小鼠普立昂蛋白在不同狀態下之結構探討

Abstract

腦海綿樣病變(Transmissible Spongiform Encephalopathies (TSE))亦稱為普立昂疾病，為一類具傳染性且致命的神經退化疾病，其主要發生原因主要為普立昂蛋白質之二級結構構型由α為主的結構轉變為β結構為主，並形成類澱粉纖維(amyloid)堆積造成腦部神經細胞死亡。然而，普立昂蛋白質之構型轉變確切的機制目前尚未被完全了解。目前普立昂蛋白結構之研究指出，N端區域之普立昂蛋白普遍認為不具有二級結構；C端區域則有三條α-helix以及兩條β-strand。目前已有研究指出，普立昂蛋白結構轉變機制，主要為普立昂蛋白在細胞膜上時，經由蛋白水解酶(protease)將N端區域水解後，剩餘之C端區域經由內吞作用之內噬體進出細胞內外，並由於內噬體內之pH值比細胞質之環境較低，造成C端區域之結構由α結構轉變為β結構，再經由內吞作用循環回到細胞膜上後，引起其他正常的普立昂蛋白結構轉變成異常的普立昂蛋白結構，便慢慢開始形成堆積(aggregation)。 在本實驗室先前之研究發現，將小鼠普立昂蛋白以胰蛋白酶進行水解反應後，第一個切位為胺基酸序列第105與106之間，因此本研究定義N端區域為胺基酸序列23-105；C端區域則是106-230。藉由利用分析式超高速離心以及圓二色光譜分析小鼠普立昂蛋白之物理特性，進而討論其結構變化。實驗結果發現，在pH4.2條件中，提高緩衝液中鹽類濃度，發現N端區域之構型開始改變，但是全長蛋白穩定度不變，因此推測N端區域與C端區域並無交互作用；而在pH5.2條件中，提高緩衝液之鹽類濃度後，發現全長小鼠普立昂蛋白之二級結構穩定性提高，因此推測可能為N端區域與C端區域產生交互作用使得其結構穩定性增加。

Transmissible Spongiform Encephalopathies (TSE), also known as prion diseases, are fatal and infectious neurodegenerative diseases. The conformational change of prion protein from α to β is considered to be the main cause of the disease. However, the structural conversion mechanism between cellular prion protein PrPC and its misfolded form PrPSc still remains unknown. It has been known that the N-terminal part of prion protein does not contain any secondary structure while the C-terminal part contains three a-helices and two b-strands. We recently found that, when full-length mouse recombinant prion protein was treated with trypsin, the first cutting site occurs between K105 and T106. It has been reported that PrPC can be partly degraded on membrane and the remained C-terminal part can enter into the cell through endocytosis, convert into b-sheet-rich structure in endosome, and then be exported out through exocytosis. Here we define the segment of 23-105 as the N-terminal part and the segment of 106-230 as the C-terminal part. We employ analytical ultracentrifugation (AUC) and circular dichroism spectroscopy (CD) to explore the structural properties of full-length prion protein and its C-terminal and N-terminal parts. We found out that at pH4.2 condition, increasing salt concentration lead to the comformational change of N-terminal domain. According to thermal stability results, the stability of full-length prion protein remain the same, we suppose that there is no interaction between N- and C-terminal domain. At pH5.2, the stability of full-length prion protein increased while salt concentration increase, we suppose that the N-terminal domain interact with the C-terminal domain.