探討小分子物質對類澱粉纖維形成之影響

Exploring the Effects of Small Molecules on the Formation of Amyloid Fibrils

洪櫻姿(Ying-Tz Hung)

指導教授 ： 王勝仕

國立臺灣大學/工學院/化學工程學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.10467

摘要

在最近幾年有關於蛋白質的研究中，有很多證據顯示蛋白質會在某些特定情況下摺疊成不正常的類澱粉纖維構型，其中有些人體內的蛋白質形成的類澱粉纖維與人類的退化疾病相關，這些蛋白質的序列幾乎都不相同，但在形成類澱粉纖維後卻都有相同的貝塔-平板構型，雖然很多研究者廣泛的研究類澱粉纖維形成的機制，但詳細的分子機構仍然未知。在本研究中，我們在生物體外以科學儀器進行實驗，研究了某些像是:變性劑、界面活性劑、短鍊磷脂質等小分子對雞蛋白溶菌酶聚集的影響，我們的結果分為三個部分：各自為溶菌酶與尿素、溶菌酶與十二烷基硫酸鈉、溶菌酶與磷脂質三個系統。以下簡述三個系統的結果。
針對第一個系統，在pH 7.4的溶液以及37，45，55度C三個溫度下，分別將2，4，8M尿素加入溶菌酶溶液中，其中除了以8M尿素在37，45和55度C下以及4M尿素在45與55度C的情況下，對溶菌酶可引誘形成類似類澱粉纖維的物質，但在這些條件下隨時間又消逝，其他的條件都沒有形成類似類澱粉纖維的物質。例如：鈉十二烷基的硫酸鹽聚丙烯酰胺凝膠電泳法也證實了在上述易轉變為規則貝塔-平板構型的條件下，持續以尿素引誘溶菌酶開展至最後面聚集消失的階段，溶菌酶轉變為較大的不規則結構。
在第二個系統裡，透過不同的光譜儀器，以及動態雷射光與穿透式電子顯微鏡，本研究證實十二烷基硫酸鈉對溶菌酶產生了兩階段的影響。溶菌酶在pH 2.0與55度C下形成類澱粉纖維的結構，然而，我們發現高濃度的十二烷基硫酸鈉中，(例如：0.25, 5.00, or 20.00 mM)可抑制溶菌酶纖維的形成，而在低濃度的十二烷基硫酸鈉中(例如: 0, 0.06, or 0.1 mM)，卻可發現溶菌酶轉變為高含量貝塔-平板構型的纖維。同時，本研究以尿素導致溶菌酶開展的熱力學平衡數據證實，在高濃度十二烷基硫酸鈉中的溶菌酶可保持較高的熱力學穩定度。最後，我們再以表面張力與恆溫滴定熱卡計的數據推導出不同十二烷基硫酸鈉濃度對溶菌酶影響的機制。
在最後一個系統裡，我們同樣透過不同的光譜儀器，以及動態雷射光與穿透式電子顯微鏡，觀察短鍊磷脂質如何對溶菌酶形成類澱粉纖維的影響，而且，此熱力學平衡數據也顯示出短鍊磷脂質可增加溶菌酶的穩定性。另外，由恆溫滴定熱卡計也可了解此系統的主要作用力由分子間或分子內的疏水作用力主導。本研究的結果可幫助了解蛋白質變性並形成類澱粉纖維的機制。

關鍵字

雞蛋白溶菌酶類澱粉纖維 ； 十二烷基硫酸鈉 ； 蛋白質聚集 ； 抑制纖維形成 ； 尿素與磷脂質

並列摘要

Recent evidence has suggested that numerous proteins can fold abnormally resulting in the formation of amyloid fibrils/aggregation. Some proteins that form amyloid fibrils are associated with human degenerative diseases. These proteins have little sequence homology, however, they can self-assemble into stable fibrils with a characteristic cross β-sheet structure. Although many research groups extensively explored the mechanism of amyloid fibrillogenesis, the detailed molecular mechanism has remained largely unknown. Here, we investigated the effects of small molecules, including denaturant, surfactant, and short-chain phospholipids, on the aggregative behavior of hen egg-white lysozymes using in vitro methods. Our results were divided into three sections by systems explored: lysozyme-urea, lysozyme-sodium dodecyl sulfate (SDS), and lysozyme-phospholipid.
Firstly, in the pH 7.4 solutions containing 8M urea at 37, 45, or 55 oC and 4M urea at 45 or 55 oC, the formation and later disappearance of amyloid fibril-like species were observed while such species were not present at all under other conditions. In addition, SDS-PAGE results further indicated that larger aggregated species with less ordered structures were formed at the later stage of the urea-induced unfolding process owing to an unidentified conformational switch.
In the second section, through various spectroscopic techniques, dynamic light scattering, and electron microscopy, we first demonstrated that SDS exhibited a biphasic effect on lysozyme fibrillation. The 129-residue enzyme, hen egg-white lysozyme, has been shown to form fibrils in vitro at pH 2.0 and 55

並列關鍵字

Hen lysozyme ； amyloid fibril ； sodium dodecyl sulfate ； aggregation ； urea ； phospholipid ； inhibition

參考文獻

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