酵母朊蛋白Sup35NM及其变异体体外淀粉样纤维形成动态及其细胞毒性作用

发布时间：2018-01-06 22:03

本文关键词：酵母朊蛋白Sup35NM及其变异体体外淀粉样纤维形成动态及其细胞毒性作用　出处：《中国协和医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 已发现人类有多种疾病和淀粉样纤维的沉积有关，包括阿尔兹海默病、帕金森病、可传播性海绵状脑病等。尽管淀粉样变相关蛋白质的氨基酸序列完全不同，但它们聚集所形成的淀粉样纤维却拥有相同的性质，即：均是高度有序的蛋白聚集体、呈现细丝状的形态、富含β折叠结构、具有抵抗蛋白酶K消化的能力、经刚果红(Congo red)染色后在偏振光显微镜下可见黄绿色双折色荧光。因此对淀粉样纤维过程的研究，不仅有助于阐明淀粉样病变的致病机理，而且有助于提出相关疾病的有效预防和治疗策略。虽然已对氨基酸序列和淀粉样蛋白形成能力之间的关系进行了大量研究，但目前淀粉样纤维的形成机制仍不清楚。 1994年，Wickner等提出了酵母Prion这个概念，认为酿酒酵母(Saccharomyces cerevisiae)中两个非孟德尔遗传元件[URE3]和[PSI~+]分别是染色体编码蛋白Ure2p和Sup35p的朊病毒形式，其行为与哺乳动物PrP相似，因此为研究淀粉样纤维的形成及Prion构象转变提供了一个较为理想的模型。Sup35p是翻译终止因子的亚单位，类似于真核细胞释放因子3(eRF3)，通过与Sup45p(eRF1)形成复合物识别终止密码子而使肽链在翻译末端释放，终止蛋白翻译。Sup35蛋白在酵母体内过表达时蛋白构象发生转变而导致聚集，丧失正常的生理功能而导致终止密码子通读，使酵母产生[PSI~+]表型。Sup35p由685个氨基酸组成，包括3个结构域：N端区(N，aa1-123)是诱导[PSI~+]产生所必需的，富含谷氨酰胺(Q)和天冬酰胺(N)，为Prion形成结构域(PrD)。C区是翻译终止功能区。M区位于N与C之间，目前功能不清。为明确N区特殊的氨基酸组成及序列在淀粉样纤维形成中的作用，我们构建了Sup35NM的变异体，分析了它们淀粉样纤维形成的动力学过程，并进一步探讨了野生型Sup35NM及其变异体聚集物的细胞毒性作用，以期为阐明淀粉样纤维形成及其致病机制提供线索。 1．酵母朊蛋白Sup35NM变异体体外淀粉样纤维形成的动态研究为了阐明氨基酸序列对纤维形成的影响，我们首先将Sup35NM片段的PrD的氨基酸序列重新随机排列同时保证氨基酸的组成及M段的氨基酸序列不变，构建了5个Sup35NM的变异体，分别称为Sup35NM-1、-2、-3、-4、-5。在E. coli中成功表达、纯化了野生型Sup35 NM及其5个变异体，进而研究了其在体外淀粉样纤维形成的动力学过程。结果显示：透射电子显微镜下观察可见Sup35NM及其5种变异体蛋白在PBS(pH7.4)缓冲液中均可发生聚集，2h时可见球形颗粒、寡聚体及少量短而细的纤维，18h时Sup35NM、Sup35NM-1、-2、-3蛋白溶液中出现大量的成熟纤维，球形颗粒消失，纤维长且边缘光滑，Sup35NM-4、-5除了长纤维以外，还可见蛋白寡聚体和球形颗粒。48h后，Sup35NM及5个变异体蛋白溶液中只有大量成熟纤维，直径约8～14 nm。用圆二色谱检测了其二级结构，显示在216nm附近出现一负峰，呈典型的β-折叠特征，提示该过程伴随蛋白结构由α-螺旋到β-折叠的转变。NM及变异体聚集所形成的纤维分别经1μg／ml和4μg／ml的蛋白酶K作用60min后仍可检测到蛋白的存在，说明纤维具有较强的抗蛋白酶K消化的特性。