淀粉样前体蛋白通过其不同N端结构域调控自身转运与加工

发布时间：2018-12-29 14:11

【摘要】：阿尔茨海默氏病(Alzheimer's disease,AD)是一种破坏性的神经退行性疾病,折磨着全球数百万的患者。越来越多的生物化学和遗传学证据显示淀粉样前体蛋白(amyloid precursor protein,APP)在AD的发病机制中奠定了举足轻重的作用。生物化学上,不断积累的APP加工产物β-淀粉样肽(beta-amyloid peptide,Aβ)形成不可溶性聚集物,它是AD独特的病理特征;遗传学上,无论是APP点突变,还是野生型APP基因重复往往都会提高Aβ生成,这些都与一部分早发性家族型AD(familial AD,FAD)和脑淀粉样血管病息息相关。APP是Ⅰ型跨膜蛋白质,它类似于一种细胞表面受体,并包含一个大的胞外N端结构域和一个短的胞质尾。β-和γ-分泌酶依次切割APP并释放出神经毒性的Aβ,相反,α-分泌酶却从Aβ中间切割阻止其产生。此外,翔实的证据表明改变APP的分泌和内吞转运途径直接影响到APP自身与这些分泌酶的相互作用以及Aβ产生。虽然APP生成Aβ被广泛的研究,但是APP N端结构域贡献于Aβ产生的问题仍然难以捉摸。利用系统性删除策略,我们已经鉴定出四种新的候选APP模体,它们可以调节APP自身加工和Aβ生成。(1)删除APP ACIDIC结构域不仅促进α-分泌酶加工APP,而且同时抑制β-分泌酶BACE1对其加工,并产生较少的胞外Aβ和胞内 iAβ(intracellular Aβ,iAβ);(2)删除 APP CAPPD 结构域能提升 BACE1 和 α-分泌酶加工APP,且伴随着Aβ和iAβ的增加。它似乎是由于缺乏APP CAPPD能增强其与BACE1的作用。与此相反,仅删除CAPPD结构域的α3螺旋将极大的减弱BACE1和α-分泌酶加工APP;(3)剔除RC结构域后,APP与BACE1的作用得到强化,并进一步提高了 BACE1和α-分泌酶对其加工。缺少RC结构域的APP加工呈现两种不同的效应,一方面增加Aβ,而另一方面却减少iAβ;(4)去除APP JMD结构域不仅抑制BACE1处理APP,而且促进非BACE1依赖性的β-分泌酶加工APP。另外,非BACE1依赖性的β-分泌酶切割APP位点在Aβ的N端之外,大概位于APPRC结构域内。正因为如此,所以能产生N端延长的长形式Aβ。这样的切割位点大概有三个,并可以被APPJMD结构域所调节。接着我们试图描绘APP N端嵌合体的转运路线。我们构建了一系列的限定APP于某特定亚细胞器加工的转运突变体。实验结果揭示分泌和内吞途径的缺陷将会削弱Aβ和iAβ的产生,而且iAβ的生成并不依赖内吞体途径。因此,我们利用iAβ作为APP亚细胞定位的指标。根据iAβ变化水平,删除APP ACIDIC或者RC结构域将阻止APP退出TGN,而删除CAPPD结构域会促进APP向内吞体定位。此外,我们还将APP整个胞外N端结构域替换成红色荧光蛋白质mCherry,并且在APP胞质尾添加增强型绿色荧光蛋白质EGFP,从而产生双色荧光蛋白质标记的APP嵌合体mC99G。mC99G能模拟α-分泌酶、β-分泌酶、γ-分泌酶以及caspase加工APP的情形。mC99G、C99G和APP695G具有类同的亚细胞定位。γ-分泌酶抑制剂DAPT处理后,尽管mC99G的mCherry信号与APP695G相似,但是mC99G的EGFP信号表现的更像C99G,即大量EGFP信号聚集在质膜上。细胞表面的生物素标记显示质膜处的聚集组分主要为CTFα-EGFP,这表明α-分泌酶介导的CTFβ-EGFP向CTFα-EGFP转化可能主要发生在质膜处。鉴于mC99G的代谢物水平接近C99G对应物,故认为APP胞外结构域对其自身转运与加工非常关键。另外,mC99G也可以作为一种APP加工研究的有用工具。作为应用,我们发现mC99G及其衍生物能通过exosome分泌至胞外环境中。
[Abstract]:Alzheimer's disease (AD) is a destructive neurodegenerative disease that afflicts millions of people around the world. More and more biochemical and genetic evidence has shown that the amyloid precursor protein (APP) has a significant role in the pathogenesis of AD. On the basis of biochemistry, the growing APP processing product of the APP-amyloid peptide (A-type) forms a non-soluble aggregate, which is a unique pathological feature of AD; in genetics, whether the APP point mutation or the wild-type APP gene repetition tends to increase the A-gene generation, These are closely related to a portion of the early-onset family-type AD (FAD) and the brain amyloid. APP is a type I transmembrane protein that is similar to a cell surface receptor and contains a large extracellular N-terminal domain and a short cytoplasmic tail. The P-and E-secreting enzymes in turn cut APP and release the A-chain of neurotoxicity, in contrast, the yeast-secreting enzyme is cut from the middle of A to prevent it from being produced. In addition, the detailed evidence suggests that the changes in the secretion of APP and the pathway of endocytosis directly affect the interaction of APP itself with these secretory enzymes and the production of A. Although APP produces a wide range of studies, the problem that the APP N-terminal domain contributes to the A-phase is still elusive. With a systematic deletion strategy, we have identified four new candidate APP dies that can adjust APP's own processing and A-generation. (1) The deletion of the APP ACIDIC domain not only promotes the production of the APP, but also inhibits the processing of the APP-secreting enzyme BACE1, and produces less extracellular