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研究生: 謝宜靜
Hsieh, I-Ching
論文名稱: 設計具有抗聚集性的人類降鈣素變異體
Design of aggregation-resistant variants of human calcitonin
指導教授: 杜玲嫻
Tu, Ling-Hsien
口試委員: 王勝仕
Wang, Sheng-Shih
李以仁
Lee, I-Ren
口試日期: 2021/07/21
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: 90
中文關鍵詞: 人類降鈣素類澱粉蛋白纖維聚集胜肽設計變異體
英文關鍵詞: human calcitonin, amyloid fibril, aggregation, peptide design, variants
DOI URL: http://doi.org/10.6345/NTNU202100795
論文種類: 學術論文
相關次數: 點閱:128下載:0
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  • 胜肽的不可逆聚集極大地限制了其做為藥物的生物利用度和治療活性,因此有效抑制胜肽聚集是一個重要的挑戰。人類降鈣素(Human calcitonin, hCT)是一種由32個胺基酸組成的的胜肽激素,經由甲狀腺中的濾泡旁細胞(C-cell)所分泌,並且hCT在生物體中具有調節血鈣水平並維持骨骼型態的生理功能,因此適合用於治療骨骼相關等疾病,如:骨質疏鬆症和佩吉特氏症。然而,hCT具有形成類澱粉蛋白纖維的高聚集傾向,這可能導致原始功能降低而阻礙其藥物潛力。目前臨床治療中,鮭魚降鈣素(Salmon calcitonine, sCT)因為較高的生物活性和極低的聚集傾向,而代替hCT成為廣泛使用的胜肽藥物,但可惜的是sCT與hCT的低序列同源性,造成患者服藥後有嚴重的副作用和免疫反應發生,因此本研究的目標是設計一種與hCT保持高序列相似性的變異體。先前已證明在hCT的特定胺基酸位點上進行修改就足以有效抑制hCT聚集,所以我們試圖找尋出調節胜肽聚集的關鍵位置和胺基酸,使hCT能以最微小的序列變化設計出具有低聚集傾向但不損失生物活性的理想策略。在這項研究中,我們使用預測軟體發現Tyr12位置在抑制hCT聚集中起關鍵作用,從而提出了三種人類降鈣素變異體(Y12E、Y12P和Y12R),並且透過多種實驗觀察聚集特性,包括ThT螢光動力學、TEM、SDS-PAGE和DLS,顯示Y12E表現出較好的抗聚集能力,以及指出三種變異體與磷脂膜相互作用和TFE誘導劑下結構幾乎不受太大影響。我們還進一步的測定細胞內被激活的cAMP來檢測這些變異體是否能與其受體結合,卻意外發現,Tyr12的胺基酸側鏈結構可能是與受體結合的重要部位。本研究證實了Tyr12突變成Glu、Pro雖然能顯著改善其類澱粉蛋白纖維形成,可是卻也嚴重影響其發揮正常的生理功能,需要更謹慎地探討Tyr12在序列中的意義。

    Irreversible aggregation greatly limits bioavailability and therapeutic activity of peptide-based drugs, so it is an important challenge to effectively inhibit peptide aggregation. Human calcitonin (hCT) is a 32-residue peptide hormone that is secreted by the parafollicular cells (C-cell) in the thyroid. hCT can regulate blood calcium levels and maintain bone formation, hence it can be used as a treatment of metabolic bone diseases, such as osteoporosis and Paget's disease. However, hCT has a relatively high propensity to form amyloid fibrils which may reduce its original function and limit pharmaceutical potential. In clinical treatment, salmon calcitonin (sCT) is the replacement of hCT as a widely therapeutic agent due to its higher biological activity and extremely low tendency to aggregate. Unfortunately, sCT has the low sequence homology with hCT that could induce severe immunoreaction in patients taking. Therefore, the goal of this study is to design a variant that maintains a high degree of sequence similarity with hCT. In previous studies, it has been demonstrated that modifying the specific residues of hCT is sufficient to effectively inhibit hCT aggregation, so we tried to find the key residue that regulate the aggregation of hCT. This minor sequence modification of hCT may be an ideal strategy to the rational design of low aggregation propensity yet without loss of bioactivity. In this work, we used amyloid propensity prediction software and found that the Tyr12 position plays a key role in regulating hCT aggregation. Thus, we proposed three human calcitonin variants (Y12E, Y12P, Y12R) and examined the aggregation characteristics by using multiple techniques including ThT fluorescence, TEM, SDS-PAGE and DLS. It was found that Y12E has better anti-aggregation ability than Y12P, but Y12R formed short fibers. It was unexpectedly discovered that the interaction of the three variants with the phospholipid membrane and the TFE-induced substructure are almost unaffected due to mutation. We further confirmed whether the variants can interact with receptors through cAMP assay. We surprisedly found that side chain of Tyr12 was a key feature upon receptor binding. This study indicated that the mutation of Tyr12 to Glu and Pro can significantly improve hCT fibril formation, but it also seriously affects its normal physiological functions. We need to more carefully explore the significance of Tyr12 in hCT sequence.

    第一章 緒論 1 1-1類澱粉蛋白與疾病的關聯 1 1-2類澱粉蛋白纖維結構與生成機制 3 1-3降鈣素(Calcitonin, CT)與其生理功能 7 1-4人類降鈣素(Human calcitonin, hCT)之聚集問題 11 1-5影響hCT聚集的關鍵胺基酸位置 19 1-6研究動機 25 第二章 實驗材料與方法 26 2-1材料與設備 26 2-2分析序列聚集率工具 29 2-3胜肽合成,純化以及鑑定 32 2-4胜肽樣品的準備與前處理 40 2-5硫磺素T螢光測定(Thioflavin T fluorescence assay)41 2-6穿透式電子顯微鏡(Transmission electron microscopy, TEM)44 2-7十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)與銀染法(Sliver staining)46 2-8染料滲漏測定(Dye leakage assay)52 2-9圓偏光二色性光譜(Circular Dichroism , CD)55 2-10動態光散射(Dynamic light scattering, DLS)58 2-11環腺苷酸活性測定(Cyclic AMP activity assay)61 第三章 結果與討論 63 3-1影響hCT聚集的最關鍵位點 63 3-2胜肽鑑定結果 66 3-3變異體對hCT聚集的影響 70 3-4透過DLS分析聚集過程 72 3-5經由核誘發(Seeding)進一步確認Y12E和Y12P的聚集傾向 75 3-6 TFE誘導hCT與其變異體的helix構象穩定 76 3-7變異體與磷脂膜之間的相互作用 78 3-8最佳抗聚集變異體Y12E抑制hCT聚集的程度 80 3-9突變Tyr12對生物活性的影響 82 第四章 結論 84 參考資料 85

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