Conformational properties of prion strains can be transmitted to recombinant prion protein fibrils in real-time quaking-induced conversion.
Conformational properties of prion strains can be transmitted to recombinant prion protein fibrils in real-time quaking-induced conversion.
复制标题
朊病毒株的构象特性可以在实时振动诱导的转化中传递给重组朊病毒蛋白原纤维。
作者：
Sano K;Atarashi R;Ishibashi D;Nakagaki T;Satoh K;Nishida N.
具有不同生物学特性的病毒株可能与异常蛋白(PrPSc)结构多样性有关。然而，菌株特性传递的分子基础仍然知之甚少。实时震颤诱导转换(RT-QuIC)是一种无细胞体系，它使用大肠杆菌衍生的重组PrP(RPrP)来灵敏地检测PrPSc。为了利用RT-Quic技术研究不同PrP菌株的特性是否能传递到由rPrP(rPrP纤维)组成的淀粉样纤维中，我们检测了来自Chandler或22L菌株的PrPSc种子的rPrP纤维的二级结构、构象稳定性和感染性。在第一轮反应中，Chandler-Seed(1-rPrP-Fibre CH)和22L-Seed(1-rPrP-Fibre 22L)rPrP纤维在二级结构和构象稳定性方面存在差异，特别是红外光谱测定的β-Sheet谱带。值得注意的是，在β-Sheet中看到的两种rPrP原纤维类型的特定识别特征与原始PrPSc的特征相似。此外，与Chandler和22L PrPSc一样，1-rPrP-fibCh1的构象稳定性显著高于1-rPrP-fib2 2 L。接种First-rPrP-fibor First-rPrP-fibor First-rPrP-fier221的小鼠的存活时间明显短于模拟第一轮RT-Quic程序混合接种的小鼠。相反，这些生化特征在接下来的几轮中不再明显，表明非特异性未感染的rPrP纤维成为优势可能是因为它们的高生长速度。综上所述，这些发现表明，至少有一些菌株特有的构象特征可以传递给rPrP纤维，可能需要未知的辅助因素或环境条件来进一步保护。重要的是，具有不同生物学特征的Pron菌株的现象被认为是异常PrPSc构象变化的结果。然而，关于构象差异和应变多样性之间的机制关系，包括应变特有构象是如何传递的，仍然存在重要的问题。在这项研究中，我们研究了不同的Prion菌株的性质是否可以传递到由大肠杆菌产生的重组PrP(RPrP)组成的淀粉样纤维中，这种重组PrP是通过实时震动诱导转换(RT-Quic)产生的，RT-Quic是最近发展起来的一种体外PrPS形成方法。我们证明，在第一轮RT-Quic中，通过检查rPrP纤维的二级结构、构象稳定性和感染性，至少可以将一些菌株特有的构象特性传递到rPrP纤维上，这些纤维来自Chandler或22L PrP菌株。我们相信，这些发现将促进我们对Pron菌株多样性的构象基础的理解。
The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrPSc). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrPSc. To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrPScderived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to β-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fibCh) and 22L-seeded (1st-rPrP-fib22L) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the β-sheets resembled those of the original PrPSc. Furthermore, the conformational stability of 1st-rPrP-fibChwas significantly higher than that of 1st-rPrP-fib22L, as with Chandler and 22L PrPSc. The survival periods of mice inoculated with 1st-rPrP-fibChor 1st-rPrP-fib22Lwere significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation.IMPORTANCEThe phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrPSc). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developedin vitroPrPScformation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrPScderived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.