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研究生: 詹璦綺
Chan, Ai-Ci
論文名稱: 胡椒酸衍生物用於加速胰島類澱粉蛋白聚集
Derivative of piperic acid accelerates the aggregation of islet amyloid polypeptide
指導教授: 杜玲嫻
Tu, Ling-Hsien
口試委員: 劉維民
Liu, Wei Min
葉怡均
Yeh, Yi Chun
口試日期: 2021/07/20
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 55
中文關鍵詞: 類澱粉纖維第二型糖尿病胡椒酸衍生物
英文關鍵詞: Amyloid fibril, Diabetes mellitus type 2, Piperic acid derivatives
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202100814
論文種類: 學術論文
相關次數: 點閱:100下載:0
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  • 蛋白質錯誤折疊形成纖維狀沉積物可能會破壞細胞並導致組織和器官的退化。這種類型的疾病被泛稱為類澱粉蛋白沉積症。例如,人類胰島類澱粉蛋白(Human islet amyloid polypeptide, hIAPP)的纖維狀沉積與第二型糖尿病的病理密切相關。過去文獻顯示聚集過程中的中間體比最終的纖維沉積物更具毒性,因此開發足以影響類澱粉蛋白聚集的化合物被視為解決類澱粉蛋白沉積症的方法之一。
    我們團隊過去已測試多種胡椒酸衍生物,其中小分子PAD-13可以加速人類胰島類澱粉蛋白聚集,我們期望此分子可以減少有毒中間體的存在,並藉以減少細胞死亡。本研究將先深入探討 PAD-13 和 hIAPP 之間的相互作用。
    由硫磺素T螢光觀察類澱粉蛋白之形成以及添加預先形成類澱粉蛋白作為核種的核誘發實驗,顯示PAD-13加速hIAPP聚集為透過促進初級(Primary nucleation)和次級成核(Secondary nucleation)來達成。hIAPP在有無PAD-13影響下所形成的類澱粉蛋白纖維於TEM 和ATR-FTIR 結果中顯示兩者具有非常相似的形態。在硫磺素T動力學中於PAD-13影響下觀察到螢光強度增加,由凝膠電泳證明其原因並非纖維數量增加,我們推測可能是參與hIAPP聚集的過程中,PAD-13與硫磺素T產生交互作用力造成。圓二色光譜時間進程(Time-course)實驗表示PAD-13 加速了hIAPP的構象變化。我們對 hIAPP 的兩個突變進行分析,確認hIAPP 中精胺酸(Arginine, Arg)和離胺酸(Lysine, Lys)皆是PAD-13與hIAPP相互作用的關鍵殘基。

    Protein misfolding and formation of fibrous deposits may destroy the cells and lead to degeneration of the tissues and organs. This type of disease is called amyloidosis. For example, amyloid deposit of human islet amyloid polypeptide (hIAPP) in pancreatic islets is closely associated with the pathology of type 2 diabetes. It has been reported that the intermediate in the aggregation process is more toxic than the final fibrous deposits, so the discovery of a compound that affects amyloid protein aggregation is regarded as a solution to amyloidosis.
    Previously a variety of piperic acid derivatives, has been tested by our team. One of a small molecule PAD-13 which can accelerate the accumulation of islet amyloid will be expected to reduce the presence of toxic intermediates and reduce the cell death. This study will get to the meat of the interaction between PAD-13 and hIAPP.
    From hIAPP amyloid fibril formation monitored by thioflavin T fluorescence and added with the condition that added pre-formed amyloid fibrils as seeds, it was shown that PAD-13 will enhance the rate of aggregation by promoting primary and secondary nucleation. Amyloid fibrils formed from hIAPP in the absence or presence of PAD-13 share very similar morphology observed in TEM and ATR-FTIR studies. The fluorescence intensity increased when hIAPP and more PAD-13 co-culture in the thioflavin T kinetic, but we have ruled out the possibility that increased fibers formed in the reaction suggested by SDS-PAGE. It seems that PAD-13 will interact with ThT while in the process of hIAPP aggregation. Time course of circular dichroism spectra imply that PAD-13 will accelerate the conformational change of hIAPP. In this study, it was also revealed that arginine and lysine of hIAPP are both key residues that PAD-13 may interacts with.

    謝辭 i 摘要 ii 目次 iv 表次 xiv 圖次 xv 第一章 緒論 1 1.1 類澱粉蛋白病變 1 1.2 類澱粉蛋白的聚集機制 3 1.3 第二型糖尿病與胰島類澱粉蛋白 4 1.4 降低類澱粉蛋白細胞毒性的機制 8 1.5 研究動機 10 第二章 實驗材料與方法 11 2.1 實驗材料 11 2.2 實驗方法 13 2.2.1 胜肽合成與純化 13 2.2.2 樣品準備與配製 19 2.2.3 吸收及螢光光譜 19 2.2.4 硫磺素T動力學測定(Thioflavin T assay) 20 2.2.5 穿透式電子顯微鏡(Transmission electron microscopy) 22 2.2.6 衰減全反射傅立葉轉換紅外光譜(Attenuated total reflection fourier-transform infrared spectroscopy) 23 2.2.7 凝膠電泳(Gel electrophoresis) 25 2.2.8 圓偏光二色性光譜圖(Circular dichroism spectroscopy) 27 2.2.9 質譜分析 29 2.2.10 細胞毒性測試 31 第三章 結果與討論 32 3.1 胜肽的合成與鑑定 32 3.2 PAD-13吸收與放光光譜 34 3.3 PAD-13對胰島類澱粉蛋白聚集的影響 36 3.4 胰島類澱粉蛋白在PAD-13存在下所形成的纖維影像 38 3.5 胰島類澱粉蛋白纖維細部結構信息 39 3.6 解析PAD-13是否增加蛋白纖維的產生 40 3.7 定時監測PAD-13對胰島類澱粉蛋白二級結構的改變 43 3.8 PAD-13與胰島類澱粉蛋白纖維交互作用力的檢測 45 3.9 PAD-13對MIN6小鼠β細胞毒性測試 48 第四章 結論 49 參考資料 51

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