Fibril conformation as the basis of species- and strain-dependent seeding specificity of mammalian prion amyloids

Spongiform encephalopathies are believed to be transmitted by self-perpetuating conformational conversion of the prion protein. It was shown recently that fundamental aspects of mammalian prion propagation can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop (PrP23-144). Here we demonstrate that PrP23-144 amyloids from different species adopt distinct secondary structures and morphologies, and that these structural differences are controlled by one or two residues in a critical region. These sequence-specific structural characteristics correlate strictly with the seeding specificity of amyloid fibrils. However, cross-seeding of PrP23-144 from one species with preformed fibrils from another species may overcome natural sequence-based structural preferences, resulting in a new amyloid strain that inherits the secondary structure and morphology of the template. These data provide direct biophysical evidence that protein conformations are transmitted in PrP amyloid strains, establishing a foundation for a structural basis of mammalian prion transmission barriers.     

Role of N-terminal familial mutations in prion protein fibrillization and prion amyloid propagation in vitro

A self-perpetuating conformational conversion of the prion protein (PrP) is believed to underlie pathology and transmission of prion diseases. Here we explore the effects of N-terminal pathogenic mutations (P102L, P105L, A117V) and the residue 129 polymorphism on amyloid fibril formation by the human PrP fragment 23-144, an in vitro conversion model that can reproduce certain characteristics of prion replication such as strains and species barriers. We find that these amino acid substitutions neither affect PrP23-144 amyloidogenicity nor introduce barriers to cross-seeding of soluble protein. However, the polymorphism strongly influences the conformation of the amyloid fibrils, as determined by infrared spectroscopy. Intriguingly, unlike conformational features governed by the critical amyloidogenic region of PrP23-144 (residues 138-139), the structural features distinguishing Met-129 and Val-129 PrP23-144 amyloid fibrils are not transmissible by cross-seeding. While based only on in vitro data, these findings provide fundamental insight into the mechanism of prion-based conformational transmission, indicating that only conformational features controlling seeding specificity (e.g. those in critical intermolecular contact sites of amyloid fibrils) are necessarily transmissible by cross-seeding; conformational traits in other parts of the PrP molecule may not be "heritable" from the amyloid template.     

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects >or=50 ag of hamster PrPSc (approximately 0.003 lethal dose) within 2-3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-microl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.     

Prion protein polymerisation triggered by manganese-generated prion protein seeds

Prion diseases are neurodegenerative diseases that can be transmitted between individuals. The exact cause of these diseases remains unknown. However, one of the key events associates with the disease is the aggregation of a cellular protein, the prion protein. The mechanism of this is still unclear. However, it is likely that the aggregation is trigged by a seeding mechanism in which an oligomer of the prion protein is able to catalyse polymerisation of further prion protein into larger aggregates. We have developed a model of this process using an oligomeric species generated from recombinant protein by exposure to manganese. On fractionation of the seeding species, we estimated that the smallest size the oligomer would be is an octomer. We analysed the catalytic mechanism of the seeding oligomer and its interaction with substrate. Different domains of the protein are necessary for the seeding ability of the prion protein as opposed to those required for it to form a substrate for the polymerisation reaction. Prion seeds formed from different sheep alleles are able to reproduce the characteristics of scrapie in terms of resistance to disease. However, we were also able to generate prion seed from chicken PrP a species where no prion disease is known. Our findings provide an insight into the aggregation process of the prion protein and its potential relation to disease progress.     

Autocatalytic conversion of recombinant prion proteins displays a species barrier

The most unorthodox feature of the prion disease is the existence of an abnormal infectious isoform of the prion protein, PrP(Sc). According to the "protein-only" hypothesis, PrP(Sc) propagates its abnormal conformation in an autocatalytic manner using the normal isoform, PrP(C), as a substrate. Because autocatalytic conversion is considered a key element of prion replication, in this study I tested whether in vitro conversion of recombinant PrP into abnormal isoform displays specific features of an autocatalytic process. I found that recombinant human PrP formed two distinct beta-sheet rich isoforms, the beta-oligomer and the amyloid fibrils. The kinetics of the fibrils formation measured at different pH values were consistent with a model in which the beta-oligomer was not on the kinetic pathway to the fibrillar form. As judged by electron microscopy, an acidic pH favored to the long fibrils, whereas short fibrils morphologically similar to "prion rods" were formed at neutral pH. At neutral pH the conversion to the fibrils can be seeded with small aliquots of preformed fibrils. As small as 0.001% aliquot displayed seeding activity. The conversion of human PrP was seeded with high efficacy only with the preformed fibrils of human but not mouse PrP and vice versa. These studies illustrate that in vitro conversion of recombinant PrP displays specific features of an autocatalytic process and mimics the transmission barrier of prion propagation observed in vivo. I speculate that this model can be used as a rapid assay for assessing the intrinsic propensities of prion transmission between different species.     

