Study of the Formation and Degradation of Protein Amyloids

Amyloids are the aggregates of misfolded proteins. Because of the concern about amyloid diseases in the society, studies on amyloids become more and more popular. In this thesis, we studied the formation and degradation of protein amyloids related to two kinds of amyloid diseases, prion diseases and Alzheimer’s disease.
Prion diseases are also called transmissible spongiform encephalopathies. It has been known that these diseases result from the conformational transition of a host-encoded prion protein to a β-sheet-rich conformer. To study the process of this structural conversion, we utilized circular dichroism and fluorescence spectroscopies to monitor the amyloid fibril formation of various synthetic prion peptides and a recombinant mouse prion protein. Previous studies have shown that an α-N-acetyl- glucosamine (α-GlcNAc) attached to the Ser135 of the hamster prion peptide 108-144 inhibits fibril formation, so we tried to interfere the fibrillogenesis of human prion peptides 108-144 with glycosylated ones. Because there is a polymorphism at residue 129 of human prion protein, either methionine or valine, and this polymorphism modulates disease susceptibility, we synthesized twoα-GlcNAc-linked human prion peptides 108-144 to examine the effect of glycosylation and polymorphism on the fibrillogenesis. The results confirmed the significant inhibitory effect of glycosylation on fibril formation, and showed that fibrils formed more easily when the residue 129 is valine. To investigate the interference between glycosylated and unglycosylated peptides, we mixed one glycosylated peptide and one unglycosylated peptide with equal molar ratio and found that the glycosylated peptides prolonged the lag time of the unglycosylated one only a little and did not inhibit fibril growth of the unglycosylated peptide.
For the purpose of mimicking the conformational transition of prion protein more vividly, we tried to express and purify the mouse full-length recombinant prion protein with an intact disulfide bond, and converted this protein into amyloid fibrils successfully. With this cell-free conversion system, we can examine whether there is interference or seeding effect between the full-length prion protein and the prion peptide.
Alzheimer’s disease (AD) is one of the most common senile dementia. Amyloid-β peptide (Aβ) has been implicated in the etiology of AD and dysfunction in Aβ clearance is crucial for the accumulation of Aβ in AD brains. There is an equilibrium of Aβ level in the brain and the blood, and removing Aβ, either in the soluble form or amyloid form, in the blood by enzymes might help decrease Aβ content in the brain. In the third part of my study, we focused on the breakdown of the amyloid fibrils formed from Aβ with nattokinase, which is well-known for its fibrinolytic activity and regarded as a healthy food, and compare the fibril-degrading efficiency of nattokinase with the other two subtilisin-like enzymes, proteinase K and PWD-1 keratinase. Our results showed that nattokinase has the potential in clearing Aβ fibrils in vitro, and all three enzymes tested have similar fibril-degrading efficiency.