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Converting the prion protein: What makes the protein infectious
Abstract
The discovery of prion disease transmission in mammals, as well as a non-Mendelian type of inheritance in yeast, has led to the establishment of a new concept in biology, the prion hypothesis. The prion hypothesis postulates that an abnormal protein conformation propagates itself in an autocatalytic manner via recruitment of the normal isoform of the same protein as a substrate, and thereby acts either as a transmissible agent of disease (in mammals) or as a heritable determinant of phenotype (in yeast and fungus). Although reconstitution of fully infectious PrP(Sc)in vitro from synthetic components has not yet been achieved, numerous lines of evidence indicate that the prion protein is the major and essential component, if not the only one, of the prion infectious agent. This article summarizes our current knowledge about the chemical nature of the prion infectious agent, describes potential strategies and challenges related to the generation of prion infectivity de novo, proposes new hypotheses to explain the apparently low infectivity observed in the first synthetic mammalian prions, and describes plausible effects of chemical modifications on prion conversion.
... Because of this particular property, it was reasonable to expect that a related prion protein (hrPrP- ) Moxanalogue could be useful for analysis of the proposed but still not clearly documented hypothetical role of Met oxidation as the main cause of the prion protein conversion to its scrapie form. 17,18 Indeed particularly Met-205 and Met-206 oxidation was suggested to be responsible for the conformational transition of the α-helix into βsheet and thus for the aggregation and the neurotoxic amyloid formation (see Figure 4). 20 The in vitro aggregation propensity of the Mox-hrPrP c - analogue was found to be significantly increased compared with that of the native Met-protein ( Figure 5). ...
... However, diselenides as additives were known to efficiently catalyze oxidative protein folding. 104,105 Correspondingly, the kinetics of oxidative folding of the μ-conotoxin SIIIA and ω-conotoxin GVIA, both containing the cystine knot ICK, were compared with the kinetics of formation of the correctly folded [Sec [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] ]-SIIIA and [Sec [8][9][10][11][12][13][14][15][16][17][18][19] ]-GVIA. The results clearly revealed an efficient catalytic effect of even intramolecular diselenides in the formation of the correct disulfides. ...
... However, diselenides as additives were known to efficiently catalyze oxidative protein folding. 104,105 Correspondingly, the kinetics of oxidative folding of the μ-conotoxin SIIIA and ω-conotoxin GVIA, both containing the cystine knot ICK, were compared with the kinetics of formation of the correctly folded [Sec [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] ]-SIIIA and [Sec [8][9][10][11][12][13][14][15][16][17][18][19] ]-GVIA. The results clearly revealed an efficient catalytic effect of even intramolecular diselenides in the formation of the correct disulfides. ...
The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur-containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl-tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl- and phenylalanyl-tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl-tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.
© 2019 European Peptide Society and John Wiley & Sons, Ltd.
... In mammals, prions causing transmissible neurodegenerative disorders are composed mainly of PrP Sc , an altered form of the cellular prion protein (PrP C ) [1, 2]. The long-standing hypothesis of prion biology is that the distinct biological and physicochemical properties separating PrP C and PrP Sc relate only to the conformational differences of the C-terminal domain, which adopts a major globular a-fold in PrP C and displays a high content of b-sheet structure in PrP Sc34567891011 . However , the populations of PrP C and PrP Sc also differ in the redox state the methionines of Helix-3, suggesting a possible role for these residues in facilitating the structural change12131415. ...
... However , the populations of PrP C and PrP Sc also differ in the redox state the methionines of Helix-3, suggesting a possible role for these residues in facilitating the structural change12131415. The chain reorganization accompanying the conversion of PrP C to PrP Sc proceeds through a complex pathway involving the obligatory initial destabilization of the PrP C a-fold for its subsequent refolding and assembly into PrP Sc [1, 9,16171819. Similarly to tau, the hyperphosphorylation of which impairs function and provokes accumulation, sulfoxidation of PrP methionines could also regulate the folding repertoire through the reorganization of intra-and intermolecular bonds [9,202122 . ...
... The chain reorganization accompanying the conversion of PrP C to PrP Sc proceeds through a complex pathway involving the obligatory initial destabilization of the PrP C a-fold for its subsequent refolding and assembly into PrP Sc [1, 9,16171819. Similarly to tau, the hyperphosphorylation of which impairs function and provokes accumulation, sulfoxidation of PrP methionines could also regulate the folding repertoire through the reorganization of intra-and intermolecular bonds [9,202122 . As opposed to the enzymatic nature of phosphorylation, methionine sulfoxidation is a chemical modification that takes place in response to oxidative insults and lacks sequence specificity23242526. ...
The conversion of the cellular prion protein (PrP(C)) into its disease-associated form (PrP(Sc)) involves a major conformational change and the accumulation of sulfoxidized methionines. Computational and synthetic approaches have shown that this change in the polarity of M206 and M213 impacts the C-terminal domain native alpha-fold allowing the flexibility required for the structural conversion. To test the effect in the full-length molecule with site-specificity, we have generated M-to-S mutations. Molecular dynamics simulations show that the replacement indeed perturbs the native state. When this mutation is placed at the conserved methionines of HaPrP(23-231), only substitutions at the Helix-3 impair the alpha-fold, stabilizing a non-native state with perturbed secondary structure, loss of native tertiary contacts, increased surface hydrophobicity, reduced thermal stability and an enhanced tendency to aggregate into protofibrillar polymers. Our work supports that M206 and M213 function as alpha-fold gatekeepers and suggests that their redox state regulate misfolding routes.
... Despite enormous investments over the last 30 years in searching for a prion virus or virion [2][3][4][5], no prion-specific nucleic acids associated with infectious prion particles have ever been identified [6]. A notable shift has occurred in the last few years from debating the question of whether a protein can be infectious to what makes a protein infectious and how many proteins are infectious [7][8][9]. Elucidating the polymerization mechanisms and structure of misfolded and aggregated isoforms of the prion protein (PrP) will help solving these long-standing research problems. ...
... Cellular cofactors participating in fragmentation of mammalian prion fibrils have yet to be identified. The intrinsic fragility (i.e. the ability of fibrils to fragment into shorter pieces) seems to be controlled by the conformational stability of amyloid fibrils and, specifically, by the stability of the cross-b-fibrillar structure [8] (Y. Sun & I. V. Baskakov, unpublished data). ...
... A similar correlation between conformational stability and infectivity was observed for synthetic mammalian prions [45,46]. Both yeast and mammalian prion studies indicated that the intrinsic infectivity of fibrils might be controlled, at least in part, by the conformational stability of the cross-b-sheet core, an unexpected lesson that we have learned [8]. If the intrinsic fragility of PrP Sc aggregates does dictate the rate of prion propagation, this property could account for substantial differences in the incubation times produces by different strains of PrP Sc . ...
The discovery of prion disease and the establishment of the protein only hypothesis of prion propagation raised substantial interest in the class of maladies referred to as conformational diseases. Although significant progress has been made in elucidating the mechanisms of polymerization for several amyloidogenic proteins and peptides linked to conformational disorders and solving their fibrillar 3D structures, studies of prion protein amyloid fibrils and their polymerization mechanism have proven to be very difficult. The present minireview introduces the mechanism of branched-chain reaction for describing the peculiar kinetics of prion polymerization and summarizes our current knowledge about the substructure of prion protein amyloid fibrils.
... Another conformational feature that is likely to be shared by both PG14 Spon and PG14 RML is an increased proportion of b-sheet structure. It is well known that experimentally induced conversion of PrP from a-helical to b-sheet-rich forms is accompanied by oligomerization of the protein (Swietnicki et al. 2000; Baskakov et al. 2002; Surewicz et al. 2006; Baskakov and Breydo 2007). Thus, PG14 Spon and PG14 RML may both represent b-rich oligomers or polymers, with the b-sheet structure endowing these assemblies with similar reactivities in the biochemical assays we have employed here. ...
... Studies of the folding and aggregation characteristics of purified, recombinant PrP provide a precedent for the existence of multiple types of PrP oligomer which differ in their degree of infectivity. In particular, Baskakov et al. have shown that PrP can adopt, in addition to its native, a-helical monomeric form, two non-native, b-rich forms: a b-oligomer and an amyloid fibril (Baskakov et al. 2002; Baskakov 2004; Baskakov and Breydo 2007). The b-oligomer consisted of approximately eight PrP molecules, while the amyloid fibril was much larger and displayed a variety of fibrillar morphologies in electron micrographs. ...
... Animal transmission No (Chiesa et al. 2003) Yes (Chiesa et al. 2003) Yes Seeding activity in PMCA reaction No Yes Yes (Castilla et al. 2005) Co-aggregation with WT PrP or C2 fragment No Yes Yes (Caughey 2001; Chen et al. 1995) Co-aggregation with WT PrP-EGFP No NT Yes (Barmada and Harris 2005) PG14, nine-octapeptide insertional mutation in prion protein; PrP Sc , scrapie isoform of prion protein; PG14 Spon , spontaneously generated form of PrP carrying the PG14 mutation; PG14 RML , Rocky Mountain Laboratory strain of scrapie (RML)-seeded form of PrP carrying the PG14 mutation; PMCA, protein misfolding cyclic amplification; WT, wild-type; NT, not tested. been applied to oligomeric assemblies generated from recombinant PrP (reviewed in Refs Baskakov and Breydo 2007; Surewicz et al. 2006). We anticipate that a similar analysis of PG14 Spon and PG14 RML particles will be particularly fruitful, as these PrP species are derived from brain tissue, and their toxic and infectious properties have been defined in vivo. ...
