Reconstitution of Prion Infectivity

描述（由申请人提供）：朊病毒蛋白（PrP）是一系列神经退行性疾病的基础，没有确定的治疗方法，对人类和经济造成破坏性后果。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。“蛋白质唯一假说”假定PrP的异常同种型（PrPSc）充当感染剂并使用正常同种型（PrPC）作为底物传播其病理构象。最终直接证明的“蛋白质唯一的假说”，重建PrPSc在体外从非感染性的PrP，尚未实现。这项研究的目的是在体外重建朊病毒的转化过程，然后在培养的细胞和动物中进行生物测定。提出了三项战略：（i）在体外操纵重组PrP（rPrP）的构象性质;（ii）增加细胞对朊病毒传播的易感性;和（iii）利用朊病毒斑块的永久性非蛋白组分来辅助体外重折叠。使用培养的神经母细胞瘤细胞而不是动物作为传播媒介的中间步骤，可以降低复杂程度，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淀粉样蛋白形成的瞬时中间产物和终产物诱导朊病毒在培养细胞中增殖的能力;（2）探索rPrP α-亚型与朊病毒斑块非蛋白组分复合物作为传播媒介的能力;（3）我们将检查糖基化抑制条件是否增加细胞对rPrP同种型诱导的朊病毒复制的易感性。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PrPSc同种型的形成。将使用生物测定法验证培养细胞中PrPSc的形成。当rPrP依赖的细胞系统的朊病毒传播的开发，将出现新的机会，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。

DESCRIPTION (provided by applicant): Prion protein (PrP) underlies a spectrum of neurodegenerative 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 (PrPSc) acts as an infectious agent and propagates its pathological conformation using normal isoform (PrPC) 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 and animals. Three strategies are suggested: (i) manipulating conformational properties of recombinant PrP (rPrP) in vitro; (ii) increasing the susceptibility of cells for prion transmission; and (iii) utilizing permanent non protein components of prion plaques to assist in vitro refolding. The use of cultured neuroblastoma cells instead of animals as an intermediate step 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 the transient intermediates and end-product of the amyloid formation of rPrP to induces prion propagation in cultured cells; (2) we will explore the capabilities of complexes of a-isoforms of rPrP with non-protein component of prion plaques as transmissible agents; (3) we will examine whether or not glycosylation-inhibiting conditions increases susceptibility of cells for prion replication induced by isoforms of rPrP. Proteinase K digestion, detergent solubility, and immunoconformational assays will be used to detect the formation of nascent PrPSc isoform in cultured cells. Bioassay will be used to validate formation of PrPSc in cultured cells. When rPrP-dependent cellular 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.