The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.

Prion diseases are associated with the conversion of the ␣-helix rich prion protein (PrP C ) into a ␤-structure-rich insoluble conformer (PrP Sc ) that is thought to be infectious. The mechanism for the PrP C 3 PrP Sc conversion and its relationship with the pathological effects of prion diseases are poorly understood, partly because of our limited knowledge of the structure of PrP Sc . In particular, the way in which mutations in the PRNP gene yield variants that confer different susceptibilities to disease needs to be clarified. We report here the 2.5-Å-resolution crystal structures of three scrapie-susceptibility ovine PrP variants complexed with an antibody that binds to PrP C and to PrP Sc ; they identify two important features of the PrP C 3 PrP Sc conversion. First, the epitope of the antibody mainly consists of the last two turns of ovine PrP second ␣-helix. We show that this is a structural invariant in the PrP C 3 PrP Sc conversion; taken together with biochemical data, this leads to a model of the conformational change in which the two PrP C Cterminal ␣-helices are conserved in PrP Sc , whereas secondary structure changes are located in the N-terminal ␣-helix. Second, comparison of the structures of scrapie-sensitivity variants defines local changes in distant parts of the protein that account for the observed differences of PrP C stability, resistant variants being destabilized compared with sensitive ones. Additive contributions of these sensitivity-modulating mutations to resistance suggest a possible causal relationship between scrapie resistance and lowered stability of the PrP protein.

The prion protein (PrP) propensity to adopt different structures is a clue to its biological role. PrP oligomers have been previously reported to bear prion infectivity or toxicity and were also found along the pathway of in vitro amyloid formation. In the present report, kinetic and structural analysis of ovine PrP (OvPrP) oligomerization showed that three distinct oligomeric species were formed in parallel, independent kinetic pathways. Only the largest oligomer gave rise to fibrillar structures at high concentration. The refolding of OvPrP into these different oligomers was investigated by analysis of hydrogen/deuterium exchange and introduction of disulfide bonds. These experiments revealed that, before oligomerization, separation of contacts in the globular part (residues 127-234) occurred between the S1-H1-S2 domain (residues 132-167) and the H2-H3 bundle (residues 174 -230), implying a conformational change of the S2-H2 loop (residues 168 -173). The type of oligomer to be formed depended on the site where the expansion of the OvPrP monomer was initiated. Our data bring a detailed insight into the earlier conformational changes during PrP oligomerization and account for the diversity of oligomeric entities. The kinetic and structural mechanisms proposed here might constitute a physicochemical basis of prion strain genesis.folding ͉ kinetics ͉ oligomers ͉ strain

The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an ␣-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display ␤-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a ␤-sheet structure. Dynamic light scattering revealed that the presence of ␤-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.

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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 ␤-rich structure.Transmissible spongiform encephalopathies are fatal neurodegenerative pathologies that affect humans as well as several other mammalian species. They are thought to be caused by the aggregation and misfolding of the prion protein (PrP).7 According to the "protein-only" hypothesis (1-3), PrP undergoes an ␣-to-␤ transition from its native state (PrP c ) to a misfolded species (PrP sc ), which is believed to act as a template to "infect" and misfold other PrP copies. As in other misfolding pathologies such as Alzheimer and Parkinson diseases, the neurotoxicity of PrP Sc is thought to be associated to an oligomeric form of the protein rather than to the mature aggregates (4).One of the crucial questions that remains unanswered concerns which region(s) of PrP promotes the polymerization process; this information would be both the key for understanding cross-species infectivity and help in decoding the bases of the aggregation process. Different regions have been proposed to be the fibrillogenic seed. PrP c consists of an unstructured N-terminal tail and a folded C-terminal domain formed by three helices (H1, H2, and H3) and a short-stranded ␤-sheet (formed by S1 and S2). H2 and H3 are connected through a disulfide bridge (5). A common view suggests the S1H1S2 region is crucial for ␤-sheet seeding and PrP Sc formation (6, 7). H1 has been implicated as a primary interaction site between PrP Sc and PrP c (8, 9), whereas the loop between S2 and H2, a rigid loop stabilized by its long range interactions with H3 (10), and the C terminus of H3 has been suggested to be recognized by a "Protein-X" that would affect the conversion of PrP c into PrP Sc (11). A study based on intrachain distance estimation performed on tagged PrP amyloid fibrils obtained under chaotropic treatment suggests the involvement of the H2H3 domain of PrP in amyloid formation (12). H/D exchange studies of the amyloid fibrils from human PrP reveal that the ␤-sheet core of PrP amyloids is formed by H2, the major part of H3, and the loop between them (13, 14).We have fol...

The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase-transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P-P and N-P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120-150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N.

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