In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features
In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features
复制标题
DOI：
10.1021/bi048322t
复制
发表时间：
2005-02-22
期刊：
影响因子：
2.9
通讯作者：
Bocharova, OV
中科院分区：
文献类型：
--
作者：
Baskakov, IV;Bocharova, OV
朊病毒传播的“仅蛋白质”假说假定朊病毒蛋白的异常同种型PrPSc作为朊病毒疾病的病原体和传播剂。为了在体外重建朊病毒感染性，我们先前开发了一种无细胞转化方案，用于从包含残基89-231（rPrP 89-230）的重组朊病毒蛋白产生淀粉样原纤维[Baskakov等（2002）J.Biol.Chem.277，21140]。当接种到转基因小鼠中时，这些淀粉样蛋白原纤维诱导朊病毒病，其可以有效地传播给野生型和转基因小鼠[Legname等人（2004）Science 305，673]。在这里，我们表明，聚合成纤维的rPrPs显示了一些独特的动力学特征，是不典型的聚合由其他淀粉样蛋白多肽。具体而言，聚合的滞后期仅显示出对蛋白质浓度的适度依赖性，并且转化反应显示出显著的体积依赖性阈值效应。为了解释这些独特的动力学特征，我们提出，转化反应是由自蔓延的纤维状异构体的增殖和失活的速率之间的动态调节。我们进一步的研究表明，表面依赖性吸附的纤维异构体是负责他们在体外失活，而原纤维碎片似乎占聚合活性中心的倍增。我们的研究结果支持这一假设，即朊病毒疾病的发展是由一个良好的自我传播和清除/失活的PrPSc之间的动态平衡控制。
The "protein only" hypothesis of prion propagation postulates that the abnormal isoform of the prion protein, PrPSc, acts as a causative and transmissible agent of prion disease. In attempt to reconstitute prion infectivity in vitro, we previously developed a cell-free conversion protocol for generating amyloid fibrils from a recombinant prion protein encompassing residues 89-231 (rPrP 89-230) [Baskakov et al. (2002) J. Biol. Chem. 277, 21140]. When inoculated into transgenic mice, these amyloid fibrils induced prion disease, which can be efficiently transmitted to both wild-type and transgenic mice [Legname et al. (2004) Science 305, 673]. Here we show that the polymerization of rPrPs into the fibrils displays a number of distinctive kinetic features that are not typical for polymerization by other amyloidogenic polypeptides. Specifically, the lag phase of polymerization showed only modest dependence on protein concentration, and the conversion reaction displayed a dramatic volurne-dependent threshold effect. To explain these unique kinetic features, we proposed that the conversion reaction is regulated by the dynamics between the rates of multiplication and deactivation of self-propagating fibrillar isoforms. Our further studies demonstrated that surface-dependent sorption of fibrillar isoforms is responsible for their deactivation in vitro, while fibril fragmentation seems to account for the multiplication of the active centers of polymerization. Our findings support the hypothesis that development of prion disease is controlled by a fine dynamic balance between self-propagation and clearance/deactivation of PrPSc.