Prion protein—Semisynthetic prion protein (PrP) variants with posttranslational modifications

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrP^C) into scrapie prion protein (PrP^Sc) that further propagates PrP^C misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrP^Sc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.     

Protocol for aerosol-free recombinant production and NMR analysis of prion proteins

The central hallmark of prion diseases is the misfolding of cellular prion protein (PrP^C) into a disease-associated aggregated isoform known as scrapie prion protein (PrP^Sc). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory.     

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein

ABSTRACT

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP^C) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP^Sc) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP^C proteins have provided evidence of a conformational equilibrium between a 3₁₀-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165–175). In vitro cell-free experiments of the seeded conversion of PrP^C indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3₁₀-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3₁₀-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.     

Paradoxical Role of Prion Protein Aggregates in Redox-Iron Induced Toxicity

Background

Imbalance of iron homeostasis has been reported in sporadic Creutzfeldt-Jakob-disease (sCJD) affected human and scrapie infected animal brains, but the contribution of this phenotype to disease associated neurotoxicity is unclear.

Methodology/Principal Findings

Using cell models of familial prion disorders, we demonstrate that exposure of cells expressing normal prion protein (PrP^C) or mutant PrP forms to a source of redox-iron induces aggregation of PrP^C and specific mutant PrP forms. Initially this response is cytoprotective, but becomes increasingly toxic with time due to accumulation of PrP-ferritin aggregates. Mutant PrP forms that do not aggregate are not cytoprotective, and cells show signs of acute toxicity. Intracellular PrP-ferritin aggregates induce the expression of LC3-II, indicating stimulation of autophagy in these cells. Similar observations are noted in sCJD and scrapie infected hamster brains, lending credence to these results. Furthermore, phagocytosis of PrP-ferritin aggregates by astrocytes is cytoprotective, while culture in astrocyte conditioned medium (CM) shows no measurable effect. Exposure to H₂O₂, on the other hand, does not cause aggregation of PrP, and cells show acute toxicity that is alleviated by CM.

Conclusions/Significance

These observations suggest that aggregation of PrP in response to redox-iron is cytoprotective. However, subsequent co-aggregation of PrP with ferritin induces intracellular toxicity unless the aggregates are degraded by autophagosomes or phagocytosed by adjacent scavenger cells. H₂O₂, on the other hand, does not cause aggregation of PrP, and induces toxicity through extra-cellular free radicals. Together with previous observations demonstrating imbalance of iron homeostasis in prion disease affected brains, these observations provide insight into the mechanism of neurotoxicity by redox-iron, and the role of PrP in this process.     

PrPST,a Soluble,Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease

While the conversion of PrP^C into PrP^Sc in the transmissible form of prion disease requires a preexisting PrP^Sc seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrP^ST), a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrP^ST as in TgMHu2ME199K mice, and “classical” PrP^Sc as in infectious prion diseases, coincide in the patient''s post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition.     

Peptide NMHRYPNQ of the Cellular Prion Protein (PrP) Inhibits Aggregation and Is a Potential Key for Understanding Prion-Prion Interactions

Pathogenesis of transmissible spongiform encephalopathies is correlated with a conversion of the normal cellular form of the prion protein (PrP^C) into the abnormal isoform (scrapie form of PrP). Contact of the normal PrP with its abnormal isoform, the scrapie form of PrP, induces the transformation. Knowledge of molecules that inhibit such contacts leads to an understanding of the mechanism of the aggregation, and these molecules may serve as leads for drugs against transmissible spongiform encephalopathies. Therefore, we screened a synthetic octapeptide library of the globular domain of the human PrP^C for binding affinity to PrP^C. Two fragments with binding affinity, 149YYRENMHR156 and 153NMHRYPNQ160, were identified with K_d values of 21 and 25 μM, respectively. A 10-fold excess of peptide 153NMHRYPNQ160 inhibits aggregation of the PrP by 99%. NMR and mass spectrometry showed that the binding region of the peptide 153NMHRYPNQ160 is located at helix 3 of the PrP.     

Anti-PrP antibodies detected at terminal stage of prion-affected mouse

The causative agent of prion diseases is the pathological isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). PrP^Sc has an identical amino acid sequence to PrP^C; thus, it has been assumed that an immune response against PrP^Sc could not be found in prion-affected animals. In this study, we found the anti-prion protein (PrP) antibody at the terminal stage of mouse scrapie. Several sera from mice in the terminal stage of scrapie reacted to the recombinant mouse PrP (rMPrP) molecules and brain homogenates of mouse prion diseases. These results indicate that mouse could recognize PrP^C or PrP^Sc as antigens by the host immune system. Furthermore, immunization with rMPrP generates high titers of anti-PrP antibodies in wild-type mice. Some anti-PrP antibodies immunized with rMPrP prevent PrP^Sc replication in vitro. The mouse sera from terminal prion disease have several wide epitopes, although mouse sera immunized with rMPrP possess narrow epitopes.     

