In JoVE (1)

Other Publications (41)

Articles by Holger Wille in JoVE

Other articles by Holger Wille on PubMed

Structural Studies of the Scrapie Prion Protein by Electron Crystallography

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2002  |  Pubmed ID: 11891310

Because the insolubility of the scrapie prion protein (PrP(Sc)) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 A resolution. By comparing projection maps of PrP 27-30 and PrP(Sc)106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP(Sc). Only models featuring parallel beta-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.

Cytosolic Prion Protein in Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2003  |  Pubmed ID: 12904479

Localizing the cellular prion protein (PrPC) in the brain is necessary for understanding the pathogenesis of prion diseases. However, the precise ultrastructural localization of PrPC still remains enigmatic. We performed the first quantitative study of the ultrastructural localization of PrPC in the mouse hippocampus using high-resolution cryoimmunogold electron microscopy. PrPC follows the standard biosynthetic trafficking pathway with a preferential localization in late endosomal compartments and on the plasma membrane of neurons and neuronal processes. PrPC is found with the same frequency within the synaptic specialization and perisynaptically, but is almost completely excluded from synaptic vesicles. Unexpectedly, PrP is also found in the cytosol in subpopulations of neurons in the hippocampus, neocortex, and thalamus but not the cerebellum. Cytosolic PrP may have altered susceptibility to aggregation, suggesting that these neurons might play a significant role in the pathogenesis of prion diseases, in particular those mammals harboring mutant PrP genes.

Evidence for Assembly of Prions with Left-handed Beta-helices into Trimers

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2004  |  Pubmed ID: 15155909

Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross-beta structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP(Sc)) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right- or left-handed beta-helical models. From a study of 119 all-beta folds observed in globular proteins, we have now determined that, if PrP(Sc) follows a known protein fold, it adopts either a beta-sandwich or parallel beta-helical architecture. With increasing evidence arguing for a parallel beta-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed beta-helical fold. Left-handed beta-helices readily form trimers, providing a natural template for a trimeric model of PrP(Sc). This trimeric model accommodates the PrP sequence from residues 89-175 in a beta-helical conformation with the C terminus (residues 176-227), retaining the disulfide-linked alpha-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed beta-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed beta-helical model for PrP(Sc) may provide important clues to the structure of filaments found in some other neurodegenerative diseases.

Engineering a 2D Protein-DNA Crystal

Angewandte Chemie (International Ed. in English). May, 2005  |  Pubmed ID: 15828044

Assembly of Natural and Recombinant Prion Protein into Fibrils

Biological Chemistry. Jun, 2005  |  Pubmed ID: 16006244

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.

Human Prions and Plasma Lipoproteins

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2006  |  Pubmed ID: 16849426

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). The polyoxometalate phosphotungstic acid has been used to separate PrP(Sc) from its precursor PrP(C) by selective precipitation; notably, native PrP(Sc) has not been solubilized by using nondenaturing detergents. Because of the similarities between PrP(Sc) and lipoproteins with respect to hydrophobicity and formation of phosphotungstic acid complexes, we asked whether these molecules are bound to each other in blood. Here we report that prions from the brains of patients with sporadic Creutzfeldt-Jakob disease (CJD) bind to very low-density (VLDL) and low-density (LDL) lipoproteins but not to high-density lipoproteins (HDL) or other plasma components, as demonstrated both by affinity assay and electron microscopy. Immunoassays demonstrated that apolipoprotein B (apoB), which is the major protein component of VLDL and LDL, bound PrP(Sc) through a highly cooperative process. Approximately 50% of the PrP(Sc) bound to LDL particles was released after exposure to 4 M guanidine hydrochloride at 80 degrees C for 20 min. The apparent binding constants of native human (Hu) PrP(Sc) or denatured recombinant HuPrP(90-231) for apoB and LDL ranged from 28 to 212 pM. Whether detection of PrP(Sc) in VLDL and LDL particles can be adapted into an antemortem diagnostic test for prions in the blood of humans, livestock, and free-ranging cervids remains to be determined.

