Role of Cellular Lipoprotein in PrPSen>PrPRes Conversion

描述(申请人提供)：本项目的长期目标是：了解PrPC和GT的分子机制；PrPSc转化，PrP与非核酸和核酸伴侣的相互作用，阐明这些细胞成分在PrPSc积累和疾病进展中的作用。本项目的主要研究工具是我们公司组装的体外三方PrP转化模型。该模型由蛋白酶K(PK)敏感的PrP(PrPSen)和BCS的高分子量成分组成，BCS与RNA结合，产生一种PK抗性形式的PrP(PrPRes)。我们建议利用这个系统来研究PrPC在小鼠体内转化的分子机制。我们将分离参与小鼠PrPC转化的高分子量BCS的活性成分，并对其进行表征，然后阐明其作用方式。为了实现这一建议的目标，将完成以下任务：(1)利用真核表达系统获得细胞性小鼠普鲁恩蛋白(MPrPC)，(2)分离促进PrPC和GT；PrPRes转换的高相对分子质量组分的活性成分(S)，(3)纯化该组分(S)，对其进行鉴定并确定其分子结构和氨基酸组成，(4)验证该组分在RQ 11+12RNA存在下从mPrPC产生相应mPrPRES亚型的能力。这项研究的成功将为进一步了解TSE的传播和进展的分子机制以及促进这一过程的细胞成分铺平道路。BCS中伴随PrPC和PrPRes转换的高分子量成分的发现和表征将导致在羊、牛和人类中识别相应的对应成分，从而开发相应的诊断和针对TSE的特定靶向治疗工具。对已确定的高分子量伴侣功能的了解应提出适当的“合理设计”的策略，这些策略可以为治疗普恩病毒疾病提供新的机会，并可能导致创建对人类血液产品和牲畜浪费疾病的死前筛查有用的灵敏的症状前检测系统。

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.