Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7–10 nm are being observed. In spite of the great clinical important of amyloidassociated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β breakage methods, is their use for the development of novel classes of amyloid formation inhibitors.Key Words: Alzheimer''s disease, amyloid disease, molecular recognition, nanostructures, protein aggregation, protein misfolding, self-assembly, type II diabetes     

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic β-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. β-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated β-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded β-sheet-rich proteins accumulate.     

Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms

Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12^undiff). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.     

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly

The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of “functional amyloids” expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.     

Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.     

An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function

Stress granules (SG) are membrane‐less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age‐related diseases, such as amyotrophic lateral sclerosis (ALS), but the molecular events triggering their formation are still unknown. Here, we find that misfolded proteins, such as ALS‐linked variants of SOD1, specifically accumulate and aggregate within SGs in human cells. This decreases the dynamics of SGs, changes SG composition, and triggers an aberrant liquid‐to‐solid transition of in vitro reconstituted compartments. We show that chaperone recruitment prevents the formation of aberrant SGs and promotes SG disassembly when the stress subsides. Moreover, we identify a backup system for SG clearance, which involves transport of aberrant SGs to the aggresome and their degradation by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.     

Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve

In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.     

Safety,specificity and immunogenicity of a PrPSc-specific prion vaccine based on the YYR disease specific epitope

Prions are a novel form of infectivity based on the misfolding of a self-protein (PrP^C) into a pathological, infectious isomer (PrP^Sc). The current uncontrolled spread of chronic wasting disease in cervids, coupled with the demonstrated zoonotic nature of select livestock prion diseases, highlights the urgent need for disease management tools. While there is proof-of-principle evidence for a prion vaccine, these efforts are complicated by the challenges and risks associated with induction of immune responses to a self-protein. Our priority is to develop a PrP^Sc-specific prion vaccine based on epitopes that are uniquely exposed upon misfolding. These disease specific epitopes (DSEs) have the potential to enable specific targeting of the pathological species through immunotherapy. Here we review outcomes of the translation of a prion DSE into a PrP^Sc-specific vaccine based on the criteria of immunogenicity, safety and specificity.     

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.     

In vitro self-propagation of recombinant PrPSc-like conformation generated in the yeast cytoplasm

Self-propagation is characteristic property for a prion conformation. Previous studies revealed that prion protein expressed in the cytoplasm gained a PrP(Sc)-like conformation. However, it remains unclear whether the PrP(Sc)-like conformation has the self-propagating property. We found that PrP partially purified from yeast cytoplasm formed amyloid fiber like structures, and we found that the PrP(Sc)-like conformation is able to convert normal PrP(C) in the brain homogenate to a proteinase K-resistant conformation. These results suggest that yeast cytoplasm expressed recombinant PrP(Sc)-like conformation has the characteristic self-propagating property of a prion, which may have implications in the pathogenesis of sporadic and inherited prion diseases.     

Antiaggregating antibody raised against human PrP 106-126 recognizes pathological and normal isoforms of the whole prion protein

Antibodies to the prion protein (PrP) have been critical to the neuropathological and biochemical characterization of PrP-related degenerative diseases in humans and animals. Although PrP is highly conserved evolutionarily, there is some sequence divergence among species; as a consequence, anti-PrP antibodies have a wide spectrum of reactivity when challenged with PrP from diverse species. We have produced an antibody [monoclonal antibody (mAb) 2-40] raised against a synthetic peptide corresponding to residues (106-126 of human PrP and have characterized it by epitope mapping, Western immunoblot analysis, and immunohistochemistry. The antibody recognizes not only human PrP isoforms but also pathological PrP from all species tested (i.e., sheep, hamsters, and mice). Together with the fact that it recognizes the whole PrP in both cellular and scrapie isoforms, mAb 2-40 may be helpful in studying conformational changes of the PrP, as well as establishing a possible connection between human and animal diseases.     

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.     