Influence of Water, Fat, and Glycerol on the Mechanism of Thermal Prion Inactivation

The Journal of Biological Chemistry. Dec, 2007  |  Pubmed ID: 17878157

Extending the recent analysis of the safety of industrial bovine fat-derived products for human consumption (Müller, H., Stitz, L., and Riesner, D. (2006) Eur. J. Lip. Sci. Technol. 108, 812-826), we investigated systematically the effects of fat, fatty acids, and glycerol on the heat destruction of prions. Prion destruction was qualitatively and quantitatively evaluated in PrP 27-30, or prion rods, by the inactivation of infectivity as well as by the degradation of the polypeptide backbone. Under all conditions analyzed, inactivation of prion infectivity was achieved more efficiently than backbone degradation by several orders of magnitude. The presence of fat enhanced prion inactivation and offers a mild treatment for prion decontamination. In contrast, the presence of fat, fatty acids, and especially glycerol protected the PrP 27-30 backbone against heat-induced degradation. Glycerol also protected against heat-induced inactivation of prion infectivity. A phase distribution analysis demonstrated that prions migrated to the interphase of a fat/water mixture at room temperature and accumulated in the water phase at higher temperatures. In a systematic study of the mechanism of prion destruction, we found an intermediate structure of PrP that has fewer fibrils in beta-sheet formation, lower resistance to protease digestion, greater aggregation, and reduced solubility compared with PrP 27-30 but retains residual infectivity. These findings suggest that prion infectivity depends on beta-sheet-rich fibrillar structure and that inactivation proceeds in a stepwise manner, which explains the tailing effect frequently observed during inactivation.

Electron Crystallography of the Scrapie Prion Protein Complexed with Heavy Metals

Archives of Biochemistry and Biophysics. Nov, 2007  |  Pubmed ID: 17935686

The insolubility of the disease-causing isoform of the prion protein (PrP(Sc)) has prevented studies of its three-dimensional structure at atomic resolution. Electron crystallography of two-dimensional crystals of N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) provided the first insights at intermediate resolution on the molecular architecture of the prion. Here, we report on the structure of PrP 27-30 and PrP(Sc)106 negatively stained with heavy metals. The interactions of the heavy metals with the crystal lattice were governed by tertiary and quaternary structural elements of the protein as well as the charge and size of the heavy metal salts. Staining with molybdate anions revealed three prominent densities near the center of the trimer that forms the unit cell, coinciding with the location of the beta-helix that was proposed for the structure of PrP(Sc). Differential staining also confirmed the location of the internal deletion of PrP(Sc)106 at or near these densities.

Small-molecule Aggregates Inhibit Amyloid Polymerization

Nature Chemical Biology. Mar, 2008  |  Pubmed ID: 18223646

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.

Mechanisms of Prion Protein Assembly into Amyloid

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2008  |  Pubmed ID: 18268326

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP(C)) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP(Sc)) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.

Genes Contributing to Prion Pathogenesis

The Journal of General Virology. Jul, 2008  |  Pubmed ID: 18559949

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Cryo-immunogold Electron Microscopy for Prions: Toward Identification of a Conversion Site

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19020041

Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).

Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change

The Journal of Biological Chemistry. Feb, 2009  |  Pubmed ID: 19010781

Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid beta, beta2-microglobulin, and prion proteins.

Surface Charge of Polyoxometalates Modulates Polymerization of the Scrapie Prion Protein

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19223590

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). N-terminally truncated PrP(Sc), denoted PrP 27-30, retains infectivity and polymerizes into rods with the ultrastructural and tinctorial properties of amyloid. We report here that some polyoxometalates (POMs) favor polymerization of PrP 27-30 into prion rods, whereas other POMs promote assembly of the protein into 2D crystals. Antibodies reacting with epitopes in denatured PrP 27-30 also bound to 2D crystals treated with 3 M urea. These same antibodies did not bind to either native PrP(Sc) or untreated 2D crystals. By using small, spherical POMs with Keggin-type structures, the central heteroatom was found to determine whether prion rods or 2D crystals were preferentially formed. An example of a Keggin-type POM with a phosphorous heteroatom is the phosphotungstate anion (PTA). Both PTA and a Keggin-type POM with a silicon heteratom have low-charge densities and favor formation of prion rods. In contrast, POMs with boron or hydrogen heteroatoms exhibiting higher negative charges encouraged 2D crystal formation. The 2D crystals of PrP 27-30 produced by selective precipitation with POMs were larger and more well ordered than those obtained by sucrose gradient centrifugation. Our findings argue that the negative charge of Keggin-type POMs determines the quaternary structure adopted by PrP 27-30. The mechanism by which POMs function in competing prion polymerization pathways--one favoring 2D crystals and the other, amyloid fibrils--remains to be established.

