臺灣博碩士論文加值系統

第一部分為阿茲海默症相關研究，γ-secretase在前類澱粉蛋白質的跨膜域區段之切位決定了Aβ40 及 Aβ42之間的比例，然而γ-secretase決定切位的機制仍未知。前類澱粉蛋白質的跨膜域區段之胺基酸序列存在有四個glycine，我們推測這些glycine也許會使跨膜域區段的結構變得類似彈簧般具有彈性且能夠隨著外在的環境變化而改變本身的結構，並且有許多研究指出細胞膜的組成很可能會影響Aβ的生成。根據這些資訊，我們提出一個假設，前類澱粉蛋白質的跨膜域區段很可能不是典型的α-helix結構，且細胞膜的組成會影響其結構，因此也影響到γ-secretase決定其切位。在本研究我們致力於合成及純化出帶有前類澱粉蛋白質的跨膜域區段之胜肽，因為帶有許多疏水胺基酸的胜肽在純化上非常不容易，成功得到純的且足夠量的胜肽之後，在未來便可利用這些胜肽進行後續的實驗來證實這個假設。
第二部分為普立昂疾病相關研究，許多研究指出普立昂蛋白序列108-144為形成類澱粉纖維及影響普立昂疾病跨種傳染的重要區段，而不同物種之普立昂蛋白108-144的胺基酸序列有高度保守性。在本研究中我們的主題為敘利亞倉鼠、小鼠及人類的普立昂蛋白序列108-144，三者的C端序列139 (編號根據敘利亞倉鼠的序列)的胺基酸差異被認為是影響類澱粉纖維形成的關鍵胺基酸，我們想去探討普立昂蛋白108-144的胺基酸序列與其所形成之類澱粉纖維的結構之間的關聯，透過合成及純化出六種不同的普立昂胜肽，分別為shaPrP(108-144)、mPrP(107-143) 與huPrP(108-144)，以及這三條帶有H140→C的突變種並進行定位自旋標記反應，我們以各物種野生型普立昂胜肽形成的類澱粉纖維為晶種進行引晶以形成帶有自旋標記的類澱粉纖維，並以電子自旋共振光譜來比較三者結構的異同。

In this paper, we undertook research into Alzheimers disease and Prion disease.
The ratio of Aβ40 to Aβ42 depends on the cleavage site of γ-secretase in the transmembrane region of APP but it is not yet clear how γ-secretase chooses its cleavage site. In the transmembrane region of APP, there are four Gly arranged as GxxxGxxxGG. We proposed that these Gly residues might provide structural flexibility, thus allowing the whole membrane spanning region to behave like a spring. The structure of this region may also vary due to environmental changes. Furthermore, many studies have suggested that the composition of lipids in the membrane might also play an important role in Aβ production. Based on above considerations, we hypothesized that the cutting site of γ-secretase may vary because the membrane-spanning region of APP doesn’t form a typical α-helical structure and lipid composition may further affect its structure. Due to the difficulties of hydrophobic peptide purification, we focused on synthesizing and purifying a peptide corresponding to the sequence in the transmembrane region of APP and successfully got enough purified peptides in the study. In the future, we can use these peptides to prove our hypothesis.
Many studies have shown that the prion protein sequence 108-144 (PrP108-144) is important to fibrillization and the cross-species infection of Prion disease as this PrP108-144 sequence is very similar amongst different mammalian prion proteins. In our study, we focused on the PrP108-144 sequence in humans, mice, and Syrian hamsters. The C-terminal residue 139 (according to the sequence from Syrian hamsters) from these three species is considered to be the key for fibrillization. To explore the relationship between the sequence and the structure of prion peptide fibrils formed by PrP108-144, we synthesized six different PrP108-144 peptides, including shaPrP(108-144), mPrP(107-143), huPrP(108-144), and the corresponding peptides with a H140→C mutation (for spin labeling). We used these wildtype peptides to form fibril seeds and used them to induce the spin-labeled peptides to form spin-labeled amyloid fibrils. We then compared their structures using Electron Spin Resonance (ESR) spectroscopy.

