Abstract
BackgroundIt is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.Methodology/Principal FindingsAs revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.Conclusions/SignificanceWe demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2) have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.
Highlights
Transmissible spongiform encephalopathies, known as prion diseases, are infectious fatal neurodegenerative diseases that affect the nervous system in humans and animals [1]
Amyloid fibrils formed by human chimera had secondary structures and proteinase K (PK)-resistant features similar to those formed by the human prion protein (PrP) Our previous studies [27,28] have shown that macromolecular crowding significantly accelerated fibril formation of the human PrP
We demonstrated that two rabbit PrP chimeras we designed did form amyloid fibrils with different structural features in absence and presence of crowding agents
Summary
Transmissible spongiform encephalopathies, known as prion diseases, are infectious fatal neurodegenerative diseases that affect the nervous system in humans and animals [1]. The Baskakov lab has provided the first demonstration that recombinant full-length prion protein (PrP) with an intact S-S bond can be folded into amyloid conformation in vitro [13]. De novo rabbit prions have been produced by rabbit brain homogenate in vitro from unseeded material [17] All of these works suggest that amyloid fibrils generated in vitro are infectious something like PrPSc and there could be some similarities between them. It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. We have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs
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