Prion Protein—Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP^C) into a β-sheet-rich disease-causing isoform (PrP^Sc) is the key molecular event in the formation of PrP^Sc aggregates. The molecular mechanisms underlying the PrP^C-to-PrP^Sc conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP^Sc in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23–230)] and N-terminally truncated recPrP(89–230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP^C into PrP^Sc.     

Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP^C and the pathological isoform PrP^Sc. The conformational change of the α-helical PrP^C into β-sheet-rich PrP^Sc is the fundamental event of prion disease. The transition of recombinant PrP from a PrP^C-like into a PrP^Sc-like conformation can be induced in vitro by submicellar concentrations of SDS. An α-helical dimer was identified that might represent either the native state of PrP^C or the first step from the monomeric PrP^C to highly aggregated PrP^Sc. In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.     

Rapid folding of the prion protein captured by pressure-jump

The conversion of the cellular form of the prion protein (PrP^C) to an altered disease state, generally denoted as scrapie isoform (PrP^Sc), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before. D. C. Jenkins and D. S. Pearson contributed equally.     

Different antigenicities of the N‐terminal region of cellular and scrapie prion proteins

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP^Sc) and cellular prion protein (PrP^C) under native conditions. To clarify conformational differences between these two isoforms, PrP‐deficient mice were immunized with brain homogenates of normal and scrapie‐infected animals. All mice generated anti‐PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP^Sc consisting of QGSPGGN (PrP41–47) at the N‐terminus. This study demonstrated a conformational dissimilarity at the N‐terminus between PrP^Sc and PrP^C, a finding that may provide novel information about conformational features of PrP^Sc.     

Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: In Vitro and In Silico Approaches

The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP^C) into the scrapie form (PrP^Sc) is the hallmark of TSEs. Once formed, PrP^Sc aggregates and catalyzes PrP^C misfolding into new PrP^Sc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed in vivo due to poor pharmacokinetic profiles. In this work we propose a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP^Sc (ScN2a) for their ability to inhibit PK-resistant PrP (PrP^Res) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP^Res in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP^Res from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP^109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP^Res in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.     

Mapping the Prion Protein Using Recombinant Antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrP^C) into a pathogenic isoform (PrP^Sc). The occurrence of PrP^C on the cell surface and PrP^Sc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp^0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrP^C on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrP^C and PrP^Sc, while epitopes associated with most of the antibodies in the panel were present on PrP^C but absent from PrP^Sc.     

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Prions are unconventional infectious agents thought to be primarily composed of PrP^Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP^Sc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP^Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP^Sc aggregates from PrP^C. The distribution of PrP^Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP^Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP^Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.     

Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.     

PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP^C, for “cellular prion protein”) into an abnormal state (PrP^Sc, for “scrapie prion protein”). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP^C. In contrast to its homologue PrP^C, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP^Sc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP^C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.     

Investigation of the molecular similarity in closely related protein systems: The PrP case study

The amyloid conversion is a massive detrimental modification affecting several proteins upon specific physical or chemical stimuli characterizing a plethora of diseases. In many cases, the amyloidogenic stimuli induce specific structural features to the protein conferring the propensity to misfold and form amyloid deposits. The investigation of mutants, structurally similar to their native isoform but inherently prone to amyloid conversion, may be a viable strategy to elucidate the structural features connected with amyloidogenesis. In this article, we present a computational protocol based on the combination of molecular dynamics (MD) and grid‐based approaches suited for the pairwise comparison of closely related protein structures. This method was applied on the cellular prion protein (PrP^C) as a case study and, in particular, addressed to the quali/quantification of the structural features conferred by either E200K mutations and treatment with CaCl₂, both able to induce the scrapie conversion of PrP. Several schemes of comparison were developed and applied to this case study, and made up suitable of application to other protein systems. At this purpose an in‐house python codes has been implemented that, together with the parallelization of the GRID force fields program, will spread the applicability of the proposed computational procedure. Proteins 2015; 83:1751–1765. © 2015 Wiley Periodicals, Inc.     

Seeded Fibrillation as Molecular Basis of the Species Barrier in Human Prion Diseases

Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and Creutzfeldt-Jakob disease (CJD) in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP^C) to its pathological isoform PrP^Sc, which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP) as substrate can be specifically seeded by PrP^Sc as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the “prion-protein-only” hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrP^C and PrP^Sc. Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD) with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrP^Sc as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

Molecular structure of amyloid fibrils controls the relationship between fibrillar size and toxicity

Background

According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.

Methodology/Principal Findings

For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP^C) at high levels confirming that cytotoxicity was in part PrP^C-dependent. Silencing of PrP^C expression by small hairpin RNAs designed to silence expression of human PrP^C (shRNA-PrP^C) deminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP^C-mediated and PrP^C-independent mechanisms depends on the structure of the aggregates.

Conclusions/Significance

This work provides a direct illustration that the relationship between an amyloid''s physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP^C expression can be exploited to reduce their deleterious effects.