臺灣博碩士論文加值系統

β-類澱粉胜肽(β-amyloid peptide; Aβ)約含有39-42個殘基，是β-類澱粉前驅蛋白(β-amyloid precursor protein; AβPP)經由水解的代謝產物。β-類澱粉胜肽的聚集(aggregation)被認為是阿茲海默症(Alzheimer's disease)發病機制的早期事件，目前對β-類澱粉胜肽聚集的分子機制尚未完全瞭解。β-類澱粉胜肽聚集成澱粉纖維的過程與其構形改變有關，這暗示β-類澱粉胜肽的構形穩定度在其聚集過程扮演重要角色。最近本實驗室研究發現，L17A/F19A循理式突變可以增加野生型Aβ40及造成家族性阿茲海默症的Aβ40(E22G)突變型(Arctic突變型)之構形的穩定性，並降低其-螺旋/β-摺疊之構形轉變和纖維的生成速率。在本研究中，利用核磁共振光譜、圓二色光譜、Thioflavin-T(ThT)螢光光譜、穿透式電子顯微鏡(TEM)等，觀察L17A/F19A循理式突變對另一個造成家族性阿茲海默症的Aβ40(D23N)突變型(Iowa突變型)之聚集行為與二級結構傾向。從聚集動力學的研究結果，發現L17A/F19A循理式突變能抑制Aβ40(D23N)之結構轉變和纖維的形成。以核磁共振光譜偵測結構之結果，發現L17A/F19A突變主要是增加Aβ40(D23N)的α/β-不穩定構形區段(α/β-discordant segment)之螺旋結構傾向。此研究結果證明α/β-不穩定構形區段之螺旋結構傾向是主導Aβ聚集傾向的關鍵之一。本研究結果所提供的訊息，可幫助我們從結構的觀點對Aβ聚集之分子機制獲得更進一步的了解。

β-amyloid peptide (Aβ), which consists of 39-42 residues, is derived from proteolysis of β-amyloid precursor protein (APP). Aggregation of β-amyloid peptide (Aβ) is thought to be an early event in the pathogenesis of Alzheimer’s disease. The molecular mechanism underlying Aβ aggregation is not entirely understood. The process of Aβ aggregation into amyloid fibril involves conformational changes, suggesting that the conformational stability of Aβ play a key role in the aggregation process. Recently, we reported that L17A/F19A replacements could increase the conformational stability of wild-type Aβ40 and familial Alzheimer's disease-linked E22G variant of Aβ40 (Arctic Aβ40 variant), and reduce their α-helix-to-β-strand conversion and fibril formation rates. In this study, the effects of L17A/F19A replacements on the aggregation behavior and secondary structure propensity of D23N variant of Aβ40 (Iowa Aβ40 variant), which is also a familial Alzheimer's disease-linked variant of Aβ40, were characterized by using Nuclear Magnetic Resonance spectroscopy (NMR spectroscopy), Circular Dichroism spectroscopy (CD spectroscopy), Transmission Electron Microscopy (TEM) and Thioflavin T fluorescence assay. The results of kinetic aggregation studies indicated that L17A/F19A replacements can inhibit conformational changes and fibril formation of Aβ40(D23N). NMR structural studies revealed that L17A/F19A replacements mainly increased the α-helical propensity of the residues located in the α/β-discordant segment of Aβ40(D23N). These findings suggested that the α-helical propensity of the α/β-discordant segment is one key factor in governing the aggregation propensity of Aβ. This result may also provide a structural basis toward understanding the molecular mechanism of Aβ aggregation.

