臺灣大學: 化學工程學研究所王勝仕林耿琦Lin, Keng-ChiKeng-ChiLin2013-03-272018-06-282013-03-272018-06-282010http://ntur.lib.ntu.edu.tw//handle/246246/252330研究證實，疏水性之β-類澱粉胜肽(β-amyloid peptide, Aβ)為老化癍塊中的主要蛋白質成份，此胜肽來自於β-類澱粉前趨蛋白(β-amyloid precursor protein, APP)，經由兩種不同蛋白質水解酵素水解後的產物，釋出之Aβ則可能因環境因子導致其聚集沉澱進而產生細胞毒性，因而形成阿滋海默症。此外，從基因的研究發現，阿滋海默症可分為偶發型(sporadic type)與家族型(familial type)兩種，且由文獻得知，家族型之β-類澱粉胜肽在分子聚集機制上與偶發型有明顯地差異。 由於β-類澱粉前趨蛋白為一種穿膜蛋白(transmembrane protein)，因此無論在經由蛋白質水解酵素切割前，於膜內之起始結構變化與切割後釋出於膜外環境下之Aβ，皆與細胞膜之間有相當密切之交互作用，同時，研究也提出，此交互作用所產生之差異性可能受細胞膜所帶電荷、組成、流動性等因素影響。 因此，本研究分別以1,2-dimyristoyl-sn-glycerol-3-phosphocholine(DMPC) 與1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)兩種不同類型之脂質當成細胞膜模型(membrane model)，以實驗方法配合分子動力學觀點探討細胞膜對β-類澱粉胜肽的影響。研究一開始嘗試以E.coli為宿主細胞(host cell)培養並純化生產目標胜肽:野生型Aβ(1-40)與突變型Arctic Aβ(1-40)，由MALDI-TOF質譜儀分析結果得知所測得之分子量與理論值符合，且產率分別約為4 mg/L及2 mg/L。接著於此兩種胜肽環境中添加不同濃度之di-C7-PC，實驗結果顯示蛋白質結構變化與類澱粉纖維形成速率隨添加di-C7-PC的提升而呈現正相關之結果，其中，當添加5 mM di-C7-PC時，我們發現蛋白質結構轉變情形與類澱粉纖維形成速率最為明顯。此外，由分子模擬數據得知，無論是野生型Aβ(1-40)與突變型Arctic Aβ(1-40)，其結構與能量變化確實與添加di-C7-PC的多寡有密切關係，結果顯示野生型Aβ(1-40)與突變型Arctic Aβ(1-40)結構皆有明顯地轉變，尤其當突變型Arctic Aβ(1-40)於較高濃度時，可發現在總模擬時間100 ns期間，其二級結構隨即轉變為β-sheet rich之構形。最後，我們更進一步嘗試以Aβ(1-60)為C99於穿膜環境下之模型，藉由分子動力學方法獲得起始結構資訊，結果發現突變型Arctic Aβ(1-60)於穿膜環境之結構較野生型Aβ(1-60)顯得不穩定，且從二級結構演化分析上發現，在模擬時間為100 ns期間內，兩者皆於殘基40-42範圍內有去折疊(unfolding)現象。 由本論文研究結果發現，細胞膜環境確實對於蛋白質結構轉變上伴演很重要的角色，因此，我們期望從實驗及理論模擬兩方面著手，進一步探討磷脂質對野生型與突變型β-類澱粉胜肽之聚集行為及相關分子機制。Previous studies have proved that the hydrophobic β-amyloid peptide (Aβ), the major proteinacious constituent of senile plaques, is the product of β-amyloid precursor protein cutting by two different enzymes. Due to the effects of various environmental factors, the released Aβ may aggregate and induce cytotoxicity to cause Alzheimer’s disease. In addition, findings from the genetic researches indicate that there are two forms of Alzheimer’s disease, sporadic and familial forms, and evidence shows that the mechanisms of aggregation behavior for these two forms are significantly different. Given that β-amyloid precursor protein is one of the transmembrane proteins, cell membrane is evidently closely correlated with either the initial structure inside the membrane before cutting by enzymes or the resultant Aβ species released into the extracellular environment. The aforesaid membrane-protein interaction is evidenced to be influenced by the charge, composition, and fluidity of membrane. With two types of phospholipids, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC), used as the model systems, the current study was aimed to explore the effects of phospholipid molecules on β-amyloid peptides experimentally and theoretically. We first used E.coli as the host cells to express and purify our target peptides, wild-type Aβ(1-40) and Arctic Aβ(1-40). The molecular mass of the purified peptide was verified by MALDI-TOF mass spectrometry. The yields for wild-type Aβ(1-40) and Arctic Aβ(1-40) were found to be ~4 mg/L and ~2 mg/L, respectively. In the second part, we found that the rate of fibrillization and the changes of protein structure were positively correlated with the concentration of added di-C7-PC. The highest rate of fibrillization and the most marked transition of protein structure were observed upon addition of 5 mM di-C7-PC. In addition, our 100ns molecular dynamic simulations results demonstrated that, in both wild-type Aβ(1-40) and Arctic Aβ(1-40), the changes in peptides structures (secondary and tertiary structures) and system energy were highly affected by the concentration of phospholipids. We also found that the pronounced influence was detected in Arctic Aβ(1-40) containing a higher concentration of di-C7-PC. Finally, we used Aβ(1-60) as a model of C99 inside the membrane to gain the initial structure information by molecular dynamics simulations. The results showed that wild-type Aβ(1-60) has higher stability than that of Arctic Aβ(1-60) when staying inside the transmembrane domain. In addition, the secondary structure analysis revealed that the unfolding behavior occurred within the range of residues 40-42 during the simulation time of 100 ns. In summary, through various experimental measurements and molecular dynamics simulations, we conclude in this study that cell membrane evidently plays an important role during the transition of protein structure. We believe the results reported here may contribute to our better understanding of the difference between β-amyloid peptide and its mutant in regards to the aggregation behaviors and the molecular mechanisms of Aβ-lipid interactions.24806835 bytesapplication/pdfen-US野生型與突變型β-類澱粉胜?類澱粉纖維磷脂質分子動力學模擬wild-type and mutation β-amyloid peptideamyloid fibrilsphospholipidsmolecular dynamics simulationsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/252330/1/ntu-99-R97524061-1.pdf