探討普利昂蛋白之八胜肽重複區對其纖維化之影響

The Effect of Octapeptide on Prion Fibril Conversion

黃玫毓(Mei-Yu Huang)

指導教授 ： 李政怡

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2015年)

摘要

普利昂疾病屬於神經退化性疾病的一種，發生於哺乳動物中，這些疾病的發生，主要歸因於正常表達在細胞膜上的普利昂蛋白 PrPC，轉變為具傳染性的普利昂蛋白 PrPSc。PrPC 結構主要以α-螺旋為主，蛋白特性為水溶性、且易於蛋白酶所切割；當 PrPC 轉變為 PrPSc 之後，PrPSc 結構為含有高度的β-摺板，蛋白為不可溶性，具有蛋白酶抗性。
根據先前研究所報導，普利昂蛋白 C 端121-230是一段具有毒性的區域，能夠誘導 PrPSc 的形成，並且對抗蛋白酶的切割。因此，彈性結構的 N 端區域，在過去不被認為與疾病有直接的相關。然而，近期的研究顯示了，家族遺傳性普利昂疾病的發生，與蛋白質單點突變，或是 N 端序列 51-91 八胜肽重複區 (OR) 的插入 (野生型普利昂蛋白含有5個 OR) 是有相關的。PrPC 八胜肽重複區含有與銅離子結合的位置。然而，銅離子結合在 PrPC 的結構和疾病上所扮演的角色，仍然是未知的。基於上述的研究，我們猜測插入八胜肽數目，能夠影響結構、纖維形成、增加 C 端區域蛋白酶的抗性。
在本篇研究，我們製備了4種不同的蛋白質：MoPrP ∆octa, MoPrP 1-OR, MoPrP 5-OR, MoPrP 8-OR, 分別在含有與不含有銅離子的結合下，比較結構的差異，與纖維形成的速率。我們發現增加八胜肽重複數後，降低了蛋白質α-螺旋的含量和 C 端結構的穩定性，影響了纖維形成的速率。另一方面，PrP 8-OR 的蛋白質和纖維與其他蛋白相比，更能抵抗蛋白酶的切割。更進一步，我們測試了纖維對細胞的毒性，隨著八胜肽重複次數的增加，降低了細胞的存活率。本研究指出，八胜肽重複數的增加影響了普利昂蛋白結構、轉化為毒性更強之纖維。這些結果提供了證據闡明八胜肽重複區域影響普利昂疾病的分子機制。

關鍵字

普利昂蛋白

並列摘要

Prion diseases are neurodegenerative disorders carried out in mammalian. These diseases are due to structural conversion of prion protein (PrP) from normal cellular isoform (PrPC) to pathogenic isoform (PrPSc). PrPC is most of α-helical with soluble and protease sensitive. After conversion to PrPSc, protein is β-sheet rich and become insoluble and protease-resistant.
According to previous studies, the C-terminal region of prion protein, PrP121-230, is the toxic component to from PrPSc and can be against protease K digestion. Therefore, the flexible N-terminal domain has not been considered to be disease-related. However, recent studies have demonstrated that the onset of familial prion diseases is associated with both point mutation and repetition of octapeptide repeats (OR) insertion in N-terminal OR region, PrP51-91 with 5 OR. OR region of PrPC has Cu2+-binding sites. However, copper binding on PrPC structure and the role in disease have not been fully clarified. Base on these studies, we speculate that the repetition of OR insertion can influence the structure, the fibril formation and protease K-resistant strength of C-terminal region.
In this study, we prepared four protein variants (MoPrP ∆octa, MoPrP 1-OR, MoPrP 5-OR (WT), MoPrP 8-OR with and without Cu2+-treatment) for the structure comparison, kinetic study of fibril formation and further characterization of the fibrils. We found that increasing number of the repeats decrease protein α-helical content and the stability of C-terminus, and also affects the kinetics of fibril formation. On the other hand, PrP-8OR proteins and fibrils are more resistant to protease K digestion than other variants. Further, we tested the cell toxicity of the fibrils converted from these protein variants. Cell viability decreased with increasing numbers of OR. This study conclusively indicates that increasing octapeptide repeat affects structure of prion and the conversion to more toxic fibrils. This result provides evidence to elucidate the molecular mechanism of prion disease.

並列關鍵字

Prion protein

參考文獻

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