探討老鼠普立昂蛋白從α至β結構轉變的機制

普立昂疾病為一類致命且具傳染力之神經退化性疾病，主要病徵為在人類或動物中樞神經系統中產生異常纖維性澱粉堆疊進而造成海綿狀病變。正常的普立昂蛋白(PrPC)以α螺旋為主要結構且執行正常生理功能；當產生普立昂疾病時，蛋白結構會轉變為以β摺板為主的致病性、堆疊型普立昂蛋白(PrPSc)，此異常的分子結構轉變即是造成細胞毒性之主要原因。但至今，我們仍然不明白有哪些二級結構以及胺基酸參與在普立昂蛋白結構轉變的過程。本實驗室先前的研究發現：移除雙硫鍵的老鼠普立昂蛋白在中性且近乎生理環境的條件下，會進行自發性的結構轉變，並且能夠利用中性溶液固定此重組普立昂蛋白在特定的結構狀態，基於先前的重要發現，使我們得以詳細分析普立昂蛋白結構轉變的機制。本篇論文主要著重於觀察普立昂蛋白中三段α螺旋在整體結構轉變時，α螺旋局部二級結構變化，並利用電子自旋共振、圓二色光譜儀、穿透式電子顯微鏡、分析級超高速離心及單分子螢光共振能量轉換等技術研究此結構轉換的過程。本篇結果顯示：helix 1以及helix 3在整體結構轉變至β型態時仍會保持α螺旋結構；然而helix 2在整體結構轉變為β型態時，α螺旋則會完全解開，顯示helix 2參與蛋白整體的結構變化。當蛋白整體結構在β型態時，helix 2會產生分子間作用的現象，說明helix 2間的作用力穩定β-oligomers結構；而普立昂蛋白形成纖維狀結構時，helix 2和helix 3更進一步參與纖維性澱粉核心(amyloid core)的形成，並搭配著helix 1或是loop上的殘基提供額外的分子間作用力穩定整體纖維狀結構。

關鍵字

普立昂蛋白；結構轉變；螺旋結構； α型態； β型態；纖維狀結構

並列摘要

Prion diseases are not only fetal but also infectious neurodegenerative disorders. The critical molecular event of prion diseases is the structural conversion of a normal cellular prion protein, PrPC, into a misfolded, infectious form, PrPSc. The overall structure of the prion protein transits from α- to β-dominant state, giving rise to formation of toxic amyloid fibrils. Up to now, the structural transition mechanism is still elusive. Recently, our lab found that disulfide-bond reduced mouse prion protein could be fixed in α-helical or β-rich structure under neutral condition. This finding provides us an opportunity to dissect the conversion process in details. To examine the role of three α-helices in mPrP during this structural conversion process, site-directed spin-labeling technique (SDSL), electron spin resonance spectroscopy (ESR), analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), circular dichroism spectroscopy (CD), and single molecule fluorescence resonance energy transfer (smFRET) were employed. In this study, we suggest that helix 1 and helix 3 are intact no matter in α- or β-state; however, helix 2 is unfolded after structural converted to β-oligomers. Only the residues in helix 2 are involved in intermolecular association in β-state, suggesting helix 2 is crucial for oligomerization process. In addition, the tertiary structural contact between helix 3 and loop is dragged open after structural transition. In fibril state, helix 2 and helix 3 cooperatively participate in association of amyloid core and helix 1 or loop supplies peripheral interaction to stabilize the fibril structure as well.

並列關鍵字

Prion protein ； structural conversion ； helical structure ； α-PrP ； β-PrP ； amyloid fibril

參考文獻

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探討老鼠普立昂蛋白從α至β結構轉變的機制

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