, and involving both the copper-co-ordinating octarepeat region and the structure conferred upon the N-terminal polybasic region by the proline motif, were essential for the correct engagement of this pathway. The phenomenon of PrP(C) post-translational modification, such as cleavage and copper co-ordination, as a molecular "switch" for activation or deactivation of certain functions provides new insight into the apparent multi-functionality of PrP(C). PMID:25391659

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

    SciTech Connect

    Coleman, Bradley M.; Nisbet, Rebecca M.; Han, Sen; Cappai, Roberto; Hatters, Danny M.; Hill, Andrew F.

    2009-03-13

    Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP{sup C}) into a disease associated form (PrP{sup Sc}). Recombinant PrP can be refolded into either an {alpha}-helical rich conformation ({alpha}-PrP) resembling PrP{sup C} or a {beta}-sheet rich, protease resistant form similar to PrP{sup Sc}. Here, we generated tetracysteine tagged recombinant PrP, folded this into {alpha}- or {beta}-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished {beta}-PrP from {alpha}-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the {alpha}-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the {beta}-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 PrP{sup Sc} from PrP{sup C}. 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.

  15. Cellular toxicity of yeast prion protein Rnq1 can be modulated by N-terminal wild type huntingtin.

    PubMed

    Sethi, Ratnika; Patel, Vishal; Saleh, Aliabbas A; Roy, Ipsita

    2016-01-15

    Aggregation of the N-terminal human mutant huntingtin and the consequent toxicity in the yeast model of Huntington's disease (HD) requires the presence of Rnq1 protein (Rnq1p) in its prion conformation [RNQ1(+)]. The understanding of interaction of wild-type huntingtin (wt-Htt) with the amyloidogenic prion has some gaps. In this work, we show that N-terminal fragment of wt-Htt (N-wt-Htt) ameliorated the toxic effect of [RNQ1(+)] depending on expression levels of both proteins. When the expression of N-wt-Htt was high, it increased the expression and delayed the aggregation of [RNQ1(+)]. As the expression of N-wt-Htt was reduced, it formed high molecular weight aggregates along with the prion. Even when sequestered by [RNQ1(+)], the beneficial effect of N-wt-Htt on expression of Rnq1p and on cell survival was evident. Huntingtin protein ameliorated toxicity due to the prion protein [RNQ1(+)] in yeast cells in a dose-dependent manner, resulting in increase in cell survival, hinting at its probable role as a component of the proteostasis network of the cell. Taking into account the earlier reports of the beneficial effect of expression of N-wt-Htt on the aggregation of mutant huntingtin, the function of wild-type huntingtin as an inhibitor of protein aggregation in the cell needs to be explored. PMID:26628321

  16. Processing of the Bovine Spongiform Encephalopathy-Specific Prion Protein by Dendritic Cells

    PubMed Central

    Rybner-Barnier, Catherine; Jacquemot, Catherine; Cuche, Céline; Doré, Grégory; Majlessi, Laleh; Gabellec, Marie-Madeleine; Moris, Arnaud; Schwartz, Olivier; Di Santo, James; Cumano, Ana; Leclerc, Claude; Lazarini, Françoise

    2006-01-01

    Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrPbse) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrPbse and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrPbse within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrPbse is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrPbse when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrPbse capture is probably specific to antigen-presenting cells since no uptake of PrPbse was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrPbse. Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 20/common cytokine γ chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings. PMID:16641258

  17. Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress.

    PubMed

    Torres, Mauricio; Castillo, Karen; Armisén, Ricardo; Stutzin, Andrés; Soto, Claudio; Hetz, Claudio

    2010-01-01

    Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES). Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES). Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES) and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models. PMID:21209925

  18. Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

    PubMed Central

    Elmallah, Mohammed IY; Borgmeyer, Uwe; Betzel, Christian; Redecke, Lars

    2013-01-01

    Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrPC into the infectious misfolded isoform PrPSc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrPSc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders. PMID:24121542

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

    PubMed Central

    2012-01-01

    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 Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies. PMID:22233534

  20. Comparison of the local structural stabilities of mammalian prion protein (PrP) by fragment molecular orbital calculations

    PubMed Central

    Hasegawa, Koji; Mohri, Shirou; Yokoyama, Takashi

    2013-01-01

    Bovine spongiform encephalopathy (BSE), a member of the prion diseases, is a fatal neurodegenerative disorder suspected to be caused by a malfunction of prion protein (PrP). Although BSE prions have been reported to be transmitted to a wide range of animal species, dogs and hamsters are known to be BSE-resistant animals. Analysis of canine and hamster PrP could elucidate the molecular mechanisms supporting the species barriers to BSE prion transmission. The structural stability of 6 mammalian PrPs, including human, cattle, mouse, hamster, dog and cat, was analyzed. We then evaluated intramolecular interactions in PrP by fragment molecular orbital (FMO) calculations. Despite similar backbone structures, the PrP side-chain orientations differed among the animal species examined. The pair interaction energies between secondary structural elements in the PrPs varied considerably, indicating that the local structural stabilities of PrP varied among the different animal species. Principal component analysis (PCA) demonstrated that different local structural stability exists in bovine PrP compared with the PrP of other animal species examined. The results of the present study suggest that differences in local structural stabilities between canine and bovine PrP link diversity in susceptibility to BSE prion infection. PMID:23232497

  1. CHRONIC WASTING DISEASE OF ELK AND DEER AND CREUTZFELDT-JAKOB DISEASE: COMPARATIVE ANALYSIS OF THE SCRAPIE PRION PROTEIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transmissible spongiform encephalopathies or prion diseases are a heterogeneous group of disorders associated with, and possibly caused by, accumulation of a neurotoxic, misfolded isoform, termed PrP-d, of a normal cellular protein, PrP-c. Primary amino acid differences and secondary conformati...

