透過人類降鈣素雙突變序列的相關片段研究其對人類降鈣素纖維化的抑制原因

Investigating the inhibitory property of DM hCT on hCT fibrillization via its relevant peptide fragments

莊雅苹(Chuang Ya-Ping)

指導教授 ： 杜玲嫻

國立臺灣師範大學/理學院/化學系/碩士(2022年)

https://doi.org/10.6345/NTNU202201118

摘要

胜肽不可逆的聚集極大地限制了其作為藥物的生物利用和治療活性，而有效抑制胜肽聚集具有很高的難度。人類降鈣素 (human calcitonin, hCT) 是一種由甲狀腺濾泡旁細胞 (C細胞) 分泌且包含32個殘基的賀爾蒙胜肽，其可以調節血鈣水平，維持骨骼形狀，因此可用於治療代謝性骨病，如骨質疏鬆症和佩吉特病。然而人類降鈣素就是因為具有高度形成澱粉樣蛋白纖維 (Amyloid) 的傾向，而降低其原始功能並限製其作為藥物的潛力。由於鮭魚降鈣素具較高的生物活性和較低的聚集傾向，目前其替代人類降鈣素作為廣泛使用的治療劑。不幸的是，鮭魚降鈣素與人類降鈣素的序列僅有一半相同 (32個胺基酸中有16個與人類降鈣素不同) ，這會使某些臨床治療中病人產生嚴重的副作用，包括厭食、嘔吐和免疫反應等。過去研究顯示，人類降鈣素中胺基酸的改變可以改善其聚集傾向。本實驗室先前的研究中，我們證明雙突變的人類降鈣素 (Y12L N17H hCT, DM hCT) 聚集能力降低許多，並可用於抑制人類降鈣素纖維的形成。
本研究基於DM hCT的序列，我們希望可以藉由拆解其片段來研究其對於人類降鈣素的抑制行為，希望可以對人類降鈣素抑制劑的設計有所幫助。由於通常認為C端不涉及聚集機制，因此我們設計4個C端截短的DM hCT變異體 (DM 1-18、DM 1-22、DM 8-22 和 DM 8-25) ，用以作進一步研究。經由硫黃素T螢光測定實驗中我們發現了四個截短的變異體與DM hCT抑制效果有所差異，爾後並使用圓二色性光譜儀測量了它們形成α-螺旋構象的傾向程度，跟抑制效果比對之後發現，α-螺旋構象對於抑制人類降鈣素聚集是有一定的重要性。DM 1-22為僅次於DM hCT最佳的抑制劑。

關鍵字

人類降鈣素 ； 類澱粉蛋白纖維 ； 胜肽藥物 ； α 螺旋

並列摘要

Irreversible aggregation greatly limits bioavailability and therapeutic activity of peptide-based drugs, so it is highly difficult 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, thus it can be used as a treatment of metabolic bone diseases, such as osteoporosis and Paget's disease. However, hCT has a high propensity to form amyloid fibrils which may reduce its original function and limit pharmaceutical potential. 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) which lead to severe side effects including anorexia, vomiting, and immune reactions in clinical therapy. It has been reported that mutation of hCT can convert its aggregation propensity. In previous study, we demonstrate that a double-mutated hCT (Y12L N17H hCT, DM hCT) has much lower ability to form amyloid and can be used to inhibit hCT amyloid formation.
Based on performance of DM hCT, we hope to understand its inhibitory behavior on hCT aggregation. We synthesize its truncated forms (DM 1-18, DM 1-22, DM 8-22, and DM 8-25) by removing C-terminal residues, and conducted further investigation using these peptide fragments. From thioflavin T fluorescence assay, we found that four truncated variants and DM hCT have different extents in self-assembly and inhibitory effect on hCT fibrillization. By using circular dichroism, we measured their propensity in forming α-helix conformation and found that formation of α-helix might be important to the inhibitory effect of DM hCT. DM 1-22 is a peptide fragment which can suppress hCT aggregation although efficiency is still less than DM hCT.

並列關鍵字

Human calcitonin ； amyloid ； peptide drug ； α-helix

參考文獻

1. Siddiqi, M. K.; Majid, N.; Malik, S.; Alam, P.; Khan, R. H., Amyloid oligomers, protofibrils and fibrils. Macromolecular protein complexes II: Structure and Function 2019, 471-503.

2. Sipe, J. D.; Benson, M. D.; Buxbaum, J. N.; Ikeda, S.-i.; Merlini, G.; Saraiva, M. J.; Westermark, P., Amyloid fibril proteins and amyloidosis: chemical identification and clinical classification International Society of Amyloidosis 2016 Nomenclature Guidelines. Amyloid 2016, 23 (4), 209-213.

3. Dobson, C. M., Protein misfolding, evolution and disease. Trends in biochemical sciences 1999, 24 (9), 329-332.

4. Goedert, M., Alzheimer’s and Parkinson’s diseases: The prion concept in relation to assembled Aβ, tau, and α-synuclein. Science 2015, 349 (6248), 1255555.

5. Antony, P. M.; Diederich, N. J.; Krüger, R.; Balling, R., The hallmarks of P arkinson's disease. The FEBS journal 2013, 280 (23), 5981-5993.