Highly promiscuous nature of prion polymerization

The primary structure of the prion protein (PrP) is believed to be the key factor in regulating the species barrier of prion transmission. Because the strength of the species barrier was found to be affected by the prion strain, the extent to which the barrier can indeed be attributed to differences in the PrP primary structures of either donor and acceptor species remains unclear. In this study, we exploited the intrinsic property of PrP to polymerize spontaneously into disease-related amyloid conformations in the absence of a strain-specified template and analyzed polymerization of mouse and hamster full-length recombinant PrPs. Unexpectedly, we found no evidence of species specificity in cross-seeding polymerization assays. Even when both recombinant PrP variants were present in mixtures, preformed mouse or hamster fibrils displayed no selectivity in elongation reactions and consumed equally well both homologous and heterologous substrates. Analysis of individual fibrils revealed that fibrils can elongate in a bidirectional or unidirectional manner. Our work revealed that, in the absence of a cellular environment, post-translational modifications, or strain-specified conformational constraints, PrP fibrils are intrinsically promiscuous and capable of utilizing heterologous PrP variants as a substrate in a highly efficient manner. This study suggests that amyloid structures are capable of accommodating local perturbations arising because of a mismatch in amino acid sequences and highlights the promiscuous nature of the self-propagating activity of amyloid fibrils.     

Mechanistic and Structural Origins of the Asymmetric Barrier to Prion-like Cross-Seeding between Tau-3R and Tau-4R

The spread and deposition of infectious fibrillar protein aggregates in the brain via a prion-like mechanism is a critical component in the patho-physiology of various neurodegenerative diseases, including the tauopathies. In tauopathies, two isoforms of tau, containing three and four microtubule binding repeats, are found to aggregate, and the type of isoform present in aggregates determines the type of tauopathy. Cross-seeding between the two tau isoforms is limited by an asymmetric barrier similar to the species barrier that restricts prion transmission across species, whose origin has remained unclear. In this study, the growth of the tau fibrils is shown to be describable by a two-step Michaelis–Menten-like model. Delineation of the mechanism as a Michaelis–Menten-like mechanism has enabled a quantitative understanding of the asymmetric seeding barrier that exists between two isoforms of tau, tau-K18 and tau-K19 (which differ in containing four and three microtubule binding repeats, respectively), wherein tau-K18 fibrils cannot seed tau-K19 monomer. Furthermore, high-resolution structural analysis of the two isoforms shows that the structural core is more ordered in tau-K19 than in tau-K18. Hence, the current work provides kinetic and structural rationales for asymmetric seeding barriers in general and for the two tau isoforms in particular.     

Annealing prion protein amyloid fibrils at high temperature results in extension of a proteinase K-resistant core

Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.     

The dominant-negative effect of the Q218K variant of the prion protein does not require protein X

Previous studies identified several single-point mutants of the prion protein that displayed dominant-negative effects on prion replication. The dominant-negative effect was assumed to be mediated by protein X, an as-yet-unknown cellular cofactor that is believed to be essential for prion replication. To gain insight into the mechanism that underlies the dominant-negative phenomena, we evaluated the effect of the Q218K variant of full-length recombinant prion protein (Q218K rPrP), one of the dominant-negative mutants, on cell-free polymerization of wild-type rPrP into amyloid fibrils. We found that both Q218K and wild-type (WT) rPrPs were incorporated into fibrils when incubated as a mixture; however, the yield of polymerization was substantially decreased in the presence of Q218K rPrP. Furthermore, in contrast to fibrils produced from WT rPrP, the fibrils generated in the mixture of WT and Q218K rPrPs did not acquire the proteinase K-resistant core of 16 kDa that was shown previously to encompass residues 97-230 and was similar to that of PrP(Sc). Our studies demonstrate that the Q218K variant exhibits the dominant-negative effect in cell-free conversion in the absence of protein X, and that this effect is, presumably, mediated by physical interaction between Q218K and WT rPrP during the polymerization process.     

