臺灣博碩士論文加值系統

阿茲海默症(Alzheimer's disease)是最為常見的神經性退化疾病，它的發病原因被認為與β-類澱粉胜肽(β-amyloid peptide; Aβ)的聚集現象有很大的關聯性。Aβ是一段短胜肽，通常由39-42個胺基酸殘基所組成，其源自於β-amyloid precursor protein (βAPP) 水解之產物。而Aβ的聚集過程包含了Aβ本身的二級結構變化以及Aβ分子和分子間的聚集反應。從結構的觀點來看，Aβ的聚集過程歷經了單體到寡聚體到纖維的形成，而在這些型態的改變中必定存在著蛋白質結構的變化。Dutch-type (E22Q)為一種家族性阿茲海默症(FAD)的類型，其在Aβ序列中第22號胺基酸，從Glutamate (E)突變為Glutamine (Q)，且此種家族性突變已被報導會促進Aβ的聚集行為。根據實驗室先前的研究報告指出，將Aβ 序列中L17以及F19置換成Alanine後可以提升野生型Aβ(wild-type Aβ40)的α-螺旋傾向性，且降低從α-helix到β-strand的結構變化速率，抑制纖維的形成。
本篇研究中，即是想瞭解L17A/F19A置換對於Dutch-type Aβ的影響，我們使用LMPG及LMPC形成之微胞(micelles)模擬細胞膜的環境，並利用核磁共振光譜儀(nuclear magnetic resonance spectroscopy)、圓二色光譜儀(circular dichroism spectroscopy)、Th-T螢光光譜(thioflavin-T fluorescence assay)及穿透式電子顯微鏡(transmission electron microscopy)觀察L17A/F19A置換後的Aβ40(L17A/F19A/E22Q)之結構特性以及聚集行為。初步的研究結果表明，不論是Aβ40(E22Q) 還是 Aβ40(L17A/F19A/E22Q)都會與兩種脂質微胞(Lipid micelles)有交互作用，而我們也發現L17A/F19A置換可以提升α-螺旋傾向性和抑制纖維的形成。而從結構上的角度來看，提升Aβ 的α-螺旋傾向性可以抑制其結構從α-helix變化到β-strand。由聚集動力學實驗得到的結果也表明L17A/F19A置換可以降低Dutch-type Aβ聚集的速率。本次研究所得到之結果可以有助於更加確認L17 和 F19 是維持Aβ本身結構穩定性的關鍵胺基酸位置。

Alzheimer’s disease (AD) is the most common neurodegenerative disease. Its pathogenesis has been thought to be associated with the aggregation of β-amyloid peptide (Aβ). Aβ is a small peptide, containing 39-42 amino-acid residues. It is derived from the cleavage of β-amyloid precursor protein (βAPP). The aggregation process of Aβ involves conformational changes and self-assembly. From the structural point of view, Aβ aggregate from monomer into oligomer and fibril must undergo conformational changes. Dutch-type (E22Q) mutations, which would cause familial Alzheimer’s disease (FAD), were located within the Aβ sequence. It has been reported that these genetic mutation would promote Aβ aggregation. Previous studies have shown that L17A/F19A replacements could increase the α-helical propensity of wild-type Aβ40, and reduce its α-helix-to-β-strand conversion and fibril formation rates. In this study, to understand the interaction mechanism, we used lyso-myristoylphosphatidylglycerol (LMPG) micelles and lyso-myristoylphosphatidylcholine (LMPC) micelles to mimic the membrane environment and the effects of L17A/F19A replacements on the structures and aggregation behaviors of Aβ40(E22Q) (Dutch-type Aβ) were characterized by using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, electron microscopy (EM) and thioflavin T fluorescence assay. Our data suggested that both Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) could bind to both types of lipid micelles, and we also obtained that the L17A/F19A replacements can increase the α-helical propensity and inhibit fibril formation of Dutch-type Aβ. From the structural point of view, the increase of α-helical propensity of Aβ would inhibit its structural transition from α-helix to β-strand. The result of aggregation kinetics showed that the aggregation rate of Dutch-type Aβ was inhibited in L17A/F19A replacements. This finding may further support the view that L17 and F19 are the key residues responsible for the conformational stability of Aβ.

