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. Different conversion efficiencies obtained with bank vole and mouse PrP C in reactions with several prion strains were due to differences at amino acid residues 155 and 170. However, the conversion efficiencies obtained with mouse and vole PrP C in reactions with sheep scrapie did not correlate with the susceptibility of the respective species to this prion strain. This discrepancy between in vitro and in vivo data may indicate that at least in the case of scrapie transmission to bank voles additional host factors can strongly modulate the species barrier. Furthermore, in vitro conversion reactions with different prion strains revealed that the degree of alteration of the conversion efficiency induced by amino acid exchanges was varying according to the prion strain. These results support the assumption that the repertoire of conformations adopted by a certain PrP C primary sequence is decisive for its convertibility to the strain-specific PrP Sc conformation.Transmissible spongiform encephalopathies (TSEs), 2 or prion diseases, are a group of neurodegenerative diseases, including CreutzfeldtJakob disease of humans, scrapie of sheep, and bovine spongiform encephalopathy (BSE) of cattle and are caused by a new class of unusual pathogens termed prions (1). Prion diseases are associated with the accumulation of an abnormal, partially protease-resistant isoform of the cellular prion protein (PrP C ) in the brain of affected individuals. This disease-related isoform, PrP Sc , is identical to PrP C with respect to amino acid sequence and chemical post-translational modifications and, according to the "protein-only" hypothesis, is the major if not the only constituent of the infectious agent (2, 3). The three-dimensional structure of PrP C is characterized by an unstructured N terminus and a globular C-terminal domain, consisting of three ␣-helices with a short stretch of -sheet (4, 5). In contrast to PrP C with its high proportion of ␣-helices, circular dichroism analysis and Fourier transform infrared spectroscopy studies revealed that the predominant structural element of PrP Sc is -sheet (6). Reduction of the -sheet content in PrP Sc preparations leads to a diminished level of infectivity, suggesting that the conversion from ␣-helices into -sheets is the fundamental event in PrP Sc formation as well as for propagating prion infectivity (7,8). PrPSc is postulated to replicate in an autocatalytic manner by acting as a conformational template that promotes the conversion of PrP C into its p...
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.clearance ͉ nitrocellulose ͉ protein misfolding cyclic amplication
15Prions are the causative infectious agents of transmissible spongiform 16 encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the 17 prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) 18 experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the 19 structural conversion of cellular prion protein (PrP C ) as efficiently as PrP Sc from the brain of 20 scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as 21 postulated by the prion hypothesis. However, the fact that PrPres generated in vitro was 22 associated with approximately ten times less infectivity than an equivalent quantity of brain-23 derived PrPSc casts doubt on the "protein-only" hypothesis of prion propagation and backs 24 theories that suggest there are additional molecular species of infectious PrP or other agent-25 associated factors. By combining sPMCA with prion delivery on suitable carrier particles we 26 were able to resolve the apparent discrepancy between the amount of PrPres and infectivity 27 which we were then able to relate to differences in the size distribution of PrP aggregates and 28 consecutive differences in regard to biological clearance. These findings demonstrate that we 29 have designed an experimental set-up yielding in vitro generated prions that are 30 indistinguishable from prions isolated from scrapie-infected hamster brain in terms of 31 proteinase K resistance, autocatalytic conversion activity, and -most notably -specific 32 biological infectivity. 33 34
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. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.
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