Evolutionary Descent of Prion Genes from the ZIP Family of Metal Ion Transporters

PloS One. Sep, 2009  |  Pubmed ID: 19784368

In the more than twenty years since its discovery, both the phylogenetic origin and cellular function of the prion protein (PrP) have remained enigmatic. Insights into a possible function of PrP may be obtained through the characterization of its molecular neighborhood in cells. Quantitative interactome data demonstrated the spatial proximity of two metal ion transporters of the ZIP family, ZIP6 and ZIP10, to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family that includes ZIP5, ZIP6 and ZIP10. Additional structural threading and orthologous sequence alignment analyses argued that the prion gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations. The phylogenetic and spatial connection to ZIP proteins is expected to open new avenues of research to elucidate the biology of the prion protein in health and disease.

Natural and Synthetic Prion Structure from X-ray Fiber Diffraction

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2009  |  Pubmed ID: 19805070

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Design and Construction of Diverse Mammalian Prion Strains

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2009  |  Pubmed ID: 19915150

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.

Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

Biophysical Journal. Dec, 2009  |  Pubmed ID: 20006956

Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly alpha-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using beta-solenoid folds, namely, the beta-helix and beta-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the beta-roll subunit model may be more stable than the beta-helix subunit model. We also constructed beta-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted beta-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies.

Colloid Formation by Drugs in Simulated Intestinal Fluid

Journal of Medicinal Chemistry. May, 2010  |  Pubmed ID: 20426472

Many organic molecules form colloidal aggregates in aqueous solution at micromolar concentrations. These aggregates promiscuously inhibit soluble proteins and are a major source of false positives in high-throughput screening. Several drugs also form colloidal aggregates, and there has been speculation that this may affect the absorption and distribution of at least one drug in vivo. Here we investigate the ability of drugs to form aggregates in simulated intestinal fluid. Thirty-three Biopharmaceutics Classification System (BCS) class II and class IV drugs, spanning multiple pharmacological activities, were tested for promiscuous aggregation in biochemical buffers. The 22 that behaved as aggregators were then tested for colloid formation in simulated intestinal fluid, a buffer mimicking conditions in the small intestine. Six formed colloids at concentrations equal to or lower than the concentrations reached in the gut, suggesting that aggregation may have an effect on the absorption and distribution of these drugs, and potentially others, in vivo.

Family Reunion--the ZIP/prion Gene Family

Progress in Neurobiology. Mar, 2011  |  Pubmed ID: 21163327

Prion diseases are fatal neurodegenerative diseases of humans and animals which, in addition to sporadic and familial modes of manifestation, can be acquired via an infectious route of propagation. In disease, the prion protein (PrP(C)) undergoes a structural transition to its disease-causing form (PrP(Sc)) with profoundly different physicochemical properties. Surprisingly, despite intense interest in the prion protein, its function in the context of other cellular activities has largely remained elusive. We recently employed quantitative mass spectrometry to characterize the interactome of the prion protein in a murine neuroblastoma cell line (N2a), an established cell model for prion replication. Extensive bioinformatic analyses subsequently established an evolutionary link between the prion gene family and the family of ZIP (Zrt-, Irt-like protein) metal ion transporters. More specifically, sequence alignments, structural threading data and multiple additional pieces of evidence placed a ZIP5/ZIP6/ZIP10-like ancestor gene at the root of the PrP gene family. In this review we examine the biology of prion proteins and ZIP transporters from the viewpoint of a shared phylogenetic origin. We summarize and compare available data that shed light on genetics, function, expression, signaling, post-translational modifications and metal binding preferences of PrP and ZIP family members. Finally, we explore data indicative of retropositional origins of the prion gene founder and discuss a possible function for the prion-like (PL) domain within ZIP transporters. While throughout the article emphasis is placed on ZIP proteins, the intent is to highlight connections between PrP and ZIP transporters and uncover promising directions for future research.

