【作者】 刘英霞；

【导师】 洪涛； 王健伟；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2007， 博士

【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。更多还原

【Abstract】 Numerous human diseases are linked to the formation of amyloid fibrils, includingAlzheimer’s disease, Parkinson’s disease, and the transmissible spongiformencephalopathies (TSEs). Although the proteins associated with amyloid diseaseshave diverse amino acid sequences, the amyloid fibers that they form seem to share acommon structure. Amyloid fibers are highly organized protein aggregatescharacterized by filamentous morphology, highβ-sheet content, protease resistance,and yellow-green birefringence upon staining with Congo red. Investigations for themechanism by which amyloid forms will no doubt provide not only the clues for theunderstanding of amyloid diseases but also the development of strategies of prevetionand treatment to such diseases. Howerver, despite considerable studies had been done,much is still unknown about the relationship between amino acid sequence andpropensity to form amyloid.In 1994, Wickner et al proposed that two non-Mendelian elements, [URE3] and[PSI~+], are prion forms of the Sacchromyces cerevisiae proteins, respectively. Since inboth PrP and yeast prions, prion formation is resulted from the conversion of nativeprotein into a self-propagating and infectious amyloid form, yeast prions provide auseful model for studying not only the mechanism of amyloid fibril formation but alsoprion-like transmission of the protein conformation. Sup35p is an essential subunit ofthe translation termination factor, which corresponds to eRF3 in mammals. It functions in termination in a complex by binding its partner Sup45p. Overexpressionof Sup35p induces protein aggregation and causes [PSI~-] cells to revert to [PSI~+].Sup35 is composed of three distinct sequence elements: N region, M region and Cregion. The N region (aa 1-123) is required for the maintenance of [PSI~+] and rich inQ/N (44%). The C region provides the termination function. The M region is highlycharged and its function is still unclear. In order to examine the role of unusual aminoacid composition and specific sequence in the prion domain and to provide clues forthe pathogenesis of amyloid diseases, in the present study, we constructed severalSup35NM variants and tested the ability and dynamics of amyloid fiber formation ofeach variant as well as their toxicity to mammalian cells.1. Dynamics of in vitro amyloid fiber formation of Sup35NM and its variantsIn order to prove the impacts of amino acid sequence on fiber formation, weprepared 5 variants of Sup35p prion domain (N) in which the order of the amino acid2-123 was randomized while without changing the amino acids composition. Thesescrambled variants were named as Sup35NM-1, -2, -3, -4, and -5, respectively. Thewild type (wt) Sup35NM and its variants which were expressed in E. coli and purifiedunder denaturing conditions respectively were subjected to the investigations on thedynamics of amyloid fibers formation. The morphological alteration of the wtSup35NM and variants in PBS (pH 7.4) during the protein aggregation was visualizedby transmission electron microscopy (TEM). The results revealed that at 2h after theinitiation of the assembly reaction, wt Sup35NM and its variants showed a mixture ofgranule, oligomer, and short filaments. After 18h, only fibers could be observed insamples of NM-1,-2, -3 and wt NM, while NM-4, -5 exhibited a mixture of structuresincluding fibers and oligomers. After 48h, all samples exhibited only fibers. Thecircular dichroism (CD) assay showed that the course of amyloid fiber formationunderwent a conformational shift fromα-helix toβ-sheet. The amyloid form wasdetectable when the fibers of either wt Sup35NM or its variants were treated with 1μg/ml or 4μg/ml (final concentration) protease K at 37℃for 60 min. The fibersshowed higher protease K resistance when compared with the native forms. ThTfluorescence assay displayed a rapid growth and a final equilibrium phase in wtSup35NM, Sup35NM-1, -2, and -3. However, the ThT fluorescence curve ofSup35NM-4, and -5 revealed a distinct decrease in conversion kinetics with aprolonged lag time of nearly 3～5 hours. The results showed that the aggregation rateof Sup35NM-4, -5 is much slower than the others. As monomeric form of wtSup35NM and its variants decreased, insoluble polymers increased gradually asmonitored by SDS-PAGE and Western blot. After the fibers were heated in boiledwater bath, ThT fluorescence intensity decreased significantly and monomers of allsamples reappeared in SDS-PAGE. The results indicated that the randomized priondomains polymerized into amyloid in vitro under native conditions, however, theaggregating speed of these variants is somewhat different. Moreover, the assembledand ordered filaments retain all the morphological, structural and biochemicalproperties reported for ex vivo Sup35p aggregate. In conclusion, the specific sequencefeatures can modulate the rate of conversion of Sup35p into amyloid in vitro, thoughit is dispensable to the ability of amyloid formation.2. Cross-seeding of wt Sup35NM and its variantsIt has been reported that Rnql, polyQ aggregates facilitated [PSI] appearance invivo, and Rnql, polyQ fibers enhance the in vitro formation of fibers formed bySup35NM. To explore the role of the heterologous Q/N-rich aggregates in fiberformation, we studied the reciprocal effect of wt Sup35NM and its variants onconformation conversion. The results showed that the rates of Sup35NM-1, -4, and -5conversion were increased when sonicated wt Sup35NM fibers were added, and viceversa. In addition, the other Q/N-rich amyloid, Ure2p, weakly augmented fiberformation of wt Sup35NM and its variants. Heterologous amyloid is much lesssufficient than its own conversion. On the contrary, non-Q/N-rich amyloid, α-synuclein did not seed conversion of wt Sup35NM and its variants. The resultssuggested that heterologous Q/N-rich amyloids promote the conversion of similarQ/N-rich domain.3. Toxicity of the amyloid aggregates formed by wt Sup35NM and its variants tomammalian cellsA number of data have confirmed that pre-fibrillar aggregates of proteins are toxicto cells. It has not been clarified whether the aggregates of Sup35p are still toxic tomammalian cells. In the initial of this study, incubation of wt Sup35NM and itsvariants (64μmol/L) for 0.5～1h at room temperature or 2d～4d at 4℃, led to theformation of early aggregates and mature fibers, respectively. Then, Vero, NIH-3T3,and SH-SY5Y cells were incubated in the aliquots of solutions containing differentmorphological types of aggregates with 5, 10, 15μmol/L final concentrations for 24h.The results revealed that cells exposed to early aggregates of wt Sup35NM and itsvariants displayed significantly reduced viability with the increase of aggregatesconcentrations, whereas mature fibrils are harmless. These results are similar to thatof other proteins reported previously, implying that the cytotoxicity isstructure-dependent. However, the cell types exposed to early aggregates alsodisplayed variable susceptibility to damage. Vero, NIH-3T3 cells are susceptible,while SH-SY5Y cells are resistant, indicating that the cytotoxicity is archived in aspecific way. Moreover, another phenomenon was notable that when Vero, NIH-3T3cells were incubated with wt Sup35NM and its variants fibers after heating in boiledwater bath for 20min, these cells expressed the similar damage just as the earlyaggregates, suggesting the structure similarity between the deaggregated maturefibrils after heating and the early aggregates.更多还原