Mechanisms of prion protein assembly into amyloid
Mechanisms of prion protein assembly into amyloid
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DOI：
10.1073/pnas.0712036105
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发表时间：
2008-02
期刊：
影响因子：
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通讯作者：
J. Stöhr;N. Weinmann;H. Wille;T. Kaimann;L. Nagel-Steger;E. Birkmann;G. Panza;S. Prusiner;M. Eigen;D. Riesner
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文献类型：
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作者：
J. Stöhr;N. Weinmann;H. Wille;T. Kaimann;L. Nagel-Steger;E. Birkmann;G. Panza;S. Prusiner;M. Eigen;D. Riesner
朊病毒蛋白（PrPC）的α-螺旋、细胞同种型转化为不溶性的、富含β-片层的、感染性的、致病的同种型（PrPSc）是朊病毒疾病的关键事件。在早期的研究中，通过使用由低浓度SDS和250 mM NaCl组成的体外转化系统，将几种形式的PrP转化为纤维状状态。在这里，我们表征的结构的原纤维前体状态，即，在纤维化条件下的可溶性状态。CD光谱、分析超离心和化学交联表明，前体状态存在于部分变性的α-螺旋PrP的单体-二聚体平衡中，二聚体中的亚基具有明确的接触位点。使用荧光与硫磺素T，我们监测和定量描述的动力学种子原纤维的形成，包括依赖于底物和种子浓度的反应。指数，种子增强生长可以在均匀的溶液中实现，这可以通过超声处理来增强。从这些数据中，我们提出了一个机制模型的fietrization，包括存在的几个中间结构。这些研究还提供了一个简化的朊病毒扩增系统。
The conversion of the α-helical, cellular isoform of the prion protein (PrPC) to the insoluble, β-sheet-rich, infectious, disease-causing isoform (PrPSc) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, α-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.