Role of PrPc Polybasic domains in prion conversion

本研究的目的是促进对朊病毒复制的分子机制的理解。对朊病毒发病机制的研究可以提高对克雅氏病和其他朊病毒疾病患者的认识。这项工作也增强了我们对蛋白质聚集机制的了解，这种机制发生在阿尔茨海默病、帕金森病和其他神经退行性疾病中。传染性朊病毒大部分或全部由PrPSc组成，PrPSc是正常细胞蛋白PrPC的错误折叠异构体。该项目旨在研究多碱基结构域（PBDs）在PrPC向PrPSc转化中的作用，PrPSc是朊病毒复制的关键事件。先前的研究表明，PrPC的pbd可能是错误折叠异构体PrPSc的结合位点。本申请旨在确定PBDs在PrPC-PrPSc结合和PrPSc对PrPC的自催化转化中的作用。具体来说，拟议的研究将确定PrPC pbd是否单独或联合介导与PrPSc的相互作用并促进转化。方法包括共免疫沉淀检测结合和体外蛋白错误折叠环扩增（PMCA）检测自催化转化。共免疫沉淀法将确定抗体结合的PrPC在各种条件下是否能与PrPSc结合。PMCA方法是一种灵敏而特异的检测方法，将测试各种PrPC制剂转化为蛋白酶抗性自催化PrPSc的能力。将采用两种技术来评估pbd的作用。首先，在培养的神经细胞中生成并纯化pbd缺失的PrPC，通过上述方法测试其与PrPSc结合并转化为新生PrPSc的能力。其次，PBD肽将被检查其竞争性抑制PrPSc结合PrPC和随后的转化过程的能力。这些研究的结果将表明pbd在PrPC转化中的作用，并有可能确定PrPC转化为PrPSc的结合位点。肽抑制实验也将评估抑制朊病毒复制的潜在治疗途径。公共卫生相关性：这项研究旨在促进我们对朊病毒自我复制导致脑感染、脑功能失调和最终死亡的方式的理解。由于死亡率一致且没有治疗，因此必须调查朊病毒在大脑中传播并引起疾病的方式。朊病毒研究还提供了有关蛋白质如何错误折叠的信息，这是一种常见的事件，为帮助了解阿尔茨海默氏症、帕金森症和其他脑部疾病的问题提供了机会。

The objective of the proposed research is to contribute to the understanding of the molecular mechanisms of prion replication. Investigation of prion pathogenesis advances knowledge that could benefit sufferers of Creutzfeldt-Jakob and other prion diseases. Such work also enhances our insight into mechanisms of protein aggregation, which occurs in Alzheimer's, Parkinson's, and other neurodegenerative conditions. The infectious prion is largely or entirely composed of PrPSc, the misfolded isoform of the normal cellular protein PrPC. This project seeks to examine the role of polybasic domains (PBDs) in conversion of PrPC to PrPSc, the pivotal event in prion replication. Previous studies indicate that PBDs of PrPC may serve as binding sites for the misfolded isoform PrPSc. This application proposes to determine the role of PBDs in both PrPC-PrPSc binding and in the autocatalytic conversion of PrPC by PrPSc. Specifically, the proposed study will determine if the PrPC PBDs, alone or in combination, mediate interaction with PrPSc and facilitate conversion. The methods involve co-immunoprecipitation to examine binding and in vitro protein misfolding cyclic amplification (PMCA) to examine autocatalytic conversion. The co-immunoprecipitation method will determine if antibody-bound PrPC can bind PrPSc under various conditions. The PMCA method, a sensitive and specific assay, will test the ability of various PrPC preparations to undergo conversion to protease-resistant autocatalytic PrPSc. Two techniques will be employed to assess the role of the PBDs. First, PBD-deleted PrPC, generated in cultured neuronal cells and purified, will be tested by the above methods for its ability to bind PrPSc and to be converted into nascent PrPSc. Second, PBD peptides will be examined for their ability to competitively inhibit PrPSc binding to PrPC and the subsequent conversion process. Results of these studies will indicate the role of PBDs in PrPC conversion, potentially identifying the binding site(s) on PrPC for its conversion to PrPSc. The peptide inhibition experiments will also evaluate a potential therapeutic avenue for inhibition of prion replication. Public Health Relevance: This research proposes to advance our understanding of the manner in which prions copy themselves to cause brain infection, improper brain function, and eventually death. With uniform fatality and no treatment, it is imperative to investigate the way that prions spread in the brain and cause disease. Prion research also generates information about how proteins can misfold, a common event which provides an opportunity to help understand what goes wrong in Alzheimer's, Parkinson's, and other brain diseases.