  2. Prion protein-deficient mice exhibit decreased CD4 T and LTi cell numbers and impaired spleen structure.

    PubMed

    Kim, Soochan; Han, Sinsuk; Lee, Ye Eun; Jung, Woong-Jae; Lee, Hyung Soo; Kim, Yong-Sun; Choi, Eun-Kyoung; Kim, Mi-Yeon

    2016-01-01

    The cellular prion protein is expressed in almost all tissues, including the central nervous system and lymphoid tissues. To investigate the effects of the prion protein in lymphoid cells and spleen structure formation, we used prion protein-deficient (Prnp(0/0)) Zürich I mice generated by inactivation of the Prnp gene. Prnp(0/0) mice had decreased lymphocytes, in particular, CD4 T cells and lymphoid tissue inducer (LTi) cells. Decreased CD4 T cells resulted from impaired expression of CCL19 and CCL21 in the spleen rather than altered chemokine receptor CCR7 expression. Importantly, some of the white pulp regions in spleens from Prnp(0/0) mice displayed impaired T zone structure as a result of decreased LTi cell numbers and altered expression of the lymphoid tissue-organizing genes lymphotoxin-α and CXCR5, although expression of the lymphatic marker podoplanin and CXCL13 by stromal cells was not affected. In addition, CD3(-)CD4(+)IL-7Rα(+) LTi cells were rarely detected in impaired white pulp in spleens of these mice. These data suggest that the prion protein is required to form the splenic white pulp structure and for development of normal levels of CD4 T and LTi cells. PMID:26299705

  3. [Prion diseases].

    PubMed

    Stoĭda, N I; Zavalishin, I A

    2012-01-01

    Prion diseases are a family of progressive neurodegenerative disorders caused by prions. There are four human prion diseases: Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal insomnia and Kuru. They can arise in three different ways: acquired, familial or sporadic. We review clinical presentations, pathophysiology, morphological picture, diagnostic procedures and available treatment options of prion diseases. PMID:23235426

  4. Free energy and hidden barriers of the ?-sheet structure of prion protein.

    PubMed

    Paz, S Alexis; Abrams, Cameron F

    2015-10-13

    On-the-fly free-energy parametrization is a new collective variable biasing approach akin to metadynamics with one important distinction: rather than acquiring an accelerated distribution via a history-dependent bias potential, sampling on this distribution is achieved from the beginning of the simulation using temperature-accelerated molecular dynamics. In the present work, we compare the performance of both approaches to compute the free-energy profile along a scalar collective variable measuring the H-bond registry of the ?-sheet structure of the mouse Prion protein. Both methods agree on the location of the free-energy minimum, but free-energy profiles from well-tempered metadynamics are subject to a much higher degree of statistical noise due to hidden barriers. The sensitivity of metadynamics to hidden barriers is shown to be a consequence of the history dependence of the bias potential, and we detail the nature of these barriers for the prion ?-sheet. In contrast, on-the-fly parametrization is much less sensitive to these barriers and thus displays improved convergence behavior relative to that of metadynamics. While hidden barriers are a frequent and central issue in free-energy methods, on-the-fly free-energy parametrization appears to be a robust and preferable method to confront this issue. PMID:26574287

  5. Prion Protein Modulates Monoaminergic Systems and Depressive-like Behavior in Mice.

    PubMed

    Beckman, Danielle; Santos, Luis E; Americo, Tatiana A; Ledo, Jose H; de Mello, Fernando G; Linden, Rafael

    2015-08-14

    We sought to examine interactions of the prion protein (PrP(C)) with monoaminergic systems due to: the role of PrP(C) in both Prion and Alzheimer diseases, which include clinical depression among their symptoms, the implication of monoamines in depression, and the hypothesis that PrP(C) serves as a scaffold for signaling systems. To that effect we compared both behavior and monoaminergic markers in wild type (WT) and PrP(C)-null (PrP(-/-)) mice. PrP(-/-) mice performed poorly when compared with WT in forced swimming, tail suspension, and novelty suppressed feeding tests, typical of depressive-like behavior, but not in the control open field nor rotarod motor tests; cyclic AMP responses to stimulation of D1 receptors by dopamine was selectively impaired in PrP(-/-) mice, and responses to serotonin, but not to norepinephrine, also differed between genotypes. Contents of dopamine, tyrosine hydroxylase, and the 5-HT5A serotonin receptor were increased in the cerebral cortex of PrP(-/-), as compared with WT mice. Microscopic colocalization, as well as binding in overlay assays were found of PrP(C) with both the 5HT5A and D1, but not D4 receptors. The data are consistent with the scaffolding of monoaminergic signaling modules by PrP(C), and may help understand the pathogenesis of clinical depression and neurodegenerative disorders. PMID:26152722

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

    PubMed Central

    2010-01-01

    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. PMID:20649983

  7. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    NASA Astrophysics Data System (ADS)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  8. Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms 'curly' intracellular aggregates.

    PubMed

    Gu, Yaping; Verghese, Susamma; Bose, Sharmila; Mohan, Maradumane; Singh, Neena

    2007-01-01

    Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann-Straussler-Scheinker disease (GSS), familial Creutzfeldt-Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrPSc) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP202N expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as 'curly' aggregates. In addition, PrP202N cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation. PMID:17873292

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

    PubMed

    Lezmi, Stphane; Baron, Thierry G M; Bencsik, Anna A

    2010-01-01

    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 available tissue blocks with the PET-Blot method as a sensitive complementary tool in case of doubtful PrP IHC results. PMID:20684771

  10. The many shades of prion strain adaptation

    PubMed Central

    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 PrPSc 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. PMID:24518385

  11. Is the prevalent human prion protein 129M/V mutation a living fossil from a Paleolithic panzootic superprion pandemic?