中文摘要 I
ABSTRACT II
目錄 III
圖目錄 V
表目錄 VII
第一章 前言 1
1-1失智症的分類 2
1-2 阿茲海默症的分類 3
1-3 阿茲海默症的臨床症狀 5
1-4 阿茲海默症的病理特徵 5
1-5 類澱粉胜肽連鎖反應假說(AMYLOID CASCADE HYPOTHESIS) 6
1-6 以結構的觀點探討A之聚集 8
1-7 研究目的與設計 10
第二章 實驗材料與方法 14
2-1 實驗藥品 14
2-2 樣品製備 16
2-2-1 質體建構(PLASMID CONSTRUCTION)與轉型作用(TRANSFORMATION) 16
2-2-2 勝任細胞(COMPETENT CELL)的製備 18
2-2-3 YEAST UBIQUITIN HYDROLASE-1(YUH-1)的製備 18
2-2-3 AΒ40(E22Q)以及AΒ40(L17A/F19A/E22Q)的製備 21
2-3 圓二色光譜分析 (CIRCULAR DICHROISM SPECTROSCOPY) 25
2-4 TH-T螢光光譜分析 (THIOFLAVIN T FLUORESCENCE SPECTROSCOPY) 26
2-5 穿透式電子顯微鏡 (TRANSMISSION ELECTRON MICROSCOPY) 27
2-6 核磁共振光譜 (NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY) 28
第三章 結果 31
3-1 樣品的製備 31
3-1-1 YUH-1的製備 31
3-1-2 A突變型AΒ40(E22Q)以及AΒ40(L17A/F19A/E22Q)的製備 34
3-2 以圓二色光譜探討L17A/F19A置換對於A40(E22Q)與脂質交互作用之影響 40
3-2-1 利用圓二色光譜探討A40(E22Q)在有無脂質環境下L17A/F19A置換之結構差異性 …………………………………………………………………………………………..40
3-2-2 利用圓二色光譜探討A40(E22Q)在L17A/F19A置換後與脂質反應強弱之差異性…………………………………………………………………………………… 43
3-3 以核磁共振光譜探討A40(E22Q)在有無脂質環境下其L17A/F19A置換之結構差異性…………………………………………………………………………………… 49
3-4 比較L17A/F19A置換對於A40(E22Q)在有無脂質環境中聚集特性之影響 63
3-4-1 利用THIOFLAVIN-T(THT) ASSAY比較A40(E22Q)以及A40(L17A/F19A/E22Q)在有無脂質環境中聚集之速率 63
3-4-2 利用圓二色光譜儀觀察A40(E22Q)以及A40(L17A/F19A/E22Q)在有無脂質環境中隨時間的二級結構變化 67
3-4-3 比較A40(E22Q)以及A40(L17A/F19A/E22Q)在有無脂質環境中纖維之生成速率…………………………………………………………………………………… 70
第四章 結論與討論 73
4-1 L17A/F19A 置換對於A40(E22Q)與LMPG、LMPC MICELLES之交互作用之影響…………………………………………………………………………………… 73

