| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 107 === Prion protein is a glycoprotein anchored on the membrane of neuron cells. The normal, cellular form (PrPC) is rich in α-helices. When PrPC is transformed to disease-causing form (PrPSc), β structures appear to dominate in prion protein. PrPSc is prone to association, leading to the formation of amyloid fibrils. This aggregation form of prion protein can induce neuronal death in the brain, which results in sponge-like holes, and finally lead to fatal consequence.
Little is known about the core regions where the structural conversion takes place and form intermolecular β structure (also known as cross-β structure). Our study aims to provide an insight in the structural features of the prion amyloid fibrils and the relationship with its amino acid sequence. Prion peptide (residue 108-144) from Syrian hamster is used as our target in this study. Seven hydrophobic, non-polar amino acid residues were picked out for substitution to cysteine in each mutant peptide respectively, serving as the spin labeling sites. The wild-type and mutant peptides were synthesized by solid-phase peptide synthesis and purified by HPLC. The mutant peptides were then labeled with MTSSL, featuring its methane thiosulfonate group and radical spin, on the side chain of cysteine residues (site-directed spin labeling). The spin-labeled peptide monomers were induced to form amyloid fibrils with or without adding seeds, which were prepared from the wild-type amyloid fibrils. The spin-labeled amyloid fibrils were further analyzed by electron spin resonance (ESR) spectroscopy for obtaining the information of relative proximity between spins. The morphologies of the amyloid fibrils were also examined by TEM to confirm the presence of fibrils.
Our ESR results revealed distinct features between peptides of different mutated sites. In the spectrograms of spontaneously formed fibrils, the linewidths could be ranked as A118R1 ≈ V121R1 > M134R1 ≈ L125R1 > A113R1 > M129R1 ≈ M138R1, and the curves of seeded fibrils were all relatively broadened except for M129R1. It is assumed that there might be two amyloid cores within the peptide 108-144, ranging from A113 to L125 and from M134 to M138 respectively. Between these two cores the residues around M129 stood out as a key region, which might be loose in structure but packed between the two cores. Our study can provide useful information to explain the location of the amyloid core in the process of prion protein propagation, and we hope to develop drugs against prion disease progression.
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