    PubMed

    Nyström, Sofie; Hammarström, Per

    2014-01-01

    Prion diseases are consistently associated with prion protein (PrP(C)) misfolding rendering a cascade of auto-catalytic self-perpetuation of misfolded PrP in an afflicted individual. The molecular process is intriguingly similar to all known amyloid diseases both local and systemic. The prion disease is also infectious by the transfer of misfolded PrP from one individual to the next. Transmissibility is surprisingly efficient in prion diseases and given the rapid disease progression following initial symptoms the prionoses stand out from other amyloidoses, which all may be transmissible under certain circumstances. The nature of the infectious prion as well as the genotype of the host is important for transmissibility. For hitherto unexplained reasons the majority of Europeans carry a missense mutation on one or both alleles of the PrP gene (PRNP), and hence express a variant of PrP with a substitution for valine (V) instead of methionine (M) in position 129. In fact the 129M/V variant is very common in all populations except for the Japanese. Sporadic Creutzfeldt-Jakob disease is a disease rarely striking people below the age of 60, where homozygosity especially 129MM is a very strong risk factor. Paradoxically, the 129M/V polymorphism suggestive of heterozygote advantage is one of the most clear cut disease associated traits of the human population, yet prion disease is extraordinarily rare. The genetic basis for how this trait spread with such prevalence within human populations is still target to investigations and deserves attention. This short essay represents a somewhat provocative hypothetical notion of a possible ancient significance of this polymorphism. PMID:24398570

  12. The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein.

    PubMed

    Torres, Mauricio; Medinas, Danilo B; Matamala, José Manuel; Woehlbier, Ute; Cornejo, Víctor Hugo; Solda, Tatiana; Andreu, Catherine; Rozas, Pablo; Matus, Soledad; Muñoz, Natalia; Vergara, Carmen; Cartier, Luis; Soto, Claudio; Molinari, Maurizio; Hetz, Claudio

    2015-09-25

    Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease. PMID:26170458

  13. Sequence repeats and protein structure

    NASA Astrophysics Data System (ADS)

    Hoang, Trinh X.; Trovato, Antonio; Seno, Flavio; Banavar, Jayanth R.; Maritan, Amos

    2012-11-01

    Repeats are frequently found in known protein sequences. The level of sequence conservation in tandem repeats correlates with their propensities to be intrinsically disordered. We employ a coarse-grained model of a protein with a two-letter amino acid alphabet, hydrophobic (H) and polar (P), to examine the sequence-structure relationship in the realm of repeated sequences. A fraction of repeated sequences comprises a distinct class of bad folders, whose folding temperatures are much lower than those of random sequences. Imperfection in sequence repetition improves the folding properties of the bad folders while deteriorating those of the good folders. Our results may explain why nature has utilized repeated sequences for their versatility and especially to design functional proteins that are intrinsically unstructured at physiological temperatures.

  14. Biology and genetics of prions causing neurodegeneration.

    PubMed

    Prusiner, Stanley B

    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 ?-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 in the prion conformation encipher the biological properties of distinct prion strains. Increasing evidence argues that prions cause many neurodegenerative diseases (NDs), including Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and Lou Gehrig's diseases, as well as the tauopathies. The majority of NDs are sporadic, and 10% to 20% are inherited. The late onset of heritable NDs, like their sporadic counterparts, may reflect the stochastic nature of prion formation; the pathogenesis of such illnesses seems to require prion accumulation to exceed some critical threshold before neurological dysfunction manifests. PMID:24274755

  15. Age-Dependent Impairment of Eyeblink Conditioning in Prion Protein-Deficient Mice

    PubMed Central

    Kishimoto, Yasushi; Hirono, Moritoshi; Atarashi, Ryuichiro; Sakaguchi, Suehiro; Yoshioka, Tohru; Katamine, Shigeru; Kirino, Yutaka

    2013-01-01

    Mice lacking the prion protein (PrPC) gene (Prnp), Ngsk Prnp0/0 mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrPC-like protein (PrPLP/Dpl). Because PrPC is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrPC and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp0/0 mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrPC and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp0/0 mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp0/0 mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrPc-deficient mice, ZrchI PrnP0/0 mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp0/0 mice. Furthermore, Ngsk Prnp0/0 mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp0/0 mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning. PMID:23593266

  16. Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions.

    PubMed

    Ramljak, Sanja; Schmitz, Matthias; Zafar, Saima; Wrede, Arne; Schenkel, Sara; Asif, Abdul R; Carimalo, Julie; Doeppner, Thorsten R; Schulz-Schaeffer, Walter J; Weise, Jens; Zerr, Inga

    2015-09-01

    Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified, a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion protein knockout mice (Prnp(0/0)) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrP(c). Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp(0/0) knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp(0/0) primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp(0/0) mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrP(c) show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrP(c) was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrP(c)-overexpression under normoxic conditions. Our data suggest LDH as a direct PrP(c) interactor with possible physiological relevance under low oxygen conditions. PMID:26024859

  17. Non-targeted Identification of Prions and Amyloid-forming Proteins from Yeast and Mammalian Cells*

    PubMed Central

    Kryndushkin, Dmitry; Pripuzova, Natalia; Burnett, Barrington G.; Shewmaker, Frank

    2013-01-01

    The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin. PMID:23926098

  18. Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells.

    PubMed

    Kryndushkin, Dmitry; Pripuzova, Natalia; Burnett, Barrington G; Shewmaker, Frank

    2013-09-20

    The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin. PMID:23926098

  19. Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform(PrPSc)of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assay...

  20. Current concepts in human prion protein (Prp) misfolding, Prnp gene polymorphisms and their contribution to Creutzfeldt-Jakob Disease (CJD).

