Structures and Activities of Prions and Prion Proteins

2009财年的成就：1)我们首次证明，引起传染性海绵状脑病（TSE或朊病毒病）的哺乳动物朊病毒可以仅由细菌表达的重组朊病毒蛋白产生。这一发现提供了迄今为止最令人信服的证据，证明了潜在的蛋白质-纯朊病毒假设是TSE感染因子的性质。然而，这些数据留下了其他分子强烈增强病理形式的朊病毒蛋白的感染滴度的可能性。2)我们扩展了PrP与聚硫酸戊聚糖结合时的柔性氨基末端结构域的结构表征，聚硫酸戊聚糖是已知最有效的抗tse治疗化合物之一。核磁共振、荧光和圆二色性实验完善了我们新的三维结构的PrP：戊聚糖聚硫酸盐配合物。3)使用改进的场流分选和朊病毒检测方法，我们改进了对最小和最具传染性的朊病毒颗粒的理解。4)我们已经寻求了新的方法来表征和可视化朊病毒蛋白与膜结合时的致病形式。5)我们检测到朊病毒蛋白与补体因子C1q之间相互作用的TSE菌株依赖性差异。6)我们使用氢-氘交换和质谱法确定细菌表达重组朊病毒蛋白的感染性朊病毒种子淀粉样蛋白原纤维与自发形成的（非种子）原纤维具有不同的结构。这项工作为感染性朊病毒独特的播种能力提供了强有力的生化证据。

Accomplishments for FY2009: 1) We have shown for the first time that mammalian prions that cause a transmissible spongiform encephalopathy (TSE or prion disease) can be generated solely from bacterially expressed recombinant prion protein. This discovery provides the most compelling evidence so far for the potential protein-only prion hypothesis for the nature of the TSE infectious agent. However, the data leave open the likelihood that other molecules strongly enhance the infectious titers of pathological forms of prion protein. 2) We have extended our characterization of the structure of the flexible amino-terminal domain of PrP when bound to pentosan polysulfate, one of the most potent known anti-TSE therapeutic compounds. Nuclear magnetic resonance, fluorescence and circular dichroism experiments have refined our new three dimensional structure of the PrP:pentosan polysulfate complex. 3)We have refined our understanding of the smallest and most infectious prion particles using improved field-flow fractionation and prion detection methodologies. 4) We have pursued new methods for characterizing and visualizing the pathogenic form of prion protein when bound to membranes. 5) We have detected TSE strain-dependent differences in the interactions between prion protein and C1q, a complement factor. 6) We have use hydrogen-deuterium exchange and mass spectrometry to determined that infectious prion-seeded amyloid fibrils of bacterially expressed recombinant prion protein have a different structure than spontaneously formed (unseeded) fibrils. This work provided strong biochemical evidence of the distinct seeding capacity of infectious prions.