Porcine prion protein amyloid

ABSTRACT

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.     

Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein

Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity.     

The Hofmeister effect on amyloid formation using yeast prion protein

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross‐β aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N‐proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to β‐sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid‐forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B‐viscosity coefficient of the Jones‐Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.     

Silent prions lying in wait: a two-hit model of prion/amyloid formation and infection

Diseases such as type 2 diabetes, Alzheimer's and Parkinson's are associated with the formation of amyloid. The transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease, are believed to result from infectious forms of amyloid proteins termed prions. The ability of amyloid to initiate spontaneously and in the case of prions, to transfer successfully from one host to another, has been hard to fully rationalize. In this paper we use a mathematical model to explore the idea that it might be a combination of the presence of the prion/amyloid form and a change in the state of the host that allows the amyloid/prion to successfully initiate and propagate itself. We raise the intriguing possibility that potentially infectious amyloid may lie dormant in an apparently healthy individual awaiting a change in the state of the host or transmittal to a new more susceptible host. On this basis we make an analogy between prion/amyloid disease development and the two-hit model of cancer progression. We additionally raise the possibility that infectious amyloid strains may be characterized by a size distribution of length or radius.     

Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion

ABSTRACT

A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP^Sc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP^Sc results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP^Sc. However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP^Sc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.     

Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.     

In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc)

The "protein only" hypothesis postulates that the infectious agent of prion diseases, PrP(Sc), is composed of the prion protein (PrP) converted into an amyloid-specific conformation. However, cell-free conversion of the full-length PrP into the amyloid conformation has not been achieved. In an effort to understand the mechanism of PrP(Sc) formation, we developed a cell-free conversion system using recombinant mouse full-length PrP with an intact disulfide bond (rPrP). We demonstrate that rPrP will convert into the beta-sheet-rich oligomeric form at highly acidic pH (<5.5) and at high concentrations, while at slightly acidic or neutral pH (>5.5) it assembles into the amyloid form. As judged from electron microscopy, the amyloid form had a ribbon-like assembly composed of two non-twisted filaments. In contrast to the formation of the beta-oligomer, the conversion to the amyloid occurred at concentrations close to physiological and displayed key features of an autocatalytic process. Moreover, using a shortened rPrP consisting of 106 residues (rPrP 106, deletions: Delta23-88 and Delta141-176), we showed that the in vitro conversion mimicked a transmission barrier observed in vivo. Furthermore, the amyloid form displayed a remarkable resistance to proteinase K (PK) and produced a PK-resistant core identical with that of PrP(Sc). Fourier transform infrared spectroscopy analyses showed that the beta-sheet-rich core of the amyloid form remained intact upon PK-digestion and accounted for the extremely high thermal stability. Electron and real-time fluorescent microscopy revealed that proteolytic digestion induces either aggregation of the amyloid ribbons into large clumps or further assembly into fibrils composed of several ribbons. Fibrils composed of ribbons were very fragile and had a tendency to fragment into short pieces. Remarkably, the amyloid form treated with PK preserved high seeding activity. Our work supports the protein only hypothesis of prion propagation and demonstrates that formation of the amyloid form that recapitulates key physical properties of PrP(Sc) can be achieved in vitro in the absence of cellular factors or a PrP(Sc) template.     

Conserved amyloid core structure of stop mutants of the human prion protein

Prion diseases are associated with misfolding of the natively α-helical prion protein into isoforms that are rich in cross β-structure. However, both the mechanism by which pathological conformations are produced and their structural properties remain unclear. Using a combination of nuclear magnetic resonance spectroscopy, computation, hydroxyl radical probing combined with mass-spectrometry and site-directed mutagenesis, we showed that prion stop mutants that accumulate in amyloidogenic plaque-forming aggregates fold into a β-helix. The polymorphic residue 129 is located in the hydrophobic core of the β-helix in line with a critical role of the 129 region in the packing of protein chains into prion particles. Together with electron microscopy our data support a trimeric left-handed β-helix model in which the trimer interface is formed by residues L125, Y128 and L130. Different prion types or strains might be related to different aggregate structures or filament assemblies.     

Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions

A soluble, oligomeric beta-sheet-rich conformational variant of recombinant full-length prion protein, PrP beta, was generated that aggregates into amyloid fibrils, PrP betaf. These fibrils have physico-chemical and structural properties closely similar to those of pathogenic PrP Sc in scrapie-associated fibrils and prion rods, including a closely similar proteinase K digestion pattern and Congo red birefringence. The conformational transition from PrP C to PrP beta occurs at pH 5.0 in bicellar solutions containing equimolar mixtures of dihexanoyl-phosphocholine and dimyristoyl-phospholipids, and a small percentage of negatively charged dimyristoyl-phosphoserine. The same protocol was applicable to human, cow, elk, pig, dog and mouse PrP. Comparison of full-length hPrP 23-230 with the N-terminally truncated human PrP fragments hPrP 90-230, hPrP 96-230, hPrP 105-230 and hPrP 121-230 showed that the flexible peptide segment 105-120 must be present for the generation of PrP beta. Dimerization of PrP C represents the rate-limiting step of the PrP C-to-PrP beta conformational transition, which is dependent on the amino acid sequence. The activation enthalpy of dimerization is about 130 kJ/mol for the recombinant full-length human and bovine prion proteins, and between 260 and 320 kJ/mol for the other species investigated. The in vitro conversion assay described here permits direct molecular characterization of processes that might be closely related to conformational transitions of the prion protein in transmissible spongiform encephalopathies.     

Dissociation of recombinant prion autocatalysis from infectivity

ABSTRACT

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication – that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain.¹ Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S. A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers. PLoS Pathog 2015; 11:e1005017; PMID:26125623; http://dx.doi.org/10.1371/journal.ppat.1005017[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor.¹ Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S. A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers. PLoS Pathog 2015; 11:e1005017; PMID:26125623; http://dx.doi.org/10.1371/journal.ppat.1005017[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP^C, and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.     

Protein misfolding and amyloid formation for the peptide GNNQQNY from yeast prion protein Sup35: simulation by reaction path annealing

We study the early steps of amyloid formation of the seven residue peptide GNNQQNY from yeast prion-like protein Sup35 by simulating the random coil to beta-sheet and alpha-helix to beta-sheet transition both in the absence and presence of a cross-beta amyloid nucleus. The simulation method at atomic resolution employs a new implementation of a Langevin dynamics "reaction path annealing" algorithm. The results indicate that the presence of amyloid-like cross-beta-sheet strands both facilitates the transition into the cross-beta conformation and substantially lowers the free energy barrier for this transition. This model systems allows us to investigate the energetic and kinetic details of this transition, which is consistent with an auto-catalyzed, nucleation-like mechanism for the formation of beta-amyloid. In particular, we find that electrostatic interactions of peptide backbone dipoles contribute significantly to the stability of the beta-amyloid state. Furthermore, we find water exclusion and interactions of polar side-chains to be driving forces of amyloid formation: the cross-beta conformation is stabilized by burial of polar side-chains and inter-residue hydrogen bonds in the presence of an amyloid-like "seed". These findings are in support of a "dry, polar zipper model" of amyloid formation.     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Seeded strain‐like transmission of β‐amyloid morphotypes in APP transgenic mice

The polymorphic β‐amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral β‐amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop β‐amyloid depositions that differ in morphology, binding of amyloid conformation‐sensitive dyes, and Aβ40/Aβ42 peptide ratio. To determine the nature of such β‐amyloid morphotypes, β‐amyloid‐containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced β‐amyloid deposition with the morphological, conformational, and Aβ40/Aβ42 ratio characteristics of β‐amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced β‐amyloid deposits with the characteristics of β‐amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced β‐amyloid deposits, although less prominent, and the induced deposits were similar to the β‐amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aβ in APP transgenic mice can be maintained by seeded conversion.     

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.     

Rapid formation of amyloid from alpha-monomeric recombinant human PrP in vitro

The infectious agent of prion diseases is identified with PrP(Sc), a beta-rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrP(C). To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low-level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro kinetics of amyloid formation from recombinant human PrP(90-231) in vitro in the context of two common allelic forms of PrP found in human populations that are associated with differences in prion disease susceptibility and pathological phenotype. We show that human PrP amyloid forms readily from its PrP(C)-like state in vitro, that the lag time of the reaction can be further shortened by the presence of a "seed" of pre-formed PrP amyloid, and that amyloid propagation is more complex than a simple crystallization process. We further show that the kinetics of amyloid formation do not differ between the Met129 and Val129 allelomorphs of human PrP, and that amyloid from each functions as an equally effective seed in heterologous, as in homologous amyloid reactions. The results could illuminate the process of amyloid formation in vivo as well as help understanding prion pathogenesis.     

Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.