Evidence That Bank Vole PrP Is a Universal Acceptor for Prions

Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates—including the size of proteinase K–resistant PrP^Sc, the pattern of cerebral PrP^Sc deposition, and the conformational stability—were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.     

MHC and Preferences for Male Odour in the Bank Vole

Highly polymorphic major histocompatibility complex (MHC) genes are thought to play a central role in the choice of genetically compatible sexual partners in some vertebrates, although the evidence is variable across species. Here, we investigate the association between similarity in the MHC region and sexual preferences in the bank vole Myodes ( =Clethrionomys ) glareolus (Arvicollinae) in a laboratory setting. Females in post-partum oestrus were given the choice between the scents of two males in a Y-maze. Both males were unrelated to the female, but differed in their MHC similarity to the female. We found that females spent more time near the scent of MHC dissimilar males than those, with whom they shared MHC alleles. This suggests that bank voles use MHC-related cues to choose compatible mates.     

蛋白质错误折叠循环扩增及其在朊病毒蛋白检测中的应用研究进展

朊病毒蛋白（prion protein,PrP）是传染性海绵状脑病的病原体,其检测是该病诊断的重要依据。该文从原理、方法、影响因素和检测应用方面对蛋白质错误折叠循环扩增（protein mis-folding cyclic amplification,PMCA）这种朊病毒蛋白新型检测技术做了介绍,旨在为朊病毒蛋白的检测和发病机制研究提供理论参考。     

Early Detection of Abnormal Prion Protein in Genetic Human Prion Diseases Now Possible Using Real-Time QUIC Assay

Introduction

The definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined.

Method/Principal Findings

56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI.

Conclusion/Significance

RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient''s family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Horse Prion Protein NMR Structure and Comparisons with Related Variants of the Mouse Prion Protein

The NMR structure of the horse (Equus caballus) cellular prion protein at 25 °C exhibits the typical PrP^C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP^Cs, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25 °C and is unique to the PrP sequences of equine species. The β2-α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, “protein X,” which would promote conversion of PrP^C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2-α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.     

Nanopore Analysis of Wild-Type and Mutant Prion Protein (PrPC): Single Molecule Discrimination and PrPC Kinetics

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP^C) in the central nervous system into the infectious isoform (PrP^Sc). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP^Sc. A number of pathogenic PrP^C mutants exist that are characterized by an increased propensity for conversion into PrP^Sc and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP^C conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP^C, (a PrP^C mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP^C capture event. Moreover, we present a four-state model to describe wild-type PrP^C kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP^C. These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.     

Multiplex PCR Assay for Detection of Vibrio vulnificus Biotype 2 and Simultaneous Discrimination of Serovar E Strains

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.     

Red-Backed Vole Brain Promotes Highly Efficient In Vitro Amplification of Abnormal Prion Protein from Macaque and Human Brains Infected with Variant Creutzfeldt-Jakob Disease Agent

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

Interaction of Prion Protein with Small Highly Structured RNAs: Detection and Characterization of PrP-Oligomers

Conformational modification of normal prion protein (PrP^c) to protease-resistant, β-sheet rich, aggregates (PrP^sc) is commonly accepted cause for prion diseases. On the other hand, several studies in recent years implicate soluble, protease-sensitive, oligomers of PrP^c in neuronal damage. Previously, our group has shown that small, highly structured RNAs (shsRNAs), in conjunction with a serum factor, facilitated the conversion of hrPrP to a protease resistant, high molecular weight isoform. In the current study we demonstrate that shsRNAs, in the absence of the serum factor, generate soluble, protease-sensitive, and potentially toxic oligomers of ovrPrP. We have isolated a 500 kD oligomer by size exclusion chromatography of the reaction mixture and identified the accessible epitopes. The soluble PrP-oligomers were present in enhanced amounts in scrapie infected sheep brain and treating extracts of normal sheep brain with shsRNA resulted in oligomerization of endogenous PrP. Isolation, characterization of PrP-oligomers and their possible implication in prion diseases is discussed.     