    PubMed

    Michalczyk, K; Ziman, M

    2007-10-01

    Transmissible spongiform encephalopathies are a group of neural degenerative diseases that may be infectious, sporadic, or hereditary and are associated with an abnormally folded prion protein. Unfortunately at the current time it is not at all clear what the normal structure of the prion protein actually is or how it is toxic to cells. Extensive research on prion diseases has led to a dramatic increase in understanding of the pathogenesis of prion disorders, which will hopefully lead to the development of effective treatments. The inability to detect the disease in blood using current technology has made screening difficult. While fortunately there has been a decline in the number of clinical cases of transmissible variant CJD, evidence indicates that very long incubation periods can occur in humans so there may be a long slow, gradual epidemic. In particular, clinical cases in genotypes other than those homozygous for methionine at codon 129 of PRNP have not yet occurred, but such cases might be expected to have longer incubation periods and show differences in pathology to those seen to date. Transgenic animal studies have shown that a large proportion of infected animals develop sub-clinical disease. Moreover, results from a large prevalence study in humans show that several cases test positive but do not develop clinical disease. It is possible therefore that further cases of secondary transmission could occur by iatrogenic spread, which could result in vCJD persisting in the UK at low levels for many years, highlighting the importance of continued vigilance. PMID:17616941

  1. Are the interactions between recombinant prion proteins and polymeric surfaces related to the hydrophilic/hydrophobic balance?

    PubMed

    Vrlinic, Tjasa; Debarnot, Dominique; Legeay, Gilbert; Coudreuse, Arnaud; El Moualij, Benaissa; Zorzi, Willy; Perret-Liaudet, Armand; Quadrio, Isabelle; Mozetic, Miran; Poncin-Epaillard, Fabienne

    2012-06-01

    New non-fouling tubes are developed and their influence on the adhesion of neuroproteins is studied. Recombinant prion proteins are considered as a single component representative of hydrophobic proteins. Samples are stored for 24 h at 4 °C in tubes coated with two different coatings: poly(N-isopropylacrylamide) as a hydrophilic surface and a plasma-fluorinated coating as a hydrophobic one. The protein adhesion is monitored by ELISA tests, XPS and confocal microscopy. It appears that the highest recovery of recombinant prion protein in the liquid phase is obtained with the hydrophilic surface while the hydrophobic character of the storage tube induces an important amount of biological loss. However, the recovery is not complete even for tubes coated with poly(N-isopropylacrylamide). PMID:22508476

  2. Identification of a Compound That Disrupts Binding of Amyloid-? to the Prion Protein Using a Novel Fluorescence-based Assay*

    PubMed Central

    Risse, Emmanuel; Nicoll, Andrew J.; Taylor, William A.; Wright, Daniel; Badoni, Mayank; Yang, Xiaofan; Farrow, Mark A.; Collinge, John

    2015-01-01

    The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease, where its monomeric cellular isoform (PrPC) is recruited into pathogenic self-propagating polymers of misfolded protein, and in Alzheimer disease, where PrPC may act as a receptor for synaptotoxic oligomeric forms of amyloid-? (A?). There has been considerable interest in identification of compounds that bind to PrPC, stabilizing its native fold and thereby acting as pharmacological chaperones to block prion propagation and pathogenesis. However, compounds binding PrPC could also inhibit the binding of toxic A? species and may have a role in treating Alzheimer disease, a highly prevalent dementia for which there are currently no disease-modifying treatments. However, the absence of a unitary, readily measurable, physiological function of PrP makes screening for ligands challenging, and the highly heterogeneous nature of A? oligomer preparations makes conventional competition binding assays difficult to interpret. We have therefore developed a high-throughput screen that utilizes site-specifically fluorescently labeled protein to identify compounds that bind to PrP and inhibit both A? binding and prion propagation. Following a screen of 1,200 approved drugs, we identified Chicago Sky Blue 6B as the first small molecule PrP ligand capable of inhibiting A? binding, demonstrating the feasibility of development of drugs to block this interaction. The interaction of Chicago Sky Blue 6B was characterized by isothermal titration calorimetry, and its ability to inhibit A? binding and reduce prion levels was established in cell-based assays. PMID:25995455

  3. Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment.

    PubMed Central

    Prez, M; Wandosell, F; Colao, C; Avila, J

    1998-01-01

    Although a number of features distinguish the disease isoform of the prion protein (PrPSc) from its normal cellular counterpart (PrPC) in the transmissible spongiform encephalopathies (TSEs), the neuropathogenesis of these diseases remains an enigma. The amyloid fibrils formed by fragments of human PrP have, however, been shown to be directly neurotoxic in vitro. We show here that sulphated polysaccharides (heparin, keratan and chondroitin) inhibit the neurotoxicity of these amyloid fibrils and this appears to be mediated via inhibition of the polymerization of the PrP peptide into fibrils. This provides a rationale for the therapeutic effects of sulphated polysaccharides and suggests a rapid in vitro functional screen for TSE therapeutics. PMID:9761736

  4. Inhibition of Protease-Resistant Prion Protein Accumulation In Vitro by Curcumin

    PubMed Central

    Caughey, Byron; Raymond, Lynne D.; Raymond, Gregory J.; Maxson, Laura; Silveira, Jay; Baron, Gerald S.

    2003-01-01

    Inhibition of the accumulation of protease-resistant prion protein (PrP-res) is a prime strategy in the development of potential transmissible spongiform encephalopathy (TSE) therapeutics. Here we show that curcumin (diferoylmethane), a major component of the spice turmeric, potently inhibits PrP-res accumulation in scrapie agent-infected neuroblastoma cells (50% inhibitory concentration, ∼10 nM) and partially inhibits the cell-free conversion of PrP to PrP-res. In vivo studies showed that dietary administration of curcumin had no significant effect on the onset of scrapie in hamsters. Nonetheless, other studies have shown that curcumin is nontoxic and can penetrate the brain, properties that give curcumin advantages over inhibitors previously identified as potential prophylactic and/or therapeutic anti-TSE compounds. PMID:12692251

  5. Induced Prion Protein Controls Immune-Activated Retroviruses in the Mouse Spleen

    PubMed Central

    Ltscher, Marius; Recher, Mike; Lang, Karl S.; Navarini, Alexander; Hunziker, Lukas; Santimaria, Roger; Glatzel, Markus; Schwarz, Petra; Bni, Jrg; Zinkernagel, Rolf M.