A-and intracellular A-(iA-); (2) the deletion of the APP CAPPD domain can enhance the BACE1 and HCO3-secreted enzyme processing APP, and is accompanied by an increase in the level of the a and the ia. It appears to be due to the lack of APP CAPPD to enhance its role with BACE1. In contrast, the deletion of the CD3 helix of the CAPPD domain will greatly reduce the BACE1 and the yeast-secreting enzyme processing APP; (3) after the RC domain is removed, the action of APP and BACE1 is enhanced, and the processing of the BACE1 and the yeast-secreting enzyme is further improved. The absence of two different effects on APP processing of the RC domain, on the one hand, increases the amount of A, while on the other hand decreases the iA ratio; (4) the removal of the APP JMD domain not only inhibits the BACE1 treatment of APP, but also promotes non-BACE1-dependent enzyme-secreting enzyme processing APP. In addition, the non-BACE1-dependent antigen-secreting enzyme cleavage APP site is located in the APPRC domain, in addition to the N-terminus of the A-chain. As such, it is possible to produce an N-terminal extended form A. Such cleavage sites are approximately three and may be regulated by the APPJMD domain. We then try to describe the transfer route of the APP N-terminal chimera. We have built a series of transport mutants that define the process of a specific subcellular organelle. The results of the experiment reveal that the defects of secretion and endocytosis will weaken the production of A-and iA-, and the generation of iA-antigen does not depend on the pathway of endocytosis. Therefore, we use iA as an indicator of the location of APP sub-cells. The deletion of the APP ACIDIC or the RC domain will prevent the APP from exiting the TGN according to the iA level of change, and the deletion of the CAPPD domain will facilitate the localization of the APP to the endocytosis. In addition, we replaced the entire extracellular N-terminal domain of APP with the red fluorescent protein mCherry, and the enhanced green fluorescent protein EGFP was added to the APP cell tail to produce the two-color fluorescent protein-labeled APP chimera mC99G. mC99G capable of simulating the enzyme-secreting enzyme, the yeast-secreting enzyme, The case of the enzyme-secreting enzyme and the caspase processing APP. mC99G, C99G and APP695G have the same subcellular localization. The mC99G 's mCherry signal was similar to that of the APP695G, but the EGFP signal of the mC99G appeared more like the C99G, that is, a large number of EGFP signals concentrated on the plasma membrane after the enzyme-secreting enzyme inhibitor DAPT treatment. The biotin-labeled display plasma membrane at the cell surface showed that the aggregation component at the plasma membrane was mainly CTF-EGFP, suggesting that the transformation of the CTF-EGFP-EGFP to the CTF-EGFP-EGFP could mainly occur at the plasma membrane. Since the metabolite level of mC99G is close to the C99G counterpart, it is considered that the extracellular domain of APP is critical to its own transport and processing. In addition, the mC99G can also be used as a useful tool for APP processing studies. As an application, we found that mC99G and its derivatives can be secreted into the extracellular environment by exosome.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

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