Role of the Lysosome in ER Associated Degradation of PrP

Prion病，如疯牛病(即疯牛病)，是与 正常细胞蛋白(PrP-C)的错误折叠异构体(PrP-SC)。最近的研究表明 错误折叠的PrP经历内质网相关降解(ERAD)，从而被反向移位到细胞质 以供蛋白酶体降解。蛋白酶体功能障碍被认为是导致Pron病的原因。 蛋白酶体抑制或定向表达后PrP在胞浆中的积累(CyPrP) 培养细胞的胞浆产生不溶性的、抗蛋白酶K(PK)的PrP，但在转基因小鼠中不产生 表达cyPrP(Tg1D4)，也不知道这些小鼠是否产生传染性普恩病毒。我们的前期工作 提供证据表明内质网中错误折叠的PrP除了被传递到溶酶体外，还被传递到溶酶体 蛋白酶体。然而，我们发现，这条途径不仅没有经历有效的降解，反而增强了 PK抗性PrP的形成是PrP-SC的标志之一。此外，我们还证明了PrP-SC是从 分泌型溶酶体，为病毒传播提供了一种新的机制。这项工作的主要目标是更好地 定义这一途径及其在Prion产生中的作用。具体来说，我们会的；1.)测试假设，早期 将PrP输送到溶酶体是ER质量控制的一个特征，2)研究的功能后果 遵循这一途径的PRP，以及3.)确定特定的信号和传递到溶酶体的性质。 使用共聚焦免疫荧光显微镜，免疫电子显微镜，溶酶体分离， CyPrP表达(Tg1D4)小鼠，[GFP标记的轻链MAPK3表达转基因(TgGFP-LC3)] 小鼠，和敲除LC3-成纤维细胞]，除了各种其他系统，我们将评估其重要性 与蛋白酶体途径相比，这一途径在普恩的从头产生和传播中起着重要的作用。 这些研究可能会提供关于ERAD的新概念，即细胞处理错误折叠的蛋白质，以及 确定治疗这些神秘且极具争议性的疾病的新途径。

Prion diseases, such as BSE (i.e. mad cow disease), are transmissible neurodegnerative diseases related to the misfolded isoform (PrP-Sc) of the normal cellular prion protein (PrP-C). Recent work suggests that misfolded PrP undergoes ER associated degradation (ERAD), whereby it is retrotranslocated to the cytosol for degradation by the proteasome. Proteasome dysfunction is hypothesized to induce prion disease. Accumulation of PrP in the cytosol (cyPrP) following proteasome inhibition or directed expression into the cytosol of cultured cells produce insoluble, Proteinase-K (PK) resistant PrP, but not in transgenic mice expressing cyPrP (Tg1D4), nor is it known if these mice produce infectious prions. Our preliminary work provides evidence that misfolded PrP within the ER is delivered to the lysosome, in addition to the proteasome. However, rather than undergoing effective degradation, we find that this pathway enhances the formation of PK-resistant PrP, a marker of PrP-Sc. In addition, we show that PrP-Sc is released from secretory lysosomes, providing a new mechanism for prion spread. The primary goal of this work is to better define this pathway and its role in prion generation. Specifically, we will; 1.) Test the hypothesis that early delivery of PrP to the lysosome is a feature of ER quality control, 2.) Study the functional consequences of PrP that follows this pathway, and 3.) Define the specific signals and nature of delivery to the lysosome. Using confocal immunohistofluorescence microscopy, immunoelectron microscopy, lysosome fractionation, cyPrP expressing (Tg1D4) mice, [GFP-tagged light chain MAP kinase 3 expressing transgenic (TgGFP-LC3) mice, and knockout LC3-fibroblasts], in addition to a variety of other systems, we will assess the importance of this pathway,in comparison with the proteasome pathway, in the de novo generation and spread of prions. These studies will likely provide new concepts about ERAD, the cellular handling of misfolded proteins, and define new avenues for treatment of these enigmatic and highly controversial diseases.