| Transmissible spongiform encephalopathies, also called prion diseases, are a large group of neurodegenerative diseases occurred widely in a variety of mammals and humans. They are sporadic, inherited, or acquired. Prion hypothesis postulates that the infectious agent is protein without nuclein acid, called prion. The pathogenicity and infectivity of such agent is that a-helical rich normal prion protein convert to a β-sheeted pathogenic isoform.Lipid appears to be an important cofactor because of its unique biophysical properties. At first, lipid binds prion protein with electrostatic interaction and it has association with prion protein hydrophobically. The interaction makes a-helical rich normal conformation lower the energy barrier to PK-resistent β-sheeted conformation. So the conformational conversion of prion protein under hydrophobic condition is an important step in folding protein to pathogenic isoform. In the thsis we first stimulated hydrophobic environment with methanol, ethanol and propanol. We checked the secondary conformational conversion of recombinant prion protein in hydrophobic environment by CD, PK-resistance by PK digestion, and aggregation by ultracentrifugation. We found that a-helical decreased with the increasing of hydrophobicity.With no difference of PK enzyme activity in different organic solvent, recombinant prion protein had PK-resistance and soluble PK-resistent conformation for the first time.In recent studies on prion conformational conversion, serial protein misfolding cyclic amplificaiton assay is widely used. With revealing the importance of lipid in conformational conversion of prion protein, it is added in sPMCA system as an important cofactor. Bacterially expressed recombinant prion protein, POPG, Triton and RNA could generated highly infectious prion with physical treatments of incubation at constant temperature and sonication. But it still remains unclear that the role of each cofactor in prion propagating. We detected the PK-resistant conformer (rPrP-res) propogated in substrate in sPMCA without each cofactor respectively. Results showed that rPrP-res conformer couldn’t be propagated as the substrate without Triton and RNA; but when the substrate was lack of POPG, rPrP-res conformer could be propagated well. It demonstrated that cofactors in sPMCA system played important roles in propagating rPrP-res conformer. Though the substrate lack of POPG could help to propagate rPrP-res conformer, it may be due to Triton form the hydrophobic environment instead of POPG.Using sPMCA system as describe above, we previously reported de novo formation of highly infectious prions in Ohio State University and our lab in East China Normal University, called rPrP-resOSU and rPrP-res17kD. The rPrP-resosu and rPrP-res17kD caused prion disease in wild-type mice with survival time around 150 days and 172 days respectively. We compared the neuropathology caused by these two recombinant prions and their biochemical properties in this study, and we found there was difference between these two separately initiated recombinant prions. Then these two prions were propageted in the same physical treatments and the latter prions were called rPrP-resOSU/ECNU and rPrP-res17kD/ECNU. We compared biochemical properties, then infected mice to compare neuropathology. The results showed that the significant biochemical properties between two prions previous disappeared gradually after propagating in the same environment; And the difference on neuropathology previous had changed, too. It revealed that not a single PrP conformer, but multiple PrP conformers were capable of supporting prion infectivity. And the conformer would change along with the environment changing.In summary, there was large influence of cofactors and physical condition in sPMCA system on conformational conversion of prion protein. The work providing plausible explanation of conformational conversion supported "prion hypothesis". |
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