ThT结合试验显示，Sup35NM-1、-2、-3与Sup35NM荧光强度上升的速度相似，经历一个快速上升期后达到平台期，未见明显的潜伏期，而Sup35NM-4、-5则能观察到一个近5h的潜伏期，提示Sup35NM-4、-5蛋白聚集明显慢于Sup35NM及其它变异体蛋白。SDS-PAGE电泳和Western blotting也进一步证明，纤维形成过程中蛋白单体逐渐减少同时多聚体则逐渐增加。淀粉样纤维经沸水浴加热后发现纤维消失，代之以大量的球形颗粒、寡聚体及少量短棒状纤维，ThT结合试验显示纤维加热后荧光强度明显下降，SDS-PAGE电泳也证实加热后蛋白单体重新出现。这些数据说明PrD的氨基酸序列被随机打乱后并没用影响其最终形成淀粉样纤维的能力，并且变异体形成的纤维具有与野生型Sup35NM的纤维具有相同的形态和生化特征，但纤维形成的速率有所不同，提示形成纤维的能力是由氨基酸组成决定的，特定的氨基酸序列在一定程度上调节纤维形成的速率。 2．野生型Sup35NM及其变异体蛋白纤维的构象诱导转变作用已有研究表明，在体内Rnq1p、polyQ等聚集体可诱导酵母菌株从[PSI~-]转变为[PSI~+]，在体外异源性的Rnq1或polyQ“种子”可促进NM形成淀粉样纤维。我们构建的变异体N区同野生型NM一样富含Q／N，为了进一步阐明富含Q／N的异源性“种子”能促进蛋白的聚集，我们研究了野生型NM与变异体的相互诱导转变以及另一个酵母朊蛋白Ure2对NM和变异体纤维形成的影响。结果显示：野生型Sup35NM“种子”可加快变异体Sup35NM-1、-4、-5的纤维形成，反之亦然。此外，异源的Ure2“种子”也能轻度增加NM及变异体的转变速率，但总的来说异源性的“种子”比自体“种子”的诱导效率相对低一些。而没有Q／N结构域的α-synuclein“种子”则对蛋白聚集的速率无影响。这些结果提示富含Q／N结构域的异源性“种子”尽管其氨基酸序列不同，能够促进同样含有Q／N结构域的蛋白的聚集，，而不含Q／N的异源“种子”则无此作用。 3．野生型Sup35NM及其变异体对哺乳动物细胞的毒性目前已有大量研究证实，在多种蛋白聚集过程中形成的中间体有细胞毒性。Sup35p是酿酒酵母中的朊蛋白，可发生构象转变形成淀粉样纤维，与哺乳动物Prion蛋白不同，不会引起酵母死亡，与人类淀粉样疾病无关，Sup35的聚集中间体是否具有细胞毒性作用还没有得到证实。为证实Sup35NM聚集体的细胞毒性，我们将纯化的蛋白(64μmol／L)于室温下放置0.5～1h或于4℃放置2d～4d后，分别获得聚集中间体和成熟纤维，然后将不同形态的蛋白分别作用于Vero、NIH-3T3、SH-SY5Y细胞系。此外，我们还进一步研究了变异体的毒性作用。结果显示：Sup35NM及其变异体的聚集中间体对细胞具有毒性作用，可明显降低细胞存活率，这种毒性作用与剂量具有相关性，成熟纤维反而无害，与其它蛋白相似。这一结果提示蛋白聚集体的细胞毒性可能与其结构相关。不同细胞系对蛋白毒性的敏感性有差异，Vero和NIH-3T3细胞比较敏感，SH-SY5Y细胞则有一定的抵抗作用，提示蛋白聚集体的细胞毒性作用是通过特定机制实现的。成熟纤维经沸水浴加热20min后作用于细胞，也表现出与聚集中间体相似的毒性作用，提示纤维经加热后出现的蛋白结构与聚集中间体的结构相似。
[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.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373

【共引文献】