臺灣博碩士論文加值系統

阿茲海默症是老年人口中最常見的失智症，為一種神經退化性疾病。目前研究阿茲海默症致病原因最主要的一個假說是類澱粉蛋白串聯假說(Amyloid cascade hypothesis)，此假說認為β-類澱粉胜肽(β-amyloid peptide; Aβ)的聚集(aggregation)是造成阿茲海默症的主要原因。Aβ約含有39-42個殘基，是β-類澱粉前驅蛋白(β-amyloid precursor protein; βAPP)經由水解的代謝產物。Aβ在聚集過程中涉及到結構的改變與自我本身的聚集，表示結構的性質在聚集過程中扮演相當重要的角色。先前研究模擬細胞膜的環境推測細胞膜會與Aβ有交互作用並且其交互作用可能會改變Aβ的結構特性與穩定度，有研究認為在細胞膜環境下會抑制Aβ的聚集，也有研究認為會促進Aβ的聚集，因此目前對β-類澱粉胜肽聚集的分子機制尚未有所定論。本篇研究主要目的是想探討Aβ在脂質環境中的結構特性。以帶負電的LMPG微胞(micelles)及造成家族性阿茲海默症的Iowa突變種(Aβ40(D23N))為模型，利用核磁共振光譜學(nuclear magnetic resonance spectroscopy)、圓二色光譜學(circular dichroism spectroscopy)、Th-T螢光光譜以及穿透式電子顯微鏡(TEM)探討Aβ與脂質的交互作用。透過比較Aβ40 (D23N)在水溶液環境中與LMPG微胞環境中的結構與聚集行為，結果發現Aβ40(D23N)會與LMPG微胞發生交互作用，而此交互作用會促進Aβ40(D23N)之螺旋傾向(helix propensity)並且會抑制Aβ40(D23N)之聚集。經由核磁共振光譜分析得知Aβ40(D23N)是以兩段螺旋結構鑲嵌於LMPG微胞中。此結果提供了Aβ與細胞膜交互作用機制的訊息，以及其對聚集行為之影響。

Alzheimer’s disease (AD) is the most common cause of dementia in elderly people. It is a chronic neurodegenerative disease. Currently, the hypothesis for the pathogenic mechanism of AD is amyloid cascade hypothesis. It states that the aggregation of β-amyloid (Aβ) is the primary cause of AD. Aβ contains 39-42 residues amino acid. It was a proteolytic product derived fromβ-amyloid precursor protein (βAPP). The aggregation process of Aβ involves conformational changes and self-assembly, indicating that the structural property plays an important role in Aβ aggregation. Previous studies also reported that the aggregation of Aβ was linked to its interaction with cell membrane-like enviroment. The interaction between Aβ and cell membrane might alter the structural property and conformational stability of Aβ. Some studies suppoted the view that cell membrane environment might inhibit the aggregation of Aβ, whereas others thought that cell membrane enviroments would promote the aggregation of Aβ. In this study, we used negatively charged LMPG micelles and familial Alzheimer’s disease-linked Iowa-type (Aβ40(D23N)) as model systems for investigating the Aβ-cell membrane interaction mechanism, and applied nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, thioflavine-T (Th-T) fluorometric assay and transmission electron microscopy (TEM) to characterize the interaction between Aβ and membrane lipid. By compairing the structure and aggregation behavior of Aβ40(D23N) in aqueous solution and LMPG micelles environment, we obtained that LMPG micelles would interact with Aβ40(D23N), leading to an increase of the α-helical propensity of Aβ40(D23N) and an inhibition of Aβ40(D23N) aggregation. Structural analysis showed that Aβ40(D23N) interacted with LMPG micelles through two short α-helical regions, L17VFFAENVGS26 and K28GAIIGLM35. These results may provide the information about the mechanism of Aβ-cell membrane interaction.

