The Cell Biology of Neurodegeneration Caused by the Prion Protein

朊病毒蛋白（PrP）是一种参与各种神经退行性疾病的脑糖蛋白，已被证明是复杂和高度调节的易位的特别有指导意义的例子。除了其作为朊病毒疾病中假定的“仅蛋白质”感染剂的恶名之外，PrP在ER的生物发生是不寻常的，因为最初同质的新生PrP链队列产生四种不同的拓扑形式：完全易位形式（称为secPrP），以相反方向跨越膜的两种跨膜形式（NtmPrP和CtmPrP）和胞质形式（cyPrP）。体内研究已经揭示，即使CtmPrP拓扑形式的轻微过度表达也会导致小鼠模型系统和自然发生的人类疾病中神经退行性疾病的发展。此外，在某些情况下，cyPrP可能既有聚集倾向又有神经毒性。 为了深入了解这些变体最初是如何产生的，我们正在解剖PrP生物合成和降解的途径。我们的研究结果表明，在避免生成的两种潜在的有害形式的PrP（CtmPrP和cyPrP）的决定性事件是信号序列介导的易位的N-末端的PrP到ER腔。通过使用组成型高效信号序列，可以显著减少CtmPrP和cyPrP的产生。在培养的神经元细胞系中这种操作的后果是PrP的细胞毒性和聚集倾向的变体的显著减少和免于凋亡细胞死亡的保护。这些结果定义了一个正常的生物合成的PrP的途径，并表明，细胞毒性形式的PrP的总细胞负荷主要是控制在其初始易位到ER。现在已经建立了转基因小鼠，以确定CtmPrP介导的神经变性和cyPrP介导的神经变性是否可以在体内通过调节PrP生物发生过程中新发现的这一步骤来避免。我们也在研究各种形式的PrP被细胞正常代谢的途径，以确定这些事件的调节是否参与神经变性的进展。预期某些形式的PrP的生物合成和/或清除中的缺陷的组合协作最终导致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缓或逆转这些疾病中的神经退行性过程。 平行生化研究采用的增溶，分馏和重建的ER膜蛋白已经证明，监管反式作用因子是绝对需要的PrP的拓扑形式的适当比例合成。我们现在已经纯化了其中两个因子，并将其鉴定为translocon相关蛋白复合物（TRAP）和蛋白质二硫键异构酶（PDI）。PrP易位中间体的分析表明，TRAP和PDI依次作用，以促进PrP的N-末端易位到ER腔，决定性的事件，在确定其拓扑结构。正在进行的研究正在调查这些新发现的因子在其他底物的生物发生中的作用及其在PrP相关神经变性发病机制中的潜在作用。

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.