Role of the Lysosome in ER Associated Degradation of PrP

描述（由申请人提供）：朊病毒疾病，如BSE（即疯牛病），是与正常细胞朊病毒蛋白（PrP-C）的错误折叠亚型（PrP-Sc）相关的传染性神经退行性疾病。最近的工作表明，错误折叠的PrP经历ER相关降解（ERAD），由此它被retrotranslocated到胞质溶胶的蛋白酶体降解。蛋白酶体功能障碍被假设为诱发朊病毒病。在蛋白酶体抑制或定向表达到培养细胞的胞质溶胶中后，胞质溶胶中PrP（cyPrP）的积累产生不溶性的蛋白酶K（PK）抗性PrP，但在表达cyPrP（Tg 1D 4）的转基因小鼠中则不然，也不知道这些小鼠是否产生感染性朊病毒。我们的初步工作提供的证据表明，错误折叠的PrP在ER被交付给溶酶体，除了蛋白酶体。然而，而不是经历有效的降解，我们发现，这一途径增强了形成的PK-抗性PrP，PrP-Sc的标记。此外，我们表明，PrP-Sc是从分泌溶酶体释放，朊病毒传播提供了一个新的机制。这项工作的主要目标是更好地确定这一途径及其在朊病毒产生中的作用。具体来说，我们会; 1.）检验PrP向溶酶体的早期递送是ER质量控制的特征的假设，2。研究遵循该途径的PrP的功能后果，以及3.）定义特定的信号和传递到溶酶体的性质。使用共聚焦荧光显微镜，免疫电子显微镜，溶酶体分离，cyPrP表达（Tg 1D 4）小鼠，[GFP标记的轻链MAP激酶3表达转基因（TgGFP-LC 3）小鼠，和敲除LC 3-成纤维细胞]，除了各种其他系统，我们将评估这一途径的重要性，与蛋白酶体途径相比，在从头产生和传播朊病毒。这些研究可能会提供有关ERAD的新概念，错误折叠蛋白质的细胞处理，并为治疗这些神秘和高度争议的疾病定义新的途径。

DESCRIPTION (provided by applicant): Prion diseases, such as BSE (i.e. mad cow disease), are transmissible neurodegenerative 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.