Role of the Lysosome in ER Associated Degradation of PrP

溶酶体在 ER 相关 PrP 降解中的作用

• 批准号: 7153538
• 负责人: JAMES A MASTRIANNI
• 金额: $ 33.54万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-05 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7153538
• 关键词: Address; Autophagocytosis; Bovine Spongiform Encephalopathy; Cells; Cultured Cells; Cytosol; Defect; Degradation Pathway; Development; Disease; Endopeptidase K; Extracellular Space; Fibroblasts; Fractionation; Functional disorder; Generations; Goals; Immunoelectron Microscopy; Knockout Mice; Light; Link; Lysosomes; Membrane; Microscopy; Mitogen-Activated Protein Kinase 3; Modeling; Mus; Nature; Neurodegenerative Disorders; Pathway interactions; Prion Diseases; Prions; Proteasome Inhibition; Protein C; Protein Isoforms; Proteins; Quality Control; Resistance; Role; Route; Signal Transduction; System; Testing; Thinking; Transgenic Mice; Transgenic Organisms; Upper arm; Work; concept; glycosylation; mouse model; multicatalytic endopeptidase complex; novel; protein misfolding; transmission process

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.

描述（由申请人提供）：朊病毒疾病，例如 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。最近的研究表明，错误折叠的 PrP 会经历内质网相关降解 (ERAD)，从而逆向转运到胞质溶胶中，被蛋白酶体降解。推测蛋白酶体功能障碍会诱发朊病毒病。蛋白酶体抑制后，PrP 在细胞质 (cyPrP) 中积累，或直接表达到培养细胞的细胞质中，产生不溶性、蛋白酶 K (PK) 抗性 PrP，但在表达 cyPrP (Tg1D4) 的转基因小鼠中则不会，也不知道这些小鼠是否产生传染性朊病毒。我们的初步工作提供的证据表明，除了蛋白酶体之外，ER 内错误折叠的 PrP 还被传递到溶酶体。然而，我们发现该途径并没有经历有效的降解，而是增强了 PK 抗性 PrP（PrP-Sc 的标志物）的形成。此外，我们还发现 PrP-Sc 从分泌性溶酶体中释放，为朊病毒传播提供了一种新机制。这项工作的主要目标是更好地定义该途径及其在朊病毒生成中的作用。具体来说，我们将； 1.) 检验这样的假设：PrP 早期递送至溶酶体是 ER 质量控制的一个特征，2.) 研究遵循该途径的 PrP 的功能后果，以及 3.) 定义递送至溶酶体的特定信号和性质。使用共聚焦免疫组织荧光显微镜、免疫电子显微镜、溶酶体分级、cyPrP表达（Tg1D4）小鼠、[GFP标记轻链MAP激酶3表达转基因（TgGFP-LC3）小鼠和敲除LC3成纤维细胞]，以及各种其他系统，我们将评估该途径的重要性，进行比较 与蛋白酶体途径一起参与朊病毒的从头生成和传播。这些研究可能会提供关于 ERAD（错误折叠蛋白的细胞处理）的新概念，并确定治疗这些神秘且高度争议的疾病的新途径。