Methionine oxidation interferes with conversion of the prion protein into the fibrillar proteinase K-resistant conformation

In recent studies, we developed a protocol for in vitro conversion of full-length mouse recombinant PrP (Mo rPrP23-230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645-659]. Because amyloid fibrils produced from recombinant Mo PrP89-230 display infectivity [Legname et al. (2004) Science 305, 673-676], polymerizatiom of rPrPs in vitro represents a valuable model for elucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol was used for hamster (Ha) rPrP23-231, we experienced substantial difficulties in forming fibrils. While searching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidation in rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that this effect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, we developed a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimal levels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methionines in preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrP(Sc)-like conformation (PrP-res). Our data are consistent with the proposition that conformational changes within the central region of the protein (residues 90-140) are essential for adopting PrP-res conformation and demonstrate that methionine oxidation interferes with this process. These studies provide new insight into the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors that modulate prion disease.     

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

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

Heterogeneous seeding of HET-s(218–289) and the mutability of prion structures

One fundamental property of prions is the formation of strains—prions that have distinct biological effects, despite a common amino acid sequence. The strain phenomenon is thought to be caused by the formation of different molecular structures, each encoding for a particular biological activity. While the precise mechanism of the formation of strains is unknown, they tend to arise following environmental changes, such as passage between different species. One possible mechanism discussed here is heterogeneous seeding; the formation of a prion nucleated by a different molecular structure. While heterogeneous seeding is not the only mechanism of prion mutation, it is consistent with some observations on species adaptation and drug resistance. Heterogeneous seeding provides a useful framework to understand how prions can adapt to new environmental conditions and change biological phenotypes.     

Structural role of glycine in amyloid fibrils formed from transmembrane alpha-helices

Amyloid fibrils associated with diseases such as Alzheimer's are often derived from the transmembrane helices of membrane proteins. It is known that the fibrils have a cross-beta-sheet structure where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. However, the structural basis for how the membrane-spanning helix is converted into a beta-sheet or how protofibrils associate into fibrils is not known. Here, we use a model peptide corresponding to a portion of the single transmembrane helix of glycophorin A to investigate the structural role of glycine in amyloid-like fibrils formed from transmembrane helices. Glycophorin A contains a GxxxG motif that is found in many transmembrane sequences including that of the amyloid precursor protein and prion protein. We propose that glycine, which mediates helix interactions in membrane proteins, also provides key packing motifs when it occurs in beta-sheets. We show that glycines in the glycophorin A transmembrane helix promote extended beta-strand formation when the helix partitions into aqueous environments and stabilize the packing of beta-sheets in the formation of amyloid-like fibrils. We demonstrate that fibrillization can be disrupted with a new class of inhibitors that target the molecular grooves created by glycine.     

Genetic and epigenetic control of the efficiency and fidelity of cross‐species prion transmission

Self‐perpetuating amyloid‐based protein isoforms (prions) transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Although mechanisms that control species specificity of prion transmission are poorly understood, studies of closely related orthologues of yeast prion protein Sup35 demonstrate that cross‐species prion transmission is modulated by both genetic (specific sequence elements) and epigenetic (prion variants, or ‘strains’) factors. Depending on the prion variant, the species barrier could be controlled at the level of either heterologous co‐aggregation or conversion of the aggregate‐associated heterologous protein into a prion polymer. Sequence divergence influences cross‐species transmission of different prion variants in opposing ways. The ability of a heterologous prion domain to either faithfully reproduce or irreversibly switch the variant‐specific prion patterns depends on both sequence divergence and the prion variant. Sequence variations within different modules of prion domains contribute to transmission barriers in different cross‐species combinations. Individual amino acid substitutions within short amyloidogenic stretches drastically alter patterns of cross‐species prion conversion, implicating these stretches as major determinants of species specificity.     