中文摘要 i
Abstract ii
縮寫表 Abbreviations iv

<PART A>
第一章 緒論 1
1.1 阿茲海默症 (Alzheimer''s Disease，簡稱AD) 1
1.2 類澱粉蛋白連鎖假說 (The amyloid cascade hypothesis) 3
1.3 Aβ的生成與清除 4
1.4 類澱粉蛋白纖絲的生成機制 (fibrillogenesis) 6
1.5 影響γ-secretase在APP蛋白上之切位 (cleavage site) 的因素 7
1.6 實驗目的與實驗設計 13
第二章 材料與方法 17
2.1 材料 17
2.1.1 水 17
2.1.2 化學藥品 17
2.1.3 儀器 19
2.2 方法 20
2.2.1 胜肽固相合成 20
2.2.2 逆相-高效液相層析純化 21
2.2.2.1 液-固相層析 21
2.2.2.1 分子篩層析 22
第三章 結果與討論 23
3.1 前類澱粉蛋白質跨膜域序列之胜肽的合成 23
3.2 前類澱粉蛋白質跨膜域序列之胜肽的純化 26
3.2.1 前類澱粉蛋白質跨膜域序列之合成胜肽混合產物對不同溶劑之溶解度測試 26
3.2.2 序列為Aβ(28-53)之胜肽在HPLC純化上的困難與改進的方法 27
3.2.3 新序列Aβ(22-54)胜肽之合成混合產物對不同溶劑之溶解度測試 29
3.2.4 序列為Aβ(22-54)之胜肽在HPLC純化上的困難與改進的方法 30
3.2.4.1 第一批Aβ(22-54)_V40C/I47C胜肽樣品之純化 30
3.2.4.2 Aβ(22-54)_V40C/I47C胜肽樣品純化產率不佳之問題的探究與解決(一) 33
3.2.4.3 Aβ(22-54)_V40C/I47C胜肽樣品純化產率不佳之問題的探究與解決(二) 36
3.2.4.4 Aβ(22-54)_V40C/I47C胜肽樣品純化產率不佳之問題的探究與解決(三) 40
3.2.5 序列為KKWK-Aβ(22-55) 之胜肽在HPLC純化上的困難與改進的方法 52
3.2.5.1 KKWK-Aβ(22-55) _V40C/I47C 胜肽之不同純化條件的比較與分析 52
3.2.5.2 胜肽序列KKWK-Aβ(22-55) 之最佳純化條件 59
3.3 前類澱粉蛋白質跨膜域序列胜肽之定位自旋標記 (SDSL) 63
3.3.1 定位自旋標記的反應條件之初步測試結果 63
第四章 結論與未來展望 69

<PART B>
第一章 緒論 73
1.1 普立昂疾病 ( Prion disease ) 73
1.2 普立昂蛋白結構相關之研究 75
1.3 普立昂蛋白之類澱粉纖維 78
1.4 普立昂蛋白序列108-144 ( PrP108-144 ) 81
1.5 實驗目的與實驗設計 85
第二章 材料與方法 87
2.1 材料 87
2.1.1 水 87
2.1.2 化學藥品 87
2.1.3 儀器 90
2.2 方法 91
2.2.1 胜肽固相合成 91
2.2.2 逆相-高效液相層析純化 92
2.2.2.1 液-固相層析 92
2.2.3 定位自旋標記 93
2.2.4 普立昂胜肽之類澱粉纖維形成與觀測 95
2.2.4.1 普立昂胜肽之自發性類澱粉纖維形成 95
2.2.4.2 以 thioflavin T 之螢光光譜記錄普立昂胜肽之類澱粉纖維形成 95
2.2.4.3 普昂胜肽類澱粉纖維之晶種製備 96
2.2.4.4 帶有MTSSL標記的普立昂胜肽之引晶實驗 96
第三章 結果與討論 97
3.1 普立昂胜肽樣品的製備 97
3.2 胜肽樣品PrP108-144_H140R1之製備 107
3.3 WT PrP108-144之自發性類澱粉纖維形成 ( spontaneous fibril formation ) 113
3.4 PrP108-144_H140R1之類澱粉纖維引晶形成 ( seeding ) 119
3.5 PrP108-144_H140R1類澱粉纖維 之ESR圖譜 126
第四章 結論與未來展望 128

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