A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of human prion disease. Transgenic (Tg) mice that express the mouse homolog of this mutation (designated PG14) spontaneously accumulate in their brains an insoluble and weakly protease-resistant form of the mutant protein. This form (designated PG14(Spon)) is highly neurotoxic, but is not infectious in animal bioassays. In contrast, when Tg(PG14) mice are inoculated with the Rocky Mountain Laboratory (RML) strain of prions, they accumulate a different form of PG14 PrP (designated PG14(RML)) that is highly protease resistant and infectious in animal transmission experiments. We have been interested in characterizing the molecular properties of PG14(Spon) and PG14(RML), with a view to identifying features that determine two, apparently distinct properties of PrP aggregates: their infectivity and their pathogenicity. In this paper, we have subjected PG14(Spon) and PG14(RML) to a panel of assays commonly used to distinguish infectious PrP (PrP(Sc)) from cellular PrP (PrP(C)), including immobilized metal affinity chromatography, precipitation with sodium phosphotungstate, and immunoprecipitation with PrP(C)- and PrP(Sc)-specific antibodies. Surprisingly, we found that aggregates of PG14(Spon) and PG14(RML) behave identically to each other, and to authentic PrP(Sc), in each of these biochemical assays. PG14(Spon) however, in contrast to PG14(RML) and PrP(Sc), was unable to seed the misfolding of PrP(C) in an in vitro protein misfolding cyclic amplification reaction. Collectively, these results suggest that infectious and non-infectious aggregates of PrP share common structural features accounting for their toxicity, and that self-propagation of PrP involves more subtle molecular differences.
... Next, the resulting PrP Sc oligomers are transported from the follicular dendritic cells to the cells of the peripheral and then central nervous system [5]. PrP Sc particles convert neuronal PrP C in the brain into an abnormal shape and cause neurodegeneration [5,6]. The prion disease called kuru was common among natives of Papua New Guinea due to ritual cannibalism [2]. ...
... Sporadic prion diseases is approximately 85%[42].5 The ratio of cases of sporadic and other forms of amyloidosis for these proteins has not been characterized.6 N/D-not determined. ...
Amyloidoses are a group of diseases associated with the formation of pathological protein fibrils with cross-β structures. Approximately 5–10% of the cases of these diseases are determined by amyloidogenic mutations, as well as by transmission of infectious amyloids (prions) between organisms. The most common group of so-called sporadic amyloidoses is associated with abnormal aggregation of wild-type proteins. Some sporadic amyloidoses are known to be induced only against the background of certain pathologies, but in some cases the cause of amyloidosis is unclear. It is assumed that these diseases often occur by accident. Here we present facts and hypotheses about the association of sporadic amyloidoses with vascular pathologies, trauma, oxidative stress, cancer, metabolic diseases, chronic infections and COVID-19. Generalization of current data shows that all sporadic amyloidoses can be regarded as a secondary event occurring against the background of diseases provoking a cellular stress response. Various factors causing the stress response provoke protein overproduction, a local increase in the concentration or modifications, which contributes to amyloidogenesis. Progress in the treatment of vascular, metabolic and infectious diseases, as well as cancers, should lead to a significant reduction in the risk of sporadic amyloidoses.
... 37,38 Conversely, neurotoxic forms may share structural descriptors with the previous forms (as the formation of insoluble aggregates, presence of proteinase K resistance, etc.) but lack the capacity to seed their amplification both in vivo and in vitro (Figure 11.2). 39,40 The structural change accompanying disease consists of the disruption of the PrP C native a-fold and the refolding into a new b-sheet rich structural state. 22,41 The secondary structure change is accompanied by the rearrangement of the tertiary structure that can be probed by the differences in the recognition by antibodies and enzymes (proteases, glycosidases and phospholipases). ...
In recent years, medical developments have resulted in an increase in human life expectancy. Some developed countries now have a larger population of individuals aged over 64 than those under 14. One consequence of the ageing population is a higher incidence of certain neurodegenerative disorders. In order to prevent these, we need to learn more about them. This book provides up-to-date information on the use of transgenic mouse models in the study of neurodegenerative disorders such as Alzheimer's and Huntington's disease. By reproducing some of the pathological aspects of the diseases, these studies could reveal the mechanism for their onset or development. Some of the transgenic mice can also be used as targets for testing new compounds with the potential to prevent or combat these disorders. The editors have extensive knowledge and experience in this field and the book is aimed at undergraduates, postgraduates and academics. The chapters cover disorders including: Alzheimer's disease, Parkinson's disease, Huntington's and other CAG diseases, amyotrophic lateral sclerosis (ALS), recessive ataxias, disease caused by prions, and ischemia.
... Prion diseases are believed to be caused by the pathogenic conversion of the cellular prion protein (PrP C ) to an abnormal and partially protease resistant PrP Sc [1,2]. The Prnp gene encodes the prion protein and this gene is located on chromosome 2 in mice and on chromosome 20 in human [3,4]. ...
Despite having therapeutic potential, anti-PrP antibodies caused a major controversy due to their neurotoxic effects. For instance, treating mice with ICSM antibodies delayed prion disease onset, but both were found to be either toxic or innocuous to neurons by researchers following cross-linking PrP C. In order to elucidate and understand the reasons that led to these contradictory outcomes, we conducted a comprehensive in silico study to assess the antibody-specific toxicity. Since most therapeutic anti-PrP antibodies were generated against human truncated recombinant PrP 91−231 or full-length mouse PrP 23−231 , we reasoned that host specificity (human vs murine) of PrP C might influence the nature of the specific epitopes recognized by these antibodies at the structural level possibly explaining the 'toxicity' discrepancies reported previously. Initially, molecular dynamics simulation and pro-motif analysis of full-length human (hu)PrP and mouse (mo)PrP 3D structure displayed conspicuous structural differences between huPrP and moPrP. We identified 10 huPrP and 6 moPrP linear B-cell epitopes from the prion protein 3D structure where 5 out of 10 huPrP and 3 out of 6 moPrP B-cell epitopes were predicted to be potentially toxic in immunoinformatics approaches. Herein, we demonstrate that some of the predicted potentially 'toxic' epitopes identified by the in silico analysis were similar to the epitopes recognized by the toxic antibodies such as ICSM18 (146-159), POM1 (138-147), D18 (133-157), ICSM35 (91-110), D13 (95-103) and POM3 (95-100). This in silico study reveals the role of host specificity of PrP C in epitope-specific anti-PrP antibody toxicity.
... These misfolded oligomers are β-sheet rich and exhibit low-level PK-resistance, however, they are likely of different structures than those of PrP Sc derived from brain, which possess an intact intramolecular disulfide bond 46,47 . However, templated in vitro conversion is much more successful in generating authentic PrP Sc material 23,28 although infectious PrP Sc formation is reported in non-templated conversion as well 22,23,29,48 . The effect of the G127V mutation has been studied on the fibril formation kinetics of the mouse prion protein (mPrP), using an in vitro conversion assay, and important differences had been revealed between the wild type and the G126V mutant mPrP (correspondent of the G127V HuPrP), such as the critical concentration being higher, the lag phase being longer and the initial effective rate constant of fibril growth being slower in the case of the mutant 49 . ...
Scrapie prion, PrP Sc , formation is the central event of all types of transmissible spongiform encephalopathies (TSEs), while the pathway with possible intermediates and their mechanism of formation from the normal isoform of prion (PrP), remains not fully understood. Recently, the G127V variant of the human PrP is reported to render the protein refractory to transmission of TSEs, via a yet unknown mechanism. Molecular dynamics studies suggested that this mutation interferes with the formation of PrP dimers. Here we analyze the dimerization of 127G and 127VPrP, in both in vitro and a mammalian cell culture system. Our results show that while molecular dynamics may capture the features affecting dimerization in vitro, G127V inhibiting dimer formation of PrP, these are not evidenced in a more complex cellular system.
... The fundamental event in the pathogenesis of TSE is not a primary structure modification but the conversion of the normal cellular prion protein (PrP c ) into the misfolded pathogenic isoform (PrP sc ) [38]. In fact, PrP c and PrP sc share the same amino acid sequence, but differences in the secondary structure originate the tertiary and quaternary structures that dictate the PrP sc with new physicochemical properties, namely, insolubility in nondenaturing detergents and partial resistance to proteolysis. ...
... Mammalian prions are unconventional infectious agents that consist of misfolded, selfreplicating states of a sialoglycoprotein called the prion protein or PrP C [1,2]. Prions replicate by recruiting and converting PrP C molecules expressed by a host into misfolded, self-replicating states referred to as PrP Sc [3,4]. While prions are unconventional pathogens, they spread from cell to cell in CNS and elicit neuroinflammatory response that resembles the response of CNS to viral infections [5][6][7]. ...
Mammalian prions are unconventional infectious agents that invade and replicate in an organism by recruiting a normal form of a prion protein (PrPC) and converting it into misfolded, disease-associated state referred to as PrPSc. PrPC is posttranslationally modified with two N-linked glycans. Prion strains replicate by selecting substrates from a large pool of PrPC sialoglycoforms expressed by a host. Brain regions have different vulnerability to prion infection, however, molecular mechanisms underlying selective vulnerability is not well understood. Toward addressing this question, the current study looked into a possibility that sialylation of PrPSc might be involved in defining selective vulnerability of brain regions. The current work found that in 22L -infected animals, PrPSc is indeed sialylated in a region dependent manner. PrPSc in hippocampus and cortex was more sialylated than PrPSc from thalamus and stem. Similar trends were also observed in brain materials from RML- and ME7-infected animals. The current study established that PrPSc sialylation status is indeed region-specific. Together with previous studies demonstrating that low sialylation status accelerates prion replication, this work suggests that high vulnerability of certain brain region to prion infection could be attributed to their low sialylation status.
... The fundamental event in the pathogenesis of TSE is not a primary structure modification but the conversion of the normal cellular prion protein (PrP c ) into the misfolded pathogenic isoform (PrP sc ) [38]. In fact, PrP c and PrP sc share the same amino acid sequence, but differences in the secondary structure originate the tertiary and quaternary structures that dictate the PrP sc with new physicochemical properties, namely, insolubility in nondenaturing detergents and partial resistance to proteolysis. ...
