Autocatalytic self-propagation of misfolded prion protein
Autocatalytic self-propagation of misfolded prion protein
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DOI：
10.1073/pnas.0404650101
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发表时间：
2004-08-17
影响因子：
11.1
通讯作者：
Kretzschmar, H
中科院分区：
文献类型：
--
作者：
Bieschke, J;Weber, P;Kretzschmar, H
朊病毒被认为在一个自催化过程中复制，该过程将细胞朊病毒蛋白（PrPC）转化为疾病相关的错误折叠PrP同种型（PrPSc）。我们的研究通过体外蛋白质错误折叠循环扩增（PMCA）来仔细检查这一假设。在连续传播PMCA实验中，将PrPSc接种到含有PrPC的健康仓鼠脑匀浆中。错误折叠的PrP通过多轮超声处理和孵育进行扩增，并每10轮PMCA重新接种到新鲜脑匀浆中。该扩增依赖于PrPC底物，并可被重组仓鼠PrP抑制。在系列稀释实验中，新形成的错误折叠和蛋白酶K抗性PrP（PrPres）催化PrPC的结构转化与来自羊瘙痒病（263K）感染仓鼠脑的PrPSc一样有效，在100轮后产生约300倍的PrPres总扩增，这证实了朊病毒假说所假设的自催化PrP错误折叠级联反应。PrPres的形成并不受生物感染性复制的影响，这似乎需要额外的因素PrP错误折叠自催化。
Prions are thought to replicate in an autocatalytic process that converts cellular prion protein (PrPC) to the disease-associated misfolded PrP isoform (PrPSc). Our study scrutinizes this hypothesis by in vitro protein misfolding cyclic amplification (PMCA). In serial transmission PMCA experiments, PrPSc was inoculated into healthy hamster brain homogenate containing PrPC. Misfolded PrP was amplified by rounds of sonication and incubation and reinoculated into fresh brain homogenate every 10 PMCA rounds. The amplification depended on PrPC substrate and could be inhibited by recombinant hamster PrP. In serial dilution experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed the structural conversion of PrPC as efficiently as PrPSc from brain of scrapie (263K)-infected hamsters, yielding an approximate to300-fold total amplification of PrPres after 100 rounds, which confirms an autocatalytic PrP-misfolding cascade as postulated by the prion hypothesis. PrPres formation was not paralleled by replication of biological infectivity, which appears to require factors additional to PrP-misfolding autocatalysis.