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Title: Recombinant PrPSc shares structural features with brain-derived PrPSc suggesting that they have a similar architecture: Insights from limited proteolysis

Author
item SEVILLANO, ALEJANDRO - University Of Santiago De Compostela
item FERNÁNDEZ-BORGES, NATALIA - Cic Biogune
item YOUNAS, NEELAM - University Of Santiago De Compostela
item WANG, FEI - University Of Santiago De Compostela
item ELEZGARAI, SAIOA - Cic Biogune
item BRAVO, SUSANA - University Of Alberta
item VÁZQUEZ-FERNÁNDEZ, ESTER - University Of Alberta
item ROSA, ISAA - University Of Santiago De Compostela
item ERAÑA, HASIER - Cic Biogune
item GIL, DAVID - Cic Biogune
item VEIGA, SONIA - University Of Santiago De Compostela
item VIDAL, ENRIC - University Of Santiago De Compostela
item Erickson-Beltran, Melissa
item GUITIÁN, ESTEBAN - Istituto Superiore Di Sanità
item Silva, Christopher - Chris
item NONNO, ROMOLO - Basque Foundation For Science
item MA, JIVAN - Van Andel Research Institute
item CASTILLA, JOAQUÍN - Basque Foundation For Science
item REQUENA, JESÚS - University Of Santiago De Compostela

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2017
Publication Date: 1/31/2018
Citation: Sevillano, A.M., Fernández-Borges, N., Younas, N., Wang, F., Elezgarai, S.R., Bravo, S., Vázquez-Fernández, E., Rosa, I., Eraña, H., Gil, D., Veiga, S., Vidal, E., Erickson-Beltran, M.L., Guitián, E., Silva, C.J., Nonno, R., Ma, J., Castilla, J., Requena, J.R. 2018. Recombinant PrPSc shares structural features with brain-derived PrPSc suggesting that they have a similar architecture: Insights from limited proteolysis. PLoS Pathogens. 14(1):e1006797. https://doi.org/10.1371/journal.ppat.1006797.
DOI: https://doi.org/10.1371/journal.ppat.1006797

Interpretive Summary: Prions are proteins that cause disease by converting a normal cellular prion protein (PrPC) into a prion (PrPSc). They are contagions that cause serious disease in agriculturally important animals, including sheep, goats, deer, elk, and cattle. Both PrPC and PrPSc have identical chemical structures and only differ in their shapes. Other researchers have shown that the shape of a prion is composed a specific chemical feature (ß-sheets). A prion is formed by four coils (like a garden hose) of these ß-sheets. The formal name for this structure is a 4-rung-ß-solenoid. These 4-rung-ß-solenoids can be stacked to form prion fibers. We prepared simplified infectious prions (recPrPSc) from the simplest protein samples possible and used molecular scissors (enzymes) to cut them into pieces. We were able to analyze those pieces to determine the diameter of the coils and which parts (amino acids) of the protein made up the individual coils. We showed that recPrPSc had the same structure as more complicated prions. We also showed that recPrPSc can be used to study the structure of prions.

Technical Abstract: An extensive body of experimental and spectroscopic evidence supports the hypothesis that PrPSc is a multimer of 4-rung ß-solenoids, and that individual PrPSc solenoids stack to form amyloid fibers. We recently used limited proteolysis to map the ß-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope mapping, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of ß-strands, and help us to predict the threading of the ß-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting both infectivity and attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments of similar length as those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest that the architecture of recPrPSc is similar to that of brain-derived PrPSc. The observed differences are due to subtle differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.