In Vitro Conversion and Seeded Fibrillization of Posttranslationally Modified Prion Protein

Biological Chemistry. May, 2011  |  Pubmed ID: 21476870

The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrP(C) into an aggregated, β-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.

Self-assembling Small Molecules Form Nanofibrils That Bind Procaspase-3 to Promote Activation

Journal of the American Chemical Society. Dec, 2011  |  Pubmed ID: 22066605

Modulating enzyme function with small-molecule activators, as opposed to inhibitors, offers new opportunities for drug discovery and allosteric regulation. We previously identified a compound, called 1541, from a high-throughput screen (HTS) that stimulates activation of a proenzyme, procaspase-3, to generate mature caspase-3. Here we further investigate the mechanism of activation and report the surprising finding that 1541 self-assembles into nanofibrils exceeding 1 μm in length. These particles are an unanticipated outcome from an HTS that have properties distinct from standard globular protein aggregators. Moreover, 1541 nanofibrils function as a unique biocatalytic material that activates procaspase-3 via induced proximity. These studies demonstrate a novel approach for proenzyme activation through binding to fibrils, which may mimic how procaspases are naturally processed on protein scaffolds.

Spontaneous Generation of Anchorless Prions in Transgenic Mice

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2011  |  Pubmed ID: 22160704

Some prion protein mutations create anchorless molecules that cause Gerstmann-Sträussler-Scheinker (GSS) disease. To model GSS, we generated transgenic mice expressing cellular prion protein (PrP(C)) lacking the glycosylphosphatidyl inositol (GPI) anchor, denoted PrP(ΔGPI). Mice overexpressing PrP(ΔGPI) developed a late-onset, spontaneous neurologic dysfunction characterized by widespread amyloid deposition in the brain and the presence of a short protease-resistant PrP fragment similar to those found in GSS patients. In Tg(PrP,ΔGPI) mice, disease onset could be accelerated either by inoculation with brain homogenate prepared from spontaneously ill animals or by coexpression of membrane-anchored, full-length PrP(C). In contrast, coexpression of N-terminally truncated PrP(Δ23-88) did not affect disease progression. Remarkably, disease from ill Tg(PrP,ΔGPI) mice transmitted to mice expressing wild-type PrP(C), indicating the spontaneous generation of prions.

Protease-resistant Prions Selectively Decrease Shadoo Protein

PLoS Pathogens. Nov, 2011  |  Pubmed ID: 22163178

The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc) causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP(C), were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc) in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc). Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc). Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc) during prion disease.

Prion Uptake in the Gut: Identification of the First Uptake and Replication Sites

PLoS Pathogens. Dec, 2011  |  Pubmed ID: 22216002

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.

Degradation of Fungal Prion HET-s(218-289) Induces Formation of a Generic Amyloid Fold

Biophysical Journal. May, 2012  |  Pubmed ID: 22677387

The prion-forming domain of the fungal prion protein HET-s, HET-s(218-289), is known from solid-state NMR studies to have a β-solenoidal structure; the β-solenoid has the cross-β structure characteristic of all amyloids, but is inherently more complex than the generic stacked β-sheets found in studies of small synthetic peptides. At low pH HET-s(218-289) has also been reported to form an alternative structure, which has not been characterized. We have confirmed by x-ray fiber diffraction that HET-s(218-289) adopts a β-solenoidal structure at neutral pH, and shown that at low pH, it forms either a β-solenoid or a stacked β-sheet structure, depending on the integrity of the protein and the conditions of fibrillization. The low pH stacked-sheet structure is usually formed only by proteolyzed HET-s(218-289), but intact HET-s(218-289) can form stacked sheets when seeded with proteolyzed stacked-sheet HET-s(218-289). The polymorphism of HET-s parallels the structural differences between the infectious brain-derived and the much less infectious recombinant mammalian prion protein PrP. Taken together, these observations suggest that the functional or pathological forms of amyloid proteins are more complex than the simple generic stacked-sheet amyloids commonly formed by short peptides.

