Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo.

Generation of genuine prion infectivity by serial PMCA.

Prions are the causative infectious agents of transmissible spongiform encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the structural conversion of cellular prion protein (PrP(C)) as efficiently as PrP(Sc) from the brain of scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as postulated by the prion hypothesis.

Cell-free formation of misfolded prion protein with authentic prion infectivity.

Prion propagation has been modeled in vitro; however, the low infectious titer of PrP(Sc) thus generated has cast doubt on the "protein-only" hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP(Sc) and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP(Sc) provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.

Photo-induced crosslinking of prion protein oligomers and prions.

Prion diseases are caused by a unique type of infectious agent, which is thought to consist of a misfolded beta-sheeted form of the alpha-helical cellular prion protein (PrPC). This misfolded isoform (PrPSc) tends to form insoluble amyloid-like aggregates, impeding classical structural analysis by X-ray crystallography or NMR. Intermolecular crosslinking may provide a means of stabilizing notoriously elusive oligomers for further analysis and may be used for analyzing aggregate architecture by characterising intermolecular contact sites.

Conversion efficiency of bank vole prion protein in vitro is determined by residues 155 and 170, but does not correlate with the high susceptibility of bank voles to sheep scrapie in vivo.

The misfolded infectious isoform of the prion protein (PrP(Sc)) is thought to replicate in an autocatalytic manner by converting the cellular form (PrP(C)) into its pathogenic folding variant. The similarity in the amino acid sequence of PrP(C) and PrP(Sc) influences the conversion efficiency and is considered as the major determinant for the species barrier. We performed in vitro conversion reactions on wild-type and mutated PrP(C) to determine the role of the primary sequence for the high susceptibility of bank voles to scrapie.

Breakage of PrP aggregates is essential for efficient autocatalytic propagation of misfolded prion protein.

The conversion of cellular prion protein (PrP(C)) to the disease-associated misfolded isoform (PrP(Sc)) is an essential process for prion replication. This structural conversion can be modelled in protein misfolding cyclic amplification (PMCA) reactions in which PrP(Sc) is inoculated into healthy hamster brain homogenate, followed by cycles of incubation and sonication. In serial transmission PMCA experiments it has recently been shown that the protease-resistant PrP obtained in vitro (PrPres) is generated by an autocatalytic mechanism.