圖目錄
圖 1，-類澱粉前驅蛋白之家族性遺傳突變胺基酸序列位置。 4
圖 2，-類澱粉胜肽連鎖反應假說(Amyloid cascade hypothesis)示意圖。 7
圖 3，以結構的角度觀察β-類澱粉胜肽在不同環境中之變化。 9
圖 4，研究設計之示意圖。 12
圖 5，實驗設計之示意圖。 13
圖 6 ，His6-Ubiquitin表現載體之建立。 17
圖 7，酵母菌泛素水解酶(YUH-1)之純化流程圖。 20
圖 8，融合蛋白Aβ40(E22Q)純化流程圖。 23
圖 9，圖2-3-1圓二色光譜儀之橢圓率(θ)。 25
圖 10 ，Thioflavin T之化學結構式。 26
圖 11，二十種胺基酸之H、13C、13Cβ與13C'二級結構傾向化學位移(chemical shift)參考值。 30
圖 12，大量表達His6-YUH-1融合蛋白之15 % SDS-PAGE。-及+分別代表有無IPTG誘導，M為marker，大約在28 kDa的位置有His6-YUH-1融合蛋白大量表達。 32
圖 13，His6-YUH-1經過鎳親和性管柱層析純化後之15 % SDS-PAGE。 32
圖 14，His6-YUH-1經由phenyl-Sepharose 管柱層析後之15 % SDS-PAGE。 33
圖 15，His6-YUH-1經由Mono Q 管柱層析後之15 % SDS-PAGE。 33
圖 16，His6-Ub-A40(E22Q)融合蛋白經過鎳親和性管柱層析純化後之15 % SDS-PAGE。使用15 % SDS-PAGE，利用分子量確認His6-Ub-Aβ40(E22Q)之位置。M代表marker，S代表supernatant，W代表wash，St代表strip。樣品收集由33 ~ 57 管。 35
圖 17，His6-Ub-A40(L17A/F19A/E22Q)融合蛋白經過鎳親和性管柱層析純化後之15 % SDS-PAGE。 36
圖 18，His6-Ub-A40(E22Q)(左圖)以及His6-Ub-A40(L17A/F19A/E22Q)(右圖)融合蛋白經過YUH-1水解前後之16.5 % Tricine-SDS-PAGE。 36
圖 19，酸性溶液純化A40(E22Q)之以及A40(L17A/F19A/E22Q)之HPLC層析圖。 37
圖 20，A40(E22Q)以及A40(L17A/F19A/E22Q)之質譜圖。 38
圖 21，LMPG微胞之TEM圖。 39
圖 22，LMPC微胞之TEM圖。 39
圖 23，A40(E22Q) 以及A40(L17A/F19A/E22Q)在水環境、LMPG micelles環境及LMPC micelles環境中的圓二色光譜。 41
圖 24，A40(E22Q)之LMPG以及LMPC濃度滴定(titration)CD光譜圖。 44
圖 25，A40(L17A/F19A/E22Q)之LMPG以及LMPC濃度滴定(titration)CD光譜圖。 45
圖 26，A40(E22Q)以及A40(L17A/F19A/E22Q)在LMPG以及LMPC濃度滴定(titration)中，波長222、208、195之曲線配適(curve fitting)。 47
圖 27，100 M A40(E22Q)在水環境中之1H15N-TROSY光譜圖。 50
圖 28，100 M A40(E22Q)在50 mM potsaaium phosphate buffer環境下278 K、283 K、288 K、293 K及298 K之1H15N-TROSY光譜圖之疊圖。 51
圖 29，100 M A40(E22Q)在水環境中之1H15N-TROSY光譜圖。 52
圖 30，200 M Dutch型β-類澱粉胜肽Aβ40(E22Q)在200 mM LMPG micelles環境中之2D 1H/15N-Trosy光譜圖。 53
圖 31，200 M Dutch型β-類澱粉胜肽Aβ40(E22Q)在200 mM LMPC micelles環境中之2D 1H/15N-Trosy光譜圖。 54
圖 32，200 M A40(E22Q)在200 mM LMPG及LMPC微胞環境中之1H15N-TROSY光譜圖之比較。 55
圖 33，100 M Aβ40(L17A/F19A/E22Q)在水溶液之2D 1H/15N-HSQC光譜圖。 56
圖 34，100 M A40(L17A/F19A/E22Q)在50 mM potsaaium phosphate buffer環境下278 K、283 K、288 K、293 K及298 K之1H15N-TROSY光譜圖之疊圖。 57
圖 35，100 M Aβ40(L17A/F19A/E22Q)在水溶液之2D 1H/15N-HSQC光譜圖。 58
圖 36，A40(E22Q)以及A40(L17A/F19A/E22Q)在LMPG微胞環境中之1H15N-TROSY光譜圖之比較。 59
圖 37，A40(E22Q)以及A40(L17A/F19A/E22Q)在LMPC微胞環境中之1H15N-TROSY光譜圖之比較。 60
圖 38，Dutch型β-類澱粉胜肽Aβ40(E22Q)在水溶液、LMPG以及LMPC micelles環境之13C二次化學位移(Secondary chemical shifts)比較圖。 61
圖 39，Aβ40(E22Q)以及Aβ40(L17A/F19A/E22Q)在LMPG以及LMPC micelles環境下之13C二次化學位移比較圖。 62
圖 40，Aβ40(E22Q) 以及A40(L17A/F19A/E22Q)在水環境、LMPG micelles環境及LMPC micelles環境中Th-T kinetics assay螢光光譜圖。 64
圖 41，Dutch型β-類澱粉胜肽Aβ40(E22Q)在水環境、LMPC micelles環境中以及Aβ40(L17A/F19A/E22Q)在LMPC micelles環境中之Th-T聚集動力學最佳曲線配適(curve fitting)。 65
圖 42，30 μM Dutch型β-類澱粉胜肽Aβ40(E22Q)在水環境、LMPG及LMPC micelles環境中之二級結構變化速率。 68
圖 43，30 μM Aβ40(L17A/F19A/E22Q)在水環境、LMPG及LMPC micelles環境中之二級結構變化速率。 69
圖 44，Dutch型β-類澱粉胜肽Aβ40(E22Q)在有無脂質環境中之TEM結果圖。 71
圖 45，Aβ40(L17A/F19A/E22Q)在有無脂質環境中之TEM結果圖。 72
圖 46，L17A/F19A 置換對於A40(E22Q)以及LMPG、LMPC micelles交互作用之影響。 74

表目錄
表 1，六種可能的排列組合以及美國人口百分比結果。 4
表 2，YUH-1純化之緩衝溶液。 19
表 3，-類澱粉胜肽純化之緩衝溶液。 22
表 4，高效液相層析(HPLC)移動相程式。 24
表 5，利用圓二色光譜網頁分析軟體DichroWeb分析Dutch型β-類澱粉胜肽Aβ40(E22Q)以及Aβ40(L17A/F19A/E22Q)於水環境、LMPG micelles及LMPC micelles環境中之二級結構組成。 42
表 6，A40(E22Q)以及A40(L17A/F19A/E22Q)在LMPG以及LMPC濃度滴定(titration)中，波長222在最佳曲線配適(curve fitting)所得出之Kd值。 48
表 7，Dutch型β-類澱粉胜肽Aβ40(E22Q)在水環境、LMPG及LMPC micelles環境中之Th-T聚集動力學時間參數。 66

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