... According to this mechanism, PrP Sc template recruits PrP C expressed by a host and converts them into a new PrP Sc with the folding pattern faithfully repeating that of the PrP Sc template [14]. While prions can propagate indefinitely via serial passaging in wild type hosts or cultured cells, generating infectious prions in vitro de novo from recombinant PrP (rPrP) has been a challenge [5]. ...
Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP.
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... Prion diseases are a group of fatal neurodegenerative diseases of humans and other mammals that can arise spontaneously or via transmission [1]. The transmissible agent of prion disease consists of a prion protein in β-sheet rich self-propagating states referred to as PrP Sc that template conversion of the same protein in its normal, cellular state (PrP C ) into disease-related pathogenic state [2][3][4][5][6]. ...
The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrPC and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.
... The crucial event in the pathogenesis of the disease is the conversion of the normal host-encoded cellular prion protein (PrP C ), to a misfolded pathogenic isoform named PrP Sc . 3 In fact, PrP C and PrP Sc share the same primary structure but, while PrP C secondary structure is rich in α-helices, PrP Sc is characterized by increased β-sheet content and decreased α-helices. As consequence of conformational change PrP Sc acquires tertiary and quaternary different feature leading to new physicochemical properties: PrP Sc is insoluble in nondenaturing detergents and partially resistant to proteolysis. ...
Prion disease or transmissible spongiform encephalopathies are characterized by the presence of the abnormal form of the prion protein (PrPSc). The pathological and transmissible properties of PrPSc are enciphered in its secondary and tertiary structures. Since it's well established that different strains of prions are linked to different conformations of PrPSc, biochemical characterization of prions seems a preliminary but reliable approach to detect, analyze, and compare prion strains.
... As was proposed previously, species-specific amino acid sequences of PrP C define the range of strain-specific folding patterns of PrP Sc accessible for individual sequences (Fig 2B). 23 Individual folding patterns govern selection of sialoglycoforms that can be accommodated within strain-specific PrP Sc structures and, as a result, define the strain-specific pattern of functional carbohydrate epitopes on PrP Sc surfaces (Fig 2B). Using this information, prion strains could be classified into 2 large categories. ...
Among a broad range of hypotheses on the molecular nature of transmissible spongiform encephalopathy or scrapie agents discussed in 1960s was a hypothesis of self-replicating polysaccharides. While the studies of the past 40 years provided unambiguous proof that this is not the case, emerging evidence suggests that carbohydrates in the form of sialylated N-linked glycans, which are a constitutive part of mammalian prions or PrPSc, are essential in determining prion fate in an organism. The current extra-view article discusses recent advancements on the role of N-linked glycans and specifically their sialylation status in controlling prion fate. In addition, this manuscript introduces a new concept on the important role of strain-specific functional carbohydrate epitopes on the PrPSc surface as main determinants of strain-specific biologic features. According to this concept, individual strain-specific folding patterns of PrPSc govern selection of PrPC sialoglycoforms expressed by a host that can be accommodated within particular PrPSc structures. Strain-specific patterns of functional carbohydrate epitopes formed by N-linked glycans on PrPSc surfaces define strain-specific biologic features. As a constitutive part of PrPSc, the individual strain-specific patterns of carbohydrate epitopes propagate faithfully within a given host as long as individual strain-specific PrPSc structures are maintained, ensuring inheritance of strain-specific biologic features.
... Prions or PrP Sc are proteinaceous infectious agents that consist of misfolded, aggregated, selfreplicating states of a sialoglycoprotein called the prion protein or PrP C (1,2). Prions replicate by recruiting and converting PrP C molecules expressed by a host into misfolded PrP Sc states (3,4). While other proteins or peptides linked to neurodegenerative diseases display certain characteristics of prion-like replication (5,6), PrP Sc is unique in several important aspects. ...
Prions or PrP(Sc) are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrP(C) The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrP(Sc) from PrP(C) using Protein Misfolding Cyclic Amplification (PMCAb), and also generated PrP(Sc) with reduced sialylation levels using the same method but with partially desialylated PrP(C) as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrP(Sc), PrP(Sc) produced in PMCAb or dsPMCAb and monitored for disease. Animals inoculated with brain- or PMCAb-derived PrP(Sc) developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrP(Sc) was detected in brains or spleen of these animals by either Western blot or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrP(Sc) to secondary lymphoid organs (SLOs) was monitored in wild type mice. PrP(Sc)c sialylation was found to be critical for effective trafficking of PrP(Sc) to SLOs. By 6 hours post-inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrP(Sc) Furthermore, this work suggests that the sialylation status of PrP(Sc) plays an important role in prion lymphotropism.
... Indeed, it has been shown that enhanced fragility of yeast prions can more than compensate for deceased templating efficiencies. Additional support for the importance of fibril brittleness in prion propagation has come from cell-free fiber formation assays where induction of fiber breakage increases rates of new fiber formation (Baskakov and Breydo 2007). Together, these data suggest that the variability in the conformational stability of prion aggregates plays a large role in dictating the rates of prion propagation and degrees of infectivity. ...
Prion diseases are a group of fatal neurodegenerative disorders caused by the misfolding of
the cellular prion protein (PrPC) into a pathogenic conformation (PrPSc). PrPSc is capable of
folding into multiple self-replicating prion strains that produce phenotypically distinct neurological
disorders. Evidence suggests that the structural heterogeneity of PrPSc is the molecular
basis of strain-specific prion properties. The self-templating of PrPSc typically ensures
that prion strains breed true upon passage. However, prion strains also have the capacity to
conformationally transform to maximize their rate of replication in a given environment.
Here, we provide an overview of the prion-strain phenomenon and describe the role of strain
adaptation in drug resistance. We also describe recent evidence that shows the presence of
distinct conformational strains in other neurodegenerative disorders.
... Given its involvement in the pathology of the prion diseases it is unlikely that PrP Sc has a cellular function, but single nucleotide polymorphisms in the human gene for PrP have been associated with differences in long term memory in healthy young adults (Papassotiropoulos et al., 2005), and aggregated, non-infectious states have been shown for PrP by numerous investigators (Baskakov and Breydo, 2007). ...
The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus.
Copyright © 2015. Published by Elsevier B.V.
... Therefore, some of the PrP Sc working models were based on the analyses of in vitro generated PrP fibrils. The difference between converted PrP structures grown in vitro and in vivo has long been discussed, mainly in terms of their infectivity, because PrP converted in vitro (in cell-free conversion systems) tended to demonstrate very low infectivity (Baskakov and Breydo, 2007;Collinge and Clarke, 2007;Prusiner, 1998). Nevertheless, several lines of evidence have suggested common structural features shared by PrP Sc and in vitro-converted PrP. ...
The structural details of the essential entity of prion disease, fibril prion protein (PrP(Sc)), are still elusive despite the large body of evidence supporting the prion hypothesis. Five major working models of PrP(Sc) structure, which are not compatible with each other, have been proposed. However, no systematic evaluation has been performed on those models. We devised a method that combined systematic point mutation with threading on knowledge-based amino acid potentials. A comprehensive mutation experiment was performed on mouse prion protein, and the PrP(Sc) conversion efficiency of each mutant was examined. The models were evaluated based on the mutation data by using the threading method. Although the data turned out to be rather more consistent with the models that assumed a conversion of the N-terminal region of core PrP into a β helix than with others, substantial modifications were also required to further improve the current model based on recent experimental results.
... Several pathologic correlates exist between prion diseases and AD. These include the presence of misfolded protein aggregates consisting of highly ordered polymeric isoforms with cross-β-sheet-rich structures, neurodegeneration, and progressively and sequentially spreading neuropathology (175)(176)(177). In addition, as noted previously, AD-like neuropathology in the form of Aβ deposits is present in most cases of Creutzfeldt-Jakob disease and kuru. ...
Late-onset Alzheimer's disease (AD) is the most prevalent cause of dementia among older adults, yet more than a century of
research has not determined why this disease develops. One prevailing hypothesis is that late-onset AD is caused by infectious
pathogens, an idea widely studied in both humans and experimental animal models. This review examines the infectious AD etiology
hypothesis and summarizes existing evidence associating infectious agents with AD in humans. The various mechanisms through
which different clinical and subclinical infections could cause or promote the progression of AD are considered, as is the
concordance between putative infectious agents and the epidemiology of AD. We searched the PubMed, Web of Science, and EBSCO
databases for research articles pertaining to infections and AD and systematically reviewed the evidence linking specific
infectious pathogens to AD. The evidence compiled from the literature linking AD to an infectious cause is inconclusive, but
the amount of evidence suggestive of an association is too substantial to ignore. Epidemiologic, clinical, and basic science
studies that could improve on current understanding of the associations between AD and infections and possibly uncover ways
to control this highly prevalent and debilitating disease are suggested.
... Hipoteza dotycząca prionów zakłada, że zmienione konformacyjnie, patologiczne białko, katalizuje reakcję przejścia fizjologicznego białka w patologiczne (ryc. 5) [4,40]. Ze względu na to, iż gen kodujący to białko (PRNP) występuje nie tylko u ludzi, ale także u wszystkich innych kręgowców, choroby prionowe mogą być przenoszone międzygatunkowo [30,53]. ...
