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

Background  

A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions.     

Heterologous cross-seeding mimics cross-species prion conversion in a yeast model

Background  

Prions are self-perpetuating, infectious, aggregated proteins that are associated with several neurodegenerative diseases in mammals and heritable traits in yeast. Sup35p, the protein determinant of the yeast prion [PSI ⁺], has a conserved C terminal domain that performs the Sup35p function and a prion domain that is highly divergent. Prions formed by chimeras of the prion domain of various species fused to the C domain of Saccharomyces cerevisiae exhibit a 'species barrier', a phenomenon first observed in mammals, and often fail to transmit the prion state to chimeras with prion domains of other species.     

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

Background  

Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrP^Sc, of the cellular prion protein, PrP^C. The conversion and thus the propensity of PrP^C 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.     

Ovine serum biomarkers of early and late phase scrapie

Background  

Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant.     

Increased expression and local accumulation of the Prion Protein,Alzheimer Aβ peptides,superoxide dismutase 1, and Nitric oxide synthases 1 &; 2 in muscle in a rabbit model of diabetes

Background  

Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP), and superoxide dismutase 1 (SOD1), as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies.     

AMYPdb: A database dedicated to amyloid precursor proteins

Background  

Misfolding and aggregation of proteins into ordered fibrillar structures is associated with a number of severe pathologies, including Alzheimer's disease, prion diseases, and type II diabetes. The rapid accumulation of knowledge about the sequences and structures of these proteins allows using of in silico methods to investigate the molecular mechanisms of their abnormal conformational changes and assembly. However, such an approach requires the collection of accurate data, which are inconveniently dispersed among several generalist databases.     

Detection of prion protein in urine-derived injectable fertility products by a targeted proteomic approach

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.     

Infectious Fold and Amyloid Propagation in Podospora anserina

Amyloid protein aggregation is involved in serious neurodegenerative disorders such as Alzheimer''s disease and transmissible encephalopathies. The concept of an infectious protein (prion) being the scrapie agent was successfully validated for several yeast and fungi proteins. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically and biochemically identified as prion proteins. Studies on these proteins have revealed critical information on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by rich β sheet content. In a previous work on the HET-s prion protein Podospora, we demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the HET-s prion domain associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review, as are relevant questions about the [Het-s] system of Podospora anserina.Key Words: prion, HET-s, Podospora, amyloid, infectious, β sheet, mutagenesis, fold, propagation     

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

Background  

The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrP^Sc) in brain tissue. Infectivity studies indicate that PrP^Sc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods.     

The contrasting effect of macromolecular crowding on amyloid fibril formation

Background

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.

Methodology/Principal Findings

As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.

Conclusions/Significance

We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.     

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

Background  

Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs.     

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

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

High CJD infectivity remains after prion protein is destroyed

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.     

Prediction of "hot spots" of aggregation in disease-linked polypeptides

Background  

The polypeptides involved in amyloidogenesis may be globular proteins with a defined 3D-structure or natively unfolded proteins. The first class includes polypeptides such as β2-microglobulin, lysozyme, transthyretin or the prion protein, whereas β-amyloid peptide, amylin or α-synuclein all belong to the second class. Recent studies suggest that specific regions in the proteins act as "hot spots" driving aggregation. This should be especially relevant for natively unfolded proteins or unfolded states of globular proteins as they lack significant secondary and tertiary structure and specific intra-chain interactions that can mask these aggregation-prone regions. Prediction of such sequence stretches is important since they are potential therapeutic targets.     

Genomic clustering and homology between HET-S and the NWD2 STAND protein in various fungal genomes

Background

Prions are infectious proteins propagating as self-perpetuating amyloid polymers. The [Het-s] prion of Podospora anserina is involved in a cell death process associated with non-self recognition. The prion forming domain (PFD) of HET-s adopts a β-solenoid amyloid structure characterized by the two fold repetition of an elementary triangular motif. [Het-s] induces cell death when interacting with HET-S, an allelic variant of HET-s. When templated by [Het-s], HET-S undergoes a trans-conformation, relocates to the cell membrane and induces toxicity.

Methodology/Principal Findings

Here, comparing HET-s homologs from different species, we devise a consensus for the HET-s elementary triangular motif. We use this motif to screen genomic databases and find a match to the N-terminus of NWD2, a STAND protein, encoded by the gene immediately adjacent to het-S. STAND proteins are signal transducing ATPases which undergo ligand-induced oligomerisation. Homology modelling predicts that the NWD2 N-terminal region adopts a HET-s-like fold. We propose that upon NWD2 oligomerisation, these N-terminal extensions adopt the β-solenoid fold and template HET-S to adopt the amyloid fold and trigger toxicity. We extend this model to a putative prion, the σ infectious element in Nectria haematococca, because the s locus controlling propagation of σ also encodes a STAND protein and displays analogous features. Comparative genomic analyses indicate evolutionary conservation of these STAND/prion-like gene pairs, identify a number of novel prion candidates and define, in addition to the HET-s PFD motif, two distinct, novel putative PFD-like motifs.

Conclusions/Significance

We suggest the existence, in the fungal kingdom, of a widespread and evolutionarily conserved mode of signal transduction based on the transmission of an amyloid-fold from a NOD-like STAND receptor protein to an effector protein.     

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP^Sc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.     

Quantum dots and prion proteins

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 (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc 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.     

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was ≥4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than ～15 nm in diameter).     

Infectious Fold and Amyloid Propagation in Podospora anserina

Aggregation of amyloid proteins is involved in serious neurodegenerative disorders such as Alzheimer disease and transmissible encephalopathies. The concept of an infectious protein (prion) proposed as the scrapie agent was successfully validated for several proteins of yeast and fungi. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically, then biochemically identified as prion proteins. Studies on these proteins have brought critical informations on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by a rich β-sheet content. In a previous work on the HET-s prion protein of Podospora we have demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the prion domain of HET-s associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review as well as relevant questions about the [Het-s] system of Podospora anserina.     

Prions are novel infectious pathogens causing scrapie and Creutzfeldt-Jakob disease

Scrapie and Creutzfeldt–Jakob disease (CJD) are caused by prions, which appear to be different from both viruses and viroids. Prions contain protein which is required for infectivity, but no nucleic acid has been found within them. Prion proteins are encoded by a cellular gene and not by a nucleic acid within the infectious prion particle. A cellular homologue of the prion protein has been IDentified. The role of this homologue in metabolism is unknown. Prion proteins, but not the cellular homologue, aggregate into rod-shaped particles that are histo-chemically and ultrastructurally IDentical to amyloid. Extracellular collections of prion proteins form amyloid plaques in scrapie- and CJD-infected rodent brains as well as CJD-infected human brains. Within the plaques, prion proteins assemble to form amyloid filaments. Elucidating the molecular differences between the prion protein and its cellular homologue may be important in understanding the chemical structure and replication of prions.