Reconstitution of Prion Transmission in Cells

朊病毒蛋白（PrP）是一系列年龄依赖性疾病的基础，这些疾病没有既定的治疗方法，并对人类和经济造成毁灭性的后果。朊病毒的传播形式将朊病毒疾病与其他神经退行性疾病区分开来。“仅蛋白质假说”假定PrP的异常同种型（PrP Sc）充当感染剂， 使用正常同种型（PrPC）作为底物传播其病理构象。最终的直接证据的“蛋白质唯一的假说”，重建prpSc在体外从非感染性PrP，尚未实现。该研究的目的是在体外重建朊病毒转化的过程，然后在培养细胞中进行生物测定。提出了三项战略：（i）在体外操纵重组PrP（rPrP）的构象性质;（ii）利用朊病毒斑块的永久性非蛋白质组分来辅助体外折叠;和（iii）增加细胞对朊病毒传播的易感性。使用培养的神经母细胞瘤细胞而不是动物作为传染性因子的靶， 的复杂程度，并确定朊病毒传播的必要因素。本研究的主要目的是：（1）确定rPrP β折叠异常亚型在培养细胞中诱导朊病毒增殖的能力;（2）研究外源性rPrP β折叠亚型在细胞内的定位和稳定性;（3）我们将研究在糖基化抑制条件下，rPrP的β-异构体是否能启动朊病毒复制。为了研究细胞内定位和稳定性，将使用免疫染色、FRET和共聚焦显微镜。将使用蛋白酶K消化、去污剂溶解度和免疫构象测定来检测培养细胞中新生PrP Sc同种型的形成。当rPrP依赖的模型系统的朊病毒传播的开发，新的机会将出现研究的详细机制的传输和传播的朊病毒在活细胞中使用非侵入性的最先进的生物物理技术。这些知识应该为治疗传染性和散发性朊病毒疾病的新分子和药理学方法奠定基础。

Prion protein (PrP) underlies a spectrum of age-dependent diseases with no established treatment and devastating human and economical consequences. The existence of transmissible forms separates prion diseases from other neurodegenerative illnesses. The "protein only hypothesis" postulates that an abnormal isoform of PrP (PrP Sc)acts as an infectious agent and propagates its pathological conformation using normal isoform (PrP C) as a substrate. The ultimate direct proof of the "protein only hypothesis', the reconstitution of prpSc in vitro from noninfectious PrP, has not been achieved yet. The proposed research aims at reconstitution of the process of prions conversion in vitro following by bioassay in cultured cells. Three strategies are suggested: (i) manipulating conformational properties of recombinant PrP (rPrP) in vitro; (ii) utilizing permanent non-protein components of prion plaques to assist in vitro folding; and (iii) increasing the susceptibility of cells for prion transmission. The use of cultured neuroblastoma cells instead of animals as a target for transmissible agent allows one to reduce the degree of complexity and to determine factors essential for prion transmission. Several hypotheses proposed by the PI are elaborated into three specific aims: (1) we will determine the capabilities of abnormal beta-sheet rich isoforms of rPrP to induce prion propagation in cultured cells; (2) we will examine intracellular localization and stability of exogenously administered beta-isoformsof rPrP; (3) we will examine whether or not prion replication in calls can be initiated by beta-isoformsof rPrP under glycosylation-inhibiting conditions. To study intracellular localization and stability, immunostaining, FRET, and confocal microscopy will be utilized. Proteinase K digestion, detergent solubility, and immunoconformational assays will be used to detect the formation of nascent PrP Sc isoform in cultured cells. When rPrP-dependent model system of prion propagation is developed, new opportunities will arise for studying detailed mechanisms of transmission and propagation of prions in living cells using non-invasive state-of-the-art biophysical techniques. Such knowledge should lay the foundation for novel molecular and pharmacological approaches of treating transmissible and sporadic prion diseases.