淀粉样蛋白（Aβ）及其前体（C99）的结构与功能研究

发布时间：2018-05-24 22:16

本文选题：阿尔兹海默病 + C99　；参考：《中国科学院上海药物研究所》2017年博士论文

【摘要】：阿尔兹海默病(Alzheimer’s disease,AD)是最常见的老年痴呆症疾病,严重危害老年人健康和生命,目前对其致病机理还没有全面的了解。淀粉样前体蛋白(amyloid precursor protein,APP)的突变以及γ-分泌酶错误地酶切C99(APP的C端99个氨基酸)导致阿尔兹海默病的发生已经广泛的被基因、细胞、动物实验证实。这使得类淀粉蛋白质致病假说越来越被科学界认可。γ-分泌酶在阿尔兹海默病致病中起着极其重要的作用,而其与底物的关系却一直没有得到科学全面的解释。目前临床针对阿尔兹海默病的药物都没有特别好的疗效。正确了解γ-分泌酶与C99底物的关系并获得C99和Aβ单独或者与γ-分泌酶的高分辨率结构将有助于科学阐述阿尔兹海默病的致病机理及正确的开发药物。为了揭示阿尔兹海默病病变机理,本实验着重于分析γ-分泌酶与阿尔兹海默病相关的C99突变底物的关系以及致力于解析C99和Aβ结构并从分子水平解释致病机制。我们建立了快速灵敏的Epsilon-酶切实验来研究γ-分泌酶对C99突变底物的酶切效率,并检测主要产物Aβ40和Aβ42产量及比列变化。我们发现这些突变能够一定程度地抵制γ-分泌酶的酶切,并且跨膜区C端在识别-结合-酶切过程中的重要性要远远高于其他区域。通过N端和C端截短实验,我们鉴定了C99片段(E22-K55)是在细胞内可以被γ-分泌酶识别并切割的最短底物。通过突变实验,我们发现最短底物N端的负电荷以及C端的正电荷簇在识别酶切过程中起着重要的作用。同时对跨膜区介导的C99二聚化的研究,我们发现一个不同于现在所报道的二聚化基序-TVIV,而这个基序是家族型阿尔兹海默病在C99上突变的热点位置。进一步分析发现,γ-分泌酶的酶切效率与跨膜区介导的二聚化具有一定的正相关性。结合酶学实验、最短底物以及跨膜区二聚化的研究,我们鉴定了γ-分泌酶识别-结合-切割底物的决定因素:(i)C99和Notch之间不同且可转移细胞外决定簇,而C99还需要胞外的负电荷,(ii)跨膜螺旋C末端一半的氨基酸序列,(iii)细胞内膜边界保守的的赖氨酸或精氨酸,(iv)包括不变赖氨酸/精氨酸的正电荷簇。因此我们提出了一个识别-结合-酶切模型。首先,C99的N端D1-A21片段以及C端Q56-N99片段对于γ-分泌酶的识别、酶切不是必需的。其次,其C端脂膜结合区域把底物锚定到γ-分泌酶附近,并促进其正确的构象。然后跨膜区C末端空间识别作用、N端的静电荷相互作用以及K53与γ-分泌酶活性中心的识别都为γ-分泌酶的酶切奠定了基础。同时采用晶体学研究,对C99和Aβ多肽进行表达尝试,运用定点突变、蛋白融合、蛋白质截短、二硫键引入、抗体共表达等蛋白质纯化、改造技术对C99和Aβ多肽进行表达优化,以及化学合成Aβ多肽并用于蛋白质晶体生长条件的筛选和优化。最终希望获得纯度高、构象均一的蛋白用于晶体生长,获得高质量晶体并解析其结构。结合结构生物学及生化细胞学方法探索阿尔兹海默病致病机制,为探讨神经退行性疾病的发病机制奠定基础。
[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.
【学位授予单位】：中国科学院上海药物研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16

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