In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrPSc
In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrPSc
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DOI：
10.1016/j.jmb.2004.11.068
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发表时间：
2005-02-18
影响因子：
5.6
通讯作者：
Baskakov, IV
中科院分区：
文献类型：
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作者：
Bocharova, OV;Breydo, L;Baskakov, IV
“仅蛋白质”假说假设朊病毒疾病的传染源 PrPSc 由转化为淀粉样蛋白特异性构象的朊病毒蛋白 (PrP) 组成。然而，全长 PrP 向淀粉样蛋白构象的无细胞转化尚未实现。为了了解 PrPSc 形成的机制，我们使用具有完整二硫键 (rPrP) 的重组小鼠全长 PrP 开发了一种无细胞转化系统。我们证明，rPrP 在高酸性 pH（< 5.5）和高浓度下会转化为富含 β 片层的寡聚形式，而在微酸性或中性 pH（> 5.5）下，它会组装成淀粉样蛋白形式。从电子显微镜观察，淀粉样蛋白形式具有由两条非扭曲的细丝组成的带状组件。与β-寡聚体的形成相反，淀粉样蛋白的转化发生在接近生理浓度的情况下，并显示出自催化过程的关键特征。此外，使用由 106 个残基组成的缩短的 rPrP（rPrP 106，缺失：Delta23-88 和 Delta141-176），我们表明体外转换模拟了体内观察到的传输屏障。此外，淀粉样蛋白形式对蛋白酶 K (PK) 表现出显着的抗性，并产生与 PrPSc 相同的 PK 抗性核心。傅里叶变换红外光谱分析表明，淀粉样蛋白形式的富含 β-折叠的核心在 PK 消化后保持完整，并具有极高的热稳定性。电子和实时荧光显微镜显示，蛋白水解消化诱导淀粉样蛋白带聚集成大团块或进一步组装成由多个带组成的原纤维。由丝带组成的原纤维非常脆弱，并且容易碎裂成短碎片。值得注意的是，经 PK 处理的淀粉样蛋白形式保留了高播种活性。我们的工作支持朊病毒增殖的蛋白质假说，并证明在没有细胞因子或 PrPSc 模板的情况下，可以在体外形成概括 PrPSc 关键物理特性的淀粉样蛋白形式。 (C) 2004 Elsevier Ltd. 保留所有权利。
The "protein only" hypothesis postulates that the infectious agent of prion diseases, PrPSc, is composed of the prion protein (PrP) converted into an amyloid-specific conformation. However, cell-free conversion of the full-length PrP into the amyloid conformation has not been achieved. In an effort to understand the mechanism of PrPSc formation, we developed a cell-free conversion system using recombinant mouse full-length PrP with an intact disulfide bond (rPrP). We demonstrate that rPrP will convert into the beta-sheet-rich oligomeric form at highly acidic pH (< 5.5) and at high concentrations, while at slightly acidic or neutral pH (> 5.5) it assembles into the amyloid form. As judged from electron microscopy, the amyloid form had a ribbon-like assembly composed of two non-twisted filaments. In contrast to the formation of the beta-oligomer, the conversion to the amyloid occurred at concentrations close to physiological and displayed key features of an autocatalytic process. Moreover, using a shortened rPrP consisting of 106 residues (rPrP 106, deletions: Delta23-88 and Delta141-176), we showed that the in vitro conversion mimicked a transmission barrier observed in vivo. Furthermore, the amyloid form displayed a remarkable resistance to proteinase K (PK) and produced a PK-resistant core identical with that of PrPSc. Fourier transform infrared spectroscopy analyses showed that the beta-sheet-rich core of the amyloid form remained intact upon PK-digestion and accounted for the extremely high thermal stability. Electron and real-time fluorescent microscopy revealed that proteolytic digestion induces either aggregation of the amyloid ribbons into large clumps or further assembly into fibrils composed of several ribbons. Fibrils composed of ribbons were very fragile and had a tendency to fragment into short pieces. Remarkably, the amyloid form treated with PK preserved high seeding activity. Our work supports the protein only hypothesis of prion propagation and demonstrates that formation of the amyloid form that recapitulates key physical properties of PrPSc can be achieved in vitro in the absence of cellular factors or a PrPSc template. (C) 2004 Elsevier Ltd. All rights reserved.