Please use this identifier to cite or link to this item: http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/111116
Title: 人類降鈣素雙突變體提升抗纖維化能力及做為胜肽藥物之潛能
Minimum Acquisition of Double Mutation in Human Calcitonin Enhances its Resistance to Fibrillization and its Use as Therapeutic Polypeptides
Authors: 杜玲嫻
Tu, Ling-Hsien
陳怡婷
Chen, Yi-Ting
Keywords: 人類降鈣素
胜肽藥物
錯誤折疊
胜肽設計
預測軟體
Human calcitonin
Peptide-based drugs
Misfolding
Peptide design
Prediction software
Issue Date: 2019
Abstract: 胜肽的不可逆聚集通常導致其生理功能的喪失。人類降鈣素為32個胺基酸組成的荷爾蒙胜肽,在人體內是由甲狀腺周圍的濾泡旁細胞所分泌,其自然態參與調節血鈣平衡和維持骨骼結構,因此可用於治療骨骼相關疾病,例如骨質疏鬆症和佩吉特氏病。然而降鈣素易形成類澱粉蛋白沉積物的高聚集傾向,導致其天然功能喪失限制做為臨床藥物的發展。由於鮭魚降鈣素具有較高的生物活性和較低的聚集傾向而替代人類降鈣素成為廣泛使用的治療藥劑。可惜的是,鮭魚降鈣素的序列只有50%與人類降鈣素相同,這種低序列同源性會導致嚴重的副作用,包括臨床治療中的厭食、嘔吐和免疫反應。已顯示針對序列做修飾可以調節人類降鈣素的聚集傾向。先前在預測軟體Waltzs幫助下所進行的研究指出,當人類降鈣素的12、17、26、27和31這五個胺基酸位置發生突變會顯著地降低聚集速率,但仍保持生理結構和功能。 在本研究中,我們嘗試對人類降鈣素序列做最小變化的修飾,成功設計並合成出抗聚集的雙突變變異體(Y12L N17H hCT)。從圓偏光二色性光譜中我們發現Y12L N17H hCT具有穩定的螺旋結構。此外,透過光誘導未修飾蛋白交聯反應和電泳分析顯示,人類降鈣素會形成高分子量寡聚物,而Y12L N17H hCT則是以單體為主。Y12L N17H hCT在緩衝溶液和脂質存在下皆能抑制原生型hCT聚集。最重要的是,Y12L N17H hCT仍具有生物活性,可以和降鈣素受體結合並能激活cAMP途徑。因此,我們推測Y12和N17在人類降鈣素形成類澱粉蛋白纖維過程中為關鍵的胺基酸位置,而Y12L N17H hCT有做為胜肽藥物之潛能。
Irreversible aggregation of polypeptides commonly results in the loss of their physiological functions. Human calcitonin (hCT) is a 32 residues peptide hormone secreted from the parafollicular cells or C cells of the thyroid gland. The native form of calcitonin is involved in mediating calcium homeostasis and maintaining bone structure, hence it can be used as a treatment of metabolic bone diseases, such as osteoporosis and Paget's disease. However, its high tendency to form amyloid aggregates limits the clinical application. Salmon calcitonin (sCT) is the replacement of hCT as a widely therapeutic agent due to its higher bioactivity and lower propensity to aggregation. Unfortunately, sCT has low sequence identity with hCT (differing from hCT in 16 of the 32 amino acids) leading to severe side effects including anorexia, vomiting, and immune reactions in clinical therapy. Modifications of hCT have been shown to modulate its aggregation propensity. The previous study with the help of Waltzs prediction software found that the mutations which occur at five residues 12, 17, 26, 27 and 31 (called phCT) significantly decreased the rate of aggregation, but maintained the structure and biological function of peptide. In this work, we try to minimize the changes on hCT and hope to generate the aggregation-resisting variant for therapeutic use. Herein, we successfully design and synthesize a double-mutated hCT(Y12L N17H hCT) that shows delayed aggregation profile compared with hCT. From circular dichroism spectra, we find that Y12L N17H hCT adapts partial α-helical structure and is as stable as phCT. Furthermore, photo-induced cross-linking of unmodified proteins (PICUP) and SDS-PAGE analysis reveal that hCT forms high molecular weight oligomers, while Y12L N17H hCT is monomer dominant. This variant is also effective in inhibiting hCT aggregation in buffer and in the presence of large unilamellar vesicles (LUVs). Most importantly, Y12L N17H hCT can still bind calcitonin receptor and activate the cAMP pathway. Therefore, we assume that Y12 and N17 play key roles in hCT amyloid aggregation and Y12L N17H hCT has the potential to be a peptide-based drug.
URI: http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060642085S%22.&%22.id.&
http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/111116
Other Identifiers: G060642085S
Appears in Collections:學位論文

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