透過將螢光信號標記嫁接於人類胰島類澱粉多肽用以偵測其寡聚體形成

Abstract

胰島類澱粉蛋白(Islet amyloid polypeptide, IAPP)是一種與胰島素共同由胰島β細胞分泌出來的多肽激素。IAPP在正常生理情況下是可溶且無結構的。然而，在某些狀態下它們往往會聚集成可溶性寡聚體，進而形成不可溶的類澱粉蛋白纖維。寡聚體具細胞毒性，多項研究指出它們會使β細胞功能失調和β細胞數量下降。因此，在IAPP纖維化的過程中，能夠即時監測寡聚體的出現顯然是一件重要的事。首先，此論文的第一部分，我們嘗試在IAPP不同位點上嫁接了對環境敏感型的螢光分子7-氮雜色胺酸(代號Wx)，此螢光分子於疏水性環境中具強放光的特性，藉此，我們用以觀察IAPP聚集過程中螢光的變化來推論是否有寡聚體的產生，雖然結果不如預期，不過我們得知將Wx嫁接於胜肽最N端時，對於IAPP整體的聚集行為影響最小。因此，我們第二個部分實驗則同樣在IAPP最N端嫁接了具有聚集誘導放光特性的四苯乙烯(Tetraphenylethylene, TPE)，當IAPP混以少量TPE標記的IAPP時，我們成功在IAPP聚集成核期就觀察到TPE螢光的上升，即時監測到IAPP寡聚體的形成。Islet amyloid polypeptide (IAPP) is a 37- residue peptide hormone that is co-secreted with insulin by pancreatic β-cells. IAPP monomers are mostly unstructured and physiologically soluble. However, they tend to aggregate into soluble oligomers, which in turn form insoluble amyloid fibrils. Studies have noted that the main cytotoxic role in the pathogenesis of type 2 diabetes is associated with the early formation of small oligomers, which could lead to β-cell dysfunction and death. Therefore, it is important to monitor the formation of oligomers during the fibrillation process of IAPP. First, an environment-sensitive fluorescent molecule 7-Azatryptophan (7-AzaTrp, noted as Wx) was utilized and grafted onto the IAPP. The fluorescence changes and aggregation properties of tagged IAPP were discussed. Although it is not able to obtain information on oligomer formation, it is known that the grafting of Wx to the most N-terminus of the peptide has the least effect on the overall aggregation process of IAPP. Next, the aggregation-induced emission molecule tetraphenylethene (TPE) was grafted also on the N-terminal of IAPP. The effect of TPE molecule on IAPP aggregation was examined and their emission which accompanies peptide aggregation was also monitored to probe the formation of IAPP oligomers.