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）的错误折叠异构体（PrP-Sc）有关的传染性神经退行性疾病。最近的研究表明，错误折叠的PrP经历内质网相关降解（ERAD），即它被蛋白酶体反转录到细胞质中降解。蛋白酶体功能障碍被推测可诱发朊病毒疾病。通过蛋白酶体抑制或直接表达到培养细胞的细胞质中，PrP在细胞质中积累会产生不溶性的、抗蛋白酶k （PK）的PrP，但在表达cypp的转基因小鼠（Tg1D4）中不会产生PrP，也不知道这些小鼠是否会产生感染性朊病毒。我们的初步工作提供了证据，证明内质网内错误折叠的PrP除了被蛋白酶体传递给溶酶体外，还被传递给溶酶体。然而，我们发现这一途径并没有进行有效的降解，而是促进了pk抗性PrP的形成，这是PrP- sc的一个标志。此外，我们发现PrP-Sc从分泌溶酶体中释放，为朊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朊病毒产生中的作用。具体来说，我们将；1.） 检验将PrP提前送到溶酶体是ER质量控制的一个特征的假设，2)。2 .研究遵循这一途径的PrP的功能后果。定义特定的信号和传递到溶酶体的性质。使用共聚焦免疫组织荧光显微镜、免疫电镜、溶酶体分离、cyrp表达（Tg1D4）小鼠、gfp标记的轻链MAP激酶3表达转基因（TgGFP-LC3）小鼠和敲除lc3成纤维细胞，除了各种其他系统外，我们将评估该途径与蛋白酶体途径相比，在朊病毒的新生和传播中的重要性。这些研究可能会提供关于ERAD的新概念，错误折叠蛋白质的细胞处理，并为治疗这些神秘且极具争议的疾病确定新的途径。

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.