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.     

Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells

A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the diphenylmethane scaffold were essential structural features responsible for the activity. Seven derivatives bearing substituents at the benzylic position exhibited an improved antiprion activity with the IC₅₀ values of 0.51-0.83 μM. Conformational analysis of model compounds suggested that the introduction of the substituent at the benzylic position restricted the conformational variability of the diphenylmethane unit.     

Structure, dynamics, and stability of assemblies of the human prion fragment SNQNNF

Misfolding of the prion protein (PrP) is associated with the development of Transmissible Spongiform Encephalopathies. The recent crystal structure of ‘steric zipper’ aggregates of the peptide SNQNNF (human PrP fragment 170-175) has highlighted its potential involvement in the misfolding process. A detailed molecular dynamics investigation on SNQNNF aggregates has been performed to analyze the behavior of the assemblies in a non-crystalline context. Stability, dynamics, and structural features suggest that SNQNNF assemblies are very good candidates to be involved in the structure of PrP fibrils. In addition, the analysis of small aggregates shows that steric zipper interfaces are able to stabilize assemblies composed of four strands per sheet. Altogether, the present findings indicate that steric zipper may play a key role in prion diseases. This suggestion is also corroborated by MD analyses of point mutations within the region 170-175.     

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.     

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.     

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

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

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.     

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.     

Fibril formation of the rabbit/human/bovine prion proteins

Prion diseases are infectious fatal neurodegenerative diseases including Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. The misfolding and conversion of cellular PrP in such mammals into pathogenic PrP is believed to be the key procedure. Rabbits are among the few mammalian species that exhibit resistance to prion diseases, but little is known about the molecular mechanism underlying such resistance. Here, we report that the crowding agents Ficoll 70 and dextran 70 have different effects on fibrillization of the recombinant full-length PrPs from different species: although these agents dramatically promote fibril formation of the proteins from human and cow, they significantly inhibit fibrillization of the rabbit protein by stabilizing its native state. We also find that fibrils formed by the rabbit protein contain less β-sheet structure and more α-helix structure than those formed by the proteins from human and cow. In addition, amyloid fibrils formed by the rabbit protein do not generate a proteinase K-resistant fragment of 15–16-kDa, but those formed by the proteins from human and cow generate such proteinase K-resistant fragments. Together, these results suggest that the strong inhibition of fibrillization of the rabbit PrP by the crowded physiological environment and the absence of such a protease-resistant fragment for the rabbit protein could be two of the reasons why rabbits are resistant to prion diseases.     

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.     

Structural dynamics of archaeal small heat shock proteins

Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.     

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.     

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.     

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

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process.     

Frequency distribution of PRNP polymorphisms in the Pakistani population

Prion diseases are neurodegenerative conditions caused by misfolding of a normal host-encoded prion protein (PrP^C) into pathogenic scrapie prion protein (PrP^Sc). In human prion diseases, the M129V prion protein polymorphism is known to confer susceptibility to the disease, determines PrP^Sc conformation and alters clinicopathological phenotypes. To date, all clinicopathologically confirmed cases of a variant form of Cruetzfeldt-Jacob disease (vCJD) have been 129MM homozygotes. There is also predominance of 129MM homozygotes in sporadic CJD (sCJD). No information regarding prion disorders is available from Pakistan. Although only invasive procedures like brain biopsy can confirm the diagnosis of prion disorders, testing a corresponding human population for variation in the prion protein gene (PRNP) may provide some insights into the presence of these disorders in a locality. The current study therefore aimed at exploring the genetic susceptibility of Pakistani population to CJD. A total of 909 unrelated individuals including 221 hemophiliacs representing all 4 major provinces of Pakistan were screened for M129V polymorphism and insertions or deletions of octapeptide repeats (OPRIs/OPRDs) using Polymerase Chain Reaction coupled with Restriction Fragment Length Polymorphism (PCR-RFLP). Concordance of the results of some PCR-RFLP reactions was also confirmed by dideoxy automated Sanger sequencing. The frequencies of M129V alleles (129M and 129V) and genotypes (129MM, 129MV and 129VV) were found in all 909 individuals to be 0.7101, 0.2899, 0.5270, 0.3663 and 0.1067, respectively. Deletion of 1 octapeptide repeat (1-OPRD) was detected in heterozygous state in PRNP of 10 individuals and in homozygous state in 1 individual. An insertion of 3 octapeptide repeats (3-OPRI) was found in 1 individual and an insertion of 1 octapeptide repeat (1-OPRI) in two individuals. Both 3-OPRI and 1-OPRI were present in heterozygous state and were linked to 129M allele. There were no significant χ² differences between M129V allelic and genotypic frequencies of healthy individuals and hemophiliacs. However, M129V allelic and genotypic frequencies differed significantly between Pakistani population and East Asian and Western populations. Non-significant χ² differences between M129V frequencies of healthy individuals and hemophiliacs suggest that individuals manifesting single gene disorders may provide naturally randomized samples for studies aiming at surveying the genetic variation. The combined excess of 129MM and 129VV homozygosity and the presence of 3-OPRI in 1 individual imply that Pakistani population is susceptible to prion disorders. Cases of prion disorders may exist in Pakistan, albeit at lower annual prevalence than other countries where life expectancy is greater than 65 years.     

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP^Sc, an abnormally misfolded, protease-resistant, and β-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP^Sc and presents similar neurotoxicity as PrP^Sc. By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.     

Inter-oligomer interactions of the human prion protein are modulated by the polymorphism at codon 129

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in β-oligomer (β^O) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the β^O N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in β^O formation. Atomic force microscopy revealed distinctive 1.8 × 15 × 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for β^O with valine at position 129. This result provides evidence for conformational differences between the β^O allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.     

Heterogeneous PrPC metabolism in skeletal muscle cells

Recent reports have shown that prions, the causative agent of transmissible spongiform encephalopathies, accumulate in the skeletal muscle of diseased animals and man. In an attempt to characterise in this tissue the prion protein (PrP(C)), whose conformational rearrangement governs the generation of prions, we have analysed the protein in primary cultured murine myocytes and in different skeletal muscle types. Our results indicate that the expression and cellular processing of PrP(C) change during myogenesis, and in muscle fibres with different contractile properties. These findings imply a potential role for PrP(C) in the skeletal muscle physiology, but may also explain the different capability of muscles to sustain prion replication.     

Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines

We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 - a monoclonal antibody recognizing active Src, we have found a 32+/-6.3% and 75+/-7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein. In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 - a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.     

Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China

Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of an abnormal isoform prion protein (PrP^Sc) encoded by a single copy prion protein gene (PRNP). Prion disease has been reported in many herbivores but not in Equus and the species barrier might be playing a role in resistance of these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may help understand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia, China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequence in GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequence analysis showed that the Xiji donkey PRNP was consisted of an open reading frame of 768 nucleotides encoding 256 amino acids. Amino acid residues unique to donkey as compared with some Equus animals, mink, cow, sheep, human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence of Xiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species and close relation to the PRNP of sika deer, sheep, dog, camel, cow, mink, rabbit and hamster with 83.1–99.7% identity. The results provided the PRNP data for an additional Equus species, which should be useful to the study of the prion disease pathogenesis, resistance and cross species transmission.