Conformational diversity in purified prions produced in vitro.
Conformational diversity in purified prions produced in vitro.
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DOI：
10.1371/journal.ppat.1011083
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发表时间：
2023-01
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
朊病毒疾病是由朊病毒蛋白（PrP）的野生型或突变形式错误折叠成自我繁殖的致病构象异构体（统称为PrPSc）引起的。野生型和突变型PrPSc分子在体内均表现出构象多样性，但通过系列蛋白质错误折叠循环扩增（sPMCA）技术产生的纯化朊病毒在体外并未表现出相同的多样性。这种差异在我们理解两种类型朊病毒在分子水平上构象多样性是如何产生的方面留下了空白。在这里，我们使用连续振荡，而不是sPMCA产生构象不同的纯化朊病毒在体外。使用这种方法，我们首次表明，野生型朊病毒最初播种不同的原生菌株可以传播作为亚稳态PrPSc构象与可区分的菌株特性在纯化的反应中含有一个单一的活性辅因子。这些亚稳态PrPSc构象的传播需要适当的振荡条件，这些条件的变化会导致所有不同的PrPSc构象不可逆地收敛到相同的单一构象中产生的sPMCA反应。我们还使用连续振荡，以显示两个突变的PrP分子与不同的致病点突变（D177 N和E199 K）采用可区分的PrPSc构象在反应中含有纯蛋白质底物没有辅因子。与野生型朊病毒不同，突变型朊病毒的构象似乎是由底物序列而不是种子构象决定的。总体而言，我们的研究使用纯化的底物在振荡反应表明，野生型和突变型朊病毒使用根本不同的机制，以产生分子水平上的构象多样性。朊病毒是一种错误折叠的蛋白质，在包括人类在内的许多动物中引起传染性、遗传性和散发性疾病。朊病毒可以错误折叠成各种不同的错误折叠形状，但我们还不知道产生这种多样性的分子机制。我们报告说，振荡试管反应与纯化的成分，可用于研究的机制，形成不同形状的朊病毒。我们的研究结果表明，传染性（野生型）朊病毒和遗传性（突变型）朊病毒使用不同的机制，以产生不同的错误折叠的形状。
Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level. Prions are misfolded proteins that cause infectious, inherited, and sporadic diseases in many animals, including humans. Prions can misfold into a variety of distinct misfolded shapes, but we do not yet know the molecular mechanisms that generate this diversity. We report that shaking test tube reactions with purified components can be used to study the mechanisms by which prions with diverse shapes are formed. Our results show that infectious (wild-type) prions and inherited (mutant) prions use different mechanisms to produce diverse misfolded shapes.
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