Polymorphism at residue 129 modulates the conformational conversion of the D178N variant of human prion protein 90-231.

One of the arguments in favor of the protein-only hypothesis of transmissible spongiform encephalopathies is the link between inherited prion diseases and specific mutations in the PRNP gene. One such mutation (Asp178 --> Asn) is associated with two distinct disorders: fatal familial insomnia or familial Creutzfeldt-Jakob disease, depending upon the presence of Met or Val at position 129, respectively. In this study, we have characterized the biophysical properties of recombinant human prion proteins (huPrP90-231) corresponding to the polymorphic variants D178N/M129 and D178N/V129. In comparison to the wild-type protein, both polymorphic forms of D178N huPrP show a greatly increased propensity for a conversion to beta-sheet-rich oligomers (at acidic pH) and thioflavine T-positive amyloid fibrils (at neutral pH). Importantly, the conversion propensity for the D178N variant is strongly dependent upon the M/V polymorphism at position 129, whereas under identical experimental conditions, no such dependence is observed for the wild-type protein. Amyloid fibrils formed by wild-type huPrP90-231 and the D178N variant are characterized by different secondary structures, and these structures are further modulated by residue 129 polymorphism. Although on the basis of only in vitro data, this study strongly suggests that polymorphism-dependent phenotypic variability of familial prion diseases may be linked to differences in biophysical properties of prion protein variants.

Molecular basis of barriers for interspecies transmissibility of mammalian prions.

Spongiform encephalopathies are believed to be transmitted by a unique mechanism involving self-propagating conformational conversion of prion protein into a misfolded form. Here we demonstrate that fundamental aspects of mammalian prion propagation, including the species barrier and strain diversity, can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop. Our data show that species-specific substitution of a single amino acid in a critical region completely changes the seeding specificity of prion protein fibrils. Furthermore, we demonstrate that sequence-based barriers that prevent cross-seeding between proteins from different species can be bypassed, and new barriers established, by a template-induced adaptation process that leads to the emergence of new strains of prion fibrils. Although the seeding barriers observed in this study do not fully match those seen in animals, the present findings provide fundamental insight into mechanistic principles of these barriers at a molecular level.

Nucleation-dependent conformational conversion of the Y145Stop variant of human prion protein: structural clues for prion propagation.

One of the most intriguing disease-related mutations in human prion protein (PrP) is the Tyr to Stop codon substitution at position 145. This mutation results in a Gerstmann-Straussler-Scheinker-like disease with extensive PrP amyloid deposits in the brain. Here, we provide evidence for a spontaneous conversion of the recombinant polypeptide corresponding to the Y145Stop variant (huPrP23-144) from a monomeric unordered state to a fibrillar form. This conversion is characterized by a protein concentration-dependent lag phase and has characteristics of a nucleation-dependent polymerization. Atomic force microscopy shows that huPrP23-144 fibrils are characterized by an apparent periodicity along the long axis, with an average period of 20 nm. Fourier-transform infrared spectra indicate that the conversion is associated with formation of beta-sheet structure. However, the infrared bands for huPrP23-144 are quite different from those for a synthetic peptide PrP106-126, suggesting conformational non-equivalence of beta-structures in the disease-associated Y145Stop variant and a frequently used short model peptide. To identify the region that is critical for the self-seeded assembly of huPrP23-144 amyloid, experiments were performed by using the recombinant polypeptides corresponding to prion protein fragments 23-114, 23-124, 23-134, 23-137, 23-139, and 23-141. Importantly, none of the fragments ending before residue 139 showed a propensity for conformational conversion to amyloid fibrils, indicating that residues within the 138-141 region are essential for this conversion.

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.