評估環境靈敏的小分子螢光探針做為偵測不同類澱粉蛋白纖維形成的可能

Environmentally Sensitive Fluorescent Probes for Detecting Different Amyloid Fibrils

陳韋伶(Chen Wei-Ling)

指導教授 ： 杜玲嫻

國立臺灣師範大學/理學院/化學系/碩士(2018年)

https://doi.org/10.6345/THE.NTNU.DC.073.2018.B05

摘要

多肽與蛋白質因錯誤摺疊而產生沉積物是許多疾病的共同病徵，例如：乙型類澱粉蛋白於腦內的沉積被認為與阿茲海默症有深的關聯性；胰島內的胰島類澱粉蛋白之沉積物被認為是第二型糖尿病的重要病徵之一，除了發展抑制蛋白聚集的藥物之外，偵測類澱粉蛋白纖維的生成是科學家致力研究的方向。目前普遍偵測類澱粉蛋白纖維生成與聚集的小分子為硫磺素T，但硫磺素T的通用性使其缺乏對特定類澱粉蛋白纖維的專一性以及會受到某些抑制小分子的影響而會有錯誤的訊號產生，因此於本研究中，我們利用對於環境靈敏度高的黃酮類化合物，在此命名為3-HF-ene-4’-OMe，作為偵測胰島類澱粉蛋白纖維的小分子螢光探針。從研究中我們確認3-HF-ene-4’-OMe對於胰島類澱粉蛋白纖維的反應性，以及透過結合親和力測試發現3-HF-ene-4’-OMe與胰島類澱粉蛋白纖維的結合力較硫磺素T高。而當3-HF-ene-4’-OMe分別與胰島類澱粉蛋白纖維及乙型類澱粉蛋白的蛋白纖維結合時，兩者的螢光波長明顯不同，表示3-HF-ene-4’-OMe與乙型類澱粉蛋白的蛋白纖維結合位置的環境不同於與胰島類澱粉蛋白纖維結合位置的環境。此外，我們利用3-HF-ene-4’-OMe來偵測胰島類澱粉蛋白聚集，聚集結果與硫磺素T偵測的結果一致。之後，再分別利用硫磺素T與3-HF-ene-4’-OMe偵測白藜蘆醇用於胰島類澱粉蛋白的抑制試驗。雖然兩者的螢光訊號在白藜蘆醇存在下皆有削弱的傾向，但由於3-HF-ene-4’-OMe與胰島類澱粉蛋白纖維的結合力較硫磺素T佳，因此在作為該抑制試驗中，3-HF-ene-4’-OMe是比較適合的探針，依舊可以觀察到蛋白質聚集過程。我們亦建議在類澱粉蛋白的抑制試驗應使用多個探針來提高實驗結果的可信度。

關鍵字

胰島類澱粉蛋白 ； 硫磺素T ； 小分子螢光探針 ； 黃酮類化合物

並列摘要

Deposition of misfolded polypeptide and protein is a hallmark of many diseases. For example, amyloid β (Aβ) was found to deposit in the brain of Alzheimer's disease (AD). Islet amyloid polypeptide (IAPP) deposition is highly associated with type 2 diabetes. Many scientists devote their efforts in finding methods to detect the formation of amyloid fibrils and the location of their deposition. Thioflavin T (ThT) is generally used as a fluorescence probe to detect amyloid fibrils formation and aggregation, however, ThT can’t tell the difference among amyloid fibrils and sometimes the fluorescence intensity is quenched by some inhibitors used in aggregation studies. Here, we used an environmentally sensitive flavone-based fluorescent probe, 3-HF-ene-4’-OMe as an alternative fluorescent probe. In this study, we showed that 3-HF-ene-4’-OMe is specific for IAPP fibrils but not monomers, and the binding affinity of 3-HF-ene-4’-OMe to IAPP fibrils is higher than ThT. In additions, 3-HF-ene-4’-OMe exhibits different emission wavelength while binding to IAPP or Aβ fibrils indicating that the binding site polarity of Aβ is different from IAPP. Moreover, 3-HF-ene-4’-OMe shows better ability in monitoring IAPP aggregation in the presence of resveratrol, an IAPP inhibitor. Although 3-HF-ene-4’-OMe was somehow replaced by resveratrol, it is still more suitable than ThT in IAPP inhibition study due to it’s better binding affinity with IAPP fibrils. As a result, our results also pointed out the experimental importance of using multiple probes for amyloid inhibition studies.

並列關鍵字

Islet amyloid polypeptide ； Thioflavin T ； fluorescence probe ； flavone

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