中文摘要 i
Abstract iii
目錄 iv
圖目錄 vi
表目錄 viii
附錄 ix
第一章 緒論 1
1-1 阿茲海默症(Alzheimer's disease) 1
1-1-1 阿茲海默症的臨床症狀 1
1-1-2 阿茲海默症的病理特徵 2
1-1-3 類澱粉斑塊(Amyloid plaques) 2
1-1-4 神經纖維糾結物(Neurofibrillary tangles) 3
1-2 阿茲海默症的分類 4
1-2-1 偶發性阿茲海默症(Sporadic Alzheimer’s disease；SAD) 4
1-2-2 家族性阿茲海默症(Familial Alzheimer’s disease；FAD) 5
1-3 類澱粉胜肽連鎖反應假說(Amyloid cascade hypothesis) 7
1-4 -類澱粉胜肽聚集之探討 9
1-4-1 野生型A 9
1-4-2 突變型Aβ 10
1-5 以結構的觀點探討Aβ之聚集 11
1-6 阿茲海默症之診斷與治療 13
1-7 研究目的與設計 15
第二章 實驗材料與方法 17
2-1 實驗藥品 17
2-2 實驗儀器 19
2-3 樣品製備 20
2-3-1 質體建構(Plasmid construction)與轉型作用(Transformation) 20
2-3-2 酵母菌泛素水解酶 (Yeast ubiquitin hydrolase-1; YUH-1)的製備 22
2-3-3 β-類澱粉胜肽融合蛋白A40(D23N)及A40(L17A/F19A/ D23N)的製備 23
2-4 圓二色光譜分析 (Circlar Dichroism) 26
2-5 Th-T螢光光譜分析 (Thioflavin T fluorescence spectroscopy) 27
2-6 穿透式電子顯微鏡 (Transmission Electron Microscopy) 28
2-7 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 野生型及突變型β-類澱粉胜肽的製備 32
3-2 Iowa突變型β-類澱粉胜肽和LMPG micelles的交互作用 37
3-2-1 Iowa突變型β-類澱粉胜肽在水溶液與LMPG micelles環境中的圓二色光譜圖 37
3-2-2 Iowa突變型β-類澱粉胜肽在水溶液與LMPG micelles環境中的核磁共振光譜圖 40
3-3 LMPG micelle環境對A40(D23N)聚集行為之影響 44
3-3-1 A40(D23N)在水溶液環境與LMPG micelles環境中聚集速率之比較 44
3-3-2 比較A40(D23N)在水溶液及LMPG micelles環境中二級結構轉變之速率 46
3-3-3 比較A40(D23N)在水溶液環境與LMPG micelles環境中形成纖維之速率 47
3-4 Iowa突變型β-類澱粉胜肽與LMPG micelles之交互作用機制 51
3-4-1 A40(D23N)在LMPG micelles環境下之二級結構 51
3-5 脂質帶電荷對Iowa突變型β-類澱粉胜肽與微胞的交互作用之影響 53
3-5-1 Iowa突變型β-類澱粉胜肽在水溶液與LMPC micelles環境中的圓二色光譜圖 53
3-5-2 Iowa突變型β-類澱粉胜肽在水溶液與LMPC micelles環境中的核磁共振光譜圖 55
3-5-3 A40(D23N)在LMPG micelles環境與LMPC micelles環境中聚集速率之比較 57
3-5-4 比較A40(D23N)在LMPG micelles與LMPC micelles環境中二級結構轉變之速率 58
第四章 結論 59
4-1 A40(D23N)與LMPG micelles之交互作用 59
4-2 A40(D23N)與LMPG micelles交互作用抑制突變型類澱粉胜肽之聚集行為 59
4-3 A40(D23N)與LMPG micelles交互作用機制 60
第五章 討論 61
參考文獻 70