Detection of Bovine Spongiform Encephalopathy-Related Prion Protein Gene Promoter Polymorphisms in Local Turkish Cattle

Polymorphisms in open reading frames of the prion protein gene (PRNP) have been shown to be associated with prion disease susceptibility in humans, sheep, and mice. Studies in recent years have demonstrated a similar effect of PRNP promoter and intron-1 polymorphisms on bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, the deletion/insertion (indel) polymorphisms of the bovine PRNP gene within the promoter sequence (23 bp) and intron 1 (12 bp) were analyzed in local Turkish cattle. For this, 150 animals belonging to three different local breeds--the South Anatolian red, the East Anatolian red, and the Turkish gray--were tested using DNA purification and polymerase chain reaction. The ins allele in the 12 bp indel, which is associated with low susceptibility to BSE, showed a high frequency in all three breeds. The low-susceptibility allele of the 23-bp indel was identified in Turkish gray cattle with a frequency of 0.80. Results of the study have shown that local Turkish cattle might have an important genetic value for selection against BSE.     

Characterisation of an Apparently Novel Soluble Protein Fraction found in Pathogen and Osmotically-stressed Cucumber Cotyledons

Cotyledons of cucumber (Cucumis sativus L. cv. Ashley) were used to study the alterations that occur in the soluble protein fraction following either tobacco necrosis virus-elicited necrosis or a period of mannitol-induced osmotic-stress. One apparently novel fraction (termed γ) was induced in the soluble protein fraction after necrosis or osmotic stress and was identified by disc-PAGE and gel staining. Smaller amounts of the γ fraction were present in apparently healthy tissue adjacent to necrotic halves of cotyledons (AH tissue), but were absent from any other leaves on these plants. The γ fraction is probably representative of at least three different proteins, two of which are constituents of control tissue (a glycoprotein and possibly one or two RNAse isozymes) while the third is a protein of unknown nature, present as the bulk of the γ fraction and possibly synthesized de novo. Following the radio-active labelling of AH tissue it would appear that an increased protein synthesis, which is sensitive to the effects of translational inhibitors may result in the production of the γ fraction, but this is speculative. The major component of the γ fraction gave a single band on SDS PAGE, with an Mr of ca. 22,000 and focussed to a single band at pl 4,1 on IEF. While it is thought the bulk of the novel portion of the γ fraction is represented in these gel systems increased amounts of the constitutive glycoprotein component should be taken into consideration. Soluble extracts of protoplasts isolated from AH tissue retained only constitutional RNAse isozyme(s), and were totally devoid of the rest of the γ fraction. This result suggests that the other component(s) of the γ fraction is (are) subcellularly located in the plasmodesmata, between the cell wall and plasma membrane or non-covalently attached to either.     

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

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 (PrP^TSE) 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 PrP^TSE 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 PrP^TSE 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 PrP^TSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.     

朊粒蛋白PrP~(Sc)寡聚体的形成与跨膜毒性

朊粒蛋白(prionprotein,PrP)传染致病机制一直是朊粒(prion)研究领域的焦点.由正常型朊粒蛋白(PrPC)向致病型朊粒蛋白(PrPSc)的转变是致病的关键步骤.本文综述了近年来PrPC向PrPSc转变的结构变化特征、PrPSc由单体形成寡聚体的组装机制、以及PrPSc寡聚体的跨膜机制与细胞毒性间的关系等方面的研究进展.     

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

Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP^C) into an abnormal isoform (PrP^Sc) that has infectious properties. The hydrophobic domain of PrP^C 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 PrP^C. Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP^C drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP^C are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP^Sc, suggesting these residues provide conformational flexibility. These data suggest that this region of PrP^C is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.     

Structural Aspects of Congo Red as an Inhibitor of Protease-Resistant Prion Protein Formation

Abstract: Congo red (CR) has been shown to inhibit the accumulation in scrapie-infected cells of prion protein (PrP) in the abnormal protease-resistant form (PrP-res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP-res or its protease-sensitive precursor (PrP-sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP-res formation in a simple cell-free reaction composed predominantly of purified PrP-res and PrP-sen. Structurally modified CR analogues were also compared in both the cell-free conversion reaction and scrapie-infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2' positions diminished the inhibitory potency by ≥10-fold. In contrast, there was little effect of 3,3' methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3'-methyl-CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl-CR and amido-CR. In contrast, 2,2'-methyl-CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP-res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP-res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP-res formation by CR and related compounds.