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研究生: 張玉蓓
Chang, Yu-Pei
論文名稱: 探究人類降鈣素雙位點突變變異體其低聚性的原因
Exploring the cause of reduced amyloidogenicity of human calcitonin double variants
指導教授: 杜玲嫻
Tu, Ling-Hsien
口試委員: 杜玲嫻
Tu, Ling-Hsien
葉怡均
Yeh, Yi-Chun
洪嘉呈
Horng, Jia-Cherng
口試日期: 2023/06/09
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 80
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202300544
論文種類: 學術論文
相關次數: 點閱:72下載:5
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  • 不可逆的聚集行為經常會大幅度地限制胜肽藥物的治療效果。舉例來說,人類降鈣素(human calcitonin, hCT)是一種由32個胺基酸所組成的胜肽激素,由甲狀腺的濾泡旁細胞(C-cells)所分泌,其在生物體中有著調節血鈣水平及維持骨骼型態的生理功能,因此能夠用於治療骨骼等相關疾病,像是骨質疏鬆症和佩吉特氏病等。但由於蛋白質聚集且形成澱粉樣蛋白纖維的傾向很高,導致其作為藥物的潛力受到限制。目前的臨床藥物則由低聚集傾向的鮭魚降鈣素(salmon calcitonin, sCT)所取代,sCT的序列中在N末端也和hCT一樣,具雙硫鍵連接而成的環形結構,但有16個胺基酸的位置與hCT不同,序列同源性很低,部分患者服藥後產生嚴重的副作用。以往的研究證實,Tyr-12和Asn-17兩個位點在誘導hCT 纖維化中起著關鍵作用,雙突變可大大地增強其抗聚集特性。在這項研究中,我們分別檢查雙突變(Y12LN17H)和單突變hCT(Y12L、N17H)形成寡聚物或α-螺旋構形的難易程度,透過比較來了解雙突變體聚性降低的原因,並幫助了解 hCT 纖維化的機制。另外,我們還參考預測軟體及其他文獻的建議繼續尋找更為優化的變異體,透過一系列實驗觀察了解新變異體的聚集特性,雖然結果不如預期,並無法超越原本的雙突變變異體使蛋白質聚性再度降低,然而有這些新變異體的實驗結果比較,協助我們更進一步推測雙突變變異體聚性很低的原因,期望我們的發現也將有助於設計治療性胜肽藥物。

    Irreversible aggregation greatly limits the therapeutic activity of peptide drugs. For example, human calcitonin (hCT) is a peptide hormone composed of 32 amino acids. It is secreted by the parafollicular cells (C-cells) located in the thyroid gland. The hormone plays a crucial role in regulating blood calcium levels and maintaining bone structure in the body. Due to its physiological functions, hCT is valuable in the treatment of conditions such as osteoporosis and Paget's disease. However, its potential for working as drug is limited due to the high propensity in forming amyloid. Current clinical drugs are replaced by low-aggregating salmon calcitonin (sCT) with the same disulfide bond at the N-terminus but different from hCT at the 16 amino acid positions. Low sequence homology of sCT sometimes causes severe side effects when patients are taking it as medicine. Previous studies have shown that Tyr-12 and Asn-17 play a crucial role in inducing hCT fiber formation, and double mutations on these two sites can significantly enhance their ability in preventing aggregation. In this study, we examined the oligomerization and helical formation of hCT double mutant (Y12LN17H) and two single mutants (Y12L, N17H) to help understand the mechanism of hCT fibrillization. In addition, we utilized prediction software and prediction results suggested from computational studies to find an even better substituent. Although the results are not satisfactory, the comparison between new and old hCT double mutants helps us to speculate the low aggregation propensity of the double mutant. We hope that our findings will also help design therapeutic peptide drugs.

    第一章 緒論 1 1.1 類澱粉蛋白與相關疾病 1 1.2 類澱粉蛋白纖維的結構與生成機制 2 1.3 降鈣素(calcitonin, CT)與其生理功能 5 1.4 人類降鈣素(human calcitonin, hCT)的聚集問題 8 1.5 影響hCT聚集的關鍵胺基酸位點 11 1.6 α-螺旋構象的穩定性對於聚集的影響 19 1.7 研究動機 22 第二章 實驗材料與流程 23 2.1 材料與設備 23 2.2 胜肽合成、純化及鑑定 26 2.2.1 胜肽合成 26 2.2.2 純化及鑑定 30 2.3 胜肽樣品的準備與前處理 34 2.4 分析蛋白質序列聚集率的軟體工具 35 2.5 ThT螢光測定(ThT fluorescence assay) 37 2.6 穿透式電子顯微鏡(Transmission electron microscopy, TEM)39 2.7 圓偏光二色性光譜(Circular Dichroism spectra, CD spectra) 40 2.8 光誘導未修飾蛋白交聯反應(Photo-induced cross-linking of unmodified proteins, PIC-UP)43 2.9 凝膠電泳法(Gel electrophoresis) 45 2.10 銀染法(Sliver staining) 47 2.11 環磷酸腺苷含量測定(cAMP Assays)48 第三章 結果與討論 50 3.1 探討DM hCT低聚性的可能原因及個別突變點的重要性 50 3.1.1 相關胜肽鑑定結果(一) 50 3.1.2 胺基酸突變對hCT聚集的影響以及其寡聚化的差異 53 3.1.3 TFE誘導hCT與其變異體形成helix構象的容易程度以及與生物活性的相關性 56 3.2 優化雙突變胜肽對於聚集的影響 59 3.2.1 相關胜肽鑑定結果(二) 59 3.2.2 探討Phe22以Tyr取代對於helix構象及聚集的影響 61 3.2.3 軟體計算結果及雙突變體(Y12LN17M、Y12MN17M)的選擇 65 3.2.4 探討17號位的胺基酸取代對於聚集的影響 67 3.2.5 TFE誘導雙突變體(Y12LN17M、Y12MN17M)形成helix構象的溶液程度以及與生物活性的相關性 70 第四章 結論 72 參考資料 75

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