All neurodegenerative diseases are related to pathology and accumulation of proteins. Proteins are basic structural and functional components of each cell and their functions are associated with their amino acid composition and spatial structure. The proper functioning of protein is necessary for the proper operation of the body system. In the case of disorders of proteins' spatial structure, the development of pathological processes may occur. Accumulation of abnormal proteins is toxic to nerve cells and causes neurodegeneration. Different disorders are characterized by abnormalities of various proteins. This type of neurodegenerative diseases includes Parkinson's disease, tauopathies, Alzheimer's disease, and prion diseases. Parkinson's disease is characterized by toxicity of α-synuclein. The pathology of tau protein is specific for tauopathies, prion protein for prion diseases. In the case of Alzheimer's disease it is β-amyloid. All proteins responsible for the pathology are present in the physiological state in the organism. Damage to the area of the brain covered by the pathological process and the clinical symptoms are characteristic for a particular type of disease. Detailed knowledge of the mechanisms of the disease can be an important element in the development of effective ways of treatment.
... These data suggest that conformational stability of rPrP fibrils appears to be more important for triggering pathogenic process than apparent structural similarity between inoculated material and PrP Sc . Conformational stability appears to be linked to the fibril's mechanical properties such as their intrinsic fragility (Sun et al., 2008; Baskakov and Breydo, 2007). One might speculate that 2M or S fibrils failed to recruit PrP C because of their low fragmentation rate. ...
The transmissible agent of prion disease consists of prion protein (PrP) in β-sheet-rich state (PrP(Sc)) that can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrP(Sc) template. Here, three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrP(Sc), whereas an alternative amyloid state, with a folding pattern different from that of PrP(Sc), induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrP(Sc) emerged. The dynamics between the two forms suggest that atypical proteinase K-resistant PrP (PrPres) gave rise to PrP(Sc). While no PrP(Sc) was found in preparations of fibrils using protein misfolding cyclic amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb, suggesting that atypical PrPres was the first product of PrP(C) misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrP(Sc)-templated or spontaneous conversion of PrP(C) into PrP(Sc) exists. This study provides compelling evidence that noninfectious amyloids with a structure different from that of PrP(Sc) could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.
... It should be noted that separation of these three categories of effects serves only to highlight individual components of a complex, dynamic scenario of signal derangement. Simultaneously, combined with the effects predicted from simple oligomerization, the conformational conversion would add to both the propensity of protein aggregation [211,212] and to changes in ligand binding, which are expected to follow modification of the secondary structure of the protein (Fig. 10). ...
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP(C)), its conformational conversion, aggregation and precipitation. We recently proposed that PrP(C) serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP(C), which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP(C) and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP(C), and that allosteric dysfunction of PrP(C) has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP(C), as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.
... They are based on the properties of recombinant PrP to polymerize into amyloid fibrils either spontaneously or upon addition of a small amount of pre-formed fibrils. Such a seeding effect is a feature of the nucleation-dependant polymerization process (for reviews see [14, 139, 187] ). Surewicz et al. elegantly modeled 'prion strain' diversity in a simple system consisting of seeded fibrilization of soluble monomeric prion protein variants (PrP23- 144). ...
Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.
... It still remains controversial whether the b-oligomer or fibrils produced from recombinant PrP are completely valid representations of PrP Sc . Oligomers and amyloid produced at low pH and moderately denaturing conditions are both toxic (Novitskaya et al. 2006) and, in at least one controversial case, have led to a report of synthetic prions with low infectivity (Legname et al. 2004; Baskakov and Breydo 2007). Another point in supporting the relevance of recombinant b-oligomer and amyloid for drawing conclusions about the in vivo systems is that their formation responds to polymorphisms and species differences in PrP sequences. ...
The term prion disease describes a group of fatal neurodegenerative diseases that are believed to be caused by the pathogenic misfolding of a host cell protein, PrP. Susceptibility to prion disease differs between species and incubation periods before symptom onset can change dramatically when infectious prion strains are transmitted between species. This effect is referred to as the species or transmission barrier. Prion strains represent different structures of PrPSc and the conformational selection model proposes that the source of theses barriers is the preferential incorporation of PrP from a given species into only a subset of PrPSc structures of another species. The basis of this preferential incorporation is predicted to reside in subtle structural differences in PrP from varying species. The overall fold of PrP is highly conserved among species, but small differences in the amino acid sequence give rise to structural variability. In particular, the loop between the second beta-strand and the second alpha-helix has shown structural variability between species, with loop mobility correlating with resistance to prion disease. Single amino acid polymorphisms in PrP within a species can also affect prion susceptibility, but do not appear to drastically alter the biophysical properties of the native form. These polymorphisms affect the propensity of self-association, in recombinant PrP, to form beta-sheet enriched, oligomeric, and amyloid-like forms. These results indicate that the major factor in determining susceptibility to prion disease is the ability of PrP to adopt these misfolded forms by promoting conformational change and self association.
... Second, rPrP fibrils and PrP Sc appear to have fundamentally different global structures [29]. Because PrP C might not be fully compatible with the structure of rPrP fibrils, seeding of PrP C conversion with rPrP fibrils might proceed through slow conformational adaptation [3] . The gradual change in conformational stability during serial transmission observed here supports the hypothesis of conformational adaptation. ...
Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrPSc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrPSc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases.
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The online version of this article (doi:10.1007/s00401-009-0633-x) contains supplementary material, which is available to authorized users.
... Since the new physical state specifies the new phenotype, distinct protein-only traits can only stably persist if their corresponding physical forms, hereafter referred to as conformers, are propagated at the expense of the other possible states. Consistent with this prediction, prion proteins "selfreplicate" both in vitro and in vivo by templating the refolding of other conformers of the same protein [21][22][23]. ...
According to the prion hypothesis, proteins may act in atypical roles as genetic elements of infectivity and inheritance by undergoing self-replicating changes in physical state. While the preponderance of evidence strongly supports this concept particularly in fungi, the detailed mechanisms by which distinct protein forms specify unique phenotypes are emerging concepts. A particularly active area of investigation is the molecular nature of the heritable species, which has been probed through genetic, biochemical, and cell biological experimentation as well as by mathematical modeling. Here, we suggest that these studies are converging to implicate small aggregates composed of prion-state conformers as the transmissible genetic determinants of protein-based phenotypes.
... 11 Recent studies have shown that the conformational stability of PrP amyloidal fibrils may have a direct impact on prion infectivity. [12][13][14][15] Indeed, a reverse correlation between the length of incubation time to prion disease and the conformational stability of prion isolates 16 has been already demonstrated. ...
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.
... Therefore, the amyloid state appears to correspond to a global energetic minimum in the PrP energy landscape, whereas the PrP Sc states occupy local energy minima (Fig. 2). The reverse correlation between the conformational stability of PrP Sc strains and the incubation time to the disease observed in recent studies [25] highlights the idea that PrP Sc structure evolved to propagate efficiently within the complex environment of a cell and cause disease within an animal's life time [26]. From a thermodynamic perspective of the prion conversion reactions, PrP Sc seeds decrease the kinetic barriers for the conversion of both substrates, PrP C and rPrP (Fig. 2). ...
Amyloid fibrils are highly ordered crystal-like structures. It is generally assumed that individual amyloid fibrils consist of conformationally uniform cross-beta-sheet structures that enable the amyloids to replicate their individual conformations via a template-dependent mechanism. Recent studies revealed that amyloids are capable of accommodating a global conformational switch from one amyloid strain to another within individual fibrils. The current review highlights the high adaptation potential of amyloid structures and discusses the implication of these findings for several emerging issues including prion strain adaptation (i.e. gradual change in strain structure). It also proposes that the catalytic activity of an amyloid structure should be separated from its templating effect, and raises the question of strain classification according to their promiscuous or species-specific nature.
... Because indiscriminative strains are capable of accommodating mismatches in primary sequence, interspecies transmission will favor indiscriminative strains. The current results support the model that outlines a hierarchical relationship between promiscuous and species-specific strains presented in the recent review article (19). ...
A key structural component of amyloid fibrils is a highly ordered, crystalline-like cross-beta-sheet core. Conformationally different amyloid structures can be formed within the same amino acid sequence. It is generally assumed that individual fibrils consist of conformationally uniform cross-beta-structures. Using mammalian recombinant prion protein (PrP), we showed that, contrary to common perception, amyloid is capable of accommodating a significant conformational switching within individual fibrils. The conformational switch occurred when the amino acid sequence of a PrP variant used as a precursor substrate in a fibrillation reaction was not compatible with the strain-specific conformation of the fibrillar template. Despite the mismatch in amino acid sequences between the substrate and template, individual fibrils recruited the heterologous PrP variant; however, the fibril elongation proceeded through a conformational adaptation, resulting in a change in amyloid strain within individual fibrils. This study illustrates the high adaptation potential of amyloid structures and suggests that conformational switching within individual fibrils may account for adaptation of amyloid strains to a heterologous substrate. This work proposes a new mechanistic explanation for the phenomenon of strain conversion and illustrates the direction in evolution of amyloid structures. This study also provides a direct illustration that catalytic activity of self-replicating amyloid structures is not ultimately coupled with their templating effect.
... In only one case, however, is there evidence that the resulting synthetic PrP aggregates may harbor infectivity (Legname et al. 2004). These results are consistent with the idea that PrP oligomerization can proceed via multiple pathways that differ in their kinetic parameters and intermediate states, only some of which result in infectious PrP Sc (Baskakov and Breydo 2007). Our findings suggest that many of these PrP oligomerization pathways generate a common set of structural rearrangements that are recognizable by both 15B3 and motif-grafted antibodies. ...
The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP(C)) into an infectious, aggregated isoform (PrP(Sc)) that has a high content of beta-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP(Sc) but not PrP(C), as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19-33, 89-112, and 136-158) have been previously reported to react specifically with infectious PrP(Sc) but not PrP(C). In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.
... As a result, PrP molecules tend to associate with each other in a concentration-dependent manner, leading to protein aggregation. If incubated under specific experimental conditions, they finally convert into abnormal isoforms presenting physicochemical characteristics of amyloid fibrils [5,6], which have long been proposed to be the toxic and infectious species. However, recently, confusion has arisen about whether the toxic and infectious properties of prions are crypted in the fibril structure of the pathogenic isoform (PrP Sc ) or are the result of other misfolded PrP conformations occurring transiently [7,8]. ...
