Mapping Molecular Pathways that Control Prion Metabolism

项目总结 哺乳动物蛋白疾病，如克雅氏病(CJD)、慢性消耗性疾病 (CWD)、牛海绵状脑病(BSE)和瘙痒病是一组传染性疾病 宿主编码的Prion的自催化转化引起的神经退行性疾病 蛋白质，PrPC，形成一组错误折叠的、有感染性的构象，统称为PrPSc。 来自生化、遗传学和细胞生物学研究的多条证据表明 PrPC和PrPSc在生物合成、运输和生物降解方面采用特定的途径 细胞。然而，由于缺乏可处理的模型，这些途径的充分阐明受到了阻碍。 用于基因筛选的生物体。为了克服这一障碍，我们最近开发了一些方法来 荧光激活细胞分选法检测脑源性CAD5细胞中PrPC和PrPSc (FACS)。我们将使用这些敏感的排序方法来执行CRISPR(定期集群 CAD5细胞中的间隔短回文重复序列)/Cas9全基因组文库筛选 感染了不同的病毒株。这些研究将是第一个完全绘制分子图谱的研究 控制PrPC和PrPSc生物合成、运输和降解的途径 分子，从而极大地提高了我们对Pron细胞生物学的基础知识。我们的 无偏筛选还将揭示Pron形成和清除的速率限制步骤， 从而确定药物治疗的最敏感目标。

Project summary Mammalian prion diseases, such as Creutzfeldt-Jakob disease (CJD), Chronic Wasting Disease (CWD), bovine spongiform encephalopathy (BSE), and scrapie, are a group of infectious neurodegenerative disorders caused by the autocatalytic conversion of the host-encoded prion protein, PrPC, into a group of misfolded, infectious conformers collectively termed PrPSc. Multiple lines of evidence from biochemical, genetic, and cell biological studies suggest that PrPC and PrPSc employ specific pathways for biosynthesis, trafficking, and degradation in living cells. However, full elucidation of these pathways has been hindered the lack of tractable model organisms for genetic screening. To overcome this obstacle, we recently developed methods to detect both PrPC and PrPSc in brain-derived CAD5 cells using fluorescence-activated cell sorting (FACS). We will use these sensitive sorting methods to perform CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 whole genome library screens in CAD5 cells infected with different prion strains. These studies will be the first to fully map the molecular pathways that control the biosynthesis, trafficking, and degradation of both PrPC and PrPSc molecules, and thereby greatly advance our fundamental knowledge of prion cell biology. Our unbiased screens will also reveal the rate limiting steps of prion formation and clearance, thereby identifying the most susceptible targets for drug therapy.