Structural Insights into Alternate Aggregated Prion Protein Forms

The conversion of the cellular form of the prion protein (PrP^C) to an abnormal, alternatively folded isoform (PrP^Sc) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies.In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.     

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.     

NMR structure of the bank vole prion protein at 20 degrees C contains a structured loop of residues 165-171

The recent introduction of bank vole (Clethrionomys glareolus) as an additional laboratory animal for research on prion diseases revealed an important difference when compared to the mouse and the Syrian hamster, since bank voles show a high susceptibility to infection by brain homogenates from a wide range of diseased species such as sheep, goats, and humans. In this context, we determined the NMR structure of the C-terminal globular domain of the recombinant bank vole prion protein (bvPrP) [bvPrP(121-231)] at 20 °C. bvPrP(121-231) has the same overall architecture as other mammalian PrPs, with three α-helices and an antiparallel β-sheet, but it differs from PrP of the mouse and most other mammalian species in that the loop connecting the second β-strand and helix α2 is precisely defined at 20 °C. This is similar to the previously described structures of elk PrP and the designed mouse PrP (mPrP) variant mPrP[S170N,N174T](121-231), whereas Syrian hamster PrP displays a structure that is in-between these limiting cases. Studies with the newly designed variant mPrP[S170N](121-231), which contains the same loop sequence as bvPrP, now also showed that the single-amino-acid substitution S170N in mPrP is sufficient for obtaining a well-defined loop, thus providing the rationale for this local structural feature in bvPrP.     

Steric zipper of the amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein

We report the results of atomic force microscopy, Fourier-transform infrared spectroscopy, solid-state nuclear magnetic resonance, and molecular dynamics (MD) calculations for amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein (H1). Our data reveal that H1 fibrils contain no more than two β-sheet layers. The peptide strands of H1 fibrils are antiparallel with the A117 residues aligned to form a linear chain in the direction of the fibril axis. The molecular structure of the H1 fibrils, which adopts the motif of steric zipper, is highly uniform in the region of the palindrome sequence AGAAAAGA. The closest distance between the two adjacent β-sheet layers is found to be about 5 Å. The structural features of the molecular model of H1 fibrils obtained by MD simulations are consistent with the experimental results. Overall, our solid-state NMR and MD simulation data indicate that a steric zipper, which was first observed in the crystals of fibril-forming peptides, can be formed in H1 fibrils near the region of the palindrome sequence.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

The pathological prion protein forms ionic conductance in lipid bilayer

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP^TSE). PrP^TSE pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP^TSE on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP^TSE isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.     

Water molecules as structural determinants among prions of low sequence identity

The nature of the factors leading to the conversion of the cellular prion protein (PrP(C)) into its amyloidogenic isoform (PrP(Sc)) is still matter of debate in the field of structural biology. The NMR structures of non-mammalian PrP(C) (non-mPrP) from frog, chicken and turtle [Calzolai, L., Lysek, D.A., Perez, D.R., Guntert, P. and Wuthrich, K. (2005) Prion protein NMR structures of chickens, turtles, and frogs. Proc. Natl. Acad. Sci. USA 102, 651-655] have provided some new and valuable information on the scaffolding elements that preserve the PrP(C) folding, despite their low sequence identity with the mammalian prions (mPrP). The present molecular dynamics study of non-mPrP(C) focuses on the hydration properties of these proteins in comparison with the mammalian ones. The data reveal new insights in the PrP hydration and focus on the implications for PrP(C) folding stability and its propensity for interactions. In addition, for the first time, a role in disfavoring the PrP(C) aggregation is suggested for a conserved beta-bulge which is stabilized by the local hydration.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.     

