Role of Cellular Lipoprotein in PrPSen>PrPRes Conversion

描述（由申请人提供）：本项目的长期目标是：了解PrPC>PrPSc转化的分子机制，PrP与非核酸和核酸分子伴侣的相互作用，阐明这些细胞组分在PrPSc蓄积和疾病进展中的作用。本项目的主要研究工具是体外三方PrP转化模型，该模型已在本公司组装。该模型由蛋白酶K（pK）敏感性PrP（PrPSen）和BCS的高分子量组分组成，BCS的高分子量组分与RNA组合产生PrP的pK抗性形式（PrPRes）。我们建议使用该系统来研究小鼠中PrPC转化的分子机制。我们将分离参与小鼠PrPC转化的BCS的高分子量活性组分，对其进行表征，然后阐明其作用方式。为实现本建议书的目标，将完成以下任务：（1）使用真核表达系统获得细胞小鼠朊病毒蛋白（mPrPC），（2）分离促进PrPC → PrPRes转变的BCS的高分子量级分的活性组分，（3）纯化该组分，表征并确定其分子结构和氨基酸组成，（4）验证这种组分在RQ 11 + 12 RNA存在下从mPrPC产生相应mPrPRes同种型的能力。这项研究的成功应该为进一步了解TSE传播和进展的分子机制以及促进这一过程的细胞成分开辟道路。BCS中高分子量组分的发现和表征，即伴侣蛋白PrPC>PrPRes转换，应导致鉴定羊、牛和人中的相应对应物，从而开发相应的诊断和特异性靶向治疗工具用于TSE。对已确定的高分子量分子伴侣功能的理解应建议适当的“合理设计”策略，这可能为治疗朊病毒疾病提供新的机会，并可能导致建立灵敏的症状前检测系统，用于人类血液制品和牲畜消耗性疾病的宰前筛查。

DESCRIPTION (provided by applicant): The long term objectives of this Project are: understanding of the molecular mechanisms of PrPC>PrPSc transformation, PrP interactions with non-nucleic acid and nucleic acid chaperones, elucidation of the role of these cellular components in PrPSc accumulation and disease progression. The major research tool of this Project is an in vitro tripartite PrP conversion model that has been assembled in our Company. This model is composed of proteinase K (pK) sensitive PrP (PrPSen) and a high molecular weight component of BCS that, in combination with RNA, generates a pK-resistant form of PrP (PrPRes). We propose to use this system to study molecular mechanisms of the PrPC conversion in the mouse. We will isolate active component of high molecular weight of BCS that participate in mouse PrPC conversion, characterize it and then elucidate the mode of its action. To achieve the Goals of this Proposal, the following Tasks will be accomplished: (1) Obtain cellular mouse prion protein (mPrPC) using eukaryotic expression systems, (2) Isolate active component(s) of a high molecular weight fraction of BCS that facilitate PrPC>PrPRes transition, (3) Purify this component(s), characterize it and determine its molecular structure and amino-acid composition, (4) Validate the ability of such component to generate corresponding mPrPRes isoforms from mPrPC in the presence of RQ 11 + 12 RNA. The success of this research should open the way for further understanding of the molecular mechanisms for TSE transmission and progression and cellular components that facilitate this process. Discovery and characterization of the high molecular weight component in BCS that chaperones PrPC>PrPRes transition should lead to the identification of corresponding counterparts in sheep, cow and humans and consequently, the development of corresponding diagnostics and specifically targeted therapeutic tools for TSE. Understanding of identified high molecular weight chaperone functions should suggest appropriate "rationally designed" strategies that could offer new opportunities for treating prion diseases and may lead to the creation of sensitive pre-symptomatic detection systems useful for human blood product and pre-mortem screening for livestock wasting diseases.