目錄

中文摘要 I
ABSTRACT II
目錄 III
圖目錄 V
表目錄 VII
附錄 VIII

第一章 緒論 1
1-1 阿茲海默症(ALZHEIMER'S DISEASE) 2
1-1-1 阿茲海默症的病理特徵 2
1-1-2 類澱粉斑塊(Amyloid plaques) 3
1-1-3 神經纖維糾結物(Neurofibrillary tangles) 3
1-2 阿茲海默症的分類 4
1-2-1 偶發型阿茲海默症(Sporadic Alzheimer’s disease；SAD) 4
1-2-2 家族型阿茲海默症(Familial Alzheimer’s disease；FAD) 5
1-3 類澱粉胜肽連鎖反應假說(AMYLOID CASCADE HYPOTHESIS) 6
1-4 -類澱粉胜肽之聚集 7
1-4-1 野生型A 7
1-4-2 突變型Aβ 8
1-5 以結構的觀點探討AΒ之聚集 9
1-6 阿茲海默症之診斷與治療 11

第二章 實驗材料與方法 15
2-1 實驗藥品 15
2-2 樣品製備 17
2-2-1 質體建構(Plasmid construction)與轉型作用(Transformation) 17
2-2-2 Yeast ubiquitin hydrolase-1(YUH-1)的製備 19
2-2-3 A40(D23N)及A40(L17A/F19A/ D23N)的製備 20
2-3 圓二色光譜分析 23
2-4 TH-T螢光光譜分析 (THIOFLAVIN T FLUORESCENCE SPECTROSCOPY) 24
2-5 穿透式電子顯微鏡 (TRANSMISSION ELECTRON MICROSCOPY, TEM) 26
2-6 核磁共振光譜 (NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY) 26

第三章 結果 29
3-1 樣品的製備 29
3-1-1 YUH-1的製備 29
3-1-2 野生型及突變型β-類澱粉胜肽的製備 32
3-2 循理式突變(L17A/F19A)對IOWA突變型Β-類澱粉胜肽聚集行為之影響 38
3-2-1 A40(D23N)與A40(L17A/F19A/D23N)聚集速率之比較 38
3-2-2 比較A40(D23N)與A40(L17A/F19A/D23N)二級結構轉變之速率 43
3-2-3 比較A40(D23N)與A40(L17A/F19A/D23N)形成纖維之速率 44
3-3 循理式突變(L17A/F19A)對IOWA突變型Β-類澱粉胜肽二級結構之影響 48
3-3-1 比較A40(D23N)與A40(L17A/F19A/D23N)在SDS溶液中之二級結構 48
3-3-2 比較A40(D23N)與A40(L17A/F19A/D23N)在水溶液中之二級結構 61
4-1 L17A/F19A突變去除K16LVFFAED23序列之/-DISCORDANCE 72
4-2 Q15-S26的-螺旋性與聚集速率之關係 73
4-3 在不同環境下之結構特性 74

