發(fā)布時(shí)間：2018-05-24 22:16

  本文選題：阿爾茲海默病 + C99��； 參考：《中國(guó)科學(xué)院上海藥物研究所》2017年博士論文

【摘要】：阿爾茲海默病(Alzheimer’s disease,AD)是最常見(jiàn)的老年癡呆癥疾病,嚴(yán)重危害老年人健康和生命,目前對(duì)其致病機(jī)理還沒(méi)有全面的了解。淀粉樣前體蛋白(amyloid precursor protein,APP)的突變以及γ-分泌酶錯(cuò)誤地酶切C99(APP的C端99個(gè)氨基酸)導(dǎo)致阿爾茲海默病的發(fā)生已經(jīng)廣泛的被基因、細(xì)胞、動(dòng)物實(shí)驗(yàn)證實(shí)。這使得類淀粉蛋白質(zhì)致病假說(shuō)越來(lái)越被科學(xué)界認(rèn)可。γ-分泌酶在阿爾茲海默病致病中起著極其重要的作用,而其與底物的關(guān)系卻一直沒(méi)有得到科學(xué)全面的解釋。目前臨床針對(duì)阿爾茲海默病的藥物都沒(méi)有特別好的療效。正確了解γ-分泌酶與C99底物的關(guān)系并獲得C99和Aβ單獨(dú)或者與γ-分泌酶的高分辨率結(jié)構(gòu)將有助于科學(xué)闡述阿爾茲海默病的致病機(jī)理及正確的開(kāi)發(fā)藥物。為了揭示阿爾茲海默病病變機(jī)理,本實(shí)驗(yàn)著重于分析γ-分泌酶與阿爾茲海默病相關(guān)的C99突變底物的關(guān)系以及致力于解析C99和Aβ結(jié)構(gòu)并從分子水平解釋致病機(jī)制。我們建立了快速靈敏的Epsilon-酶切實(shí)驗(yàn)來(lái)研究γ-分泌酶對(duì)C99突變底物的酶切效率,并檢測(cè)主要產(chǎn)物Aβ40和Aβ42產(chǎn)量及比列變化。我們發(fā)現(xiàn)這些突變能夠一定程度地抵制γ-分泌酶的酶切,并且跨膜區(qū)C端在識(shí)別-結(jié)合-酶切過(guò)程中的重要性要遠(yuǎn)遠(yuǎn)高于其他區(qū)域。通過(guò)N端和C端截短實(shí)驗(yàn),我們鑒定了C99片段(E22-K55)是在細(xì)胞內(nèi)可以被γ-分泌酶識(shí)別并切割的最短底物。通過(guò)突變實(shí)驗(yàn),我們發(fā)現(xiàn)最短底物N端的負(fù)電荷以及C端的正電荷簇在識(shí)別酶切過(guò)程中起著重要的作用。同時(shí)對(duì)跨膜區(qū)介導(dǎo)的C99二聚化的研究,我們發(fā)現(xiàn)一個(gè)不同于現(xiàn)在所報(bào)道的二聚化基序-TVIV,而這個(gè)基序是家族型阿爾茲海默病在C99上突變的熱點(diǎn)位置。進(jìn)一步分析發(fā)現(xiàn),γ-分泌酶的酶切效率與跨膜區(qū)介導(dǎo)的二聚化具有一定的正相關(guān)性。結(jié)合酶學(xué)實(shí)驗(yàn)、最短底物以及跨膜區(qū)二聚化的研究,我們鑒定了γ-分泌酶識(shí)別-結(jié)合-切割底物的決定因素:(i)C99和Notch之間不同且可轉(zhuǎn)移細(xì)胞外決定簇,而C99還需要胞外的負(fù)電荷,(ii)跨膜螺旋C末端一半的氨基酸序列,(iii)細(xì)胞內(nèi)膜邊界保守的的賴氨酸或精氨酸,(iv)包括不變賴氨酸/精氨酸的正電荷簇。因此我們提出了一個(gè)識(shí)別-結(jié)合-酶切模型。首先,C99的N端D1-A21片段以及C端Q56-N99片段對(duì)于γ-分泌酶的識(shí)別、酶切不是必需的。其次,其C端脂膜結(jié)合區(qū)域把底物錨定到γ-分泌酶附近,并促進(jìn)其正確的構(gòu)象。然后跨膜區(qū)C末端空間識(shí)別作用、N端的靜電荷相互作用以及K53與γ-分泌酶活性中心的識(shí)別都為γ-分泌酶的酶切奠定了基礎(chǔ)。同時(shí)采用晶體學(xué)研究,對(duì)C99和Aβ多肽進(jìn)行表達(dá)嘗試,運(yùn)用定點(diǎn)突變、蛋白融合、蛋白質(zhì)截短、二硫鍵引入、抗體共表達(dá)等蛋白質(zhì)純化、改造技術(shù)對(duì)C99和Aβ多肽進(jìn)行表達(dá)優(yōu)化,以及化學(xué)合成Aβ多肽并用于蛋白質(zhì)晶體生長(zhǎng)條件的篩選和優(yōu)化。最終希望獲得純度高、構(gòu)象均一的蛋白用于晶體生長(zhǎng),獲得高質(zhì)量晶體并解析其結(jié)構(gòu)。結(jié)合結(jié)構(gòu)生物學(xué)及生化細(xì)胞學(xué)方法探索阿爾茲海默病致病機(jī)制,為探討神經(jīng)退行性疾病的發(fā)病機(jī)制奠定基礎(chǔ)。
[Abstract]:Alzheimer 's disease (AD) is the most common Alzheimer's disease, which seriously endangers the health and life of the elderly. There is no comprehensive understanding of the pathogenesis of Alzheimer's disease. The mutation of amyloid precursor protein (amyloid precursor protein, APP) and the error of gamma secretase in C99 (APP C, 99 amino acids) lead to opioid. The occurrence of Alzheimer's disease has been widely confirmed by gene, cell, and animal experiments. This makes the sick say of amyloid protein more and more recognized by the scientific community. Gamma secretase plays an extremely important role in the pathogenesis of Alzheimer's disease, but its relationship with the substrate has not been explained scientifically and comprehensively. A correct understanding of the relationship between gamma secretase and C99 substrates and obtaining a high resolution structure of C99 and A beta alone or with gamma secretase will help to scientifically explain the pathogenesis of Alzheimer's disease and the correct development of drugs. In order to reveal the pathogenesis of Alzheimer's disease, it is true that the pathogenesis of Alzheimer's disease is revealed. The test focuses on the analysis of the relationship between gamma secretase and Alzheimer's disease related