Indolicidin及其類似物的聚集行為及其與仿生細胞膜間之交互作用; Oligomerization behavior of Indolicidin and its analogues and its interaction with bio-mimic membranes

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Title:	Indolicidin及其類似物的聚集行為及其與仿生細胞膜間之交互作用;Oligomerization behavior of Indolicidin and its analogues and its interaction with bio-mimic membranes
Authors:	林達翰;Da-han Lin
Contributors:	化學工程與材料工程研究所
Keywords:	磷脂微脂粒滲漏;仿生細胞膜;分子動態模擬;凝膠電泳;胜肽寡聚;鹼性抗生胜肽;Indolicidin;antimicrobial peptide;electrophoresis;molecular dynamics simulations;dye leakage;peptide oligomerization
Date:	2011-07-27
Issue Date:	2012-01-05 11:22:48 (UTC+8)
Abstract:	鹼性抗生胜肽Indolicidin (IL) 因具有廣泛且迅速之抗菌活性，甚至可以對抗病毒與癌細胞，也不易造成微生物抗藥性，因此被認為是極具有潛力的抗生藥物。然而，Indolicidin對人體紅血球的溶血活性，限制了其在臨床應用上之發展。本實驗室曾利用分子動態模擬設計了一連串的Indolicidin類似物：IL-K7、IL-F89、IL-K7F89，設計出具有高抗菌、低溶血之抗生胜肽，但對於此類胜肽何以導致溶血又何以能抗菌並不能完全了解。先期的研究發現，Indolicidin及其類似物在水溶液中似乎有寡聚現象，而其寡聚程度與其溶血活性正相關，因此本論文之研究目的在於探討Indolicidin及其類似物的寡聚現象以及寡聚現象對於仿生物細胞膜的影響。我們利用凝膠電泳與分子模擬探討Indolicidin及其類似物的寡聚行為，結果發現Indolicidin及其類似物最多由三條胜肽形成聚集。然後我們測試Indolicidin及其類似物與仿生微脂粒間的作用。我們使用兩種微脂粒，一種完全由POPC組成，表面呈現電中性；另一種由POPC與POPG混合物形成，表面帶負電荷。結果發現這些胜肽對兩種仿生物細胞微脂粒作用情形類似，皆能使其產生破洞，破洞程度也隨著濃度的增加而增加。然而當我們使用文獻中所載低溶血性的SAP胜肽時，則發現SAP並不會造成帶負電荷的POPC/POPG微脂粒的破壞，由此可說明SAP的抗菌性不是經由破壞細胞膜而產生。我們又以分子模擬觀察Indolicidin寡聚體在兩種仿生細胞膜上不同位置造成膜雙層擾動的情形。由結果發現，Indolicidin寡聚體確實會會對磷脂雙層膜造成擾動，甚至在膜中心有些微水分子滲入，說明可能有短暫孔洞的產生。由以上研究，我們猜測Indolicidin及其類似物所形成之寡聚物，在吸附於紅血球細胞膜後，會造成短暫的孔洞，而此短暫孔洞的形成可能與其溶血活性有關。 Cationic antimicrobial peptide –Indolicidin (IL) has been considered to be a potential antibiotic drug by its broad-spectrum of antibiotic activity against bacteria, fungi and even viruses. However, the hemolytic activity limits its clinical applications. To reduce its hemolytic activity, we had designed several less hemolytic Indolicidin analogues, IL-K7, IL-F89, IL-K7F89, through MD simulations in our previous work. But the mechanisms of its antibiotic and hemolytic behaviors are stIndolicidinl not clear. We had found that these peptides might oligomerized in aqueous solution and the degree of peptide oligomerization was consisted with their hemolytic activity. Therefore, we try to first study the possible structure of peptide oligomerization. And then the interactions between peptide oligomers and bio-mimic membranes are under investigation. We studied the size of oligomer by gel electrophoresis and molecular dynamic simulation. The results showed that Indolicidin and its analogues might form dimer or trimer. We then studied the interaction between peptides and bio-mimic membranes by calcium dye leakage experiment. Two types of bio-mimic liposomes were made. One was made of pure POPC lipid of which the surface was neutral, the other was made of the mixture of POPC and POPG of which the surface was negatively charged. We found that both types of liposomes were perturbed by the peptides and the degree of dye release was increased with the amount of peptide added. Surprisingly, no dye leakage was observed when we test the peptide SAP, a well-known antimicrobial peptide of low hemolytic activity. The result indicated that the antimicrobial activity of SAP was not caused by cell membrane perturbation. We further investigate the interaction between Indolicidin trimmers and bio-mimic membranes by all-atom molecular dynamic simulation. It was found that the Indolicidin trimmer dissociated and then re-associated in the POPC/POPG membrane. Simultaneously, we found that water molecules entered the hydrophobic core of membrane. The result supported the possibility of transient pore formation. All the results indicated that Indolicidin and its derivatives might form dimmers or trimmers in the aqueous solution. The oligomers adsorbed onto the membrane and perturb the membrane structure. We considered that the membrane perturbation by peptide oligomers was related to the hemolytic behavior of these peptides.
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