以電子自旋共振光譜探討小鼠普立昂胜肽片段108-144所生成的澱粉樣纖維之結構特性

Abstract

普立昂疾病是一種因普立昂蛋白不正常堆疊形成澱粉樣纖維所造成的神經退化性疾病。其臨床上的症狀包含失智，肌肉抽搐，共濟失調…等等。而因目前對於此疾病的相關知識仍嚴重不足，導致目前並無任何方式可以醫治此種疾病。對此，我們針對現有大眾所普遍接受的普立昂蛋白假說進行深入研究。普立昂蛋白在正常形態下(PrPC)以膜蛋白的形式存在於細胞膜上，但當它經過了一個未知原因的構型轉變後，即會轉變為致病型態的普立昂蛋白(PrPSc)。此結構轉變會導致普立昂蛋白產生了許多特性像是不易被蛋白酶水解、不易溶於水…而這些特性則導致了其無法藉由X射線晶體學或是核磁共振等通俗方式解出或看出結構。 在本研究中我們選取了小鼠普立昂蛋白108-144這個普遍被認為是形成澱粉樣纖維的中心片段的胜肽，其後我們選取了五個胺基酸位點做了突變以利於我們接上可以被電子自旋共振所偵測到的含有自由電子的(1-氧-2,2,5,5-四甲基吡咯口林-3-甲基)甲基硫代硫磺酸酯(MTSSL)自旋標記。接著我們利用野生型普立昂蛋白所形成的澱粉樣纖維為晶種去誘使各胜肽形成澱粉樣纖維，最後再以電子自旋共振光譜ESR來解析各突變胜肽的自旋標記之間的相對距離為何，藉此探討致病型普立昂蛋白在形成澱粉樣纖維後之結構特性。 根據得到的結果我們推測出了一個模型,那就是在中性環境下，澱粉樣纖維的產生可能是先以一條胜肽折兩折形成一個S型的形態,再以這個形態整齊排列而成。而在酸性環境下所得到的ESR跟CD圖譜則跟中性有差別,此發現亦代表普立昂的澱粉樣纖維可能會因環境不同而以不同的形態存在。本研究希望能藉由電子自旋共振光譜來找出小鼠的致病型普立昂蛋白在形成澱粉樣纖維的關鍵位點及區域，其後推展到人類的普立昂蛋白上，希望能以此發展有利於針對普立昂疾病的深度研究，疾病預防或藥物開發…等等。

Prion diseases are a group of neurodegenerative diseases caused by abnormal aggregations of prion protein into amyloid fibrils. The clinical symptoms of these diseases include dementia, muscle twitching, ataxia…etc. Knowledge relating to prion diseases is still severely insufficient, which results in the lack of effective treatments for these diseases. To this extend, we looked deeper into the protein-only hypothesis of prion which is generally accepted by the community. Prion protein exists as a membrane protein on the cellular membrane when it is in its normal cellular form (PrPC). However, when PrPC undergoes an unknown-reasoned structural conversion, it becomes the disease-causing form of prion protein (PrPSc). This major structural conversion not only renders PrPSc more prone to aggregate, but also creates a lot of additional features of PrPSc such as difficult to be hydrolyzed by proteases, hard to be dissolved in water…etc. These additional features further impede the application of conventional protein structural studies such as X-ray crystallization or NMR on PrPSc. Here in this study, we chose mouse prion peptide (108-144) as our target of interest since this region is believed to be of the critical region regarding amyloid fibril formation. We synthesized 5 different mutant peptides separately and added spin-labels named (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate (MTSSL) onto the cysteine mutations of these peptides. These MTSSL labels contained free radicals, which can be detected by ESR spectroscopy. After labeling, fibrils were formed by these peptides in vitro by adding buffers containing salt which facilitates fibril formation. Seeds formed by the sonication of wildtype fibrils were also added as templates to further increase the propensity of different mutant peptides to form wildtype-like fibrils. Finally, by doing ESR spectroscopy, we could detect the intensity of interactions between labels, and by doing spin-dilutions, we could further estimate the distance between two labels in amyloid fibrils. This information could help us better understand the structural nature of PrPSc. According to our results, we proposed a structural model of fibrils formed by mPrP(108-144), which is that the peptide monomers first self-folds into an S-like structure before stacking on one another uniformly to build the whole fibril. As for the results done under the acidic condition, the ESR spectra obtained were different than the ones done under the neutral condition, which indicate that prion peptide might adopt different conformations of amyloid fibrils under different environment. This study aims to find out the critical sites and regions of the amyloid core formed by mouse prion peptide, and hopefully, one day to further extend the research to the level of human prion. The information obtained by this study could be useful in prion disease prevention or therapy.