Geoghegan James C, Miller Michael B, Kwak Aimee H, Harris Brent T, Supattapone Surachai
Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA.
PLoS Pathog. 2009 Jul;5(7):e1000535. doi: 10.1371/journal.ppat.1000535. Epub 2009 Jul 31.
Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrP(C) molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrP(C) is pre-incubated with poly(A) RNA and PrP(Sc) template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP(Sc) and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrP(Sc) molecules, most likely through an epitope containing residue 172.
先前的研究鉴定出了朊病毒蛋白（PrP）突变体，这些突变体通过一种假说机制作为朊病毒形成的显性负性抑制剂，该机制被认为需要一种未知的物种特异性辅因子，称为蛋白X。为了在体外研究显性负性抑制的机制，我们将在中国仓鼠卵巢细胞中表达的重组PrP（C）分子用作连续蛋白错误折叠循环扩增（sPMCA）反应的底物。生物测定证实这些反应的产物具有传染性。利用该系统，我们发现：（1）反式显性抑制可以与转化活性分离，（2）即使野生型（WT）PrP（C）与聚（A）RNA和PrP（Sc）模板预孵育，仅使用纯化的底物就能在体外重建朊病毒形成的显性负性抑制，并且（3）Q172R是所测试的唯一不能转化为PrP（Sc）且能在亚化学计量水平上显性抑制WT PrP转化的仓鼠PrP突变体。这些结果反驳了蛋白X是介导朊病毒传播显性抑制所必需的这一假说，并表明PrP分子最有可能通过包含残基172的表位竞争结合新形成的PrP（Sc）分子上的新生种子位点。