Plasma Membrane Invaginations Containing Clusters of Full-length PrPSc Are an Early Form of Prion-associated Neuropathology in Vivo

Neurobiology of Aging. Jun, 2013  |  Pubmed ID: 23481568

During prion disease, cellular prion protein (PrP(C)) is refolded into a pathogenic isoform (PrP(Sc)) that accumulates in the central nervous system and causes neurodegeneration and death. We used immunofluorescence, quantitative cryo-immunogold EM, and tomography to detect nascent, full-length PrP(Sc) in the hippocampus of prion-infected mice from early preclinical disease stages onward. Comparison of uninfected and infected brains showed that sites containing full-length PrP(Sc) could be recognized in the neuropil by bright spots and streaks of immunofluorescence on semi-thin (200-nm) sections, and by clusters of cryo-immunogold EM labeling. PrP(Sc) was found mainly on neuronal plasma membranes, most strikingly on membrane invaginations and sites of cell-to-cell contact, and was evident by 65 days postinoculation, or 54% of the incubation period to terminal disease. Both axons and dendrites in the neuropil were affected. We hypothesize that closely apposed plasma membranes provide a favorable environment for prion conversion and intercellular prion transfer. Only a small proportion of clustered PrP immunogold labeling was found at synapses, indicating that synapses are not targeted specifically in prion disease.

The ZIP5 Ectodomain Co-localizes with PrP and May Acquire a PrP-like Fold That Assembles into a Dimer

PloS One. 2013  |  Pubmed ID: 24039764

The cellular prion protein (PrP(C)) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrP(C) and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein. When recombinantly expressed, the PrP-like domain of ZIP5 could be obtained with yields and levels of purity sufficient for structural analyses but it tended to aggregate, thereby precluding attempts to study its structure. These obstacles were overcome by moving to a mammalian cell expression system. The subsequent biophysical characterization of a homogeneous preparation of the ZIP5 PrP-like ectodomain shows that this protein acquires a dimeric, largely globular fold with an α-helical content similar to that of mammalian PrP(C). The use of a mammalian cell expression system also allowed for the expression and purification of stable preparations of Takifugu rubripes PrP-1, thereby overcoming a key hindrance to high-resolution work on a fish PrP(C).

The Structure of the Infectious Prion Protein: Experimental Data and Molecular Models

Prion. Jan-Feb, 2014  |  Pubmed ID: 24583975

The structures of the infectious prion protein, PrP(Sc), and that of its proteolytically truncated variant, PrP 27-30, have evaded experimental determination due to their insolubility and propensity to aggregate. Molecular modeling has been used to fill this void and to predict their structures, but various modeling approaches have produced significantly different models. The disagreement between the different modeling solutions indicates the limitations of this method. Over the years, in absence of a three-dimensional (3D) structure, a variety of experimental techniques have been used to gain insights into the structure of this biologically, medically, and agriculturally important isoform. Here, we present an overview of experimental results that were published in recent years, and which provided new insights into the molecular architecture of PrP(Sc) and PrP 27-30. Furthermore, we evaluate all published models in light of these recent, experimental data, and come to the conclusion that none of the models can accommodate all of the experimental constraints. Moreover, this conclusion constitutes an open invitation for renewed efforts to model the structure of PrP(Sc).

The Structure of Human Prions: from Biology to Structural Models-considerations and Pitfalls

Viruses. Oct, 2014  |  Pubmed ID: 25333467

Prion diseases are a family of transmissible, progressive, and uniformly fatal neurodegenerative disorders that affect humans and animals. Although cross-species transmissions of prions are usually limited by an apparent “species barrier”, the spread ofa prion disease to humans by ingestion of contaminated food, or via other routes of exposure, indicates that animal prions can pose a significant public health risk. The infectious agent responsible for the transmission of prion diseases is a misfolded conformer of the prion protein, PrPSc, a pathogenic isoform of the host-encoded, cellular prion protein,PrPC. The detailed mechanisms of prion conversion and replication, as well as the high-resolution structure of PrPSc, are unknown. This review will discuss the general background related to prion biology and assess the structural models proposed to date,while highlighting the experimental challenges of elucidating the structure of PrPSc.

