The Cell Biology of Neurodegeneration Caused by the Prion Protein

朊病毒蛋白引起的神经变性的细胞生物学

• 批准号: 7594283
• 负责人: Ramanujan S Hegde
• 金额: $ 57.04万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594283
• 关键词: Anabolism; Animals; Apoptotic; Biochemical; Biogenesis; Biological Models; Bovine Spongiform Encephalopathy; Brain; Cell Death; Cell Line; Cells; Cellular biology; Cessation of life; Complex; Cultured Cells; Defect; Development; Disease; Event; Fractionation; Functional disorder; Future; Generations; Genes; Glycoproteins; Goals; Infectious Agent; Inherited; Lead; Mediating; Membrane; Membrane Proteins; Metabolism; Molecular; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Prion Diseases; Prions; Process; Protein Disulfide Isomerase; Protein translocation; Proteins; Role; Site; Therapeutic Intervention; Trans-Activators; Transgenic Mice; Variant; cohort; cytotoxic; disease-causing mutation; human disease; in vivo; insight; interest; mouse model; neuron loss; neurotoxic; protein metabolism; reconstitution; signal sequence receptor

The prion protein (PrP), a brain glycoprotein involved in various neurodegenerative diseases, has proven to be a particularly instructive example of complex and highly regulated translocation. In addition to its notoriety as the putative "protein-only" infectious agent in prion diseases, the biogenesis of PrP at the ER is unusual in that an initially homogeneous cohort of nascent PrP chains gives rise to four distinct topologic forms: a fully translocated form (termed secPrP), two transmembrane forms that span the membrane in opposite orientations (NtmPrP and CtmPrP), and a cytosolic form (cyPrP). In vivo studies have revealed that even a slight overrepresentation of the CtmPrP topologic form results in the development of neurodegenerative disease in both mouse model systems and naturally occurring human disease. Furthermore, cyPrP can be both aggregation-prone and neurotoxic under some circumstances. To gain insight into how these variants are initially generated, we are dissecting the pathways of PrP biogenesis and degradation. Our results indicate that the decisive event in avoiding the generation of both of the potentially harmful forms of PrP (CtmPrP and cyPrP) is the signal sequence-mediated translocation of the N-terminus of PrP into the ER lumen. By using a constitutive, highly efficient signal sequence, the generation of CtmPrP and cyPrP can be substantially reduced. The consequences of this manipulation in a cultured neuronal cell line are a marked reduction of cytotoxic and aggregation-prone variants of PrP and protection from apoptotic cell death. These results define a pathway for the normal biogenesis of PrP, and demonstrate that the total cellular burden of cytotoxic forms of PrP is controlled primarily during its initial translocation into the ER. Transgenic mice have now been created to determine whether CtmPrP-mediated neurodegeneration and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered step during PrP biogenesis. We are also investigating the pathways by which the various forms of PrP are normally metabolized by the cell to determine whether modulation of these events are involved in the progression of neurodegeneration. It is anticipated that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborate to eventually cause neuronal dysfunction and death. Conversely, manipulation of these events may be able to slow or reverse the neurodegenerative process in these diseases. Parallel biochemical studies employing the solubilization, fractionation and reconstitution of ER membrane proteins have demonstrated that regulatory trans-acting factors are absolutely required for PrP to be synthesized in the proper ratio of its topologic forms. We have now purified two of these factors and identified them as the translocon-associated protein complex (TRAP) and protein disulfide isomerase (PDI). Analysis of PrP translocation intermediates suggests that TRAP and PDI act sequentially to facilitate translocation of PrP's N-terminus into the ER lumen, the decisive event in determining its topology. Ongoing studies are investigating the role of these newly discovered factors in the biogenesis of other substrates and their potential role in the pathogenesis of PrP-associated neurodegeneration.

事实证明，参与各种神经退行性疾病的大脑糖蛋白（PRP）是一种特别有启发性的复杂且高度调节的易位例子。 In addition to its notoriety as the putative "protein-only" infectious agent in prion diseases, the biogenesis of PrP at the ER is unusual in that an initially homogeneous cohort of nascent PrP chains gives rise to four distinct topologic forms: a fully translocated form (termed secPrP), two transmembrane forms that span the membrane in opposite orientations (NtmPrP and CTMPRP）和胞质形式（CYPRP）。体内研究表明，即使CTMPRP拓扑形式的略有代表性略有分量也会导致小鼠模型系统和自然发生的人类疾病的神经退行性疾病的发展。此外，在某些情况下，CYPRP可以易于聚集，且神经毒性。 为了了解这些变体最初如何产生，我们正在解剖PRP生物发生和降解的途径。我们的结果表明，避免产生两种潜在有害形式的PRP（CTMPRP和CYPRP）的决定性事件是信号序列介导的PRP N端迁移到ER管腔中。通过使用本构，高效的信号序列，可以大大降低CTMPRP和CYPRP的产生。这种操纵在培养的神经元细胞系中的后果是明显减少了PRP的细胞毒性和易受聚集的变体，并保护了凋亡细胞死亡。这些结果定义了PRP正常生物发生的途径，并证明了PRP的细胞毒性形式的总细胞负担主要在其初始易位到ER期间受到控制。现在已经创建了转基因小鼠来确定CTMPRP介导的神经变性和CyPRP介导的神经变性是否可以通过调节PRP生物发生过程中的这一新发现的步骤来避免体内。我们还正在研究细胞通常对各种形式的PRP代谢的途径，以确定这些事件的调节是否涉及神经变性的进展。预计生物合成和/或清除某些形式的PRP合作的缺陷的组合最终引起神经元功能障碍和死亡。相反，对这些事件的操纵可能能够减慢或扭转这些疾病中的神经退行性过程。 使用ER膜蛋白的溶解，分馏和重构的平行生化研究表明，PRP绝对需要以其拓扑形式的适当比率合成调节性跨性别因子。现在，我们已经纯化了其中两个因素，并将其确定为与易位相关的蛋白质复合物（TRAP）和蛋白二硫化物异构酶（PDI）。 PRP易位中间体的分析表明，陷阱和PDI依次起作用，以促进PRP的N末端转移到ER腔内，这是确定其拓扑结构的决定性事件。正在进行的研究正在研究这些新发现的因素在其他底物的生物发生中的作用及其在与PRP相关的神经变性的发病机理中的潜在作用。