本文關(guān)鍵詞：酵母朊蛋白Sup35NM及其變異體體外淀粉樣纖維形成動(dòng)態(tài)及其細(xì)胞毒性作用　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2007年博士論文　論文類型：學(xué)位論文

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【摘要】： 已發(fā)現(xiàn)人類有多種疾病和淀粉樣纖維的沉積有關(guān)，包括阿爾茲海默病、帕金森病、可傳播性海綿狀腦病等。盡管淀粉樣變相關(guān)蛋白質(zhì)的氨基酸序列完全不同，但它們聚集所形成的淀粉樣纖維卻擁有相同的性質(zhì)，即：均是高度有序的蛋白聚集體、呈現(xiàn)細(xì)絲狀的形態(tài)、富含β折疊結(jié)構(gòu)、具有抵抗蛋白酶K消化的能力、經(jīng)剛果紅(Congo red)染色后在偏振光顯微鏡下可見(jiàn)黃綠色雙折色熒光。因此對(duì)淀粉樣纖維過(guò)程的研究，不僅有助于闡明淀粉樣病變的致病機(jī)理，而且有助于提出相關(guān)疾病的有效預(yù)防和治療策略。雖然已對(duì)氨基酸序列和淀粉樣蛋白形成能力之間的關(guān)系進(jìn)行了大量研究，但目前淀粉樣纖維的形成機(jī)制仍不清楚。 1994年，Wickner等提出了酵母Prion這個(gè)概念，認(rèn)為釀酒酵母(Saccharomyces cerevisiae)中兩個(gè)非孟德?tīng)栠z傳元件[URE3]和[PSI~+]分別是染色體編碼蛋白Ure2p和Sup35p的朊病毒形式，其行為與哺乳動(dòng)物PrP相似，因此為研究淀粉樣纖維的形成及Prion構(gòu)象轉(zhuǎn)變提供了一個(gè)較為理想的模型。Sup35p是翻譯終止因子的亞單位，類似于真核細(xì)胞釋放因子3(eRF3)，通過(guò)與Sup45p(eRF1)形成復(fù)合物識(shí)別終止密碼子而使肽鏈在翻譯末端釋放，終止蛋白翻譯。Sup35蛋白在酵母體內(nèi)過(guò)表達(dá)時(shí)蛋白構(gòu)象發(fā)生轉(zhuǎn)變而導(dǎo)致聚集，喪失正常的生理功能而導(dǎo)致終止密碼子通讀，使酵母產(chǎn)生[PSI~+]表型。Sup35p由685個(gè)氨基酸組成，包括3個(gè)結(jié)構(gòu)域：N端區(qū)(N，aa1-123)是誘導(dǎo)[PSI~+]產(chǎn)生所必需的，富含谷氨酰胺(Q)和天冬酰胺(N)，為Prion形成結(jié)構(gòu)域(PrD)。C區(qū)是翻譯終止功能區(qū)。M區(qū)位于N與C之間，目前功能不清。為明確N區(qū)特殊的氨基酸組成及序列在淀粉樣纖維形成中的作用，我們構(gòu)建了Sup35NM的變異體，分析了它們淀粉樣纖維形成的動(dòng)力學(xué)過(guò)程，并進(jìn)一步探討了野生型Sup35NM及其變異體聚集物的細(xì)胞毒性作用，以期為闡明淀粉樣纖維形成及其致病機(jī)制提供線索。 1．酵母朊蛋白Sup35NM變異體體外淀粉樣纖維形成的動(dòng)態(tài)研究 為了闡明氨基酸序列對(duì)纖維形成的影響，我們首先將Sup35NM片段的PrD的氨基酸序列重新隨機(jī)排列同時(shí)保證氨基酸的組成及M段的氨基酸序列不變，構(gòu)建了5個(gè)Sup35NM的變異體，分別稱為Sup35NM-1、-2、-3、-4、-5。在E. coli中成功表達(dá)、純化了野生型Sup35 NM及其5個(gè)變異體，進(jìn)而研究了其在體外淀粉樣纖維形成的動(dòng)力學(xué)過(guò)程。結(jié)果顯示：透射電子顯微鏡下觀察可見(jiàn)Sup35NM及其5種變異體蛋白在PBS(pH7.4)緩沖液中均可發(fā)生聚集，2h時(shí)可見(jiàn)球形顆粒、寡聚體及少量短而細(xì)的纖維，18h時(shí)Sup35NM、Sup35NM-1、-2、-3蛋白溶液中出現(xiàn)大量的成熟纖維，球形顆粒消失，纖維長(zhǎng)且邊緣光滑，Sup35NM-4、-5除了長(zhǎng)纖維以外，還可見(jiàn)蛋白寡聚體和球形顆粒。48h后，Sup35NM及5個(gè)變異體蛋白溶液中只有大量成熟纖維，直徑約8～14 nm。用圓二色譜檢測(cè)了其二級(jí)結(jié)構(gòu)，顯示在216nm附近出現(xiàn)一負(fù)峰，呈典型的β-折疊特征，提示該過(guò)程伴隨蛋白結(jié)構(gòu)由α-螺旋到β-折疊的轉(zhuǎn)變。NM及變異體聚集所形成的纖維分別經(jīng)1μg／ml和4μg／ml的蛋白酶K作用60min后仍可檢測(cè)到蛋白的存在，說(shuō)明纖維具有較強(qiáng)的抗蛋白酶K消化的特性。ThT結(jié)合試驗(yàn)顯示，Sup35NM-1、-2、-3與Sup35NM熒光強(qiáng)度上升的速度相似，經(jīng)歷一個(gè)快速上升期后達(dá)到平臺(tái)期，未見(jiàn)明顯的潛伏期，而Sup35NM-4、-5則能觀察到一個(gè)近5h的潛伏期，提示Sup35NM-4、-5蛋白聚集明顯慢于Sup35NM及其它變異體蛋白。