Disulfide mapping reveals the domain swapping as the crucial process of the structural conversion of prion protein

Prion diseases are infectious conformational diseases. Despite the determination of many native prion protein (PrP) structures and in vitro production of infectious prions from recombinant PrP the structural background of PrP conversion remains the largest unsolved problem. The aggregated state of PrP^Sc makes it inaccessible to high resolution techniques, therefore indirect methods have to be used to investigate the conversion process. We engineered disulfide bridges into the structured domain of PrP in order to determine the secondary structure elements that remain conserved upon conversion. Rather surprisingly, introduction of disulfides into each or both of the subdomains B1-H1-B2 and H2-H3 of the C-terminal globular domain retained the robust ability to convert into fibrils with increased content of β-structure, indistinguishable from the wild-type PrP. On the other hand disulfide bridges tethering the two subdomains completely prevented conversion, while their reduction reversed their conversion ability. The same conversion propensity was replicated also in prion infected cell lines. Experiments with combinations of engineered cysteine residues further support that domain swapping, centered on the B2-H2 loop, previously associated to species barrier, leads to PrP swapped dimers as the building block of prion fibrils.     

PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain

[PSI(+)], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI(+)] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI(+)] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI(+)] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI(+)]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI(+)] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.     

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.     

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.     

Comparative computational analysis of prion proteins reveals two fragments with unusual structural properties and a pattern of increase in hydrophobicity associated with disease-promoting mutations

Prion diseases are a group of neurodegenerative disorders associated with conversion of a normal prion protein, PrPC, into a pathogenic conformation, PrPSc. The PrPSc is thought to promote the conversion of PrPC. The structure and stability of PrPC are well characterized, whereas little is known about the structure of PrPSc, what parts of PrPC undergo conformational transition, or how mutations facilitate this transition. We use a computational knowledge-based approach to analyze the intrinsic structural propensities of the C-terminal domain of PrP and gain insights into possible mechanisms of structural conversion. We compare the properties of PrP sequences to those of a PrP paralog, Doppel, and to the distributions of structural propensities observed in known protein structures from the Protein Data Bank. We show that the prion protein contains at least two sequence fragments with highly unusual intrinsic propensities, PrP(114-125) and helix B. No segments with unusual properties were found in Doppel protein, which is topologically identical to PrP but does not undergo structural rearrangements. Known disease-promoting PrP mutations form a statistically significant cluster in the region comprising helices B and C. Due to their unusual properties, PrP(114-125) and the C terminus of helix B may be considered as primary candidates for sites involved in conformational transition from PrPC to PrPSc. The results of our study also show that most PrP mutations associated with neurodegenerative disorders increase local hydrophobicity. We suggest that the observed increase in hydrophobicity may facilitate PrP-to-PrP or/and PrP-to-cofactor interactions, and thus promote structural conversion.     

Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Biochemical and structural studies of the prion protein polymorphism.

A hallmark event in transmissible spongiform encephalopathies is the conversion of the physiological prion protein into the disease-associated isoform. A natural polymorphism at codon 129 of the human prion gene, resulting in either methionine or valine, has profound influence on susceptibility and phenotypic expression of the disease in humans. In this study, we investigated the local propensity of synthetic peptides, corresponding to the region of the polymorphism and containing either methionine or valine, to adopt a beta-sheet-rich structure similar to the pathological protein. Circular dichroism studies showed that the methionine-containing peptide has a greater propensity to adopt a beta-sheet conformation in a variety of experimental conditions. The higher beta-sheet tendency of this peptide was also associated with an increased ability to aggregate into amyloid-like fibrils. These results suggest that methionine at position 129 of the prion protein increases its susceptibility to switch to the abnormal conformation, in comparison with the presence of valine at the same position.     

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides.     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Slow spontaneous ��-to-�� structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP^C structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP^C nor for the formation of a stable prion structure.     

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.     

Atypical effect of salts on the thermodynamic stability of human prion protein

Prion diseases are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Previous studies indicate that salts promote conformational conversion of the recombinant prion protein into a PrPSc-like form. To gain insight into the mechanism of this effect, here we have studied the influence of a number of salts (sodium sulfate, sodium fluoride, sodium acetate, and sodium chloride) on the thermodynamic stability of the recombinant human prion protein. Chemical unfolding studies in urea show that at low concentrations (below approximately 50 mm), all salts tested significantly reduced the thermodynamic stability of the protein. This highly unusual response to salts was observed for both the full-length prion protein as well as the N-truncated fragments huPrP90-231 and huPrP122-231. At higher salt concentrations, the destabilizing effect was gradually reversed, and salts behaved according to their ranking in the Hofmeister series. The present data indicate that electrostatic interactions play an unusually important role in the stability of the prion protein. The abnormal effect of salts is likely because of the ion-induced destabilization of salt bridges (Asp144-Arg148 and/or Asp147-Arg151) in the extremely hydrophilic helix 1. Contrary to previous suggestions, this effect is not due to the interaction of ions with the glycine-rich flexible N-terminal region of the prion protein. The results of this study suggest that ionic species present in the cellular environment may control the PrPC to PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.     

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.     