    2007-01-01

    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. PMID:17987132

  6. BSE-associated polymorphisms in the prion protein gene: an investigation.

    PubMed

    Vernerova, K; Tothova, L; Mikova, A; Vodrazka, P; Simek, B; Hanusova, L; Citek, J

    2014-10-01

    The aim of this study was to determine the frequency of the 12-bp and 23-bp indel polymorphisms in the prion protein gene (PRNP) in cattle and to investigate the association between these frequencies and the occurrence of bovine spongiform encephalopathy (BSE). There was no significant difference in the 12-bp indel frequency between the BSE animals and control group. For the 23-bp indel, the BSE animals had a significantly lower + + (insins) genotype frequency and + allele frequency compared with the control animals. The - - / - - genotype frequency in the BSE animals was not significantly higher when compared with the control animals. One - allele increased the risk of BSE by a factor of 1.55 (i.e. by 55%) for the 12-bp indel and by a factor of 2.10 for the 23-bp indel. When both indels are considered, one - allele increased the risk of BSE by a factor of 1.54. PMID:24720684

  7. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1.

    PubMed

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-09-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

  8. Variant-specific prion interactions

    PubMed Central

    Sharma, Jaya; Liebman, Susan W

    2013-01-01

    Prions are protein conformations that self-seed the misfolding of their non-prion iso-forms into prion, often amyloid, conformations. The most famous prion is the mammalian PrP protein that in its prion form causes transmissible spongiform encephalopathy. Curiously there can be distinct conformational differences even between prions of the same protein propagated in the same host species. These are called prion strains or variants. For example, different PrP variants are faithfully transmitted during self-seeding and are associated with distinct disease characteristics. Variant-specific PrP prion differences include the length of the incubation period before the disease appears and the deposition of prion aggregates in distinct regions of the brain.1 Other more common neurodegenerative diseases (e.g., Alzheimer disease, Parkinson disease, type 2 diabetes and ALS) are likewise caused by the misfolding of a normal protein into a self-seeding aggregate.2-4 One of the most important unanswered questions is how the first prion-like seed arises de novo, resulting in the pathological cascade. PMID:24475372

  9. The crystal structure of the globular domain of sheep prion protein.

    PubMed

    Haire, L F; Whyte, S M; Vasisht, N; Gill, A C; Verma, C; Dodson, E J; Dodson, G G; Bayley, P M

    2004-03-01

    The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure. PMID:15037077

  10. Dextran sulfate sodium inhibits amyloid-β oligomer binding to cellular prion protein.

    PubMed

    Aimi, Takahiro; Suzuki, Koichiro; Hoshino, Tatsuya; Mizushima, Tohru

    2015-08-01

    Amyloid-β peptide (Aβ), especially its oligomeric form, is believed to play an important role in the pathogenesis of Alzheimer's disease (AD). To this end, the binding of Aβ oligomer to cellular prion protein (PrP(C)) plays an important role in synaptic dysfunction in a mouse model of AD. Here, we have screened for compounds that inhibit Aβ oligomer binding to PrP(C) from medicines already used clinically (Mizushima Approved Medicine Library 1), and identified dextran sulfate sodium (DSS) as a candidate. In a cell-free assay, DSS inhibited Aβ oligomer binding to PrP(C) but not to ephrin receptor B2, another endogenous receptor for Aβ oligomers, suggesting that the drug's action is specific to the binding of Aβ oligomer to PrP(C) . Dextran on the other hand did not affect this binding. DSS also suppressed Aβ oligomer binding to cells expressing PrP(C) but not to control cells. Furthermore, while incubation of mouse hippocampal slices with Aβ oligomers inhibited the induction of long-term potentiation, simultaneous treatment with DSS restored the long-term potentiation. As DSS has already been approved for use in patients with hypertriglyceridemia, and its safety in humans has been confirmed, we propose further analysis of this drug as a candidate for AD treatment. Amyloid-β peptide (Aβ) oligomer-binding to cellular prion protein (PrP(C) ) is important in synaptic dysfunction in Alzheimer's disease (AD). We found here that dextran sulfate sodium (DSS) inhibits Aβ oligomer binding to PrP(C) . Simultaneous treatment of hippocampal slices with DSS restored long-term potentiation (LTP) in the presence of Aβ oligomers. Since DSS has already been approved for clinical use, we propose this drug is a candidate drug for AD treatment. PMID:25963375

  11. Both Met(109) and Met(112) are Utilized for Cu(II) Coordination to the Amyloidogenic Fragment of the Human Prion Protein

    SciTech Connect

    Shearer, J.; Soh, P; Lentz, S

    2008-01-01

    The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five Cu{sup II} ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured 'amyloidogenic' domain between residues 91 and 126 PrP(91-126). Herein we expand upon a previous study (J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710-719) where we demonstrated that the physiologically relevant high affinity Cu{sup II} coordination site within PrP(91-126) is found between residues 106 and 114. It was shown that Cu{sup II} is contained within a square planar (N/O){sub 3}S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the Cu{sup II} coordination environment within the PrP fragment containing residues 106-114 (PrP(106-114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106-114) with systematic Met {yields} Ile 'mutations' we show that the Cu{sup II} coordination environment within PrP(106-114) is actually comprised of a mixture of two major species; one CuII(N/O){sub 3}S center with the M(109) thioether coordinated to Cu{sup II} and another Cu{sup II}(N/O){sub 3}S center with the M(112) thioether coordinated to Cu{sup II}. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106-114) both reduces the Cu{sup II} affinity of the peptide by two to seven fold, and renders the resulting Cu{sup II} metallopeptides redox inactive. The biological implications of these findings are discussed.

  12. Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines.

    PubMed

    Chasseigneaux, Stphanie; Pastore, Manuela; Britton-Davidian, Janice; Mani, Elodie; Stern, Marc-Henri; Callebert, Jacques; Catalan, Josette; Casanova, Danielle; Belondrade, Maxime; Provansal, Monique; Zhang, Yonghua; Brkle, Alexander; Laplanche, Jean-Louis; Svenet, Nicolas; Lehmann, Sylvain

    2008-01-01

    The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection. PMID:18696008

  13. Solid-state NMR studies of the secondary structure of a mutant prion protein fragment of 55 residues that induces neurodegeneration.

