Role of PrPc Polybasic domains in prion conversion

这项拟议的研究的目的是为了帮助理解Pron复制的分子机制。对Prion发病机制的研究促进了人们对CreutzFeldt-Jakob和其他Prion疾病患者的认识。这些工作还增强了我们对蛋白质聚集机制的洞察，蛋白质聚集发生在阿尔茨海默氏症、帕金森氏症和其他神经退行性疾病中。具有感染性的普恩蛋白主要或全部由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性结构域(PBD)在PrPC到PrPSc转换中的作用，PrPC是Prion复制中的关键事件。以往的研究表明，PrPC的PBD可能是错误折叠的异构体PrPSc的结合位点。本申请建议确定多溴二苯醚在PrPC-PrPSc结合和PrPSc对PrPC的自催化转化中的作用。具体地说，拟议的研究将确定PrPC多溴二苯醚是单独还是结合在一起，调解与PrPSc的相互作用并促进转换。这些方法包括免疫共沉淀检测结合和体外蛋白质错误折叠循环扩增(PMCA)检测自催化转化。免疫共沉淀法将确定抗体结合PrPC在不同条件下是否能与PrPSc结合。PMCA方法是一种灵敏而特异的检测方法，它将测试各种PrPC制剂转化为抗蛋白酶的自催化PrPSc的能力。将使用两种技术来评估PBD的作用。首先，从培养的神经细胞中产生并纯化的PBD缺失PrPC将通过上述方法检测其与PrPSc结合并转化为新生PrPSc的能力。其次，将检测PBD多肽是否能够竞争性地抑制PrPSc与PrPC的结合以及随后的转换过程。这些研究的结果将表明多溴二苯醚在PrPC转化中的作用，可能确定PrPC上转换为PrPSc的结合位点(S)。肽抑制实验还将评估抑制PrN复制的潜在治疗途径。与公共卫生的相关性：这项研究建议促进我们对Pron复制自身导致大脑感染、大脑功能不正常以及最终死亡的方式的理解。在没有统一的死亡和治疗的情况下，调查普恩病毒在大脑中传播并导致疾病的方式是当务之急。Prion研究还产生了关于蛋白质如何错误折叠的信息，这是一种常见的事件，为帮助理解阿尔茨海默氏症、帕金森氏症和其他大脑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.