As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes beta-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, Agg(HP), which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, Agg(HP) transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially beta-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, I(HP), is formed under high pressure. After pressure release, I(HP) relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, I(HP), constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids.
In recent years, there has been a lot of interest in using nanotechnology in medicine to diagnose and cure various infectious diseases. Although the precise mechanism of action is frequently challenged, many nanosized materials have been extensively explored for this purpose, using their innately unique features. Their functionality has also been enhanced by the addition of various coatings, such as those that increase chemical stability and stealth properties or contain targeting agents that lessen off-target effects while producing effective "smart" nanoplatforms for the early detection, treatment, and possibly resolution of diseases.
This book aims to compile research and review articles that concentrate on the synthesis, characterization, and applications of nanomaterials, such as nanoparticles, with a focus on investigating novel ways to improve their properties and create new efficient diagnostic, therapeutic, or theranostic approaches with a clear understanding of how they work.
The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms (“strains”) and synthetic prions, was outlined.
Posttranslational modifications are a common feature of proteins associated with neurodegenerative diseases including prion protein (PrPC), tau and α-synuclein. Alternative self-propagating protein states or strains give rise to different disease phenotypes and display strain-specific subsets of posttranslational modifications. The relationships between strain-specific structure, posttranslational modifications and disease phenotype are poorly understood. We previously reported that among hundreds of PrPC sialoglycoforms expressed by a cell, individual prion strains recruited PrPC molecules selectively, according to the sialylation status of their N-linked glycans. Here we report that transmission of a prion strain to a new host is accompanied by a dramatic shift in the selectivity of recruitment of PrPC sialoglycoforms giving rise to PrPSc with a unique sialoglycoform signature and disease phenotype. The newly emerged strain has the shortest incubation time to disease, is characterized by a colocalization of PrPSc with microglia and a very profound proinflammatory response, features that are linked to a unique sialoglycoform composition of PrPSc. The current work provides experimental support for a hypothesis that strain-specific patterns of PrPSc sialoglycoforms formed as a result of selective recruitment dictate strain-specific disease phenotypes. This work suggests a causative relationship between a strain-specific structure, posttranslational modifications and disease phenotype.
The degree of hydrophobicity and net-charge-per-residue are physical properties that enable discriminating folded from intrinsically disordered proteins (IDPs) based solely on amino-acid sequence. Here, we improve upon the existing classification of proteins and IDPs based on the above parameters by adopting the scale of nonpolar content by Rose et al. and by taking amino-acid side-chain acidity and basicity into account. The resulting algorithm, denoted here as net-charge-non-polar or NECNOP, enables the facile prediction of the folded and disordered status of proteins under physiologically relevant conditions with > 95 % accuracy. The NECNOP approach displays a much-enhanced performance for proteins with > 140 residues, suggesting that small proteins are more likely to have irregular charge and hydrophobicity features. NECNOP analysis of the entire E. coli proteome identifies specific net-charge and nonpolar regions peculiar to soluble, integral-membrane and non-integral-membrane proteins. Surprisingly, protein net charge and hydrophobicity are found to converge to specific values as chain length increases, across the E. coli proteome. In addition, NECNOP plots enable the straightforward identification of protein sequences corresponding to prion proteins and, due to the observed convergence characteristics, promise to serve as a powerful predictive tool for the design of large proteins. In summary, NECNOP plots are a straightforward approach that improves our understanding of the relation between amino-acid sequence and three-dimensional structure of proteins as a function of molecular mass.
Neurodegenerative disorders and type 2 diabetes are global epidemics compromising the quality of life of millions worldwide, with profound social and economic implications. Despite the significant differences in pathology – much of which are poorly understood – these diseases are commonly characterized by the presence of cross-β amyloid fibrils as well as the loss of neuronal or pancreatic β-cells. In this review, we document research progress on the molecular and mesoscopic self-assembly of amyloid-beta, alpha synuclein, human islet amyloid polypeptide and prions, the peptides and proteins associated with Alzheimer’s, Parkinson’s, type 2 diabetes and prion diseases. In addition, we discuss the toxicities of these amyloid proteins based on their self-assembly as well as their interactions with membranes, metal ions, small molecules and engineered nanoparticles. Through this presentation we show the remarkable similarities and differences in the structural transitions of the amyloid proteins through primary and secondary nucleation, the common evolution from disordered monomers to alpha-helices and then to β-sheets when the proteins encounter the cell membrane, and, the consensus (with a few exceptions) that off-pathway oligomers, rather than amyloid fibrils, are the toxic species regardless of the pathogenic protein sequence or physicochemical properties. In addition, we highlight the crucial role of molecular self-assembly in eliciting the biological and pathological consequences of the amyloid proteins within the context of their cellular environments and their spreading between cells and organs. Exploiting such structure-function-toxicity relationship may prove pivotal for the detection and mitigation of amyloid diseases.
Programmed assembly and self-assembly of soft materials offers significant promise for the generation of new types of materials with useful properties. Through evolutionary processes occurring over billions of years, nature has produced numerous optimised building blocks for the controlled assembly of a wide range of complex architectures. Our challenge now is to imitate these naturally occurring processes for technological applications, either using biological molecules such as DNA and proteins, or macromolecular mimics that retain many of the important features of biological molecules while introducing new functionalities. We focus on a single example of biomolecular self-assembly - the self-assembly of polypeptides, including polypeptide mimics, into quasi-one-dimensional fibres - to provide a flavour of the utility of soft biological materials for construction purposes. This journal is
Prion-like transmission of protein aggregates or amyloid in several neurodegenerative diseases, such as Parkinson's disease, Huntington's disease and Alzheimer's disease, in addition to the transmissible spongiform encephalopathies (or prion diseases), has been proposed recently. This is a controversial idea and, in this paper, we consider what we mean by a "prion", and by "amyloid", and present some biochemical investigations of cerebral prion amyloidosis in mice.
Converting normal prion protein (PrP(C)) to the pathogenic PrP(Sc) isoform is central to prion disease. We previously showed that, in the presence of lipids, recombinant mouse PrP (rPrP) can be converted into the highly infectious conformation, suggesting a crucial role of lipid-rPrP interaction in PrP conversion. To understand the mechanism of lipid-rPrP interaction, we analyzed the ability of various rPrP mutants to bind anionic lipids and to gain lipid-induced proteinase K (PK) resistance. We found that the N-terminal positively charged region contributes to electrostatic rPrP-lipid binding but does not affect lipid-induced PK resistance. In contrast, the highly conserved middle region of PrP, consisting of a positively charged region and a hydrophobic domain, is essential for lipid-induced rPrP conversion. The hydrophobic domain deletion mutant significantly weakened the hydrophobic rPrP-lipid interaction and abolished the lipid-induced C-terminal PK resistance. The rPrP mutant without positive charges in the middle region reduced the amount of the lipid-induced PK-resistant rPrP form. Consistent with a critical role of the middle region in lipid-induced rPrP conversion, both disease-associated P105L and P102L mutations, localized between lysine residues in the positively charged region, significantly affected lipid-induced rPrP conversion. The hydrophobic domain-localized 129 polymorphism altered the strength of hydrophobic rPrP-lipid interaction. Collectively, our results suggest that the interaction between the middle region of PrP and lipids is essential for the formation of the PK-resistant conformation. Moreover, the influence of disease-associated PrP mutations and the 129 polymorphism on PrP-lipid interaction supports the relevance of PrP-lipid interaction to the pathogenesis of prion disease.
We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, [SWI+], demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational
switch influences Swi1 function and to define the sequence and structural requirements for [SWI+] formation and propagation, we functionally dissected the Swi1 molecule. We show here that the [SWI+] prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was
aggregated in [SWI+] cells but diffuse in [swi−] cells; chromosomal deletion of the N-coding region resulted in [SWI+] loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro, enabling [SWI+] de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate
in [SWI+] cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also
show that excessive Swi1 incurred Li+/Na+ sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the
final proof of “protein-only” transmission of [SWI+] and demonstrate that the widely distributed “dispensable” glutamine/asparagine-rich regions/motifs might have important
and divergent biological functions.
Specific, quantitative, and sensitive protein detection with minimal sample preparation is an enduring need in biology and medicine. Protein detection assays ideally provide quick, definitive measurements that use only small amounts of material. Fluorescence cross-correlation spectroscopy (FCCS) has been proposed and developed as a protein detection assay for several years. Here, we combine several recent advances in FCCS apparatus and analysis to demonstrate it as an important method for sensitive, quantitative, information-rich protein detection with low false positives. The addition of alternating laser excitation (ALEX) to FCCS along with a method to exclude signals from occasional aggregates leads to a very low rate of false positives, allowing the detection and quantification of the concentrations of a wide variety of proteins. We detect human chorionic gonadotropin (hCG) using an antibody-based sandwich assay and quantitatively compare our results with calculations based on binding equilibrium equations. Furthermore, using our aggregate exclusion method, we detect smaller oligomers of the prion protein PrP by excluding bright signals from large aggregates.
Prion disease is characterized by the alpha-->beta structural conversion of the cellular prion protein (PrP(C)) into the misfolded and aggregated "scrapie" (PrP(Sc)) isoform. It has been speculated that methionine (Met) oxidation in PrP(C) may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrP(C) [rhPrP(C)(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrP(C) variant is an alpha-helix rich protein (like Met-rhPrP(C)) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrP(C) variant is a beta-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrP(C), the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the alpha-->beta secondary structure conversion in rhPrP(C) with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.