    PubMed

    Laws, D D; Bitter, H M; Liu, K; Ball, H L; Kaneko, K; Wille, H; Cohen, F E; Prusiner, S B; Pines, A; Wemmer, D E

    2001-09-25

    The secondary structure of a 55-residue fragment of the mouse prion protein, MoPrP(89-143), was studied in randomly aggregated (dried from water) and fibrillar (precipitated from water/acetonitrile) forms by (13)C solid-state NMR. Recent studies have shown that the fibrillar form of the P101L mutant of MoPrP(89-143) is capable of inducing prion disease in transgenic mice, whereas unaggregated or randomly aggregated samples do not provoke disease. Through analysis of (13)C chemical shifts, we have determined that both wild-type and mutant sequence MoPrP(89-143) form a mixture of beta-sheet and alpha-helical conformations in the randomly aggregated state although the beta-sheet content in MoPrP(89-143, P101L) is significantly higher than in the wild-type peptide. In a fibrillar state, MoPrP(89-143, P101L) is completely converted into beta-sheet, suggesting that the formation of a specific beta-sheet structure may be required for the peptide to induce disease. Studies of an analogous peptide from Syrian hamster PrP verify that sequence alterations in residues 101-117 affect the conformation of aggregated forms of the peptides. PMID:11562491

  14. Roles of the cellular prion protein in the regulation of cell-cell junctions and barrier function

    PubMed Central

    Petit, Constance S.V.; Besnier, Laura; Morel, Etienne; Rousset, Monique; Thenet, Sophie

    2013-01-01

    The cellular prion protein was historically characterized owing to its misfolding in prion disease. Although its physiological role remains incompletely understood, PrPC has emerged as an evolutionary conserved, multifaceted protein involved in a wide-range of biological processes. PrPC is a GPI-anchored protein targeted to the plasma membrane, in raft microdomains, where its interaction with a repertoire of binding partners, which differ depending on cell models, mediates its functions. Among identified PrPC partners are cell adhesion molecules. This review will focus on the multiple implications of PrPC in cell adhesion processes, mainly the regulation of cell-cell junctions in epithelial and endothelial cells and the consequences on barrier properties. We will show how recent findings argue for a role of PrPC in the recruitment of signaling molecules, which in turn control the targeting or the stability of adhesion complexes at the plasma membrane. PMID:24665391

  15. In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

    PubMed Central

    Fernandez-Funez, Pedro; Casas-Tinto, Sergio; Zhang, Yan; Gmez-Velazquez, Melisa; Morales-Garza, Marco A.; Cepeda-Nieto, Ana C.; Castilla, Joaqun; Soto, Claudio; Rincon-Limas, Diego E.

    2009-01-01

    Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded isoform (PrPSc) 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 PrPC; in older flies, PrP misfolds, acquires biochemical and structural properties of PrPSc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrPSc-specific conformational epitopes. In contrast to PrPSc 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 PrPSc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrPSc-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 PrPSc 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 the potential therapeutic role of Hsp70 in treating these devastating disorders. PMID:19503596

  16. Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity.

    PubMed

    Müller-Schiffmann, Andreas; Petsch, Benjamin; Leliveld, S Rutger; Muyrers, Janine; Salwierz, Agnieska; Mangels, Christian; Schwarzinger, Stephan; Riesner, Detlev; Stitz, Lothar; Korth, Carsten

    2009-02-01

    The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrP(Sc), associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrP(Sc) that recognizes both PrP(C) and PrP(Sc). This scFv fragment had a dissociation constant (K(D)) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrP(Sc) but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrP(Sc) and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs. PMID:18973947

  17. Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues.

    PubMed Central

    Robakis, N K; Sawh, P R; Wolfe, G C; Rubenstein, R; Carp, R I; Innis, M A

    1986-01-01

    We have isolated a hamster cDNA clone representing the coding sequences for the entire precursor of prion protein (PrP) 27-30. This clone encodes a protein of 254 residues and contains an in-frame ATG codon 42 bases upstream from the one previously reported. Analysis of the predicted amino acid sequence suggests that the PrP precursor protein contains an amino-terminal signal sequence, and a membrane-spanning domain in the carboxyl terminus. Cleavage of the signal peptide would produce a mature protein of 232 amino acids. Sequences homologous to the hamster PrP cDNA were detected in hamster, mouse, sheep, human, and rabbit genomes. A related 2.5-kilobase transcript was present in the brain of normal and scrapie-infected rodents. Two homologous transcripts, 2.5 and 1.1 kilobases, were detected in the lung and heart of normal animals. No PrP mRNA was detected in spleen stroma, a tissue known to contain high titers of scrapie. Antisera raised to the 27- to 30-kDa polypeptide detected the PrP in both normal and infected brains but failed to detect this protein in either normal or infected spleens. Homologous mRNA species were detected in human, sheep, and rabbit brain, even though the latter is resistant to scrapie infection. Our data suggest that PrP is not a necessary component of the infectious agent. Images PMID:3529083

