SUMMARYFibril 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 smallest possible fibrillar fragments. Using amyloid fibrils produced from fulllength mammalian PrP under three different growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable was found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires cross-β structure. Using atomic force microscopy imaging we found that fibril fragmentation occurred in both directions, perpendicular to and along of fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins including α-B-crystalline, 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 primary structure of the prion protein (PrP) is believed to be the key factor in regulating the species barrier of prion transmission. Because the strength of the species barrier was found to be affected by the prion strain, the extent to which the barrier can indeed be attributed to differences in the PrP primary structures of either donor and acceptor species remains unclear. In this study, we exploited the intrinsic property of PrP to polymerize spontaneously into disease-related amyloid conformations in the absence of a strain-specified template and analyzed polymerization of mouse and hamster full-length recombinant PrPs. Unexpectedly, we found no evidence of species specificity in cross-seeding polymerization assays. Even when both recombinant PrP variants were present in mixtures, preformed mouse or hamster fibrils displayed no selectivity in elongation reactions and consumed equally well both homologous and heterologous substrates. Analysis of individual fibrils revealed that fibrils can elongate in a bidirectional or unidirectional manner. Our work revealed that, in the absence of a cellular environment, post-translational modifications, or strain-specified conformational constraints, PrP fibrils are intrinsically promiscuous and capable of utilizing heterologous PrP variants as a substrate in a highly efficient manner. This study suggests that amyloid structures are capable of accommodating local perturbations arising because of a mismatch in amino acid sequences and highlights the promiscuous nature of the self-propagating activity of amyloid fibrils.

In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttranslational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant nonpeptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with a N-myristoylamido-maleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to offpathway aggregation, and (ii) it prohibited assembly of filaments into higher-order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher-order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure, and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core, but controls the mode of lateral assembly of filaments into higher-order fibrils.Conversion of proteins and peptides into amyloid fibrils is linked to several neurodegenerative or conformational maladies including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion diseases [1;2]. Most amyloidogenic proteins involved in these diseases are cytosolic and do not contain any significant post-translational modifications. Prion protein is unique in this respect. PrP is modified with either one or two polysaccharides [3] and a GPI anchor, which attaches PrP to the plasma membrane [4]. However, like other amyloidogenic proteins, PrP adopts a fibrillar shape, referred to as scrapie associated fibrils or prion rods, when converted into the disease-associated form [5][6][7]. Therefore, PrP fibril *To whom correspondence should be addressed: 725 W. Lombard St., Baltimore, MD 21201. Phone: 410-706-4562; FAX: 410-706-8184. Email: Baskakov@umbi.umd formation offers a unique opportunity to evaluate the effect of biologically relevant natural modifications consisting of polysaccharides and/or hydrophobic anchor, both of non-peptide nature, on acquiring the amyloid conformation.GPI anchor can impact prion po...