全長小鼠普昂蛋白與短片段小鼠普昂胜肽類澱粉纖維形成之研究

Abstract

類澱粉纖維為蛋白質發生錯誤摺疊後並堆疊所產生；當類澱粉纖維於生物個體內形成並累積時，則可能會造成疾病的產生，而這些疾病則被泛稱為類澱粉病變，普昂疾病便屬於其中之一。普昂疾病又稱為傳播性海綿樣腦病變，係因哺乳動物體內之普昂蛋白發生構形變化，由富含 α-螺旋之結構轉為富含 β-摺板之結構後，堆疊累積成類澱粉纖維之結構所致。 為了解普昂蛋白發生構形變化而形成類澱粉纖維的過程以及能夠影響此過程進行之因子，本研究團隊利用大腸桿菌表現出全長小鼠普昂蛋白（簡稱作 mPrP(23-231)）並進行純化以作為研究主角；純化後的 mPrP(23-231) 於試管內進行類澱粉纖維形成之試驗，並以螢光分子 thioflavin T（ThT）與類澱粉纖維結合後之螢光放射行為觀察其類澱粉纖維之形成動力學。在輕度變性及劇烈震盪環境下，mPrP(23-231) 在經過約一天的遲滯期後，能夠自發地形成類澱粉纖維；若於培養環境中加入已形成的普昂類澱粉纖維作為晶種，則類澱粉纖維形成過程中之遲滯期時間有縮短之現象。此外，本研究團隊亦使用化學合成之小鼠普昂胜肽片段 108-144（簡稱作 mPrP(108-144)），配合 mPrP(23-231) 進行交叉引晶之試驗，結果發現由 mPrP(23-231) 所形成之類澱粉纖維可作為晶種加速 mPrP(108-144) 單體之類澱粉纖維形成，而當二者角色對調時，亦可得到相同的結果，隱含著二者在形成類澱粉纖維時，皆是以序列 108-144 堆疊成為纖維主體之資訊。 而為了探討 mPrP(23-231) 與 mPrP(108-144) 所形成之類澱粉纖維在於其核心結構以及外在形態上之異同，則分別使用到 ThT 進行其與類澱粉纖維複合物之螢光壽命測定以及穿透式電子顯微鏡觀察；二者皆在以上試驗中具有相似之結果，顯示出 mPrP(23-231) 與 mPrP(108-144) 在形成類澱粉之纖維過程具有相同之核心結構，且形成之序列即在 108-144 之中。

Amyloids are aggregates of misfolded proteins. Many diseases are caused by amyloids accumulation. The prion disease, also called transmissible spongiform encephalopathy (TSE), is caused by conformational change of host-encoded prion protein to a β-sheet-rich conformer. When prion protein changes its conformation, it will aggregate and form fibril-like structure. To know how prion protein forms amyloid fibrils and the factors affecting its formation, we expressed full-length mouse prion protein [mPrP(23-231)] in Escherichia coli and purified it. We studied the amyloid fibril formation in vitro by thioflavin T (ThT) fluorescence spectroscopy. In mild denaturing condition with vigorous shaking, mPrP(23-231) forms amyloid fibrils after one-day incubation. Adding pre-formed amyloid fibrils as seeds may shorten the lag time of amyloid fibril formation. In addition, we synthesized mouse prion peptide (mPrP(108-144)) to do cross seeding experiment. Full-length mPrP(23-231) can function as seeds to accelerate the fibril formation of chemically synthetic mPrP(108-144), and vice versa. Amyloid fibrils formed from mPrP(23-231) and mPrP(108-144) with share very similar morphology in TEM and fluorescence lifetime assay using ThT as dye, suggesting that mPrP(23-231) and mPrP(108-144) has the same amyloid core.