Formation of native prions from minimal components in vitro - PubMed (original) (raw)

Comparative Study

. 2007 Jun 5;104(23):9741-6.

doi: 10.1073/pnas.0702662104. Epub 2007 May 29.

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Comparative Study

Formation of native prions from minimal components in vitro

Nathan R Deleault et al. Proc Natl Acad Sci U S A. 2007.

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Abstract

The conformational change of a host protein, PrP(C), into a disease-associated isoform, PrP(Sc), appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt-Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP(C) and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP(Sc) molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP(Sc) molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP(Sc) molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP(C) molecules, copurified lipid molecules, and a synthetic polyanion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Silver stain analysis of purified PrPC substrate. Twelve percent SDS/PAGE showing (from left to right): crude, detergent-solubilized brain supernatant; preparation mock-purified from Prnp0/0 mouse brains; preparation purified from normal hamster brains; and molecular weight markers.

Fig. 2.

Fig. 2.

Seeded PrPSc propagation by using purified substrates. (A) Schematic diagram of serial dilution and propagation paradigm used in subsequent experiments, adapted from Castilla et al. (22). (B and C) Western blots showing samples subjected to 16 rounds of PMCA, serial dilution, and propagation as depicted in A. (B) Samples originally seeded with Sc237 PrP27-30 were propagated in substrate containing either poly(A) RNA alone (top gel), purified PrPC alone (middle gel), or PrPC plus poly(A) RNA (bottom gel). (C) Samples originally seeded with 139H PrP27-30 were propagated in substrate containing either purified PrPC alone (top gel), or poly(A) PrPC plus poly(A) RNA (bottom gel). In all gels, a sample containing PrPC not subjected to proteinase K digestion is shown in the first lane as a reference for comparison of electrophoretic mobility (PrPC-PK). All other samples were subjected to limited proteolysis with 50 μg/ml proteinase K (+PK).

Fig. 3.

Fig. 3.

Formation of PrPSc molecules de novo during serial PMCA propagation of unseeded purified substrates. (A) Western blot showing unseeded samples containing PrPC plus poly(A) RNA subjected to 16 rounds of PMCA, serial dilution, and propagation. A sample containing PrPC not subjected to proteinase K digestion is shown in the first lane as a reference for comparison of electrophoretic mobility (PrPC-PK). All other samples were subjected to limited proteolysis with 50 μg/ml proteinase K (+PK). (B) Representative slot blot showing the formation of protease-resistant PrP molecules de novo in multiple propagation experiments. Unseeded samples were propagated for 15 rounds in either the presence or absence of poly(A) RNA, as indicated by plus (+) and minus (−) symbols, respectively. In experiments designated by asterisks, propagation was not carried beyond the 12th round because a preliminary assay performed at that stage already detected PrPSc in several preceding rounds. The experiment designated by the # symbol was performed entirely in a prion-free laboratory. (C) Histogram showing the temporal distribution of the first detectable PrPSc signals during serial propagation of unseeded PrPC plus poly(A) RNA substrate mixtures in 10 separate propagation experiments, 7 of which were carried out simultaneously in the same sonicator.

Fig. 4.

Fig. 4.

Representative histological fields of the CA2 hippocampus region in control animals and animals inoculated with _in vitro_-generated PrPSc molecules. Rows from top to bottom: (top row) uninoculated, normal 190 day old hamster; (second row) terminally ill hamster inoculated with Sc237-seeded, serially propagated PrPSc molecules; (third row) terminally ill hamster inoculated with 139H-seeded, serially propagated PrPSc molecules; (bottom row) terminally ill hamster inoculated with spontaneously generated, serially propagated PrPSc molecules. Hematoxylin and eosin (H&E) staining, as well as glial fibrillary acidic protein (GFAP) and PrP immunohistochemical staining results are shown for each group. (Scale bar, 100 μm.)

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