The same primary structure of the prion protein yields two distinct self-propagating states

The question of whether distinct self-propagating structures could be formed within the same amino acid sequence in the absence of external cofactors or templates has important implications for a number of issues, including the origin of prion strains and the engineering of smart, self-assembling peptide-based biomaterials. In the current study, we showed that chemically identical prion protein can give rise to conformationally distinct, self-propagating amyloid structures in the absence of cellular cofactors, post-translational modification, or PrP(Sc)-specified templates. Even more surprising, two self-replicating states were produced under identical solvent conditions, but under different shaking modes. Individual prion conformations were inherited by daughter fibrils in seeding experiments conducted under alternative shaking modes, illustrating the high fidelity of fibrillation reactions. Our study showed that the ability to acquire conformationally different self-propagating structures is an intrinsic ability of protein fibrillation and strongly supports the hypothesis that conformational variation in self-propagating protein states underlies prion strain diversity.     

Prion and Nonprion Amyloids

Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI+] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI+] and stability of its inheritance. Besides [PSI+], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI+] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion amyloids. In yeast, these phenomena are distinguished by the frequency of polymer fragmentation. We argue that in mammals the fragmentation frequency also represents a key factor defining differing properties of prion and nonprion amyloids, including infectivity. By analogy with the Rnq1 seeding of nonheritable Sup35 polymers, the “species barrier” in prion transmission may be due to seeding by heterologous prion of nontransmissible type of amyloid, rather than due to the lack of seeding.     

Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease

The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrP(Sc)), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP(Sc) template. Here we report that authentic PrP(Sc) and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrP(Sc) and lack any detectable PrP(Sc) particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrP(Sc) evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP(Sc) and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as "deformed templating" postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrP(Sc) can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.     

Leu138 in bovine prion peptide fibrils is involved in seeding discrimination related to codon 129 M/V polymorphism in the prion peptide seeding experiment

The risk of acquiring variant Creutzfeldt-Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt-Jakob disease patients are Met/Met homozygotes. Although animal transmission experiments corroborated this seeding discrimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previously developed 'seed-titration method' and time-resolved CD spectroscopy to compare sequence-dependent seeding efficiency regarding codon 129 polymorphism. Our results showed that the Met→Val substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and structural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M/V polymorphism and the interference effect of huPrP129V in the huPrP129M/V mixture. We propose a 'surface competition hypothesis' to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment.     

酿酒酵母Sup35朊蛋白结构域的自组装机理及其应用的研究进展

Sup35是酿酒酵母的翻译终止因子,其朊蛋白结构域在体内外都能形成淀粉样蛋白纤维。由于其高度有序的交叉β-片层构象与其他物种中的淀粉样蛋白纤维相似,因此,Sup35的分子自组装机理的研究可以作为蛋白质错误折叠性疾病及朊病毒生物学等相关研究的理想模型。而Sup35朊蛋白结构域自组装成纳米线的能力在生物技术和纳米材料等方面已得到广泛的应用。     

In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features

The "protein only" hypothesis of prion propagation postulates that the abnormal isoform of the prion protein, PrP(Sc), acts as a causative and transmissible agent of prion disease. In attempt to reconstitute prion infectivity in vitro, we previously developed a cell-free conversion protocol for generating amyloid fibrils from a recombinant prion protein encompassing residues 89-231 (rPrP 89-230) [Baskakov et al. (2002) J. Biol. Chem. 277, 21140]. When inoculated into transgenic mice, these amyloid fibrils induced prion disease, which can be efficiently transmitted to both wild-type and transgenic mice [Legname et al. (2004) Science 305, 673]. Here we show that the polymerization of rPrPs into the fibrils displays a number of distinctive kinetic features that are not typical for polymerization by other amyloidogenic polypeptides. Specifically, the lag phase of polymerization showed only modest dependence on protein concentration, and the conversion reaction displayed a dramatic volume-dependent threshold effect. To explain these unique kinetic features, we proposed that the conversion reaction is regulated by the dynamics between the rates of multiplication and deactivation of self-propagating fibrillar isoforms. Our further studies demonstrated that surface-dependent sorption of fibrillar isoforms is responsible for their deactivation in vitro, while fibril fragmentation seems to account for the multiplication of the active centers of polymerization. Our findings support the hypothesis that development of prion disease is controlled by a fine dynamic balance between self-propagation and clearance/deactivation of PrP(Sc).