Prions are proteins that convert between structurally and functionally distinct states, one or more of which is transmissible. In yeast, this ability allows them to act as non-Mendelian elements of phenotypic inheritance. To further our understanding of prion biology, we conducted a bioinformatic proteome-wide survey for prionogenic proteins in S. cerevisiae, followed by experimental investigations of 100 prion candidates. We found an unexpected amino acid bias in aggregation-prone candidates and discovered that 19 of these could also form prions. At least one of these prion proteins, Mot3, produces a bona fide prion in its natural context that increases population-level phenotypic heterogeneity. The self-perpetuating states of these proteins present a vast source of heritable phenotypic variation that increases the adaptability of yeast populations to diverse environments.
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.
Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-beta-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions--perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including alpha B-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.
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.
Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met(35) is associated with altered Abeta assembly, we examined the role of Met(35) in controlling Abeta oligomerization. Oxidation of Met(35) in Abeta42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by Abeta40. Systematic structural alterations of the C(gamma)(35)-substituent group revealed that its electronic nature, rather than its size (van der Waals volume), was the factor controlling oligomerization pathway choice. Preventing assembly of toxic Abeta42 paranuclei through selective oxidation of Met(35) thus represents a potential therapeutic approach for AD.
Bovine spongiform encephalopathy (BSE) has become a public health issue because a recently evolved BSE agent has infected people, yielding an unusual form of Creutzfeld- Jakob disease (CJD). A new CJD agent that provokes similar amyloid plaques and cerebellar pathology was serially propagated. First-passage rats showed obvious clinical signs and activated microglia but had negligible PrP-res (the more protease-resistant form of host PrP) or cerebellar lesions. Microglia and astrocytes may participate in strain selection because the agent evolved, stabilized, and reproducibly provoked BSE-like disease in subsequent passages. Early vacuolar change involving activated microglia and astrocytes preceded significant PrP-res accumulation by more than 50 days. These studies reveal several inflammatory host reactions to an exogenous agent.
An extensive loss of a selected population of neurons in Alzheimer’s disease is closely related to the formation of paired
helical filaments (PHFs). The most striking characteristic of PHFs upon Western blotting is their smearing. According to a
previously described protocol (Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993)
Neuron10, 1151–1160), smeared tau was purified, and its peptide map was compared with that of soluble (normal) tau. A CNBr fragment
from soluble tau (CN5; residues 251–419 according to the 441-residue isoform) containing the microtubule-binding domain migrated
at 15 and 18 kDa on SDS-polyacrylamide gel electrophoresis, whereas that from smeared tau exhibited two larger, unusually
broad bands at ∼30 and ∼45 kDa, presumably representing dimers and trimers of CN5. In the peptide map of smeared tau-derived
CN5, distinct peaks eluting at unusual locations were noted. Amino acid sequence and mass spectrometric analyses revealed
that these distinct peptides bear isoaspartate at Asn-381 and Asp-387. Because no unusual peptides other than aspartyl or
isoaspartyl peptide were found in the digests of smeared tau-derived CN5, it is likely that site-specific deamidation and
isoaspartate formation are involved in its dimerization and trimerization and thus in PHF formation in vivo.
The main hypothesis for prion diseases proposes that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc), which in most cases undergoes aggregation. In an organism infected with PrPSc, PrPC is converted into the β-sheet form, generating more PrPSc. We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23–231)) converts it from an α-helical
conformation (cellular isoform) into a soluble, β-sheet isoform similar to that found in the fibrillar state. The recombinant
murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in
molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering,
fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue
the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of
prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate
the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the
protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein
bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation,
but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.
The only component of the infectious scrapie prion identified to date is a protein designated PrPSc. A posttranslational process converts the cellular PrP isoform (PrPC) into PrPSc. Denatured PrPSc was digested with endoproteases, and the resulting fragments were isolated by HPLC. By both mass spectrometry and Edman sequencing, the primary structure of PrPSc was found to be the same as that deduced from the PrP gene sequence, arguing that neither RNA editing nor protein splicing feature in the synthesis of PrPSc. Mass spectrometry also was used to search for posttranslational chemical modifications other than the glycosylinositol phospholipid anchor attached to the C-terminus and two Asn-linked oligosaccharides already known to occur on both PrPSc and PrPC. These results contend that PrPSc molecules do not differ from PrPC at the level of an amino acid substitution or a posttranslational chemical modification; however, we cannot eliminate the possibility that a small fraction of PrPSc is modified by an as yet unidentified posttranslational process or that PrPC carries a modification that is removed in the formation of PrPSc. It seems likely that PrPSc differs from PrPC in its secondary and tertiary structure, but the possibility of a tightly bound, disease-specific molecule which purifies with PrPSc must also be considered.
Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrPSc). To explore this postulate, we examined the properties of PrPSc from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrPSc molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrPSc as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have ∼75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a ∼170-d incubation period and ∼1.25 M for the SHa(RML) and 139H isolates with ∼180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a ∼320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrPSc conformers encipher the biological properties of prion strains.
Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.
According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has
Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10(8.7) ID50 units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (less than 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.
During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.
The incubation of lens proteins with reducing sugars leads to the formation of fluorescent yellow pigments and cross-like
similar to those reported in aging and cataractous human lenses. Called nonenzymatic browning or the Maillard reaction, this
aging process also occurs in stored foods. Reducing sugars condense with the free amino group of proteins, then rearrange
and dehydrate to form unsaturated pigments and cross-linked products. Although most proteins in living systems turn over with
sufficient rapidity to avoid nonenzymatic browning, some, such as lens crystallins and skin collagen, are exceptionally long-lived
and may be vulnerable.
Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.
Experimental infection of transmissible mink encephalopathy (TME) in two closely related mustelids, black ferret (Mustela putorius furo) and mink (Mustela visa), revealed differences in their susceptibility to the TME agent. When challenged with the Stetsonville TME agent, a longer incubation period was observed in ferrets (28 to 38 months) than mink (4 months). Western blot analysis of ferret and mink prion proteins (PrP) demonstrated no detectable differences between the proteins. Northern blot analysis of ferret brain RNA indicated that PrP mRNA abundance is similar in infected and uninfected individuals. We amplified the PrP coding region from ferret DNA using the polymerase chain reaction and compared the deduced amino acid sequence of the ferret PrP gene with the mink PrP gene. This comparison revealed six silent base changes and two amino acid changes between mink and ferret: Phe-->Lys at codon 179 and Arg-->Gln at codon 224, respectively. These changes may indicate the region of PrP that is responsible for the species barrier effect between mink and ferret.
Amyloid plaques, associated with argyrophilic dystrophic neurites, and cerebral amyloid angiopathy (CAA), but no neurofibrillary tangles, were found in the brains of three middle-aged marmoset monkeys that had been injected intracerebrally (ic) 6-7 yr earlier with brain tissue from a patient with early-onset Alzheimer's disease. Such changes were not found in the brains of three age-matched control marmosets. Immunochemically the amyloid plaques and CAA stained with antibody to beta (A4)-protein. The plaques and CAA displayed dichroic birefringence when stained with Congo red and viewed under polarized light. beta (A4)-amyloid plaques and CAA were also found in the brain of one of two marmosets injected ic 6 yr previously with brain tissue from a patient with prion disease with concomitant beta (A4)-amyloid plaques and CAA. An occasional beta (A4)-amyloid plaque was found in the brains of two of four marmosets injected ic > 4.5 yr previously with brain tissue from three elderly patients, two of whom had suspected (but untransmitted) CJD. No beta (A4)-amyloid plaques or CAA were found in six marmosets who were older than the injected animals, in four marmosets that had not developed spongiform encephalopathy (SE) having been injected several years previously with human brain tissue from three younger patients with suspected or atypical prion disease, or in 10 younger marmosets who had undergone various neurosurgical procedures. Seventeen marmosets injected in the same way with brain tissue from patients or animals with SE developed SE 17-49 mo after injection. These results suggest that beta (A4)-amyloidosis is a transmissible process comparable to the transmissibility of SE.
Alzheimer disease (AD) is characterized by deposits of an aggregated 42-amino-acid beta-amyloid peptide (beta AP) in the brain and cerebrovasculature. After a concentration-dependent lag period during in vitro incubations, soluble preparations of synthetic beta AP slowly form fibrillar aggregates that resemble natural amyloid and are measurable by sedimentation and thioflavin T-based fluorescence. Aggregation of soluble beta AP in these in vitro assays is enhanced by addition of small amounts of pre-aggregated beta-amyloid "seed" material. We also have prepared these seeds by using a naturally occurring reaction between glucose and protein amino groups resulting in the formation of advanced "glycosylation" end products (AGEs) which chemically crosslink proteins. AGE-modified beta AP-nucleation seeds further accelerated aggregation of soluble beta AP compared to non-modified "seed" material. Over time, nonenzymatic advanced glycation also results in the gradual accumulation of a set of posttranslational covalent adducts on long-lived proteins in vivo. In a standardized competitive ELISA, plaque fractions of AD brains were found to contain about 3-fold more AGE adducts per mg of protein than preparations from healthy, age-matched controls. These results suggest that the in vivo half-life of beta-amyloid is prolonged in AD, resulting in greater accumulation of AGE modifications which in turn may act to promote accumulation of additional amyloid.
beta 2-Microglobulin (beta 2M) is a major constituent of amyloid fibrils in hemodialysis-associated amyloidosis, a complication of long-term hemodialysis patients. Amyloid fibril proteins were isolated from connective tissues forming carpal tunnels in hemodialysis patients with carpal tunnel syndrome. Two-dimensional polyacrylamide gel electrophoresis and Western blotting demonstrated that most of the beta 2M forming amyloid fibrils exhibited a more acidic pI value than normal beta 2M. This acidic beta 2M was also found in a small fraction of beta 2M in sera and urine from these patients, whereas heterogeneity was not observed in healthy individuals. We purified acidic and normal beta 2M from the urine of long-term hemodialysis patients and compared their physicochemical and immunochemical properties. Acidic beta 2M, but not normal beta 2M, was brown in color and fluoresced, both of which are characteristics of advanced glycation end products (AGEs) of the Maillard reaction. Immunochemical studies showed that acidic beta 2M reacted with anti-AGE antibody and also with an antibody against an Amadori product, an early product of the Maillard reaction, but normal beta 2M did not react with either antibody. Incubating normal beta 2M with glucose in vitro resulted in a shift to a more acidic pI, generation of fluorescence, and immunoreactivity to the anti-AGE antibody. The beta 2M forming amyloid fibrils also reacted with anti-AGE antibody. These data provided evidence that AGE-modified beta 2M is a dominant constituent of the amyloid deposits in hemodialysis-associated amyloidosis.
