探討修飾胰島類澱粉蛋白以增強其抗菌活性的可能性

Abstract

人類胰島類澱粉蛋白(Islet amyloid polypeptide, IAPP)是由37個胺基酸所組成，和胰島素共同由胰腺β細胞所分泌的賀爾蒙胜肽，由於IAPP聚集形成纖維狀沉積物的過程會造成細胞死亡，所以過去曾有文獻報導，IAPP可以作為一種對抗金黃色葡萄球菌的抗菌胜肽使用，起因可能是AMP與膜互相作用後形成孔隙，導致細菌膜破裂進而死亡，這與類澱粉蛋白聚集造成細胞死亡被推測的機制之一類似，因此類澱粉蛋白被認為有作為AMP的潛力，本研究試將以胺基酸替換的方式設計比IAPP抗菌效果更好的AMP。由於AMP多為帶正電荷具兩親性的大分子，我們先以理論計算工具FoldAmyloid計算以帶正電荷的離胺酸(K)取代IAPP不同位點之後的聚集傾向，結果發現將24和33號位的甘胺酸(G)取代為離胺酸後可能不會改變IAPP聚集的特性，並且可能可以增加跟細菌膜的作用，因此我們合成了G33K-IAPP和G24K-IAPP，並透過硫磺素T螢光實驗、圓偏光二色性光譜和穿透式電子顯微鏡驗證這兩條胜肽確實會聚集形成纖維狀沉積物。由抗菌實驗我們發現G33K-IAPP和G24K-IAPP均會抑制金黃色葡萄球菌的生長，並且抗菌效果比IAPP更好。Human islet amyloid polypeptide (IAPP) is a 37-residue peptide and it is co-secreted with insulin from the pancreatic β-cells. The formation of fibrillar deposits by IAPP may cause β-cell death. In the past, it was also reported that IAPP can be used as antimicrobial peptides (AMP) against Staphylococcus aureus. The mechanism of action of AMP accepted by most people is that they interact with bacterial membranes to form pores, leading to membrane rupture and death. This mechanism of action is similar to the mechanism by which amyloidogenic proteins cause cell death, so amyloidogenic proteins are regarded as AMP potentially. This study aims to design new AMP with better antibacterial effects by appropriate amino acid substitution. AMP are usually cationic (positively charged) and amphipathic. Therefore, we used the theoretical calculation tool FoldAmyloid to calculate the aggregation tendency of IAPP after it was substituted by positively charged lysine (Lys, K) at each residue. It was found that glycine (Gly, G) substitution by Lys at residues 24 and 33 made IAPP retain aggregation tendency. Here, we synthesized two peptides, G33K-IAPP and G24K-IAPP, and verified the aggregation properties of G33K and G24K by using the thioflavin-T fluorescence assay, circular dichroism spectroscopy, and transmission electron microscopy. Through antibacterial experiments, we found that both G33K-IAPP and G24K-IAPP can inhibit the growth of Gram-positive Staphylococcus aureus, and the antibacterial effect is better than IAPP.