SDS-PAGE電泳和Western blotting也進(jìn)一步證明，纖維形成過(guò)程中蛋白單體逐漸減少同時(shí)多聚體則逐漸增加。淀粉樣纖維經(jīng)沸水浴加熱后發(fā)現(xiàn)纖維消失，代之以大量的球形顆粒、寡聚體及少量短棒狀纖維，ThT結(jié)合試驗(yàn)顯示纖維加熱后熒光強(qiáng)度明顯下降，SDS-PAGE電泳也證實(shí)加熱后蛋白單體重新出現(xiàn)。這些數(shù)據(jù)說(shuō)明PrD的氨基酸序列被隨機(jī)打亂后并沒(méi)用影響其最終形成淀粉樣纖維的能力，并且變異體形成的纖維具有與野生型Sup35NM的纖維具有相同的形態(tài)和生化特征，但纖維形成的速率有所不同，提示形成纖維的能力是由氨基酸組成決定的，特定的氨基酸序列在一定程度上調(diào)節(jié)纖維形成的速率。 2．野生型Sup35NM及其變異體蛋白纖維的構(gòu)象誘導(dǎo)轉(zhuǎn)變作用 已有研究表明，在體內(nèi)Rnq1p、polyQ等聚集體可誘導(dǎo)酵母菌株從[PSI~-]轉(zhuǎn)變?yōu)閇PSI~+]，在體外異源性的Rnq1或polyQ“種子”可促進(jìn)NM形成淀粉樣纖維。我們構(gòu)建的變異體N區(qū)同野生型NM一樣富含Q／N，為了進(jìn)一步闡明富含Q／N的異源性“種子”能促進(jìn)蛋白的聚集，我們研究了野生型NM與變異體的相互誘導(dǎo)轉(zhuǎn)變以及另一個(gè)酵母朊蛋白Ure2對(duì)NM和變異體纖維形成的影響。結(jié)果顯示：野生型Sup35NM“種子”可加快變異體Sup35NM-1、-4、-5的纖維形成，反之亦然。此外，異源的Ure2“種子”也能輕度增加NM及變異體的轉(zhuǎn)變速率，但總的來(lái)說(shuō)異源性的“種子”比自體“種子”的誘導(dǎo)效率相對(duì)低一些。而沒(méi)有Q／N結(jié)構(gòu)域的α-synuclein“種子”則對(duì)蛋白聚集的速率無(wú)影響。這些結(jié)果提示富含Q／N結(jié)構(gòu)域的異源性“種子”盡管其氨基酸序列不同，能夠促進(jìn)同樣含有Q／N結(jié)構(gòu)域的蛋白的聚集，，而不含Q／N的異源“種子”則無(wú)此作用。 3．野生型Sup35NM及其變異體對(duì)哺乳動(dòng)物細(xì)胞的毒性 目前已有大量研究證實(shí)，在多種蛋白聚集過(guò)程中形成的中間體有細(xì)胞毒性。Sup35p是釀酒酵母中的朊蛋白，可發(fā)生構(gòu)象轉(zhuǎn)變形成淀粉樣纖維，與哺乳動(dòng)物Prion蛋白不同，不會(huì)引起酵母死亡，與人類淀粉樣疾病無(wú)關(guān)，Sup35的聚集中間體是否具有細(xì)胞毒性作用還沒(méi)有得到證實(shí)。為證實(shí)Sup35NM聚集體的細(xì)胞毒性，我們將純化的蛋白(64μmol／L)于室溫下放置0.5～1h或于4℃放置2d～4d后，分別獲得聚集中間體和成熟纖維，然后將不同形態(tài)的蛋白分別作用于Vero、NIH-3T3、SH-SY5Y細(xì)胞系。此外，我們還進(jìn)一步研究了變異體的毒性作用。結(jié)果顯示：Sup35NM及其變異體的聚集中間體對(duì)細(xì)胞具有毒性作用，可明顯降低細(xì)胞存活率，這種毒性作用與劑量具有相關(guān)性，成熟纖維反而無(wú)害，與其它蛋白相似。這一結(jié)果提示蛋白聚集體的細(xì)胞毒性可能與其結(jié)構(gòu)相關(guān)。不同細(xì)胞系對(duì)蛋白毒性的敏感性有差異，Vero和NIH-3T3細(xì)胞比較敏感，SH-SY5Y細(xì)胞則有一定的抵抗作用，提示蛋白聚集體的細(xì)胞毒性作用是通過(guò)特定機(jī)制實(shí)現(xiàn)的。成熟纖維經(jīng)沸水浴加熱20min后作用于細(xì)胞，也表現(xiàn)出與聚集中間體相似的毒性作用，提示纖維經(jīng)加熱后出現(xiàn)的蛋白結(jié)構(gòu)與聚集中間體的結(jié)構(gòu)相似。