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 μg ml⁻¹) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (β(1–3),β(1–6)-glucan) and β(1–3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.Hydrophobins are a class of surface active proteins that play diverse roles in fungal growth and development. For instance, they allow fungi to escape an aqueous environment, confer hydrophobicity to fungal surfaces in contact with air, and mediate attachment of fungi to hydrophobic surfaces (1, 2). They also play a role in the architecture of the cell wall (3).Hydrophobins share eight conserved cysteine residues, but otherwise their sequences are diverse (4). Class I and II hydrophobins are distinguished on the basis of differences in hydropathy patterns and biophysical properties (5). SC3 of Schizophyllum commune is the best characterized class I hydrophobin. It self-assembles at interfaces between water and air, water and oil, and water and hydrophobic solids (6–8). The four disulfide bridges of SC3 prevent spontaneous self-assembly in solution and thus account for the controlled assembly at hydrophobic-hydrophilic interfaces (9).The water-soluble form of SC3 is oligomeric (10) and rich in β-sheet (11). Upon assembly at the water-air interface, SC3 proceeds via an intermediate form that has increased α-helical structure (α-helical state) to a stable end form that has increased β-sheet structure (β-sheet state) (11–13). SC3 in the β-sheet state initially has no clear ultrastructure (β-sheet I state) (12), but after prolonged incubation, the protein forms 10-nm wide amyloid-like fibrils (β-sheet II state) (12–14) that are called rodlets (6, 15). Like other amyloid fibrils (16), rodlets of the hydrophobins SC3 of S. commune and EAS of Neurospora crassa increase fluorescence of thioflavin T and bind Congo red (14, 17, 18). Moreover, x-ray diffraction of rodlets of EAS showed reflections at 4.8 Å (distance between strands in a β-sheet) and 10–12 Å (spacing between β-sheets stacked perpendicular to the fibril long axis) (19), which are indicative for amyloid fibrils.Notably, SC3 does not spontaneously self-assemble into amyloid fibrils at an interface between water and a hydrophobic solid. Instead, SC3 is arrested in the intermediate α-helical state. Transition to the β-sheet state is observed only by heating the sample in the presence of detergent (11, 12). These observations prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that amyloid formation of SC3 is promoted by increasing its concentration or by the presence of cell wall polysaccharides.     

Susceptibility of cell substrates to PrPSc infection and safety control measures related to biological and biotherapeutical products

Concerns over the potential for infectious prion proteins to contaminate human biologics and biotherapeutics have been raised from time to time. Transmission of the pathogenic form of prion protein (PrPSc) through veterinary vaccines has been observed, yet no human case through the use of vaccine products has been reported. However, iatrogenic transmissions of PrPSc in humans through blood components, tissues, and growth hormone have been reported. These findings underscore the importance of reliable detection or diagnostic methods to prevent the transmission of prion diseases, given that the number of asymptomatic infected individuals remains unknown, the perceived incubation time for human prion diseases could be decades, and no cure of the diseases has been found yet. A variety of biochemical and molecular methods can selectively concentrate PrPSc to facilitate its detection in tissues and cells. Furthermore, some methods routinely used in the manufacturing process of biological products have been found to be effective in reducing PrPSc from the products. Questions remain unanswered as to the validation criteria of these methods, the minimal infectious dose of the PrPSc required to cause infection and the susceptibility of cells used in gene therapy or the manufacturing process of biological products to PrPSc infections. Here, we discuss some of these challenging issues.     

Molecular Biology and Transgenetics of Prion Diseases

Abstract

Considerable progress has been made deciphering the role of an abnormal isoform of the prion protein (PrP) in scrapie of animals and Gerstmann-Sträussler syndrome (GSS) of humans. Some transgenic (Tg) mouse (Mo) lines that carry and express a Syrian hamster (Ha) PrP gene developed scrapie 75 d after inoculation with Ha prions; non-Tg mice failed to show symptoms after 500 d. Brains of these infected Tg(HaPrP) mice featured protease-resistant HaPrP^sc, amyloid plaques characteristic for Ha scrapie, and 10⁹ ID₅₀ units of Ha-specific prions upon bioassay. Studies on Syrian, Armenian, and Chinese hamsters suggest that the domain of the PrP molecule between codons 100 and 120 controls both the length of the incubation time and the deposition of PrP in amyloid plaques. Ataxic GSS in families shows genetic linkage to a mutation in the PrP gene, leading to the substitution of Leu for Pro at codon 102. Discovery of a point mutation in the Prp gene from humans with GSS established that GSS is unique among human diseases it is both genetic and infectious. These results have revised thinking about sporadic Creutzfeldt-Jakob disease, suggesting it may arise from a somatic mutation. These findings combined with those from many other studies assert that PrP^sc is a component of the transmissible particle, and the PrP amino acid sequence controls the neuropathology and species specificity of prion infectivity. The precise mechanism of PrP^& formation remains to be established. Attempts to demonstrate a scrapie-specific nucleic acid within highly purified preparations of prions have been unrewarding to date. Whether transmissible prions are composed only of PrP^sc molecules or do they also contain a second component such as small polynucleotide remains uncertain.     

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

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.     

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP^Sc. Studies showed that recombinant cellular PrP, PrP^C, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V₁₃₆L₁₄₁R₁₅₄Q₁₇₁ and A₁₃₆F₁₄₁R₁₅₄Q₁₇₁, and one genotype of white-tailed deer (Q₉₅G₉₆, X₁₃₂,Y₂₁₆) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP^C shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP^C and supports amplification of PrP^Sc and PrP^CWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP^Sc or PrP^CWD production. Addition of 2 to 20 μM of manganese chloride (MnCl₂) to unseeded PMCA resulted in conversion of recombinant PrP^C to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.