附圖 75
參考文獻 91

圖目錄
圖 1-1-1 β-類澱粉前驅蛋白(APP)水解途徑及家族性阿茲海默症突變位.………….….5
圖 1-1-2 Aβ在各個環境下的結構。………………………………………………………10
圖 2-2-1 His6-ubiquitin表現載體之建立。………………………………………………18
圖 2-2-2 酵母菌泛素水解酶(YUH-1)之純化流程圖。…………………………………..19
圖 2-2-3 融合蛋白A40(D23N)及A40(L17A/F19A/D23N)純化流程圖。……………21
圖 3-1-1 表達酵母菌泛素水解酶(Yeast Ubiquitin Hydrolase；YUH-1)之SDS-PAGE。...29
圖 3-1-2 YUH-1經鎳親和性管柱層析(Immobilized Metal Affinity Chromatography)純化後以15% SDS-PAGE分析之電泳圖。………………………………………………………30
圖 3-1-3 YUH-1經phenyl-Sepharose 管柱純化後以15% SDS-PAGE分析之電泳圖。..30
圖 3-1-4 YUH-1經Mono Q 管柱純化分析之層析光譜。………………………………31
圖 3-1-5 YUH-1經Mono Q 管柱純化後以15% SDS-PAGE分析之電泳圖。………..31
圖 3-1-6 表達-類澱粉胜肽融合蛋白之SDS-PAGE。………………………………..32
圖 3-1-7 (A) His6-ubiquitin-A40(D23N)融合蛋白經鎳親和性管柱層析純化後以15% SDS-PAGE分析之電泳圖。…………………………………………………………………32
圖 3-1-7 (B) His6-ubiquitin-A40(L17A/F19A/D23N)融合蛋白經鎳親和性管柱層析純化後以15% SDS-PAGE分析之電泳圖。……………………………………………………..33
圖 3-1-8 (A) H6Ub-A40(D23N)水解前後之15% Tricine-SDS-PAGE。………………..34
圖 3-1-8 (B)H6Ub-A40(L17A/F19A/D23N)水解前後之15%Tricine-SDS-PAGE。……34
圖 3-1-9 (A) A40(D23N)經HPLC純化後之15%Tricine-SDS-PAGE。………………..34
圖 3-1-9 (B) A40(L17A/F19A/D23N)經HPLC純化後之15%Tricine-SDS-PAGE。….34
圖 3-1-10 鹼性溶液純化A40(D23N)之HPLC層析圖。…………………………………35
圖 3-1-11 酸性溶液純化A40(D23N)之HPLC層析圖。…………………………………35
圖 3-1-12 鹼性溶液純化A40(L17A/F19A/D23N)之HPLC層析圖。……………………36
圖 3-1-13 酸性溶液純化A40(L17A/F19A/D23N)之HPLC層析圖。……………………36
圖 3-1-14 A40(D23N)之質譜圖。…………………………………….…………………..37
圖 3-1-15 A40(L17A/F19A/23N)之質譜圖。…...….……………………………………..37
圖 3-2-1 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下凝聚機制動力學Th-T螢光光譜圖。…………………………………………………………………………………39
圖 3-2-2 A40(D23N)Ⅰ之最佳曲線配適(curve fitting)。………………………………40
圖 3-2-3 A40(L17A/F19A/D23N)Ⅰ之最佳曲線配適(curve fitting)。…….…………..40
圖 3-2-4 A40(D23N)Ⅱ之最佳曲線配適(curve fitting)。………………………………41
圖 3-2-5 A40(L17A/F19A/D23N)Ⅱ之最佳曲線配適(curve fitting)。…….…………..41
圖 3-2-6 A40(D23N)Ⅲ之最佳曲線配適(curve fitting)。………………………………42
圖 3-2-7 A40(L17A/F19A/D23N)Ⅲ之最佳曲線配適(curve fitting)。…….…………..42
圖 3-2-8 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下的二級結構變化。..43
圖 3-2-9 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下的纖維型態比較。..44
圖 3-2-10 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下隨著時間對纖維型態及纖維形成速率之比較。重覆一。…..………………………………………………………45
圖 3-2-11 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下隨著時間對纖維型態及纖維形成速率之比較。重覆二。…..………………………………………………………46
圖 3-2-12 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下隨著時間對纖維型態及纖維形成速率之比較。重覆三。…..………………………………………………………47
圖 3-3-1 A40(D23N)及A40(L17A/F19A/D23N)在SDS環境下的初始二級結構。.....49
圖 3-3-2 A40(D23N)在SDS環境下的1H-HSQC光譜圖。………..……………………..51
圖 3-3-3 A40(L17A/F19A/D23N)在SDS環境下的1H-HSQC光譜圖。……………….52
圖 3-3-4 A40(D23N)及A40(L17A/F19A/D23N)在SDS環境下之1H-HSQC重疊光譜圖
。…………………………………………………………………………………………53
圖 3-3-5 A40(L17A/F19A/D23N)在SDS環境下13C、13C、13C’和1H之化學位移比較圖。…………………………………………………………………………………………54
圖 3-3-6 A40(D23N)及A40(L17A/F19A/D23N)在SDS環境下的13C化學位移比較圖。………………………………………………………………………………………...55
圖 3-3-7 A40(D23N)及A40(L17A/F19A/D23N)在SDS環境下的之CSI比較圖。……56
圖 3-3-8 A40(D23N)在水溶液環境下的1H-HSQC光譜圖。…………………………..62
圖 3-3-9 A40(L17A/F19A/D23N)在水溶液環境下的1H-HSQC光譜圖。…..………..63
圖 3-3-10 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下的1H-HSQC重疊光譜
圖。……………………………………………………………………………………………64
圖 3-3-11 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下之13C、13C、13C’和1H之化學位移比較圖。……………………………………………………………………..65
圖 3-3-12 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下的13C化學位移比較。…………………………………………………………………………………………66
圖 3-3-13 A40(D23N)及A40(L17A/F19A/D23N)在水溶液環境下的之CSI比較圖。67
圖 4-1 -類澱粉胜肽Q15-D23區段之-螺旋螺旋結構傾向。………………….73