 
圖目錄
圖 1-2-1 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置。…………..6
圖 1-3-1 類澱粉胜肽連鎖反應假說(Amyloid cascade hypothesis)示意圖。………….....8
圖 1-5-1 研究目的與設計示意圖。…......................……………………………………...16
圖 2-3-1 His6-ubiquitin表現載體之建立。….……………………….………..………….21
圖 2-3-2 融合蛋白A40(D23N)純化流程圖。……….………………………....…………24
圖 3-1-1 表達酵母菌泛素水解酶(Yeast Ubiquitin Hydrolase；YUH-1)之SDS-PAGE。...31
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。………………………………………………………32
圖 3-1-3 表達β-類澱粉胜肽融合蛋白之SDS-PAGE。……………..…………..………..33
圖 3-1-4 His6-ubiquitin-Aβ40(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。…………………………………….……………………………34
圖 3-1-5 H6Ub-Aβ40(D23N)水解前後之15% Tricine-SDS-PAGE。……………………..34
圖 3-1-6 鹼性溶液純化Aβ40(D23N)之HPLC層析圖。…………………….……………34
圖 3-1-7 酸性溶液純化Aβ40(D23N)之HPLC層析圖。……….…………………………34
圖 3-1-8 Aβ40(D23N)之質譜圖。…………..………………………….…………………..36
圖 3-1-9 Aβ40(D23N)經HPLC純化後之15%Tricine-SDS-PAGE。……………………...36
圖 3-2-1 Aβ40(D23N) 在水溶液及LMPG micelles環境下的初始二級結構。….………38
圖 3-2-2 Aβ40(D23N) 在水溶液環境下的初始二級結構三重複。……………………39
圖 3-2-3 Aβ40(D23N) 在50 mM LMPG micelles環境下的初始二級結構三重複。……39
圖 3-2-4 Aβ40(D23N) 在水溶液環境下的1H-HSQC光譜圖。…………………………41
圖 3-2-5 Aβ40(D23N) 在LMPG micelles環境下的1H-HSQC光譜圖。………………42
圖 3-2-6 Aβ40(D23N)在水溶液與LMPG micelles環境下的1H-HSQC光譜圖比較。..43
圖 3-3-1 Aβ40(D23N) 在水溶液與LMPG micelles環境下凝聚機制動力學之Th-T螢光光譜圖。………………………………………………………………………………………45
圖 3-3-2 Aβ40(D23N) 在水溶液環境及LMPG micelle環境下的二級結構變化。……..46
圖 3-3-3 Aβ40(D23N) 在水溶液環境與LMPG micelles環境下的纖維型態比較。.........47
圖 3-3-4 Aβ40(D23N)水溶液與LMPG micelles環境下隨著時間對纖維型態及纖維形成速率之比較。重複一。…………………..…………………………………………………....48
圖 3-3-5 Aβ40(D23N)水溶液與LMPG micelles環境下隨著時間對纖維型態及纖維形成速率之比較。重複二。…………………..…………………………………………………....49
圖 3-3-6 Aβ40(D23N)水溶液與LMPG micelles環境下隨著時間對纖維型態及纖維形成速率之比較。重複三。…………………..…………………………………………………....50
圖 3-4-1 Aβ40(D23N)在水溶液與LMPG micelles環境下的13C化學位移比較圖。...….52
圖 3-5-1 Aβ40(D23N) 在LMPG及LMPC環境下的1H-HSQC重疊光譜圖。………….54
圖 3-5-2 Aβ40(D23N) 與LMPG micelles交互作用機制。…………………….…………56
圖 3-5-3 Aβ40(D23N) 在LMPG micelles與LMPC micelles環境下凝聚機制動力學之Th-T螢光光譜圖。…………………………...………………………………………………57
圖 3-5-4 Aβ40(D23N)在LMPG micelle 及LMPC micelles環境下的二級結構變化。…58
圖 5-1 Aβ40(D23N) 在5 mM、20 mM與100 mM LMPG環境下的1H-HSQC重疊光譜圖。…………………………………………………………………………………………63
圖 5-2 Aβ40(D23N) 在100 % LMPG、75 % LMPG 25 % LMPC與50 % LMPG 50 % LMPC環境下的1H-HSQC重疊光譜圖。…………………………………………………64
圖 5-3 Aβ40(L17A/F19A/D23N) 在水溶液、LMPG micelles與LMPC micelles環境下的1H-HSQC光譜比較圖。…………………………………………………………….….…65
圖 5-4 Aβ40(L17A/F19A/D23N) 在水溶液、LMPG及LMPC micelles環境下的初始二級結構。……………………………………………………………………………………66
圖 5-5 Aβ40(L17A/F19A/D23N) 在水溶液、LMPG與LMPC micelles環境下凝聚機制動力學之Th-T螢光光譜圖。………...………………………………………………………67

 
表目錄
表 2-3-1 酵母菌泛素水解酶純化之緩衝溶液。…………………………………....…….22
表 2-3-2 β-類澱粉胜肽純化之緩衝溶液………….………………………………………24
表 2-3-3高效液相層析(HPLC)移動相程式。...........……………………………………...25
表 2-4-1圓二色光譜之蛋白質二級結構之波長特徵。……………………………..…….26
表 2-7-1二十種胺基酸之H、13C、13C與13C'二級結構傾向化學位移(chemical shift)參考值。…...……………………………………………………………………………30
表 3-2-1 Aβ40(D23N)在H2O及LMPG micelles環境中二級結構分析。………………......38
表 3-5-1 Aβ40(D23N)在LMPG及LMPC micelles環境中二級結構分析。………………..54
表 5-1 Aβ40(L17A/F19A/D23N)在H2O、LMPG及LMPC micelles環境中二級結構分析。……………………………………………………………………………………………66


























附錄
附圖1 Aβ40(D23N) 在200 mM LMPG micelles中骨架原子化學位移。………………..68

 

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