C99 mutant substrates and is committed to analyzing C99 and A beta structures and explaining the pathogenesis from molecular level. We have established a rapid and sensitive Epsilon- enzyme cutting experiment to study the enzyme cutting efficiency of gamma secretase on C99 mutant substrates, and to detect the production of major products A beta 40 and A beta 42. We found that these mutations can resist the enzyme digestion of gamma secretase to a certain extent, and the importance of the C end of the transmembrane region in the identification binding enzyme cutting process is far higher than that in other regions. By the truncation of the N end and C ends, we identified that the C99 fragment (E22-K55) can be identified and cut by the gamma secretase within the cell. The shortest substrate. Through the mutation experiment, we found that the negative charge at the N end of the shortest substrate and the positive charge cluster at the C end play an important role in identifying the enzyme cutting process. At the same time, the study of the C99 dimerization mediated by the transmembrane region was found to be different from the dimerization -TVIV, which is now reported, and this order is family type. Further analysis shows that there is a positive correlation between the enzyme cutting efficiency of gamma secretase and the dimerization mediated by the transmembrane region. Combining enzymology experiments, the shortest substrate and the dimerization of transmembrane regions, we identified the determinants of gamma secretase identification and junction cutting substrates: (I) C99 and Notch Different and transferable extracellular determinants, and C99 also needs an extracellular negative charge, (II) the amino acid sequence of half of the end of the transmembrane spiral C, (III) the conserved lysine or arginine on the boundary of the cell intima, (IV) including the positive charge cluster of the unaltered lysine / arginine. The end D1-A21 fragment and the C terminal Q56-N99 fragment are not necessary for the identification of gamma secretase. Secondly, the C terminal binding region anchors the substrate to the gamma secretase, and promotes its correct conformation. Then, the spatial recognition of the C terminal in the transmembrane region, the interaction of the electrostatic charge in the N terminal and the identification of the active center of the K53 and gamma secretase All of them lay the foundation for the enzyme digestion of gamma secretase. At the same time, the expression of C99 and A beta peptides was expressed by crystallography, and the protein purification of C99 and A beta peptides was purified by fixed-point mutation, protein fusion, protein truncation, the introduction of two sulfur bonds and the co expression of antibody, and the chemical synthesis of A beta polypeptide was used in the chemical synthesis of protein and the protein was used in protein. In the end, we hope to obtain high purity and homogeneous protein for crystal growth, obtain high quality crystal and analyze its structure, and combine structural biology and biochemical cytology to explore the pathogenesis of Alzheimer's disease, which lays the foundation for exploring the pathogenesis of neurodegenerative diseases.
【學(xué)位授予單位】：中國(guó)科學(xué)院上海藥物研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

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