[Abstract]:Have been found in a variety of diseases and deposition of human amyloid fibrils, including Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathy. Although the amino acid sequence of amyloid related protein is completely different, but they formed amyloid fibrils but have the same properties, which are highly ordered the protein aggregates, showing filamentous morphology, including beta folding structure, has the ability to resist proteinase K digestion, the Congo red (Congo red) after staining with polarized light microscopy showed yellow green color fluorescence. So the double amyloid fiber process research, not only help to elucidate the pathogenesis of amyloid diseases effective prevention and treatment strategies, but also helps to put forward related diseases. Although has formed a relationship between the ability of amino acid sequence and amyloid has been studied, but the amyloid fiber The mechanism of the formation of vitamins is still unclear.
In 1994, Wickner proposed the concept of Prion in yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae) that in two non Mendel genetic element [URE3] and [PSI~+] are respectively Ure2p and Sup35p chromosome encoding protein prion form, its behavior and mammals was similar to that of PrP, so as to form and study the conformation of Prion amyloid transition provides a an ideal model of.Sup35p subunit translation termination factor, similar to eukaryotic release factor 3 (eRF3), with Sup45p (eRF1) complex formation recognition stop codon and the peptide chain release at the end of translation, translation termination protein overexpression of.Sup35 in yeast protein conformational change and cause together, the loss of the normal physiological function and lead to stop codon readthrough, the yeast [PSI~+] phenotype of.Sup35p is composed of 685 amino acids, including 3 domains: N The end (N, aa1-123) is necessary to induce the production of [PSI~+] (Q), rich in glutamine and asparagine (N), the formation of domain of Prion (PrD).C area is between the termination function is located in zone.M, N and C, the function is not clear. For the amino acid composition and sequence specific N special region is formed in the role of amyloid fibrils, we constructed the variant of Sup35NM, analyzes the dynamic process of their amyloid fibril formation, and further discusses the cytotoxic effect of wild type Sup35NM and its mutant aggregates, in order to provide clues for elucidating the amyloid fibril formation and pathogenesis.