  18. Highly infectious CJD particles lack prion protein but contain many viral-linked peptides by LC-MS/MS.

    PubMed

    Kipkorir, Terry; Tittman, Sarah; Botsios, Sotirios; Manuelidis, Laura

    2014-11-01

    It is widely believed that host prion protein (PrP), without nucleic acid, converts itself into an infectious form (PrP-res) that causes transmissible encephalopathies (TSEs), such as human sporadic CJD (sCJD), endemic sheep scrapie, and epidemic BSE. There are many detailed investigations of PrP, but proteomic studies of other proteins in verified infectious TSE particles have not been pursued, even though brain homogenates without PrP retain their complete infectious titer. To define proteins that may be integral to, process, or protect an agent genome, we developed a streamlined, high-yield purification of infectious FU-CJD mouse brain particles with minimal PrP. Proteinase K (PK) abolished all residual particle PrP, but did not reduce infectivity, and viral-size particles lacking PrP were ?70S (vs. 90-120S without PK). Furthermore, over 1,500 non-PrP proteins were still present and positively identified in high titer FU-CJD particles without detectable PrP by mass spectrometry (LC-MS/MS); 114 of these peptides were linked to viral motifs in the environmental-viral database, and not evident in parallel uninfected controls. Host components were also identified in both PK and non-PK treated particles from FU-CJD mouse brain and human sCJD brain. This abundant cellular data had several surprises, including finding Huntingtin in the sCJD but not normal human brain samples. Similarly, the neural Wiskott-Aldrich sequence and multivesicular and endosome components associated with retromer APP (Alzheimer amyloid) processing were only in sCJD. These cellular findings suggest that new therapies directed at retromer-vesicular trafficking in other neurodegenerative diseases may also counteract late-onset sCJD PrP amyloid pathology. PMID:24933657

  19. Physiological and environmental control of yeast prions

    PubMed Central

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

    2014-01-01

    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 formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions. PMID:24236638

  20. Biochemical typing of pathological prion protein in aging cattle with BSE

    PubMed Central

    Tester, Seraina; Juillerat, Valerie; Doherr, Marcus G; Haase, Bianca; Polak, Miroslaw; Ehrensperger, Felix; Leeb, Tosso; Zurbriggen, Andreas; Seuberlich, Torsten

    2009-01-01

    Background The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland. Results To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated. Conclusion Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities. PMID:19470160

  1. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey

    PubMed Central

    2013-01-01

    Objectives To carry out a further survey of archived appendix samples to understand better the differences between existing estimates of the prevalence of subclinical infection with prions after the bovine spongiform encephalopathy epizootic and to see whether a broader birth cohort was affected, and to understand better the implications for the management of blood and blood products and for the handling of surgical instruments. Design Irreversibly unlinked and anonymised large scale survey of archived appendix samples. Setting Archived appendix samples from the pathology departments of 41 UK hospitals participating in the earlier survey, and additional hospitals in regions with lower levels of participation in that survey. Sample 32?441 archived appendix samples fixed in formalin and embedded in paraffin and tested for the presence of abnormal prion protein (PrP). Results Of the 32?441 appendix samples 16 were positive for abnormal PrP, indicating an overall prevalence of 493 per million population (95% confidence interval 282 to 801 per million). The prevalence in those born in 1941-60 (733 per million, 269 to 1596 per million) did not differ significantly from those born between 1961 and 1985 (412 per million, 198 to 758 per million) and was similar in both sexes and across the three broad geographical areas sampled. Genetic testing of the positive specimens for the genotype at PRNP codon 129 revealed a high proportion that were valine homozygous compared with the frequency in the normal population, and in stark contrast with confirmed clinical cases of vCJD, all of which were methionine homozygous at PRNP codon 129. Conclusions This study corroborates previous studies and suggests a high prevalence of infection with abnormal PrP, indicating vCJD carrier status in the population compared with the 177 vCJD cases to date. These findings have important implications for the management of blood and blood products and for the handling of surgical instruments. PMID:24129059

  2. Neutron Reflectometry Studies Define Prion Protein N-terminal Peptide Membrane Binding

    PubMed Central

    Le Brun, Anton P.; Haigh, Cathryn L.; Drew, Simon C.; James, Michael; Boland, Martin P.; Collins, Steven J.

    2014-01-01

    The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational 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. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. 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 our previous studies, interaction was 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. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions. PMID:25418300

  3. Structural facets of disease-linked human prion protein mutants: a molecular dynamic study.

    PubMed

    Rossetti, Giulia; Giachin, Gabriele; Legname, Giuseppe; Carloni, Paolo

    2010-12-01

    Prion propagation in transmissible spongiform encephalopathies involves the conversion of the cellular prion protein, PrPC, into the pathogenic conformer PrPSc. Human familial forms of the disease are linked to specific mutations in the PrP gene, PRNP, and include Gerstmann-Strussler-Scheinker syndrome (GSS), familial Creutzfeldt-Jakob disease (fCJD), and fatal familial insomnia. To gain insights into the molecular basis of these disorders, we performed 200 ns of classical molecular dynamic simulations in aqueous solution on wild type (WT) human PrP (HuPrP), and on three HuPrP variants located in the globular HuPrP domain: two pathological mutations, HuPrP(Q212P) and HuPrP(E200K), linked to GSS and to fCJD respectively, and one protective polymorphism, HuPrP(E219K) (total time-scale simulated 800 ns). A comparison between the predicted structural determinants of WT HuPrP and HuPrP(E200K) with their NMR structures established the accuracy of the methods used. Strikingly, the analyzed disease-linked variants produced their major effect on the α2-α3 region and the β2-α2 loop, regardless of the mutation position. The conformational change of the latter might affect the interactions with cellular partners in the fibrillation process. The protocol proposed here represents a powerful approach for reproducing the structural effects of genetic mutations located in the globular domain of HuPrP, such as the GSS-related HuPrP(Q212P) and the protective polymorphism HuPrP(E219K). PMID:20806222

  4. Creutzfeldt-Jakob Disease (CJD) with a Mutation at Codon 148 of Prion Protein Gene

    PubMed Central

    Pastore, Manuela; Chin, Steven S.; Bell, Karen L.; Dong, Zhiqian; Yang, Qiwei; Yang, Lizhu; Yuan, Jue; Chen, Shu G.; Gambetti, Pierluigi; Zou, Wen-Quan

