淀粉樣前體蛋白通過其不同N端結(jié)構(gòu)域調(diào)控自身轉(zhuǎn)運(yùn)與加工
發(fā)布時(shí)間：2018-12-29 14:11
【摘要】：阿爾茨海默氏病(Alzheimer's disease,AD)是一種破壞性的神經(jīng)退行性疾病,折磨著全球數(shù)百萬的患者。越來越多的生物化學(xué)和遺傳學(xué)證據(jù)顯示淀粉樣前體蛋白(amyloid precursor protein,APP)在AD的發(fā)病機(jī)制中奠定了舉足輕重的作用。生物化學(xué)上,不斷積累的APP加工產(chǎn)物β-淀粉樣肽(beta-amyloid peptide,Aβ)形成不可溶性聚集物,它是AD獨(dú)特的病理特征;遺傳學(xué)上,無論是APP點(diǎn)突變,還是野生型APP基因重復(fù)往往都會(huì)提高Aβ生成,這些都與一部分早發(fā)性家族型AD(familial AD,FAD)和腦淀粉樣血管病息息相關(guān)。APP是Ⅰ型跨膜蛋白質(zhì),它類似于一種細(xì)胞表面受體,并包含一個(gè)大的胞外N端結(jié)構(gòu)域和一個(gè)短的胞質(zhì)尾。β-和γ-分泌酶依次切割A(yù)PP并釋放出神經(jīng)毒性的Aβ,相反,α-分泌酶卻從Aβ中間切割阻止其產(chǎn)生。此外,翔實(shí)的證據(jù)表明改變APP的分泌和內(nèi)吞轉(zhuǎn)運(yùn)途徑直接影響到APP自身與這些分泌酶的相互作用以及Aβ產(chǎn)生。雖然APP生成Aβ被廣泛的研究,但是APP N端結(jié)構(gòu)域貢獻(xiàn)于Aβ產(chǎn)生的問題仍然難以捉摸。利用系統(tǒng)性刪除策略,我們已經(jīng)鑒定出四種新的候選APP模體,它們可以調(diào)節(jié)APP自身加工和Aβ生成。(1)刪除APP ACIDIC結(jié)構(gòu)域不僅促進(jìn)α-分泌酶加工APP,而且同時(shí)抑制β-分泌酶BACE1對(duì)其加工,并產(chǎn)生較少的胞外Aβ和胞內(nèi) iAβ(intracellular Aβ,iAβ);(2)刪除 APP CAPPD 結(jié)構(gòu)域能提升 BACE1 和 α-分泌酶加工APP,且伴隨著Aβ和iAβ的增加。它似乎是由于缺乏APP CAPPD能增強(qiáng)其與BACE1的作用。與此相反,僅刪除CAPPD結(jié)構(gòu)域的α3螺旋將極大的減弱BACE1和α-分泌酶加工APP;(3)剔除RC結(jié)構(gòu)域后,APP與BACE1的作用得到強(qiáng)化,并進(jìn)一步提高了 BACE1和α-分泌酶對(duì)其加工。缺少RC結(jié)構(gòu)域的APP加工呈現(xiàn)兩種不同的效應(yīng),一方面增加Aβ,而另一方面卻減少iAβ;(4)去除APP JMD結(jié)構(gòu)域不僅抑制BACE1處理APP,而且促進(jìn)非BACE1依賴性的β-分泌酶加工APP。另外,非BACE1依賴性的β-分泌酶切割A(yù)PP位點(diǎn)在Aβ的N端之外,大概位于APPRC結(jié)構(gòu)域內(nèi)。正因?yàn)槿绱?所以能產(chǎn)生N端延長的長形式Aβ。這樣的切割位點(diǎn)大概有三個(gè),并可以被APPJMD結(jié)構(gòu)域所調(diào)節(jié)。接著我們?cè)噲D描繪APP N端嵌合體的轉(zhuǎn)運(yùn)路線。我們構(gòu)建了一系列的限定APP于某特定亞細(xì)胞器加工的轉(zhuǎn)運(yùn)突變體。實(shí)驗(yàn)結(jié)果揭示分泌和內(nèi)吞途徑的缺陷將會(huì)削弱Aβ和iAβ的產(chǎn)生,而且iAβ的生成并不依賴內(nèi)吞體途徑。因此,我們利用iAβ作為APP亞細(xì)胞定位的指標(biāo)。根據(jù)iAβ變化水平,刪除APP ACIDIC或者RC結(jié)構(gòu)域?qū)⒆柚笰PP退出TGN,而刪除CAPPD結(jié)構(gòu)域會(huì)促進(jìn)APP向內(nèi)吞體定位。此外,我們還將APP整個(gè)胞外N端結(jié)構(gòu)域替換成紅色熒光蛋白質(zhì)mCherry,并且在APP胞質(zhì)尾添加增強(qiáng)型綠色熒光蛋白質(zhì)EGFP,從而產(chǎn)生雙色熒光蛋白質(zhì)標(biāo)記的APP嵌合體mC99G。mC99G能模擬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具有類同的亞細(xì)胞定位。γ-分泌酶抑制劑DAPT處理后,盡管mC99G的mCherry信號(hào)與APP695G相似,但是mC99G的EGFP信號(hào)表現(xiàn)的更像C99G,即大量EGFP信號(hào)聚集在質(zhì)膜上。細(xì)胞表面的生物素標(biāo)記顯示質(zhì)膜處的聚集組分主要為CTFα-EGFP,這表明α-分泌酶介導(dǎo)的CTFβ-EGFP向CTFα-EGFP轉(zhuǎn)化可能主要發(fā)生在質(zhì)膜處。鑒于mC99G的代謝物水平接近C99G對(duì)應(yīng)物,故認(rèn)為APP胞外結(jié)構(gòu)域?qū)ζ渥陨磙D(zhuǎn)運(yùn)與加工非常關(guān)鍵。另外,mC99G也可以作為一種APP加工研究的有用工具。作為應(yīng)用,我們發(fā)現(xiàn)mC99G及其衍生物能通過exosome分泌至胞外環(huán)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16
本文編號(hào)：2394932
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16
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