Spontaneous generation of distinct prion variants with recombinant prion protein from a baculovirus-insect cell expression system
Spontaneous generation of distinct prion variants with recombinant prion protein from a baculovirus-insect cell expression system
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利用杆状病毒-昆虫细胞表达系统的重组朊病毒蛋白自发产生不同的朊病毒变体
DOI：
10.1016/j.bbrc.2022.04.137
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Atarashi R.
中科院分区：
文献类型：
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作者：
Imamura M;Tabeta N;Iwamaru Y;Takatsuki H;Mori T;Atarashi R.
朊病毒病是一种以朊病毒蛋白（PrPSc）在中枢神经系统异常聚集为特征的进行性神经退行性疾病。合成PrPScin无细胞转化系统的产生及其对动物的可传递性的检查将有助于测试仅蛋白质假说和理解散发性朊病毒疾病的分子基础。我们使用来自杆状病毒-昆虫细胞表达系统的重组朊病毒蛋白，（Bac-rPrP）和昆虫细胞衍生的辅因子，以确定Bac-rPrPScis是否在间歇超声反应中自发产生。在37 °C下没有观察到Bac-rPrPSc的自发产生，但是当反应温度升高到45 °C时，在所有试验中均产生Bac-rPrPSc。某些Bac-rPrPSc变异体可传递给小鼠，但当反应重复40轮时，传递性丧失。值得注意的是，在相同的实验条件下，包括相同的超声处理设置和辅因子，产生了各种Bac-rPrPSc变体，包括非传染性的，不同的耐蛋白酶K和辅因子的依赖性在扩增过程中。然而，经过40轮反应后，它们的特征也消失了，各种各样的变异体汇聚成一个单一的变异体。这些结果表明，产生了对小鼠具有不同传递性和结构特性的各种Bac-rPrPSc变体，它们彼此竞争并逐渐收敛到具有稍快扩增速率的变体上。
Prion diseases are transmissible and progressive neurodegenerative disorders characterized by abnormal prion protein (PrPSc) accumulation in the central nervous system. Generation of synthetic PrPScin a cell-free conversion system and examination of its transmissibility to animals would facilitate testing of the protein-only hypothesis and the understanding of the molecular basis of sporadic prion diseases.In this study, we used recombinant prion protein from a baculovirus-insect cell expression system (Bac-rPrP) and insect cell-derived cofactors to determine whether Bac-rPrPScis spontaneously produced in intermittent ultrasonic reactions. No spontaneous generation of Bac-rPrPScwas observed at 37 °C, but when the reaction temperature was increased to 45 °C, Bac-rPrPScwas generated in all trials. Some Bac-rPrPScvariants were transmissible to mice, but when the reaction was repeated for 40 rounds, the transmissibility was lost. Notably, a variety of Bac-rPrPScvariants, including non-transmissible ones, differing in resistance to proteinase K and cofactor dependence during amplification, was generated under the same experimental conditions, including the same sonication settings and cofactors. However, their characteristics also disappeared after 40 reaction rounds and the variety converged onto a single variant. These results indicate that various Bac-rPrPScvariants with different transmissibility to mice and structural properties are generated, which compete with each other and gradually converge onto a variant with a slightly faster amplification rate.