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: 7560376
• 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/7560376
• 关键词: Address; Autophagocytosis; Bovine Spongiform Encephalopathy; Cell Culture Techniques; 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; glycosylation; mouse model; multicatalytic endopeptidase complex; novel; protein misfolding; transmission process

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.

朊病毒疾病，如BSE（即疯牛病），是与下列疾病有关的可传播的神经变性疾病： 正常细胞朊病毒蛋白（PrP-C）的错误折叠同种型（PrP-Sc）。最近的研究表明 错误折叠的PrP经历ER相关降解（ERAD），由此它被逆向转运到胞质溶胶 被蛋白酶体降解。蛋白酶体功能障碍被假设会诱发朊病毒病。 蛋白酶体抑制或定向表达到细胞质中后PrP在细胞质中的积累（cyPrP） 培养细胞的胞质溶胶产生不溶性的、抗蛋白酶K（PK）的PrP，但在转基因小鼠中不产生 表达cyPrP（Tg 1D 4）的小鼠，也不知道这些小鼠是否产生感染性朊病毒。我们的前期工作 提供了证据表明，错误折叠的PrP在ER内被传递到溶酶体，除了 蛋白酶体然而，我们发现，这种途径并没有经历有效的降解，而是增强了 此外，我们还发现，PrP-Sc的一个标志物，即PK抗性PrP的形成， 分泌溶酶体，为朊病毒传播提供了新的机制。这项工作的主要目标是更好地 定义这个途径及其在朊病毒产生中的作用。具体而言，我们将：1。检验早期 PrP向溶酶体的递送是ER质量控制的特征，2.）研究的功能后果 PrP遵循这一途径，和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，我们将评估的重要性 与蛋白酶体途径相比，这一途径在朊病毒的从头产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， 为治疗这些神秘且极具争议的疾病开辟了新的途径。