| Transmissible spongiform encephalopathies, also known as prion diseases, is a infectious fatal neurodegenerative disease exiting in most mammal animals and including BSE, Scrapie, CJD and FFI, but rabbits are among the few animal species that have resistance to prions from other animal species. The key event of such diseases is the conversion from cellular prion protein (PrPc) to scrapie isoform of prion protein (PrP Se), but no one can clarify it. There is mounting evidence that amyloid fibrils formed by recomibinant prion protein (PrP) can induce the prion diseases in animals, and we can figure that there are some commonality between amyloid fibrils in vitro and PrPSc in vivo. Our previous work indicates the structure of amyloid fibris formd by human/bovine PrP is different from that formed by rabbit PrP and macromolecular crowding can accelerate amyloid formation of the human/bovine PrP while inhibit that of rabbit prion protein. However, the mechanism of this phenomenon is not clear.In this study, we constructed two PrP chimeras, in one of which the H2H3domain of the human PrP was replaced with that of the rabbit PrP (termed rabbit chimera) and in another of which the H2H3domain of the rabbit PrP is replaced with that of the human PrP (termed human chimera), to analysis how domain replacement affects fibril formation of the rabbit/human PrPs, according to many similarities in their amino sequence and three-dimensional structure. As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent (Ficoll70or Ficoll400) dramatically promoted fibril formation of rabbit chimera and human chimera. The results of circular dichroism (CD), PK digestion assays and Fourier transform infrared (FTIR) show that 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. The results from transmission electron microscopy (TEM) show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.Then we investigate the fibrillization of human and rabbit PrP-H2H3fragment in vitro. CD, FTIR and thioflavin T binding assays showed that human and rabbit PrP-H2H3both formed amyloid fibrils with P-sheet-rich conformation, and the addtion of macromolecular crowding agent can shorten their lag time but inhibit their fibril elongation steps. There is little difference between the fibrillization of human and rabbit PrP-H2H3fragment in vitro.We also study the effect of B1H1B2domains (β-strand1, α-helix1, and β-strand2) beyond PrP-H2H3on fibrillization of the rabbit PrP and human PrP and the results demonstrate that the exchange of B1H1B2domain have almost no effect on fibrillization of the human PrP but a remarkable effect on fibrillization of the rabbit PrP. The evidence that the fibrillization of chimera BR, in which the H2H3domain of the bovine PrP was replaced with that of the rabbit PrP, is similar to human/bovine PrP fibrillization indicate the fibrillization of the rabbit PrP is not only influnced by its H2H3domain.These findings may help to better understand why human PrP in vivo tends to form fibril deposits associated with serious infectious diseases while the rabbit PrP does not and thus is unlikely that will cause prion diseases... |
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