    2005-01-01

    Creutzfeldt-Jakob disease (CJD), the most common human prion disease, includes sporadic (s) and familial (f) forms. Regardless of etiology, both forms are thought to share the pathogenic mechanism whereby the cellular prion protein (PrPC) converts into its pathogenic isoform (PrPSc). While PrPC conversion is thought to be random in sCJD, conversion in fCJD is facilitated by the congenital presence of mutated PrP. Differences in PrP genotype (PRNP) and in conversion circumstances lead to PrPSc with distinct characteristics that elicit different disease phenotypes. Here, we describe a case of fCJD with a substitution of histidine (H) for arginine (R) at codon 148 (R148H) and heterozygosity of the methionine/valine (M/V) polymorphic codon 129, with the 129M allele coupled with the mutation. The disease phenotype and all major characteristics of PrPSc of fCJDR148H were virtually indistinguishable from those of sCJDMV2, which has features different from those of any other sCJD. Therefore, despite the differences in etiology, PRNP, and conversion process, the two forms of PrPSc had similar characteristics. Furthermore, comparison of fCJDR148H with a recently reported case carrying R148H and homozygosity at codon 129 suggests that codon 129 coupled with the mutation as well as that located on the normal allele can modify major phenotypic and PrPSc features of fCJDR148H. PMID:16314483

  5. Mass Spectrometric Detection of Attomole Amounts of the Prion Protein by nanoLC-MS-MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitation of prions in biological samples other than brain or spinal cord is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases in animal and humans known as Transmissible Spongiform Encephalopathies (TSEs). At present there...

  6. INSIGHTS ON SCRAPIE PRION PROTEIN (PrPSc) STRUCTURE OBTAINED BY LIMITED PROTEOLYSIS AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Establishing homologies in protein sequences

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.; Barker, W. C.; Hunt, L. T.

    1983-01-01

    Computer-based statistical techniques used to determine homologies between proteins occurring in different species are reviewed. The technique is based on comparison of two protein sequences, either by relating all segments of a given length in one sequence to all segments of the second or by finding the best alignment of the two sequences. Approaches discussed include selection using printed tabulations, identification of very similar sequences, and computer searches of a database. The use of the SEARCH, RELATE, and ALIGN programs (Dayhoff, 1979) is explained; sample data are presented in graphs, diagrams, and tables and the construction of scoring matrices is considered.

  8. Induction of cellular prion protein (PrPc) under hypoxia inhibits apoptosis caused by TRAIL treatment.

    PubMed

    Park, Jin-Young; Jeong, Jae-Kyo; Lee, Ju-Hee; Moon, Ji-Hong; Kim, Sung-Wook; Lee, You-Jin; Park, Sang-Youel

    2015-03-10

    Hypoxia decreases cytotoxic responses to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. Cellular prion protein (PrPc) is regulated by HIF-1? in neurons. We hypothesized that PrPc is involved in hypoxia-mediated resistance to TRAIL-induced apoptosis. We found that hypoxia induced PrPc protein and inhibited TRAIL-induced apoptosis. Thus silencing of PrPc increased TRAIL-induced apoptosis under hypoxia. Overexpression of PrPc protein using an adenoviral vector inhibited TRAIL-induced apoptosis. In xenograft model in vivo, shPrPc transfected cells were more sensitive to TRAIL-induced apoptosis than in shMock transfected cells. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. PMID:25742790

  9. Assessing the Role of Oxidized Methionine at Position 213 in the Formation of Prions in Hamsters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prions are infectious proteins that are able to recruit a normal cellular prion protein and convert it into a prion. The mechanism of this conversion is unknown. Detailed mass spectrometric analysis of the normal cellular prion protein and a corresponding prion has shown they possess identical post-...

  10. Prion Protein-Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering.

    PubMed

    Carter, Lester; Kim, Seung Joong; Schneidman-Duhovny, Dina; Sthr, Jan; Poncet-Montange, Guillaume; Weiss, Thomas M; Tsuruta, Hiro; Prusiner, Stanley B; Sali, Andrej

    2015-08-18

    Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly ?-helical cellular prion protein (PrP(C)) into a ?-sheet-rich disease-causing isoform (PrP(Sc)) is the key molecular event in the formation of PrP(Sc) aggregates. The molecular mechanisms underlying the PrP(C)-to-PrP(Sc) conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP(Sc) in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23-230)] and N-terminally truncated recPrP(89-230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP(C) into PrP(Sc). PMID:26287631

  11. Zebrafish prion protein PrP2 controls collective migration process during lateral line sensory system development.

    PubMed

    Huc-Brandt, Sylvaine; Hieu, Nelson; Imberdis, Thibaut; Cubedo, Nicolas; Silhol, Michelle; Leighton, Patricia L A; Domaschke, Thomas; Allison, W Ted; Perrier, Vronique; Rossel, Mireille

    2014-01-01

    Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2-/- mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and -catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion. PMID:25436888

  12. Zebrafish Prion Protein PrP2 Controls Collective Migration Process during Lateral Line Sensory System Development

    PubMed Central

    Huc-Brandt, Sylvaine; Hieu, Nelson; Imberdis, Thibaut; Cubedo, Nicolas; Silhol, Michelle; Leighton, Patricia L. A.; Domaschke, Thomas; Allison, W. Ted; Perrier, Vronique; Rossel, Mireille

    2014-01-01

    Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2?/? mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and -catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion. PMID:25436888

  13. Evolutionary Descent of Prion Genes from the ZIP Family of Metal Ion Transporters

    PubMed Central

    Schmitt-Ulms, Gerold; Ehsani, Sepehr; Watts, Joel C.; Westaway, David; Wille, Holger

    2009-01-01

    In the more than twenty years since its discovery, both the phylogenetic origin and cellular function of the prion protein (PrP) have remained enigmatic. Insights into a possible function of PrP may be obtained through the characterization of its molecular neighborhood in cells. Quantitative interactome data demonstrated the spatial proximity of two metal ion transporters of the ZIP family, ZIP6 and ZIP10, to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family that includes ZIP5, ZIP6 and ZIP10. Additional structural threading and orthologous sequence alignment analyses argued that the prion gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations. The phylogenetic and spatial connection to ZIP proteins is expected to open new avenues of research to elucidate the biology of the prion protein in health and disease. PMID:19784368

  14. Mass Spectrometric Approaches to Detecting and Quantifying Prions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...