陳佩燁臺灣大學：生化科學研究所徐瑞璘Hsu, Ruei-LinRuei-LinHsu2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52753類澱粉蛋白是錯誤摺疊的蛋白質聚集而成。由於社會對類澱粉疾病的關注，類澱粉蛋白的研究也愈加熱烈。本論文有兩個主軸，分別在探討普昂疾病與阿茲海默症兩種類澱粉疾病之致病類澱粉蛋白的形成與分解。 普昂疾病是一種傳染性海綿樣腦病變，此病是由內生的普昂蛋白發生構形變異、形成富含β結構的構形而造成。為了探討此構形變異的過程，我們以普昂胜肽和小鼠重組普昂蛋白為材料，以圓二色光譜及螢光光譜來偵測其類澱粉纖維的形成。有鑑於先前研究發現將α-N-乙醯葡萄醣胺接上大頰鼠普昂胜肽108-144的135號殘基絲胺酸會抑制類澱粉纖維的形成，我們試圖以擁有同樣醣化修飾的人類普昂胜肽來干擾纖維形成。由於人類普昂蛋白的129號殘基具有胺基酸多型性（甲硫胺酸或纈胺酸），而經統計顯示此多型性會影響人類罹患普昂疾病的機率，故我們合成了兩種含上述單醣的人類普昂胜肽108-144，觀察醣化及129號殘基多型性對類澱粉纖維形成的影響。結果發現醣化會大大的抑制自發性纖維形成，而且以含醣胜肽的129號殘基為纈胺酸時，纖維的形成較容易。為了進一步觀察干擾現象，我們將含單醣和不含單醣的胜肽依等比例混合來培養纖維，結果含醣胜肽可以延長無醣胜肽形成纖維的遲滯期，但不影響無醣胜肽的纖維延長。 　　為了更真切地模擬普昂蛋白的構形轉變，我們也嘗試表現及純化帶有一對雙硫鍵的全長小鼠普昂蛋白，並且成功令其形成類澱粉纖維。以此為起點，我們可以進一步將此試管內形成纖維的系統用於研究全長普昂蛋白和普昂胜肽間的干擾現象。 　　阿茲海默症是一種好發於老年人的神經退化疾病。此病的發生與β類澱粉胜肽（簡稱Aβ）在腦部累積有關，而阿茲海默症患者通常有腦部清除Aβ的功能不彰的問題。研究發現腦中與血液中的Aβ含量存在一種平衡關係，因此依靠酵素分解血液中的可溶性Aβ或Aβ類澱粉纖維將可減少腦中Aβ的含量。納豆激酶（nattokinase）具有血纖維分解活性而且可以食用，因此本研究的第三部份將著重於以納豆激酶分解Aβ所形成的纖維，並比較納豆激酶和另外兩種酵素（PWD-1角質素酶和蛋白酶K）的纖維水解能力。實驗結果顯示納豆激酶有能力在試管中分解Aβ纖維，而且3種受測酵素的纖維水解能力相近。Amyloids are the aggregates of misfolded proteins. Because of the concern about amyloid diseases in the society, studies on amyloids become more and more popular. In this thesis, we studied the formation and degradation of protein amyloids related to two kinds of amyloid diseases, prion diseases and Alzheimer’s disease. Prion diseases are also called transmissible spongiform encephalopathies. It has been known that these diseases result from the conformational transition of a host-encoded prion protein to a β-sheet-rich conformer. To study the process of this structural conversion, we utilized circular dichroism and fluorescence spectroscopies to monitor the amyloid fibril formation of various synthetic prion peptides and a recombinant mouse prion protein. Previous studies have shown that an α-N-acetyl- glucosamine (α-GlcNAc) attached to the Ser135 of the hamster prion peptide 108-144 inhibits fibril formation, so we tried to interfere the fibrillogenesis of human prion peptides 108-144 with glycosylated ones. Because there is a polymorphism at residue 129 of human prion protein, either methionine or valine, and this polymorphism modulates disease susceptibility, we synthesized twoα-GlcNAc-linked human prion peptides 108-144 to examine the effect of glycosylation and polymorphism on the fibrillogenesis. The results confirmed the significant inhibitory effect of glycosylation on fibril formation, and showed that fibrils formed more easily when the residue 129 is valine. To investigate the interference between glycosylated and unglycosylated peptides, we mixed one glycosylated peptide and one unglycosylated peptide with equal molar ratio and found that the glycosylated peptides prolonged the lag time of the unglycosylated one only a little and did not inhibit fibril growth of the unglycosylated peptide. For the purpose of mimicking the conformational transition of prion protein more vividly, we tried to express and purify the mouse full-length recombinant prion protein with an intact disulfide bond, and converted this protein into amyloid fibrils successfully. With this cell-free conversion system, we can examine whether