Concentration of Disease-Associated Prion Protein with Silicon Dioxide

Reagents that can precipitate the disease-associated prion protein (PrP^Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP^C) and ovine scrapie PrP^Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP^Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP^Sc (PrP^res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO₂ precipitation step into the immunological detection of PrP^res increased detection sensitivity by over 1,500-fold. Minerals such as SiO₂ are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

Functionally Relevant Domains of the Prion Protein Identified In Vivo

The prion consists essentially of PrP^Sc, a misfolded and aggregated conformer of the cellular protein PrP^C. Whereas PrP^C deficient mice are clinically healthy, expression of PrP^C variants lacking its central domain (PrP_ΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrP^C (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrP_ΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrP_ΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrP_ΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants.     

Human Prion Diseases in the United States

Background

Prion diseases are a family of rare, progressive, neurodegenerative disorders that affect humans and animals. The most common form of human prion disease, Creutzfeldt-Jakob disease (CJD), occurs worldwide. Variant CJD (vCJD), a recently emerged human prion disease, is a zoonotic foodborne disorder that occurs almost exclusively in countries with outbreaks of bovine spongiform encephalopathy.This study describes the occurrence and epidemiology of CJD and vCJD in the United States.

Methodology/Principal Findings

Analysis of CJD and vCJD deaths using death certificates of US residents for 1979–2006, and those identified through other surveillance mechanisms during 1996–2008. Since CJD is invariably fatal and illness duration is usually less than one year, the CJD incidence is estimated as the death rate. During 1979 through 2006, an estimated 6,917 deaths with CJD as a cause of death were reported in the United States, an annual average of approximately 247 deaths (range 172–304 deaths). The average annual age-adjusted incidence for CJD was 0.97 per 1,000,000 persons. Most (61.8%) of the CJD deaths occurred among persons ≥65 years of age for an average annual incidence of 4.8 per 1,000,000 persons in this population. Most deaths were among whites (94.6%); the age-adjusted incidence for whites was 2.7 times higher than that for blacks (1.04 and 0.40, respectively). Three patients who died since 2004 were reported with vCJD; epidemiologic evidence indicated that their infection was acquired outside of the United States.

Conclusion/Significance

Surveillance continues to show an annual CJD incidence rate of about 1 case per 1,000,000 persons and marked differences in CJD rates by age and race in the United States. Ongoing surveillance remains important for monitoring the stability of the CJD incidence rates, and detecting occurrences of vCJD and possibly other novel prion diseases in the United States.     

Structural Determinants in Prion Protein Folding and Stability

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.     

Co-existence of Distinct Prion Types Enables Conformational Evolution of Human PrPSc by Competitive Selection

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrP^Sc). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrP^Sc particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrP^Sc particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrP^C substrate, the dominant PrP^Sc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrP^Sc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrP^Sc conformers.     

Characterization of the Prion Protein in Human Urine

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.     

Protein Interaction Network-based Deep Learning Framework for Identifying Disease-Associated Human Proteins

Infectious diseases in humans appear to be one of the most primary public health issues. Identification of novel disease-associated proteins will furnish an efficient recognition of the novel therapeutic targets. Here, we develop a Graph Convolutional Network (GCN)-based model called PINDeL to identify the disease-associated host proteins by integrating the human Protein Locality Graph and its corresponding topological features. Because of the amalgamation of GCN with the protein interaction network, PINDeL achieves the highest accuracy of $83.45\%$ while AUROC and AUPRC values are $0.90$ and $0.88$, respectively. With high accuracy, recall, F1-score, specificity, AUROC, and AUPRC, PINDeL outperforms other existing machine-learning and deep-learning techniques for disease gene/protein identification in humans. Application of PINDeL on an independent dataset of 24320 proteins, which are not used for training, validation, or testing purposes, predicts 6448 new disease-protein associations of which we verify 3196 disease-proteins through experimental evidence like disease ontology, Gene Ontology, and KEGG pathway enrichment analyses. Our investigation informs that experimentally-verified 748 proteins are indeed responsible for pathogen-host protein interactions of which 22 disease-proteins share their association with multiple diseases such as cancer, aging, chem-dependency, pharmacogenomics, normal variation, infection, and immune-related diseases. This unique Graph Convolution Network-based prediction model is of utmost use in large-scale disease-protein association prediction and hence, will provide crucial insights on disease pathogenesis and will further aid in developing novel therapeutics.     

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

Background

Cellular prion-related protein (PrP^c) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP^c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypotesize that PrP^c could exert antimicrobial activity.

Methodology and Principal Findings

Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the “classical” human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-α in vitro.

Conclusions

The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.     

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

Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrP^Sc) converted from a normal host cellular prion protein (PrP^C). Experimental studies suggest that PrP^C is enriched with α-helical structure, whereas PrP^Sc contains a high proportion of β-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 PrP^C 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.     

Structural and Dynamic Properties of the Human Prion Protein

Prion diseases involve the conformational conversion of the cellular prion protein (PrP^C) to its misfolded pathogenic form (PrP^Sc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrP^C to PrP^Sc.     

Structural and Dynamic Properties of the Human Prion Protein

Prion diseases involve the conformational conversion of the cellular prion protein (PrP^C) to its misfolded pathogenic form (PrP^Sc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrP^C to PrP^Sc.     

Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations

Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression.     

PrPc介导的细胞信号转导

朊病毒病的发生是由于细胞正常朊蛋白PrPc转变成了异常构象的PrPc形式。PrPc的生理学功能目前尚不完全明确,可能与铜离子代谢、脂质摄取以及细胞信号传递有关。PrPc可以与小窝蛋白相互作用而活化Fyn非受体酪氨酸激酶从而引起下游信号通路的转导;可以作为受体与PrPc键合多肽结合后激活cAMP／PKA信号通路;以及引起细胞内钙离子浓度变化而活化信号通路。     

Two Amyloid States of the Prion Protein Display Significantly Different Folding Patterns

It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question, we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein. Several biophysical techniques including X-ray diffraction, CD, Fourier transform infrared spectroscopy (FTIR), hydrogen-deuterium exchange, proteinase K digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary, and quaternary structures. While both states displayed a 4. 8-Å meridional X-ray diffraction typical for amyloid cross-β-spines, they showed markedly different equatorial profiles, suggesting different folding pattern of β-strands. The experiments on hydrogen-deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-β-structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 °C, only local unfolding was revealed, while individual state-specific cross-β features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-β-structure. Both S- and R-fibrils, however, shared high conformational stability, arguing that the energy landscape for protein folding and aggregation can contain several deep free-energy minima.     

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 Unfolded State of the Murine Prion Protein and Properties of Single-point Mutants Related to Human Prion Diseases

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.