Dynamic study on the formation of amyloid fiber in vitro of 1. yeast prion Sup35NM variants
In order to elucidate the effects of amino acid sequence on fiber formation, we first divide the amino acid sequence of Sup35NM fragment of PrD to random arrangement and ensure the amino acid composition and M segment change, build 5 variants of Sup35NM, called Sup35NM-1, -2, -3, -4, -5. in E. coli in the successful expression of the wild Sup35 NM and its 5 variants were purified, and then studied the dynamical process in the formation of amyloid fibrils in vitro. The results showed that under a transmission electron microscope and 5 variants in the PBS protein showed Sup35NM (pH7.4) can buffer aggregation of 2H visible spherical particles, and a small amount of short oligomers fine fibers, 18h Sup35NM, Sup35NM-1, -2, the emergence of a large number of mature fiber -3 protein solution, spherical particles disappear, fiber long and smooth edge, Sup35NM-4 -5, in addition to long fiber, also visible oligomeric protein The body and the spherical particles of.48h, Sup35NM and 5 variants of the protein concentration in only a large number of mature fiber, a diameter of about 8 ~ 14 nm. with round two determination of secondary structure, showed a negative peak near 216nm, a typical beta folding feature, suggesting that the process along with the role of 60min protein structure by K protease alpha helix beta and.NM fiber to change the aggregation formed by folding variants respectively by 1 g / ml and 4 g / ml can be detected the presence of protein, fiber that has strong anti proteinase K digestion characteristics of.ThT binding test showed that Sup35NM-1, -2, -3 and Sup35NM fluorescence rise the strength of a rate similar to that experienced a period of rapid rise after reaching the plateau, there is no obvious latency, and Sup35NM-4, -5 is able to observe a nearly 5h incubation period, suggesting that Sup35NM-4, -5 protein accumulation was slower than that of Sup35NM and other variants of.SDS-PAGE protein Blotting electrophoresis and Western is further proved that the fiber formation process of protein monomers and multimers decreased gradually increased. Amyloid fibrils by boiling water after heating fiber was disappeared, replaced by a large number of spherical particles, oligomer and a few short rod like fibers, ThT binding test showed that the fluorescence intensity of the fiber decreased significantly after heating, SDS-PAGE electrophoresis confirmed after heating protein monomer again. These data indicated that the amino acid sequence of PrD were disrupted after did not affect the ability of the final formation of amyloid fibers, fiber and the formation of variants with the same morphological and biochemical characteristics and wild type Sup35NM fiber has, but the rate of fiber formation the different, suggesting the ability forming fiber is determined by amino acid composition, amino acid sequence specific regulation rate of fiber formation to a certain extent.
Conformation induced transformation of 2. wild type Sup35NM and its variant protein fibers
Studies have shown that Rnq1p in vivo, polyQ aggregates induced by yeast strains from [PSI~-] into [PSI~+], the in vitro heterologous Rnq1 or polyQ "seed" NM can promote the formation of amyloid fibrils. We construct the N variant with the wild type NM rich Q / N, in order to further illustrate the rich Q / N heterologous "seed" can promote protein aggregation, we studied the change effect of wild type NM and mutant induced and another yeast prion protein Ure2 of NM variants and fiber formation. The results showed that wild type Sup35NM seeds with mutant Sup35NM-1, -4, -5 fiber formation vice versa. In addition, the heterologous Ure2 "seeds" can also increase the rate of conversion of mild NM and variants, but overall heterogeneity of "seed" than the induction efficiency of autologous "seed" is relatively low. And no Q / N domains. No effect on the rate of alpha -synuclein "seeds" of aggregation. These results suggest that in Q / N domain of heterologous seed while its amino acid sequence, can promote the same with Q / N domain protein aggregation, but not containing Q / N heterologous "seed" is not the role.
The toxicity of 3. wild type Sup35NM and its variants to mammalian cells
Many studies have shown that, in a variety of protein aggregation intermediates formed in the process of cell toxicity of.Sup35p is yeast prion protein conformational transition can occur, the formation of amyloid fibrils, unlike mammalian Prion protein, yeast does not cause death, has nothing to do with human starch like disease, Sup35 concentration between the body of cells toxicity has not been confirmed. Cytotoxicity confirmed that Sup35NM aggregates, we purified protein (64 mol / L) at room temperature for 0.5 ~ 1H or 2D ~ 4D placed at 4 DEG C, respectively, obtained aggregation intermediates and mature fiber, and then different forms of protein respectively for Vero, NIH-3T3 SH-SY5Y, cell line. In addition, we also further study the toxic effect of variants. The results showed that aggregation intermediates Sup35NM and its variants have toxic effects on the cells, can significantly reduce the fine Cell survival was correlated with the dose of the toxic effects of mature fiber but harmless, similar to other proteins. These results suggest that the cytotoxicity of protein aggregates and its structure. The sensitivity of different cell lines to protein toxicity are different, Vero and NIH-3T3 cells are more sensitive, while SH-SY5Y cells are resistant. Indicating that the cytotoxicity of protein aggregates is achieved by a specific mechanism. The mature fibers were heated in boiling water bath for 20min after exposure of cells also showed a concentration between the similar toxicity, suggesting that protein structure of fiber after heating and the focus between the similar structure.

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373

【共引文獻(xiàn)】

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