there is interference or seeding effect between the full-length prion protein and the prion peptide. Alzheimer’s disease (AD) is one of the most common senile dementia. Amyloid-β peptide (Aβ) has been implicated in the etiology of AD and dysfunction in Aβ clearance is crucial for the accumulation of Aβ in AD brains. There is an equilibrium of Aβ level in the brain and the blood, and removing Aβ, either in the soluble form or amyloid form, in the blood by enzymes might help decrease Aβ content in the brain. In the third part of my study, we focused on the breakdown of the amyloid fibrils formed from Aβ with nattokinase, which is well-known for its fibrinolytic activity and regarded as a healthy food, and compare the fibril-degrading efficiency of nattokinase with the other two subtilisin-like enzymes, proteinase K and PWD-1 keratinase. Our results showed that nattokinase has the potential in clearing Aβ fibrils in vitro, and all three enzymes tested have similar fibril-degrading efficiency.縮寫表…………………………………….……….i 中文摘要　…………………………………….……….iv Abstract …………………………………….………. V 第一章 緒論 1.1 關於類澱粉纖維…..…..………………..…………………..…….…. 1 1.2 簡介普昂疾病…………………………………….………..……….. 2 1.3 普昂蛋白的結構研究………………………………………………... 4 1.4 普昂蛋白的類澱粉纖維及其形成之模型……………………………… 7 1.5 普昂蛋白構形轉變的相關研究………………………………………. 9 1.6 普昂蛋白的醣化及其對普昂蛋白構形轉變的影響…………………….. 9 1.7 人類普昂疾病與普昂蛋白129號殘基多型性的關係………………….. 11 1.8 β類澱粉胜肽的相關研究…………………………………………... 13 1.8.1 簡介阿茲海默症…………………………………………….….. 13 1.8.2 β類澱粉胜肽的清除策略………………………………………. 15 1.9 Nattokinase…………………………………………………………. 16 1.10 Proteinase K……………………………………………………….. 17 1.11 PWD-1 keratinase………………………………………………….. 17 1.12 本論文的研究動機………………………………………………... 18 第二章 材料與方法 2.1 材料………………………………………………………………. 20 2.1.1 水……………………………………………………………... 20 2.1.2 化學藥品………………………………………………………. 20 2.1.3 載體…………………………………………………………... 22 2.1.4 菌株…………………………………………………………... 22 2.1.5 培養基………………………………………………………… 22 2.1.6 酵素…………………………………………………………… 22 2.2 儀器……...……………………………………………………...… 23 2.3 方法……………………………………………………………….. 23 2.3.1 普昂胜肽的合成、純化與鑑定………………………………….. 23 2.3.2 以光譜記錄普昂胜肽的纖維形成………………………………... 25 2.3.3 穿透式電子顯微鏡……………………………………………... 27 2.3.4 時間相依單光子探測螢光光譜………………………………….. 27 2.3.5 重組普昂蛋白的表現…………………………………………… 28 2.3.6 普昂蛋白的純化與鑑定………………………………………… 28 2.3.7 普昂蛋白的纖維形成…………………………………………… 32 2.3.8 β類澱粉胜肽纖維的水解…………………………………….… 32 第三章 結果與討論 (一) 3.1 O-linked α-GlcNAc對人類普昂胜肽形成類澱粉纖維的影響……...….. 34 3.1.1 α-GlcNAc的修飾抑制人類普昂胜肽形成類澱粉纖維…………… 36 3.1.2 醣化普昂胜肽對普昂胜肽類澱粉纖維形成的干擾作用…………… 42 3.1.3 醣化普昂胜肽可以用普昂胜肽纖維作為纖維形成的晶種………… 47 3.2 從普昂胜肽纖維的thioflavin T (ThT) 螢光壽命探討纖維構造……..…. 49 第四章 結果與討論 (二) 4.1 全長小鼠普昂蛋白的表現與純化…………………………………… 54 4.1.1 全長小鼠普昂蛋白的表現……………………………………… 54 4.1.2全長小鼠普昂蛋白之粗萃取……………………………………. 55 4.1.3以親和層析純化全長小鼠普昂蛋白……………………………... 57 4.1.4以逆相高效液相層析純化全長小鼠普昂蛋白……………………. 60 4.1.5純化之全長小鼠普昂蛋白的身分鑑定…………………………... 63 4.2 全長小鼠普昂蛋白類澱粉纖維的形成…………………………........ 64 第五章 結果與討論 (三) 5.1比較不同蛋白酶對β類澱粉胜肽纖維的水解效力……………………. 66 5.1.1測定蛋白酶活性……………………………………………….. 66 5.1.2比較proteinase K、PWD-1 keratinase與nattokinase對β類澱粉胜肽 纖維的水解效力………………………………………………… 70 第六章 結論與未來展望………………………………………... 73 參考文獻……………………..…………………………….………..… 751800144 bytesapplication/pdfen-US普昂類澱粉纖維prionamyloidfibril[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52753/1/ntu-96-R94b46023-1.pdf