Mapping molecular pathways that control prion metabolism

项目摘要 哺乳动物朊病毒疾病，如克雅氏病（CJD）、慢性消耗性疾病 (CWD)牛海绵状脑病（BSE）和羊瘙痒症是一组传染性疾病， 由宿主编码的朊病毒的自催化转化引起的神经变性疾病 蛋白质PrPC转化为一组错误折叠的感染性构象异构体，统称为PrPSc。 来自生物化学、遗传学和细胞生物学研究的多条证据表明， PrPC和PrPSc采用生物合成、运输和降解的特定途径， 细胞然而，由于缺乏易处理的模型，这些途径的充分阐明受到阻碍 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， 使用荧光激活细胞分选检测脑源性CAD 5细胞中的PrPC和PrPSc （FACS）。我们将使用这些敏感的排序方法来执行CRISPR（定期聚类 间隔短回文重复序列）/Cas9全基因组文库筛选 感染了不同的朊病毒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，从而极大地推进了我们对朊病毒细胞生物学的基础知识。我们 无偏筛选也将揭示朊病毒形成和清除的限速步骤， 从而鉴定药物治疗的最敏感靶点。

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.