Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
Department of Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
J Neurochem. 2023 Sep;166(5):875-884. doi: 10.1111/jnc.15930. Epub 2023 Aug 7.
Cofactor molecules are required to generate infectious mammalian prions in vitro. Mouse and hamster prions appear to have different cofactor preferences: Whereas both mouse and hamster prions can use phosphatidylethanolamine (PE) as a prion cofactor, only hamster prions can also use single-stranded RNA as an alternative cofactor. Here, we investigated the effect of detergent solubilization on rodent prion formation in vitro. We discovered that detergents that can solubilize PE (n-octylglucoside, n-octylgalactoside, and CHAPS) inhibit mouse prion formation in serial protein misfolding cyclic amplification (sPMCA) reactions using bank vole brain homogenate substrate, whereas detergents that are unable to solubilize PE (Triton X-100 and IPEGAL) have no effect. For all three PE-solubilizing detergents, inhibition of RML mouse prion formation was only observed above the critical micellar concentration (CMC). Two other mouse prion strains, Me7 and 301C, were also inhibited by the three PE-solubilizing detergents but not by Triton X-100 or IPEGAL. In contrast, none of the detergents inhibited hamster prion formation in parallel sPMCA reactions using the same bank vole brain homogenate substrate. In reconstituted sPMCA reactions using purified substrates, n-octylglucoside inhibited hamster prion formation when immunopurified bank vole PrP substrate was supplemented with brain phospholipid but not with RNA. Interestingly, phospholipid cofactor solubilization had no effect in sPMCA reactions using bacterially expressed recombinant PrP substrate, indicating that the inhibitory effect of solubilization requires PrP post-translational modifications. Overall, these in vitro results show that the ability of PE to facilitate the formation of native but not recombinant prions requires phospholipid bilayer integrity, suggesting that membrane structure may play an important role in prion formation in vivo.
辅助因子分子是在体外产生感染性哺乳动物朊病毒所必需的。鼠和仓鼠朊病毒似乎具有不同的辅助因子偏好：尽管鼠和仓鼠朊病毒都可以使用磷脂酰乙醇胺（PE）作为朊病毒辅助因子，但只有仓鼠朊病毒也可以使用单链 RNA 作为替代辅助因子。在这里，我们研究了去污剂溶解对体外啮齿动物朊病毒形成的影响。我们发现，能够溶解 PE 的去污剂（辛基葡萄糖苷、辛基半乳糖苷和 CHAPS）抑制使用 bank vole 脑匀浆底物的串行蛋白错误折叠循环扩增（sPMCA）反应中的鼠朊病毒形成，而不能溶解 PE 的去污剂（Triton X-100 和 IPEGAL）则没有影响。对于所有三种能够溶解 PE 的去污剂，只有在临界胶束浓度（CMC）以上才能观察到对 RML 鼠朊病毒形成的抑制作用。另外两种鼠朊病毒株 Me7 和 301C 也被三种能够溶解 PE 的去污剂抑制，但不受 Triton X-100 或 IPEGAL 的影响。相比之下，在使用相同的 bank vole 脑匀浆底物的平行 sPMCA 反应中，没有一种去污剂抑制仓鼠朊病毒的形成。在使用纯化底物的重建 sPMCA 反应中，当用免疫纯化的 bank vole PrP 底物补充脑磷脂而不是 RNA 时，辛基葡萄糖苷抑制了仓鼠朊病毒的形成。有趣的是，磷脂辅助因子的溶解在使用细菌表达的重组 PrP 底物的 sPMCA 反应中没有影响，这表明溶解的抑制作用需要 PrP 的翻译后修饰。总的来说，这些体外结果表明，PE 促进天然而不是重组朊病毒形成的能力需要磷脂双层的完整性，这表明膜结构可能在体内朊病毒形成中发挥重要作用。