Octarepeat Region Flexibility Impacts Prion Function, Endoproteolysis and Disease Manifestation

EMBO Molecular Medicine. Mar, 2015  |  Pubmed ID: 25661904

The cellular prion protein (PrP(C)) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrP(S) (c) in Creutzfeldt-Jakob disease. PrP(C) β-endoproteolysis to the C2 fragment allows PrP(S) (c) formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, 'S1' and 'S3', locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrP(S) (c) and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrP(S) (c) and infectivity can either uncouple or engage to drive the onset of clinical disease.

Subcellular Distribution of the Prion Protein in Sickness and in Health

Virus Research. Sep, 2015  |  Pubmed ID: 25683509

The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus.

Mechanism of Scrapie Prion Precipitation with Phosphotungstate Anions

ACS Chemical Biology. May, 2015  |  Pubmed ID: 25695325

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions.

Structural Studies of Truncated Forms of the Prion Protein PrP

Biophysical Journal. Mar, 2015  |  Pubmed ID: 25809267

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrP(Sc).

Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains

Journal of Virology. Dec, 2015  |  Pubmed ID: 26423950

Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrP(C)). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrP(C) modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95(+)) with distinct biological properties. Transmission of first-passage tg60CWD-H95(+) isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrP(C) primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrP(C) molecules results in the generation of novel CWD strains.

Toll-like Receptor-mediated Immune Response Inhibits Prion Propagation

Glia. Jun, 2016  |  Pubmed ID: 26880394

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-β). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity.

The Detection of Infectious Prions: In Vitro Conversion Assays Change the (Folding) Landscape

The Journal of Molecular Diagnostics : JMD. May, 2016  |  Pubmed ID: 27020948

This commentary highlights the article by Gray et al describing a novel diagnostic protocol for rapidly testing transmissible spongiform encephalopathies.

Secretory Pathway Retention of Mutant Prion Protein Induces P38-MAPK Activation and Lethal Disease in Mice

Scientific Reports. Apr, 2016  |  Pubmed ID: 27117504

Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrP(C)) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrP(Sc) leading to prion diseases. We show that a deletion in the C-terminal domain of PrP(C) (PrPΔ214-229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrP(C). Transgenic (Tg(PrPΔ214-229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214-229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice.

A Neuronal Culture System to Detect Prion Synaptotoxicity

PLoS Pathogens. May, 2016  |  Pubmed ID: 27227882

Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrPSc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrPC, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrPC molecule. Both protease-resistant and protease-sensitive forms of PrPSc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrPSc and testing potential therapeutic agents.

Neuroinvasion of α-Synuclein Prionoids After Intraperitoneal and Intraglossal Inoculation

Journal of Virology. Oct, 2016  |  Pubmed ID: 27489279

α-Synuclein is a soluble, cellular protein that in a number of neurodegenerative diseases, including Parkinson's disease and multiple system atrophy, forms pathological deposits of protein aggregates. Because misfolded α-synuclein has some characteristics that resemble those of prions, we investigated its potential to induce disease after intraperitoneal or intraglossal challenge injection into bigenic Tg(M83(+/-):Gfap-luc(+/-)) mice, which express the A53T mutant of human α-synuclein and firefly luciferase. After a single intraperitoneal injection with α-synuclein fibrils, four of five mice developed paralysis and α-synuclein pathology in the central nervous system, with a median incubation time of 229 ± 17 days. Diseased mice accumulated aggregates of Sarkosyl-insoluble and phosphorylated α-synuclein in the brain and spinal cord, which colocalized with ubiquitin and p62 and were accompanied by gliosis. In contrast, only one of five mice developed α-synuclein pathology in the central nervous system after intraglossal injection with α-synuclein fibrils, after 285 days. These findings are novel and important because they show that, similar to prions, α-synuclein prionoids can neuroinvade the central nervous system after intraperitoneal or intraglossal injection and can cause neuropathology and disease.

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy

PLoS Pathogens. Sep, 2016  |  Pubmed ID: 27606840

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27-30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

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