(壹)探討胰島類澱粉蛋白序列位置一號離胺酸的角色 (貳)探討胡椒鹼及其衍生物對胰島類澱粉蛋白聚集的影響

Abstract

主題一 探討胰島類澱粉蛋白序列位置一號離胺酸的角色 胰島類澱粉蛋白(Islet amyloid polypeptide，IAPP)是由37個胺基酸所組成的胜肽荷爾蒙，其不正常堆積會誘導胰島β細胞喪失功能甚至凋亡，因此被認為與第二型糖尿病有相當重要的關聯。IAPP在人體內的聚集機制相當複雜，科學家尚未完全了解其聚集機制為何。IAPP的聚集傾向很容易受到序列改變而有明顯差異，即使只有改變其中一個胺基酸。在過去，許多研究提出了IAPP特定區域或是特定胺基酸位置對其聚集之重要性，然而在這些研究中鮮少指出IAPP第一號胺基酸離胺酸的影響。本實驗室先前的研究中探討IAPP經過醣化修飾後得到的最終醣化產物AGE-IAPP之聚集情形，發現AGE-IAPP聚集傾向較IAPP高許多，而且會誘導IAPP更快速的聚集。而AGE-IAPP與IAPP差異僅在第一號位的胺基酸為羧甲基離胺酸。 為了能更了解IAPP第一號位離胺酸在IAPP聚集過程以及IAPP纖維的構型中所扮演的角色，我們額外設計了兩條IAPP突變體—K1E IAPP與K1Nle IAPP，它們分別是將第一號位的離胺酸換成麩胺酸與正亮胺酸。從硫磺素T動力學實驗中，我們發現帶負電或是不帶電的胺基酸側鏈會使IAPP單體之間有更高的聚集傾向。結合TEM、ATR-FTIR與CD實驗，我們也發現第一號位胺基酸的改變不會讓成熟纖維結構改變，但是會改變IAPP單體與纖維之間的動態平衡。除此之外，我們還測試了常見的IAPP聚集抑制劑應用於K1E IAPP與AGE-IAPP的聚集中，我們發現部分黃酮類抑制劑的抑制聚集機制可能需要離胺酸側鏈的胺基參與反應。總結上述，我們點出了IAPP第一號位也是參與IAPP聚集的一個非常重要的角色，其影響不容忽略。第一號位胺基酸若經由體內的修飾機制而改變其側鏈官能基，將有機會影響到用於降低類澱粉蛋白聚集藥物的效果。