The intrinsic stability of the human prion β-sheet region investigated by molecular dynamics

Human prion diseases are neurodegenerative disorders associated to the misfolding of the prion protein (PrP). Common features of prion disorders are the fibrillar amyloid deposits and the formation of prefibrillar oligomeric species also suggested as the origin of cytotoxicity associated with diseases. Although the process of PrP misfolding has been extensively investigated, many crucial aspects of this process remain unclear. We have here carried out a molecular dynamics study to evaluate the intrinsic dynamics of PrP β-sheet, a region that is believed to play a crucial role in prion aggregation. Moreover, as this region mediates protein association in dimeric assemblies frequently observed in prion crystallographic investigations, we also analyzed the dynamics of these intermolecular interactions. The extensive sampling of replica exchange shows that the native antiparallel β-structure of the prion is endowed with a remarkable stability. Therefore, upon unfolding, the persistence of a structured β-region may seed molecular association and influence the subsequent phases of the aggregation process. The analysis of the four-stranded β-sheet detected in the dimeric assemblies of PrP shows a tendency of this region to form dynamical structured states. The impact on the β-sheet structure and dynamics of disease associated point mutations has also been evaluated.     

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.     

Prion Protein mPrP[F175A](121-231): Structure and Stability in Solution

The three-dimensional structures of prion proteins (PrPs) in the cellular form (PrP^C) include a stacking interaction between the aromatic rings of the residues Y169 and F175, where F175 is conserved in all but two so far analyzed mammalian PrP sequences and where Y169 is strictly conserved. To investigate the structural role of F175, we characterized the variant mouse prion protein mPrP[F175A](121-231). The NMR solution structure represents a typical PrP^C-fold, and it contains a 3₁₀-helical β2-α2 loop conformation, which is well defined because all amide group signals in this loop are observed at 20 °C. With this “rigid‐loop PrP^C” behavior, mPrP[F175A](121-231) differs from the previously studied mPrP[Y169A](121-231), which contains a type I β-turn β2-α2 loop structure. When compared to other rigid‐loop variants of mPrP(121-231), mPrP[F175A](121-231) is unique in that the thermal unfolding temperature is lowered by 8 °C. These observations enable further refined dissection of the effects of different single-residue exchanges on the PrP^C conformation and their implications for the PrP^C physiological function.     

Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.     

Influence of the N-terminal domain on the aggregation properties of the prion protein

Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrP(C)) into an infectious form denoted PrP(Sc). The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrP(Sc) is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein because it contains a largely structured, alpha-helical, C-terminal region in addition to an N terminus that is unstructured in the absence of metal ion binding. Herein, the in vitro aggregation of a truncated portion of the prion protein (PrP 90-231) and a full-length version (PrP 23-231) were compared. In each case, concentration-dependent aggregation was analyzed to discern whether it proceeds by a nucleation-dependent pathway. Both protein constructs appear to aggregate via a nucleated polymerization with a small nucleus size, yet the later steps differ. The full-length protein forms larger aggregates than the truncated protein, indicating that the N terminus may mediate higher-order aggregation processes. In addition, the N terminus has an influence on the assembly state of PrP before aggregation begins, causing the full-length protein to adopt several oligomeric forms in a neutral pH buffer. Our results emphasize the importance of studying the full-length protein in addition to the truncated forms for in vitro aggregation studies in order to make valid hypotheses about the mechanisms of prion aggregation and the distribution of aggregates in vivo.     

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.     

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.     

Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases.     

Transgenic mice recapitulate the phenotypic heterogeneity of genetic prion diseases without developing prion infectivity: Role of intracellular PrP retention in neurotoxicity

Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome and fatal familial insomnia (FFI). The reason for this variability is not known. It has been suggested that prion strains with unique self-replicating and neurotoxic properties emerge spontaneously in individuals carrying PrP mutations, dictating the phenotypic expression of disease. We generated transgenic mice expressing the FFI mutation, and found that they developed a fatal neurological illness highly reminiscent of FFI, and different from those of similarly generated mice modeling genetic CJD and GSS. Thus transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs expressed in these mice are misfolded but unable to self-replicate. They accumulate in different compartments of the neuronal secretory pathway, impairing the membrane delivery of ion channels essential for neuronal function. Our results indicate that conversion of mutant PrP into an infectious isoform is not required for pathogenesis, and suggest that the phenotypic variability may be due to different effects of mutant PrP on intracellular transport.     

Structure of the Flexible Amino-Terminal Domain of Prion Protein Bound to a Sulfated Glycan

The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear (¹H, ¹³C, ¹⁵N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations.     

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice: application to Prnp

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.