Tanaka Motomasa, Chien Peter, Naber Nariman, Cooke Roger, Weissman Jonathan S
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, California 94143, USA.
Nature. 2004 Mar 18;428(6980):323-8. doi: 10.1038/nature02392.
A remarkable feature of prion biology is the strain phenomenon wherein prion particles apparently composed of the same protein lead to phenotypically distinct transmissible states. To reconcile the existence of strains with the 'protein-only' hypothesis of prion transmission, it has been proposed that a single protein can misfold into multiple distinct infectious forms, one for each different strain. Several studies have found correlations between strain phenotypes and conformations of prion particles; however, whether such differences cause or are simply a secondary manifestation of prion strains remains unclear, largely due to the difficulty of creating infectious material from pure protein. Here we report a high-efficiency protocol for infecting yeast with the [PSI+] prion using amyloids composed of a recombinant Sup35 fragment (Sup-NM). Using thermal stability and electron paramagnetic resonance spectroscopy, we demonstrate that Sup-NM amyloids formed at different temperatures adopt distinct, stably propagating conformations. Infection of yeast with these different amyloid conformations leads to different [PSI+] strains. These results establish that Sup-NM adopts an infectious conformation before entering the cell--fulfilling a key prediction of the prion hypothesis--and directly demonstrate that differences in the conformation of the infectious protein determine prion strain variation.
朊病毒生物学的一个显著特征是毒株现象，即明显由相同蛋白质组成的朊病毒颗粒会导致表型上不同的可传播状态。为了使毒株的存在与朊病毒传播的“仅蛋白质”假说来协调一致，有人提出单一蛋白质可以错误折叠成多种不同的感染形式，每种不同的毒株对应一种形式。多项研究已经发现毒株表型与朊病毒颗粒构象之间存在关联；然而，这种差异是导致朊病毒毒株产生的原因还是仅仅是其一种次要表现，目前仍不清楚，这主要是由于难以从纯蛋白质制备感染性物质。在此，我们报告了一种使用由重组Sup35片段（Sup-NM）组成的淀粉样蛋白高效感染酵母使其产生[PSI+]朊病毒的方法。利用热稳定性和电子顺磁共振光谱，我们证明在不同温度下形成的Sup-NM淀粉样蛋白具有不同的、能稳定传播的构象。用这些不同的淀粉样蛋白构象感染酵母会导致不同的[PSI+]毒株。这些结果表明Sup-NM在进入细胞之前就采用了感染性构象——这符合朊病毒假说的一个关键预测——并直接证明了感染性蛋白质构象的差异决定了朊病毒毒株的变异。
