The Cell Biology of Neurodegeneration Caused by the Prion Protein

Prion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控的易位的一个特别有启发性的例子。除了PrP在PrP疾病中被认为是“纯蛋白质”感染性病原体之外，PrP在内质网的生物发生也是不寻常的，因为最初同源的新生PrP链产生了四种不同的拓扑形式：完全移位的形式(称为secPrP)，两种以相反方向跨越膜的跨膜形式(NtmPrP和CtmPrP)，以及胞浆形式(CyPrP)。体内研究表明，即使CtmPrP拓扑形式略有过度代表，也会导致在小鼠模型系统和自然发生的人类疾病中发生神经退行性疾病。此外，在某些情况下，cyPrP既容易聚集，又具有神经毒性。 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的生物发生和降解途径。我们的结果表明，避免两种潜在有害形式PrP(CtmPrP和cyPrP)的产生的决定性事件是信号序列介导的PrP的N端移位到内质网腔内。通过使用构成的、高效的信号序列，可以大大减少CtmPrP和CyPrP的产生。在培养的神经细胞系中，这种操作的结果是显着减少了PrP的细胞毒性和易于聚集的变体，并保护了细胞免受凋亡细胞的死亡。这些结果为PrP的正常生物发生定义了一条途径，并表明细胞毒性形式的PrP的总细胞负荷主要在其最初移位到内质网期间受到控制。现在已经建立了转基因小鼠来确定在体内是否可以通过调节PrP生物发生过程中新发现的这一步骤来避免CtmPrP介导的神经变性和CyPrP介导的神经变性。我们还在研究各种形式的PrP通常由细胞代谢的途径，以确定这些事件的调制是否参与了神经退行性变的进展。据预测，某些形式PrP的生物合成和/或清除方面的缺陷共同作用，最终导致神经元功能障碍和死亡。相反，操纵这些事件可能能够减缓或逆转这些疾病的神经退化过程。 利用内质网膜蛋白的增溶、分离和重组的平行生化研究表明，以适当的拓扑形式比例合成PrP是绝对必要的调节反式作用因子。我们现在已经纯化了其中的两个因子，并将它们鉴定为转位相关蛋白复合体(TRAP)和蛋白质二硫键异构酶(PDI)。对PrP转位中间产物的分析表明，TRAP和PDI依次作用于促进PrP的N端移位到内质网管腔，这是决定其拓扑结构的决定性事件。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，以及它们在PrP相关神经变性的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.