主題二 探討胡椒鹼及其衍生物對胰島類澱粉蛋白聚集的影響 胰島類澱粉蛋白(Islet amyloid polypeptide，IAPP)是由37個胺基酸所組成的胜肽荷爾蒙，其不正常聚集是第二型糖尿病的病理特徵，許多細胞實驗已指出其聚集的過程或是形成的沉積物與β細胞質量減少甚至凋亡有很大關係。所以科學家致力於尋找影響IAPP聚集的小分子。過去專家認為IAPP聚集的過程所產生之寡聚體會和細胞膜產生交互作用而誘發細胞毒性，因此希望能透過藥物添加減少其聚集傾向，或是加快其聚集以跳過寡聚體生成的階段，形成較低細胞毒性的沉積物。然而可惜的是，IAPP本身聚集的機制並不清楚，加上過去眾多研究能影響IAPP聚集的小分子中，無法說明小分子影響聚集的機制，故尋找能針對IAPP影響其聚集的藥物是非常龐大的工程。 近年來，薑黃素和胡椒鹼的組合藥物已被證明可減少第二型糖尿病患者的血糖和相關病症。薑黃素過去已被認為可以減少IAPP誘導的細胞毒性，然而尚未有人探討胡椒鹼對IAPP聚集的作用效果。本篇研究中發現胡椒鹼的水解產物胡椒酸具有加速IAPP聚集的功能，因此我們想深入了解甚麼是胡椒酸影響IAPP聚集的重要結構，故以胡椒鹼結構為基礎合成一系列衍生物，將衍生物添加進IAPP並以硫磺素T動力學實驗測試衍生物的影響。我們發現結構中必須要含有重要官能基如羧酸、苯環上的取代基應以疏水的取代基如亞甲基二氧基或甲氧基為主。結構中間的雙鍵數量也會影響到加速效果。本篇研究期望能為開發影響IAPP聚集的小分子藥物提供一個參考方向，並發展出能針對IAPP聚集的藥物應用於IAPP相關類澱粉變性症。主題一 探討胰島類澱粉蛋白序列位置一號離胺酸的角色 Islet amyloid polypeptide (IAPP) is a peptide hormone consists of 37 amino acids. The abnormal deposition of IAPP, known as IAPP amyloid, in pancreas may induce β-cell dysfunction or cell apoptosis. Therefore, it is considered that there is a close relationship between amyloid formation of IAPP and type 2 diabetes. However, the mechanism of IAPP amyloid formation in human body has not been fully discovered. Different primary sequence of IAPP may affect their aggregation tendency. In the past, the influence of specific regions or certain residue has been examined in IAPP aggregation, but few studies have discussed the effect of lysine residue at position 1 of IAPP. In our previous study, we examined the impact of protein glycation of IAPP. Glycated IAPP, named AGE-IAPP, formed amyloid faster than normal IAPP and can enhance IAPP aggregation process. The only difference in sequence between IAPP and AGE-IAPP is the first residue which is carboxymethyllysine in AGE-IAPP. To gain more insight into the role of Lys 1 in kinetics of fibril formation and fibril morphology, we synthesized IAPP and three IAPP variants, K1E-IAPP, K1Nle-IAPP and AGE-IAPP. Lysine was mutated to glutamate and norleucine respectively for K1E IAPP and K1Nle IAPP. We found that the negative or neutral charged side chain at position 1 may induce higher aggregation propensity between monomers. We also found that the replacement of the first reside will not change the secondary structure of mature fibers, but may affect the monomer-fibril equilibrium state. Besides, we also tested some common IAPP aggregation inhibitors on K1E IAPP and AGE-IAPP. We found that some flavonoid inhibitors may first need to react with the lysine residue then exhibit their inhibitory effect. Our study emphasizes the importance of the lysine residue in regulating IAPP aggregation, and modifications on this residue may affect the inhibitory effect of some potential drugs in amyloidosis treatment.

主題二 探討胡椒鹼及其衍生物對胰島類澱粉蛋白聚集的影響 Unusual fibrillar accumulation of 37-residue peptide hormone islet amyloid polypeptide (IAPP) is a pathological hallmark of type 2 diabetes (T2D). Many biological evidences have shown that aggregation of IAPP is closely associated with mass reduction or apoptosis of β-cell in pancreas. A common strategy to prevent β-cell disorders is to inhibit the IAPP aggregation process by small molecular inhibitors. In contrast, threreis another fashion to deal with such problem by accelerating the aggregation process to reduce the formation of toxic oligomers. Unfortunately, the inhibition mechanism of some present inhibitors on IAPP aggregation is not clear, and it is a difficult task to find effective and specific molecules to modulate IAPP aggregation. In recent years, the usage of curcumin and piperine combination has been shown to reduce glycemia and related complications in T2D. Curcumin is thought to be capable of reducing amyloid-induced cytotoxicity. However, the effect of piperine on IAPP aggregation has yet been explored. In our research, we found the hydrolyzed product of piperine, piperic acid, may enhance IAPP aggregation. Therefore, piperine is used as a core stucuture and a series of derivatives have been synthesized to further understand the structure activity relationship of piperine in IAPP aggregation by using thioflavin T (ThT) assay. We found several essential characteristics, such as carboxylic acid, and the substituents on the benzene ring should mainly be hydrophobic. The number of double bonds in the center of the structure may also affect its accelerating effect. We expected to provide some information for the development of small molecule drugs that can affect IAPP aggregation.