表目錄
表 2-2-1 高效液相層析(HPLC)移動相程式。…………………………………….………22
表 2-3-1 遠紫外光圓二色光譜之蛋白質二級結構之波長特徵。………………….……..23
表 2-6-1 二十種胺基酸之H、13C、13C與13C'二級結構傾向化學位移(chemical shift)參考值。………………………………………………………………………………………28
表 3-2-1 三組實驗之A40(D23N)及A40(L17A/F19A/D23N)時間與速率常數之關係。.39
表 3-3-1 A40(D23N)及A40(L17A/F19A/D23N)在100 mM SDS-d25二級結構分析。.49
表 3-3-2 A40(D23N)在100 mM SDS-d25緩衝溶液中骨架原子化學位移。…...………59
表 3-3-3 A40(L17A/F19A/D23N)在100 mM SDS-d25緩衝溶液中骨架原子化學位移。60
表 3-3-4 A40(D23N)在50 mM phosphate buffer中骨架原子化學位移。……………..70
表 3-3-5 A40(L17A/F19A/D23N)在50 mM phosphate buffer中骨架原子化學位移。..71

附錄
附圖1 A40(D23N)在100 mM SDS-d25之3D HNCA條狀圖譜。………………………..75
附圖2 A40(D23N)在100 mM SDS-d25之3D HNCO條狀圖譜。………………………..76
附圖3 A40(D23N)在100 mM SDS-d25之3D CBCACONH條狀圖譜。………….……..77
附圖4 A40(D23N)在100 mM SDS-d25之3D HBHACONH條狀圖譜。…………….…..78
附圖5 A40(L17A/F19A/D23N)在100 mM SDS-d25之3D HNCA條狀圖譜。………..79
附圖6 A40(L17A/F19A/D23N)在100 mM SDS-d25之3D HNCO條狀圖譜。………..80
附圖7 A40(L17A/F19A/D23N)在100 mM SDS-d25之3D CBCACONH條狀圖譜。…..81
附圖8 A40(L17A/F19A/D23N)在100 mM SDS-d25之3D HBHACONH條狀圖譜。…..82
附圖9 A40(D23N)在10 mM磷酸緩衝溶液之3D HNCA條狀圖譜。………………..83
附圖10 A40(D23N)在10 mM磷酸緩衝溶液之3D HNCO條狀圖譜。……….…….…..84
附圖11 A40(D23N)在10 mM磷酸緩衝溶液之3D CBCACONH條狀圖譜。….……..85
附圖12 A40(D23N)在10 mM磷酸緩衝溶液之3D HBHACONH條狀圖譜。…….…..86
附圖13 A40(L17A/F19A/D23N)在10 mM磷酸緩衝溶液之3D HNCA條狀圖譜。…..87
附圖14 A40(L17A/F19A/D23N)在10 mM磷酸緩衝溶液之3D HNCO條狀圖譜。…..88
附圖15 A40(L17A/F19A/D23N)在10 mM磷酸緩衝溶液之3D CBCACONH條狀圖譜。……………………………………………………………………………………………89
附圖16 A40(L17A/F19A/D23N)在10 mM磷酸緩衝溶液之3D HBHACONH條狀圖譜。……………………………………………………………………………………….…..90

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