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We illustrate the use of statistical tools (asymptotic theories of standard error quantification using appropriate statistical models, bootstrapping, and model comparison techniques) in addition to sensitivity analysis that may be employed to determine the information content in data sets. We do this in the context of recent models [S. Prigent, A. Ballesta, F. Charles, N. Lenuzza, P. Gabriel, L.M. Tine, H. Rezaei, and M. Doumic, An efficient kinetic model for assemblies of amyloid fibrils and its application to polyglutamine aggregation, PLoS ONE 7 (2012), e43273. doi:10.1371/journal.pone.0043273.] for nucleated polymerization in proteins, about which very little is known regarding the underlying mechanisms; thus, the methodology we develop here may be of great help to experimentalists. We conclude that the investigated data sets will support with reasonable levels of uncertainty only the estimation of the parameters related to the early steps of the aggregation process.

Neurodegenerative diseases constitute a large group of affections characterized by brain dysfunction and degeneration, often occurring with advancing age. Among them, prion diseases are fatal transmissible spongiform encephalopathies (TSEs) affecting Humans (Creutzfeldt-Jacob disease) and animals (BSE or the "Mad cow" disease). The causes of prion diseases have long been debated and it is widely accepted by the scientific community that a protein, namely, the prion protein (PrP), plays a critical role in the disease development. The prion protein or PrP is present in two structurally distinct forms: the natural cellular form (PrPc) and an abnormal polymeric form (PrPsc), which is at the origin of fibrillar deposits found in affected brains. PrPc has been shown to convert into small toxic oligomeric species but the mechanisms of oligomerization are not fully understood. In this study, we have combined Molecular Dynamics (MD) simulations and Small Angle X-ray Scattering (SAXS) to investigate the critical unfolding steps of the ovine prion protein (OvPrP) and to determine the low resolution structure of some of the oligomeric species obtained from various truncated forms of the OvPrP.We show that the domain formed by Helix2 and Helix3 (H2H3) is critical in the unfolding of OvPrP and its conversion of into a beta-rich conformer. The analysis also suggests the incorporation of 9-10 monomer units for all monomeric species studied. We therefore propose that the minimal β-rich core of OvPrP-O3 oligomer is formed by the H2H3 domain while the N-terminal domain either combines with other N-terminal domains to form a second domain or surrounds the oligomeric core.

International audience; Prion diseases are fatal neurodegenerative diseases characterized by the formation of β-rich oligomers and the accumulation of amyloid fibrillar deposits in the central nervous system. Understanding the conversion of the cellular prion protein into its β-rich polymeric conformers is fundamental to tackling the early stages of the development of prion diseases. In this paper, we have identified unfolding and refolding steps critical to the conversion into a β-rich conformer for different constructs of the ovine prion protein by molecular dynamics simulations. By combining our results with in vitro experiments, we show that the folded C-terminus of the ovine prion protein is able to recurrently undergo a drastic conformational change by displacement of the H1 helix, uncovering of the H2H3 domain, and formation of persistent β-sheets between H2 and H3 residues. The observed β-sheets refold toward the C-terminus exposing what we call a "bending region" comprising residues 204-214. This is strikingly coincident with the region harboring mutations determining the fate of the prion oligomerization process. The β-rich intermediate is used here for the construction of a putative model for the assembly into an oligomeric aggregate. The results presented here confirm the importance of the H2H3 domain for prion oligomer formation and therefore its potential use as molecular target in the design of novel prion inhibitors.

International audience; The concept of prion is applied to protein modules that share the ability to switch between at least two conformational states and transmit one of these through intermolecular interaction and change of conformation. Although much progress has been achieved through the understanding of prions from organisms such as Saccharomyces cerevisiae, Podospora anserina, or Aplysia californica, the criteria that qualify a protein module as a prion are still unclear. In addition, the functionality of known prion domains fails to provide clues to understand the first identified prion, the mammalian infectious prion protein, PrP. To address these issues, we generated mammalian cellular models of expression of the C-terminal two helices of PrP, H2 and H3, which have been hypothesized, among other models, to hold the replication and conversion properties of the infectious PrP. We found that the H2H3 domain is an independent folding unit that undergoes glycosylations and glycosylphosphatidylinositol anchoring similar to full-length PrP. Surprisingly, in some conditions the normally folded H2H3 was able to systematically go through a conversion process and generate insoluble proteinase K-resistant aggregates. This structural switch involves the assembly of amyloid structures that bind thioflavin S and oligomers that are reactive to A11 antibody, which specifically detects protein oligomers from neurological disorders. Overall, we show that H2H3 is a conformational switch in a cellular context and is thus suggested to be a candidate for the conversion domain of PrP.

International audience; The "protein only" hypothesis states that the key phenomenon in prion pathogenesis is the conversion of the host protein (PrPC) into a beta-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.

Aggregation and misfolding of the prion protein (PrP) are thought to be the cause of a family of lethal neurodegenerative diseases affecting humans and other animals. Although the structures of PrP from several species have been solved, still little is known about the mechanisms that lead to the misfolded species. Here, we show that the region of PrP comprising the hairpin formed by the helices H2 and H3 is a stable independently folded unit able to retain its secondary and tertiary structure also in the absence of the rest of the sequence. We also prove that the isolated H2H3 is highly fibrillogenic and forms amyloid fibers morphologically similar to those obtained for the full-length protein. Fibrillization of H2H3 but not of full-length PrP is concomitant with formation of aggregates. These observations suggest a "banana-peeling" mechanism for misfolding of PrP in which H2H3 is the aggregation seed that needs to be first exposed to promote conversion from a helical to a beta-rich structure.