Embedding the Amyloid β‐Peptide Sequence in Green Fluorescent Protein Inhibits Aβ Oligomerization
Embedding the Amyloid β‐Peptide Sequence in Green Fluorescent Protein Inhibits Aβ Oligomerization
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DOI：
10.1002/cbic.200700108
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发表时间：
2007-06
期刊：
影响因子：
3.2
通讯作者：
Tsuyoshi Takahashi;K. Ohta;H. Mihara
中科院分区：
文献类型：
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作者：
Tsuyoshi Takahashi;K. Ohta;H. Mihara
蛋白质原纤维的形成与致命性疾病有关。例如，阿尔茨海默病(AD)是一种进行性神经退行性疾病，其特征是淀粉样蛋白B肽(Ab)的纤维化，积聚在老年斑中。AB多肽可以自组装成可溶性低聚物、原纤维和淀粉样纤维。所有这些聚集形式都包含重要的b-折叠结构；成熟的纤维具有特别有序的超分子b结构。AB在ITS序列的中心(17-21个残基)和C末端(30-42个残基)具有两个疏水区，可能促进纤维的形成。各种模拟抗体中心序列的多肽已被设计为纤化抑制剂。事实上，这些短肽可以与抗体结合并抑制其纤维形成，含有五个氨基酸残基的脯氨酸已被证明可以部分减少淀粉样蛋白的沉积。虽然淀粉样蛋白纤维最初被认为是导致AD的有毒物质，但最近的研究发现，可溶性寡聚体抗体被证明可以杀死培养的海马脑片中的神经元。9]然而，上述短肽是否能抑制毒性的、可溶性的抗体寡聚体的产生尚不清楚。最近有报道称，抗单抗多肽抗体可以减少单抗低聚体及其毒性，这支持了通过与单抗紧密结合的分子来靶向单抗的治疗策略。与抗体不同的是，从水母Aequorea Victoria中分离得到的绿色荧光蛋白(GFP)是一种可溶的球状蛋白，可以自动催化产生荧光团。赫克特和他的同事构建了抗体与绿色荧光蛋白的融合，以筛选抑制抗体聚集的化合物。GFP折叠成由11条b链组成的b桶结构。荧光团位于一个埋在桶中央的螺旋上。在GFP结构中包括平行和反平行b片。通过用抗体取代GFP中的部分b链，该蛋白不仅含有抗体序列，而且呈现出抗体的b-折叠结构。因此，蛋白质结构应该通过与抗体结合来抑制抗体的寡聚。此外，由于GFP具有非常稳定的结构，因此蛋白质结构不应该非特异性地展开或聚集。在本研究中，我们设计并合成了GFP突变体，其中从抗体序列衍生的两条b链作为抗体结构模拟物嵌入到GFP序列中。这些GFP变异体可以与抗体结合并抑制其寡聚。利用平行和反平行b-折叠结构的模型来构建GFP变体。AB1-40和AB1-42纤维的模型结构由平行的b-折叠结构组成。相反，在该模型中，由抗体(AB11-25)的11-25残基形成的纤维由反平行的板状结构组成。GFP含有11条b链，其中10条反平行排列，1条平行排列。GFP的第一和第六链是平行对，因此被选择用于模拟抗体结构的平行b-折叠模型。构建了GFP突变体P13H，该突变体含有P13H、L15K、E17V、D19F、D21E、T118H、V120K、R122V、E124F和K126E等突变。前5个突变位于链1上，其他突变位于链6上。
Protein fibril formation is related to fatal diseases. For example, Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is characterized by the fibrillization of amyloid bpeptide (Ab), which accumulates in senile plaques. Ab peptides can self-assemble into soluble oligomers, protofibrils, and amyloid fibrils. All of these aggregated forms contain significant b-sheet structure; the mature fibrils have a particularly well-organized supramolecular b structure. Ab possesses two hydrophobic regions in the center (residues 17–21) and at the C terminus (residues 30–42) of its sequence, which might promote fibril formation. Various peptides that mimic the central sequence of Ab have been designed as fibrillization inhibitors. Indeed, these short peptides can bind to Ab and inhibit its fibrillogenesis, and a five-residue peptide containing proline has been shown to partially decrease amyloid deposition. Although amyloid fibrils were initially hypothesized to be the toxic species that drives AD, recent studies have imACHTUNGTRENNUNGplicated the soluble oligomeric species of Ab, which has been shown to kill neurons in cultured hippocampal brain slices. 9] However, it is not known whether the short peptides mentioned above can inhibit the generation of the toxic, soluble Ab oligomers. Recently, it was reported that antibodies against the Ab peptide can reduce Ab oligomers and their toxicity, which lends support to the therapeutic strategy of targeting Ab with molecules that bind it tightly. In contrast to Ab, green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria, is a soluble globular protein that generates a fluorophore autocatalytically. Hecht and co-workers constructed a fusion of Ab to GFP for screening compounds that inhibit Ab aggregation. GFP folds into a b-barrel structure composed of eleven b strands. The fluorophore is on an a helix that is buried in the center of the barrel. Both parallel and antiparallel b sheets are included in the GFP structure. By replacing some of the b strands in GFP with those from Ab, the protein not only contains the Ab sequence but also presents the b-sheet structure of Ab. Therefore, the protein construct should inhibit the oligomerization of Ab by binding to it. Further, since GFP has a very stable structure, the protein construct should not unfold or aggregate nonspecifically. In the present study, we designed and synthesized GFP variants in which two b strands derived from the Ab sequence were embedded in the sequence of GFP as an Ab structural mimic. These GFP variants can bind to Ab and inhibit its oligomerization. Models of both parallel and antiparallel b-sheet structures were utilized to construct the GFP variants. The model structures of Ab1–40 and Ab1–42 fibrils are composed of a parallel b-sheet organization. In contrast, the fibrils formed by residues 11–25 of Ab (Ab11–25) are composed of an antiparallel bsheet structure in the model. GFP contains eleven b strands, ten of which are aligned antiparallel and one that is oriented in a parallel fashion. The first and sixth strands of GFP are the parallel pair and thus were chosen for making a mimic of the parallel b-sheet model of Ab structure. A GFP variant named P13H was constructed, which contained the following mutations: P13H, L15K, E17V, D19F, D21E, T118H, V120K, R122V, E124F, and K126E. The first five mutations are located on strand 1 and the others on strand 6. The mutations were de-