Role of PrPc Polybasic domains in prion conversion

PrPc 多碱结构域在朊病毒转化中的作用

• 批准号: 8206564
• 负责人: Michael B Miller
• 金额: $ 4.72万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206564
• 关键词: Alzheimer' s Disease; Amino Acids; Antibodies; Binding; Binding Sites; Biological Assay; Bovine Spongiform Encephalopathy; Brain; Brain Diseases; Cattle; Cells; Cessation of life; Charge; Co-Immunoprecipitations; Creutzfeldt-Jakob Syndrome; Detergents; Disease; Docking; Electrostatics; Event; Human; In Vitro; Infection; Investigation; Knowledge; Length; Mammals; Mediating; Methods; Molecular; Molecular Conformation; Molecular Models; Mutation; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenesis; Peptide Hydrolases; Peptides; PrP; PrPC Proteins; PrPSc Proteins; Preparation; Prion Diseases; Prions; Process; Protein Isoforms; Proteins; Research; Resistance; Role; Site; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Work; in vivo; insight; molecular modeling; particle; protein aggregation; protein misfolding cyclic amplification; public health relevance; research study

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）： 拟议研究的目的是为理解prion复制的分子机制做出贡献。对王室发病机理的调查可以使知识有益，这可能使Creutzfeldt-Jakob和其他prion疾病的患者受益。此类工作还增强了我们对蛋白质聚集机制的见解，该机理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。感染性prion主要或完全由PRPSC组成，即正常细胞蛋白PRPC的错误折叠同工型。该项目旨在检查多重域（PBD）在PRPC转换为PRPSC的作用，PRPC是Prion Replication中的关键事件。先前的研究表明，PRPC的PBD可以作为错误折叠的同工型PRPSC的结合位点。该应用程序建议确定PBD在PRPC-PRPSC结合以及PRPSC对PRPC的自催化转化中的作用。具体而言，拟议的研究将确定PRPC PBD单独或合并是否介导与PRPSC的相互作用并促进转化。该方法涉及共免疫沉淀，以检查结合和体外蛋白质错误折叠循环扩增（PMCA）以检查自催化转化率。共免疫沉淀方法将确定在各种条件下抗体结合的PRPC是否可以结合PRPSC。 PMCA方法是一种敏感且特定的测定法，将测试各种PRPC制剂进行转化为抗蛋白酶的自催化PRPSC的能力。将采用两种技术来评估PBD的作用。首先，在培养的神经元细胞中产生并纯化的PBD删除的PRPC将通过上述方法测试其结合PRPSC并将其转换为新生的PRPSC的能力。其次，将检查PBD肽的竞争能力抑制PRPSC与PRPC的结合以及随后的转化过程。这些研究的结果将表明PBD在PRPC转化中的作用，有可能识别PRPC上的结合位点的转化为PRPSC。肽抑制实验还将评估潜在的治疗途径，以抑制prion复制。公共卫生相关性：这项研究建议促进我们对prions王复制自己的方式的理解，以引起大脑感染，大脑功能不当以及最终死亡。由于死亡统一，没有治疗，必须研究王室在大脑中传播并引起疾病的方式。 Prion Research还生成了有关蛋白质如何错误折叠的信息，这是一个常见的事件，提供了一个机会，可以帮助了解阿尔茨海默氏症，帕金森氏症和其他脑部疾病中出了问题。