Bovine spongiform encephalopathy (BSE) has become a public health issue because a recently evolved BSE agent has infected people, yielding an unusual form of Creutzfeld-Jakob disease (CJD). A new CJD agent that provokes similar amyloid plaques and cerebellar pathology was serially propagated. First-passage rats showed obvious clinical signs and activated microglia but had negligible PrP-res (the more protease-resistant form of host PrP) or cerebellar lesions. Microglia and astrocytes may participate in strain selection because the agent evolved, stabilized, and reproducibly provoked BSE-like disease in subsequent passages. Early vacuolar change involving activated microglia and astrocytes preceded significant PrP-res accumulation by more than 50 days. These studies reveal several inflammatory host reactions to an exogenous agent.
The ‘protein only’ hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the ‘protein only’ hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).
Endogenous proteins are highly susceptible to modification by ROS produced as byproducts of normal metabolic processes, or upon exposure to oxidative stress and atmospheric pollutants. The ROS-mediated modification of proteins may lead to loss of biological function and to conversion of the proteins to forms that are rapidly degraded by endogenous proteases, especially by the multicatalytic protease. One of many different kinds of protein modification elicited by ROS is the oxidation of some amino acid side chains to carbonyl derivatives. The carbonyl content of protein is therefore a convenient marker of ROS-mediated protein damage. By means of highly sensitive methods for the detection and quantitation of protein carbonyl groups, it has been established in several different animal models that there is an exponential increase in the level of oxidized protein during aging and that elevated levels of oxidized proteins are associated with a number of diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, rheumatoid arthritis, diabetes, muscular dystrophy, and cataractogenesis, to name a few. Although protein oxidation probably contributes to the biological dysfunction associated with these diseases, a causal relationship between protein oxidation and the etiology or progression of a disease has not been established. Nevertheless, in the case of some neurological disorders, there is a positive correlation between the loss of a particular biological function and an elevation of the level of oxidized protein in the brain (153) and in specific regions of the brain that control that function (154). Finally, because oxidized proteins are readily degraded by endogenous proteases, the steady state intracellular level of oxidized proteins is a complex function of a multiplicity of factors that govern the generation of ROS, the antioxidant systems that scavenge ROS, the susceptibility of proteins to oxidative modification, and the activities of the proteases that degrade oxidized proteins. Accordingly, the accumulation of oxidized protein that occurs during aging and in various diseases is likely attributable to the accumulated genetic damage (ROS-mediated mutations?) that affects one or more of the numerous factors that determine the balance between protein oxidation on the one hand and the degradation of oxidized proteins on the other.
In this study, we compared the neuronal induction of the antioxidant heme oxygenase-1 (HO-1) in Alzheimer's disease with abnormalities in marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO-1-containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO-1. These findings suggest that phosphorylation precedes the HO-1 response and that HO-1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal and induces the Alz50 epitope in . It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of but also requires phosphorylation because neither methylated, recombinant, nor dephosphorylated reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament- isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE-lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of and neuronal oxidative stress-induced pathology is important in the formation of neurofibrillary tangles.
It is believed that the critical event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the prion protein from an α-helical form, PrPC, to β-sheet-rich conformer, PrPSc. Recently, we have shown that incubation of the recombinant prion protein under mildly acidic conditions (pH 5 or below) in the presence of low concentrations of guanidine hydrochloride results in a transition to PrPSc-like β-sheet-rich oligomers that show fibrillar morphology and an increased resistance to proteinase K digestion [Swietnicki, W., Morillas, M, Chen, S., Gambetti, P., and Surewicz, W. K. (2000) Biochemistry 39, 424-431]. To gain insight into the mechanism of this transition, in the present study we have characterized the biophysical properties of the recombinant human prion protein (huPrP) at acidic pH in the presence of urea and salt. Urea alone induces unfolding of the protein but does not result in protein self-association or a conversion to β-sheet structure. However, a time-dependent transition to β-sheet structure occurs upon addition of both urea and NaCl to huPrP, even at a sodium chloride concentration as low as 50 mM. This transition occurs concomitantly with oligomerization of the protein. At a given protein and sodium chloride concentration, the rate of monomeric α-helix to oligomeric β-sheet transition is strongly dependent on the concentration of urea. Low and medium concentrations of the denaturant accelerate the reaction, whereas strongly unfolding conditions are not conducive to the conversion of huPrP into an oligomeric β-sheet-rich structure. The present data strongly suggest that partially unfolded intermediates may be involved in the transition of the monomeric recombinant prion protein into the oligomeric scrapie-like form.
The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27–30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27–30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27–30. Denaturation of PrP 27–30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27–30.
A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.
A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrPC) to a protease-resistant form (PrP-res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.
According to the “protein-only” hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrPC) and pathological (PrPSc) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90−231 of the human prion protein (huPrP90−231). Incubation of the protein under acidic conditions (pH 3.6−5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an α-helical conformation to a β-sheet structure and oligomerization of huPrP90−231 into large molecular weight aggregates. No stable monomeric β-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of β-sheet structure and protein oligomerization likely occur concomitantly. The β-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrPSc). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrPSc-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90−231 to an oligomeric β-sheet structure is the presence of salt.
Proteins are a major target for oxidants as a result of their abundance in biological systems, and their high rate constants for reaction. Kinetic data for a number of radicals and non-radical oxidants (e.g. singlet oxygen and hypochlorous acid) are consistent with proteins consuming the majority of these species generated within cells. Oxidation can occur at both the protein backbone and on the amino acid side-chains, with the ratio of attack dependent on a number of factors. With some oxidants, damage is limited and specific to certain residues, whereas other species, such as the hydroxyl radical, give rise to widespread, relatively non-specific damage. Some of the major oxidation pathways, and products formed, are reviewed. The latter include reactive species, such as peroxides, which can induce further oxidation and chain reactions (within proteins, and via damage transfer to other molecules) and stable products. Particular emphasis is given to the oxidation of methionine residues, as this species is readily oxidised by a wide range of oxidants. Some side-chain oxidation products, including methionine sulfoxide, can be employed as sensitive, specific, markers of oxidative damage. The product profile can, in some cases, provide valuable information on the species involved; selected examples of this approach are discussed. Most protein damage is non-repairable, and has deleterious consequences on protein structure and function; methionine sulfoxide formation can however be reversed in some circumstances. The major fate of oxidised proteins is catabolism by proteosomal and lysosomal pathways, but some materials appear to be poorly degraded and accumulate within cells. The accumulation of such damaged material may contribute to a range of human pathologies.
Transgenic mice expressing chimeric prion protein (PrP) genes derived from Syrian hamster (SHa) and mouse (Mo) PrP genes were constructed. One SHa/MoPrP gene, designated MH2M PrP, contains five amino acid substitutions encoded by SHaPrP, while another construct, designated MHM2 PrP, has two substitutions. Transgenic (Tg) (MH2M PrP) mice were susceptible to both Syrian hamster and mouse prions, whereas three lines expressing MHM2 PrP were resistant to Syrian hamster prions. The brains of Tg(MH2M PrP) mice dying of scrapie contained chimeric PrPSc and prions with an artificial host range favoring propagation in mice that express the corresponding chimeric PrP and were also transmissible, at reduced efficiency, to nontransgenic mice and hamsters. Our findings provide genetic evidence for homophilic interactions between PrPSc in the inoculum and PrPC synthesized by the host.
The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the β-form of this peptide developed signs of central nervous system dysfunction at ∼360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Sträussler-Scheinker disease. By contrast, eight of eight mice receiving a non-β-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.
A polysaccharide consisting of mainly 1,4-linked glucose units was found associated with prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein (PrP 27-30) exhibiting the ultrastructural and tinctorial properties of amyloid. The polysaccharide differs in composition from the Asn-linked oligosaccharides and the GPI-anchor of the prion protein. Prion rods were prepared from scrapie-infected hamster brains using two different purification protocols. Prolonged digestion of rods with proteinase K reduced PrP by a factor of at least 500, leaving about 10% (w/w) of the sample as an insoluble remnant. Only glucose was obtained by acid hydrolysis of the remnant and methylation analysis showed 80% 1,4-, 15% 1,6- and 5% 1,4,6-linked glucose units. The physical and chemical properties as well as the absence of terminal glucose units indicate a very high molecular mass of the polysaccharide. No evidence was found for covalent bonds between PrP and the polysaccharide. The polysaccharide certainly contributes to the unusual chemical and physical stability of prion rods, acting like a scaffold. A potential structural and/or functional relevance of the polysaccharide scaffold is discussed.
A previous paper (Kimberlin & Walker, 1977) described an experimental model of scrapie in hamsters in which the incubation period decreased progressively over the first 4 passages before becoming stable at the 5th and subsequent passes. Studies have been made of some of the agent strains present in brains taken from the 2nd, 3rd, 4th and 6th hamster passes. The results indicate the presence of at least two strains of agent at the 3rd passage level. One of these (431K) is highly pathogenic for mice and the other (263K) has an extremely low pathogenicity for mice. However only one of these strains (263K) is present in hamster brain after the 6th serial passage. It is suggested that the 'adaptation' of scrapie to hamsters may involve the selection, from a mixture, of a single strain which is highly pathogenic for hamsters. The possibility of modification of the properties of agent strains on passage discussed.