PrP assemblies

The “protein only” hypothesis states that the key phenomenon in prion pathogenesis is the conversion of the host protein (PrPC) into a b-sheet enriched polymeric and pathogenic conformer (PrPSc). However the region of PrP bearing the information for structural transfer is still controversial. In a recent report, we highlighted the role of the C terminal part i.e. the helixes H2 and H3, using mutation approaches on recombinant PrP. The H2H3 was shown to be the minimal region necessary to reproduce the oligomerisation pattern of the full-length protein. The oligomers produced from isolated H2H3 domain presented the same structural characteristics as the oligomers formed from the full-length PrP. Combining other groups’ results, this paper further discusses the relative, direct or indirect role of different PrP regions in assembly. The H2H3 region represents the core of PrP oligomers and fibrils, whereas the N terminus could explain divergences among different aggregates. Finally this review evocates the possibility to separate the domain involved in prion information transference (i.e. prion replication) from the domain bearing the cytotoxicity properties.     

The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.     

The C-terminal globular domain of the prion protein is necessary and sufficient for import into the endoplasmic reticulum

The mammalian prion protein (PrP) is composed of an unstructured flexible N-terminal region and a C-terminal globular domain. We examined the import of PrP into the endoplasmic reticulum (ER) of neuronal cells and show that information present in the C-terminal globular domain is required for ER import of the N terminus. N-terminal fragments of PrP, devoid of structural domains located in the C terminus, remained in the cytosol with an uncleaved signal peptide and were rapidly degraded by the proteasome. Conversely, the separate C-terminal domain of PrP, comprising the highly ordered helix 2-loop-helix 3 motif, was entirely imported into the ER. As a consequence, two PrP mutants linked to inherited prion disease in humans, PrP-W145Stop and PrP-Q160Stop, were partially retained in the cytosol. The cytosolic fraction was characterized by an uncleaved N-terminal signal peptide and was degraded by the proteasome. Our study identified a new regulatory element in the C-terminal globular domain of PrP necessary and sufficient to promote import of PrP into the ER.     

Structural and Dynamic Properties of the Human Prion Protein

Prion diseases involve the conformational conversion of the cellular prion protein (PrP^C) to its misfolded pathogenic form (PrP^Sc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrP^C to PrP^Sc.     

The effect of β2-α2 loop mutation on amyloidogenic properties of the prion protein

Recent studies revealed that elk-like S170N/N174T mutation in mouse prion protein (moPrP), which results in an increased rigidity of β2-α2 loop, leads to a prion disease in transgenic mice. Here we characterized the effect of this mutation on biophysical properties of moPrP. Despite similar thermodynamic stabilities of wild type and mutant proteins, the latter was found to have markedly higher propensity to form amyloid fibrils. Importantly, this effect was observed even under fully denaturing conditions, indicating that the increased conversion propensity of the mutant protein is not due to loop rigidity but rather results from greater amyloidogenic potential of the amino acid sequence within the loop region of S170N/N174T moPrP.     

PrP assemblies: Spotting the responsible regions in prion propagation

The “protein only” hypothesis states that the key phenomenon in prion pathogenesis is the conversion of the host protein (PrP^C) into a β-sheet enriched polymeric and pathogenic conformer (PrP^Sc). However the region of PrP bearing the information for structural transfer is still controversial. In a recent report, we highlighted the role of the C terminal part i.e., the helixes H2 and H3, using mutation approaches on recombinant PrP. The H2H3 was shown to be the minimal region necessary to reproduce the oligomerization pattern of the full-length protein. The oligomers produced from isolated H2H3 domain presented the same structural characteristics as the oligomers formed from the full-length PrP. Combining other groups'' results, this paper further discusses the relative, direct or indirect role of different PrP regions in assembly. The H2H3 region represents the core of PrP oligomers and fibrils, whereas the N terminus could explain divergences among different aggregates. Finally this review evocates the possibility to separate the domain involved in prion information transference (i.e., prion replication) from the domain bearing the cytotoxicity properties.Key words: prion, H2H3, amyloid, domain of replication, unfolding, strain, polymer, fibersTransmissible spongiform encephalopathies (TSE), fatal neurodegenerative diseases affecting humans and other mammalians, induce in most cases loss of motor control and dementia. PrP is a protein physiologically present in parts of the animal kingdom (in mammals, birds, reptiles and fishes). According to the “protein-only” hypothesis,^1,2 the key phenomenon in the pathogenesis is the conversion of the α-helix rich host-encoded PrP form (PrP^C) into a pathogenic conformer (PrP^Sc) characterized by a higher content in β-sheet and a polymeric state. The conversion to an enriched β-sheet structure is supposed to be due to the modification—induced only by a PrP^Sc-like state acting as a template- of PrP^C into the PrP^Sc conformer. This hypothesis was first proposed by Griffith in 1967³ and revisited by Lansbury et al. in 1993.⁴ The prion hypothesis has now found increasing support from experimental evidence based on the synthetic production of β-sheeted recombinant PrP which shows pathogenic properties in a wide variety of physico-chemical conditions.^5–7 However, the molecular basis of prion conversion remains unclear, especially the various structural landscape of the PrP^Sc, which is the basis of the strain phenomenon.⁸To understand the mechanisms of transfer of the structural information, two mains issues have to be addressed: (1) we need to understand which region(s) of the protein act as template for conversion and (2) what is the “pathogenic” state of this domain. In this review, we shall assume that the region bearing the infectious information for replication and the region responsible for polymerization are identical. However, the link between the propensity of a domain to form aggregates and the ability to contain the necessary information for prion replication is far from being trivial. Generally the formation of amyloid assemblies results from the aggregation of disordered peptides or in some cases from disordered regions of a folded protein.⁸ If we consider that prion replication is only supported by the globular part of PrP⁹ the currently available model involves the folded domain. Since all structural transitions need at least a partial unfolding and refolding process, pre-required structural events should be considered prior to the conversion process.