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標題: | 以電子自旋共振光譜儀探索類澱粉前驅蛋白於跨膜區之結構 Exploring the Structure of Membrane-spanning Region of Amyloid Precursor Protein by Electron Spin Resonance Spectroscopy |
作者: | Chun-Ting Kuo 郭俊廷 |
指導教授: | 陳佩燁(Rita Pei-Yeh Chen) |
關鍵字: | 乙型類澱粉胜肽,類澱粉前驅蛋白,阿茲海默症,脂質體,電子自旋共振, Amyloid β,Amyloid precursor protein,Alzheimer’s disease,Liposome,Electron spin resonance, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 阿茲海默症是最常見的一種失智症。根據類澱粉連鎖假說,過多乙型類澱粉胜肽的累積會形成寡聚體或纖維,而這些胜肽的凝集會進一步地對腦部的神經細胞造成傷害。乙型類澱粉胜肽是由兩個坐落在細胞膜上的蛋白酶β-secretase與γ-secretase依序裁切類澱粉前驅蛋白而得到的代謝產物。此外,脂質組成一直都被認為是造成阿茲海默症的危險因子之一,例如膽固醇。而細胞膜脂質組成是否會影響類澱粉前驅蛋白在跨膜區的結構,並進而影響到γ-secretase對其的裁切位點的選擇尚待釐清,所以我們合成了含有類澱粉前驅蛋白跨膜區序列的胜肽,並在其中特定位點標記上含有自由基的化合物:MTSSL。將帶有標記的胜肽與DOPC這種脂質混合形成脂質體。利用圓二色儀觀察標記胜肽位於膜上的二級結構。最後,透過電子自旋共振光譜儀觀察標記胜肽的兩個標記位點之間的距離。本研究發現降低胜肽對脂質的比例以及搭配適當的緩衝溶液可以得到較好電子自旋共振訊號。無論是何種脂質組成(DOPC以及有或無添加膽固醇的混合脂質(POPC、POPE和POPS))中,我們合成的穿膜蛋白G29R1和V36R1之間以及G29R1和V40R1之間的距離相近,大約是12至13 Å。而第32和第46個位點之間在DOPC中的距離約為26 Å。另外,本實驗也嘗試製作nanodisc當作另一個脂質的系統,目前已能成功組裝由DPPC所組成的nanodisc。我們的結果顯示穿膜蛋白在29至40之間的結果不會受到脂質組成的改變,但我們得到的距離資訊與現有文獻的結構模型仍有一些出入。因此,我們需要做更多的結構研究去驗證我們提出的假說。 Alzheimer’s disease (AD) is the most common form of dementia. According to the Amyloid cascade hypothesis, accumulation of Amyloid β (Aβ) peptide leads to the fibril formation or oligomerization further damages the neuron cell in the brain. Aβ peptide is a catabolic metabolite from the processing of amyloid precursor protein (APP) by two integral membrane proteins, β-secretase, and γ-secretase. It is known that lipid composition, such as cholesterol, is a risk factor of AD. Whether lipid composition alters the structure of APP’s transmembrane region, and further influences the cutting site of γ-secretase remains elusive. We synthesized a peptide containing the transmembrane domain (TMD) of APP and label it with MTSSL, a radical carrier, at different positions of the peptide. The spin-labeled peptide was mixed with lipids to form liposomes. Circular dichroism spectroscopy was used to measure the secondary structure of the spin-labeled peptide in the liposome. The spin-spin distance of the peptide in the liposomes with different lipid compositions was measured by electron spin resonance spectroscopy. I found that decreasing the ratio of peptide to lipid and using PBS to prepare liposomes can obtain better electron spin resonance signals. In our transmembrane peptide, the distances between G29R1 and V36R1 and that between G29R1 and V40R1 are approximately 12-13 Å regardless of lipid composition (in DOPC or mix lipid (POPC, POPE, and POPS) with or without cholesterol). The distance between I32R1 and V46R1 is approximately 26 Å in DOPC liposome. Moreover, we have successfully made a nanodisc with DPPC. Our data suggested that the structure of our peptide is not affected by lipid composition in the regions of 29 to 40. The distance we obtained does not match the structural models proposed in the literature. More structural studies should be done to prove our hypothesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55784 |
DOI: | 10.6342/NTU202002001 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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