Repeated passage of the "Chandler" strain of scrapie in female golden hamsters using the intracerebral route of inoculation reduces the minimum incubation period to 60 days, about half of the minimum incubation period so far found in any of the mouse models of scrapie. The infectivity titres in brain in the clinical stage of the disease are considerably higher (greater than 8-0 -log10 LD50 i.c. units/0-05 g) than those found in mouse scrapie. The biological characteristics of this model of hamster scrapie are reported, including the effects on incubation period of route of inoculation, dose of agent, sex of hamster, ambient temperature (hibernation) and splenectomy. Some general and specific applications of this experimental model of scrapie are discussed.
This chapter focuses on the biochemical basis of advanced glycosylation and discusses the diverse effects of advanced glycosylation products in biology and medicine. The chemical and biological conception of the advanced glycosylation process has evolved considerably since early studies on the formation of HbA1c. Despite difficulties in the structural elucidation of advanced glycosylation products, the ensuing years have yielded much insights into the biochemistry of AGEs, in large part assisted by consideration of related pathways in the Maillard reaction. Studies of biological processes have been motivated by the multiorgan pathology that occurs during chronic hyperglycemia. The basis of much of this pathology is still poorly understood. Related investigations of normal, age-related processes are only now beginning to bear insight into some of the clinicopathological sequalae that characterize normal aging. Given the slow, progressive nature of AGE accumulation in vivo and the active cell-mediated processes that appear to be required for AGE removal, it is likely that the investigation of advanced glycosylation mechanisms will continue to provide insight into a variety of additional biological and pathological processes that are characterized by long-term, age-related, and degenerative changes.
A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.
Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.
The interspecies transmission of scrapie is frequently associated with exceptionally long incubation periods at first passage in the new host compared to later passages (the species barrier effect). The basis of this was investigated using the 139A strain of scrapie which had been cloned by three serial passages in mice at limiting infectious doses. Cloned scrapie was passaged through hamsters (twice) or rats (thrice) and then reisolated in mice. Large species barrier effects were encountered on mouse-to-hamster and hamster-to-mouse passage resulting in the isolation of a mutant strain, 139-H/M, with properties very different from 139A. In contrast, the strain reisolated from rats was indistinguishable from 139A. However, a large species barrier was encountered at the mouse-to-rat passage but not at the rat-to-mouse passage. It is suggested that the transmission of scrapie between species may be associated with no change in properties or a permanent change in the scrapie genome due to the selection of mutants. A third possibility, the donor species effect, is a temporary change occurring only at first passage in the new host species which is largely or entirely caused by the introduction of material from the previous host. We speculate that the donor species effect could be explained if some host protein forms a functional part of the infectious agent.
The infectious agent (or 'prion') of the transmissible spongiform encephalopathies (TSEs) such as scrapie resembles a virus in that it replicates in vivo and has distinct strains, but it was postulated long ago to contain only protein. More recently, PrPSc, a pathogenic, scrapie-associated form of the host-encoded prion protein (PrP), was identified as a possible primary TSE agent protein. PrPSc is defined biochemically by its insolubility and resistance to proteases and is derived post-translationally from normal, protease-sensitive PrP (PrPc). The conversion seems to involve conformational change rather than covalent modification. However, the conversion mechanism and the relationship of PrPSc formation to TSE agent replication remain unclear. Here we report the conversion of PrPc to protease-resistant forms similar to PrPSc in a cell-free system composed of substantially purified constituents. This conversion was selective and required the presence of preexisting PrPSc, providing direct evidence that PrPSc derives from specific PrPc-PrPSc interactions.
Prion diseases are assumed to be caused by the infectious isoform, PrPsc, of a single cellular surface protein, PrPc. PrPsc is an insoluble form of PrPc and is believed to possess a different three-dimensional fold. It may propagate by causing PrPc to adopt its own infectious conformation by an unknown mechanism. Studies on folding and thermodynamic stability of prion proteins are essential for understanding the processes underlying the conversion from PrPc to PrPsc, but have so far been hampered by the low solubility of prion proteins in the absence of detergents. Here, we show that the amino-terminally truncated segment of mouse PrP comprising residues 121 to 231 is an autonomous folding unit. It consists predominantly of alpha-helical secondary structure and is soluble at high concentrations up to 1 mM in distilled water. PrP(121-231) undergoes a cooperative and completely reversible unfolding/refolding transition in the presence of guanidinium chloride with a free energy of folding of -22 kJ/mol at pH 7. The intrinsic stability of segment 121-231 is not in accordance with present models of the structure of PrPc and PrPsc PrP(121-231) may represent the only part of PrPc with defined three-dimensional structure.
Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPC) into a protease-resistant form (PrPSc) by an unknown mechanism. In vitro studies have indicated that PrPSc can induce this conversion, consistent with proposals that PrPSc itself might be the infectious scrapie agent. Using this cell-free model of the PrPC to PrPSc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrPSc-derived species that has converting activity.
The cell-free conversion of 35S PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (above an apparent minimum threshold concentration) and 35S PrPC. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrPSc and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of approximately 2000 kDa.
The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.
The conversion of the normal form of prion protein (PrPC) to a disease-specific form (PrPSc) is a central event in scrapie and other transmissible spongiform encephalopathies. PrPSc is distinguished from PrPC by its insolubility and its resistance to proteolysis. PrPSc is also capable of converting 35S-PrPC, in vitro, into a form which is indistinguishable from PrPSc with respect to its protease-sensitivity. Both the "converting activity" and the protease-resistance of isolated hamster PrPSc can be at least partially eliminated by denaturation and recovered by renaturation, provided that the concentration of denaturant does not exceed a threshhold. This study was undertaken in order to localize the regions of native PrPSc structure that must remain intact to allow refolding. Proteinase K was used to digest exposed, denatured PrPSc sequences, and the residual fragments were characterized using anti-PrP antibodies directed toward four PrP epitopes. A 16-kDa fragment marked by an epitope within residues 143-156 remained protease-resistant under conditions which at least partially unfolded epitopes within residues 90-115 and 217-232. However, dilution of denaturant restored protease-resistance to these epitopes. This reversible unfolding was observed with both purified PrPSc and PrPSc in crude brain homogenates. Size fractionation of partially GdnHCl-solubilized PrPSc revealed that only the insoluble aggregates retained the ability to refold, consistent with the hypothesis that native PrPSc is an ordered aggregate. When the threshold denaturant concentration was exceeded, both protease-resistance of the 16-kDa C-terminal domain and converting activity were irreversibly destroyed. These results suggest that the in vitro converting activity requires ordered, protease-resistant PrPSc aggregates and that a critical aspect of the PrPSc structure is the folding of a particularly stable approximately 16-kDa C-terminal domain.
The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.
Nucleation-dependent polymerization of beta-amyloid peptide, the major component of plaques in patients with Alzheimer's disease, is significantly accelerated by crosslinking through Advanced Glycation End-products (AGEs) in vitro. During the polymerization process, both nucleus formation and aggregate growth are accelerated by AGE-mediated crosslinking. Formation of the AGE-crosslinked amyloid peptide aggregates could be attenuated by the AGE-inhibitors Tenilsetam, aminoguanidine and carnosine. These experimental data, and clinical studies, reporting a marked improvement in cognition and memory in Alzheimer's disease patients after Tenilsetam treatment, suggest that AGEs might play an important role in the etiology or progression of the disease. Thus AGE-inhibitors may generally become a promising drug class for the treatment of Alzheimer's disease.
Several diverse disorders, including the prevalent dementias and encephalopathies, are now believed to arise from the same general disease mechanism. In each, there is abnormal unfolding and then aggregation of an underlying protein. The gradual accumulation of these aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the clinical disease. The understanding of these processes at the molecular level is opening prospects of more rational approaches to investigation and therapy.
Highly reactive oxygen species that are formed during normal metabolism and under conditions of oxidative stress are able to oxidize proteins or convert lipid and carbohydrate derivatives to compounds that react with functional groups on proteins. Among other changes, these ROS-mediated reactions lead to the formation of protein carbonyl derivatives, which serves as a marker of ROS-mediated protein damage. On the basis of this marker, it is established that oxidatively damaged protein is associated with aging and some diseases. The accumulation of oxidatively damaged protein reflects the balance among a myriad of factors that govern the rates of ROS generation and the rate at which damaged protein is degraded. Peroxynitrite, which is formed under normal physiological conditions, is able to oxidize methionine residues in proteins and to nitrate tyrosine residues; however, its ability to do so is dependent on the availability of CO2, which stimulates the nitration of tyrosine residues but inhibits the oxidation of methionine residues. Nitration of tyrosine residues may contribute to peroxynitrite toxicity, as nitration precludes the phosphorylation or nucleotidylation of tyrosine residues and thereby seriously compromises one of the most important mechanisms of cellular regulation and signal transduction.
In transmissible spongiform encephalopathies (TSE), the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to a pathologic form (PrP-res) that has greatly enhanced proteinase K resistance, insolubility, and beta sheet content. To investigate the possibility that alterations at aspartyl or asparaginyl residues in the form of D-aspartate and/or L-isoaspartate could play a role in either the formation or stabilization of PrP-res in TSE-infected animals, we assayed for the presence of these abnormal residues in PrP-res. Protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) was used to methylate and radiolabel altered aspartyl residues, which were detected in PrP-res, but at low levels (0.5 mole%). The scarcity of D-aspartyl and/or L-isoaspartyl groups in PrP-res suggests that this modification is unlikely to be primarily responsible for the differences between PrP-res and PrP-sen. However, it remains possible that such modifications in substoichiometric numbers of PrP molecules could help to initiate the PrP-res formation or stabilize PrP-res polymers in vivo.
Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30. Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species. Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS. From the two alternatives, that the sphingolipids are either required for prion function or are relics from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.