Mapping Molecular Pathways that Control Prion Metabolism

绘制控制朊病毒代谢的分子途径

基本信息

批准号：
10670437
负责人：
Surachai Supattapone
金额：
$ 67.84万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10670437
关键词：
Anabolism Atlases Biochemical Biochemical Pathway Biological Bovine Spongiform Encephalopathy Brain Brain Diseases Brain region Cell Line Cells Cellular biology Chronic Wasting Disease Clustered Regularly Interspaced Short Palindromic Repeats Creutzfeldt-Jakob Syndrome Fluorescence-Activated Cell Sorting Genes Genetic Genetic Screening Genomic Library Genomic approach Genomics Human Incubated Individual Induced pluripotent stem cell derived neurons Infection Knock-out Knowledge Libraries Maps Metabolic Metabolic Pathway Metabolism Methods Modeling Molecular Molecular Conformation Mus Neurodegenerative Disorders Neuronal Differentiation Neurons Neurotropism Pathway interactions Pattern PrP PrPSc Proteins Predisposition Prion Diseases Prions Scrapie Sorting Surface Techniques Testing Time Undifferentiated Work cofactor conformer experimental study metabolic profile misfolded protein model organism new therapeutic target next generation sequencing novel novel strategies targeted treatment trafficking whole genome

项目摘要

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.

项目概要哺乳动物朊病毒疾病，例如克雅氏病 (CJD)、慢性消耗性疾病 (CWD)、牛海绵状脑病 (BSE) 和痒病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退行性疾病蛋白质 PrPC 转化为一组错误折叠的传染性构象异构体，统称为 PrPSc。来自生化、遗传和细胞生物学研究的多种证据表明 PrPC 和 PrPSc 在生物体中采用特定的生物合成、运输和降解途径细胞。然而，由于缺乏易于处理的模型，对这些途径的全面阐明受到了阻碍用于基因筛选的生物体。为了克服这个障碍，我们最近开发了方法使用荧光激活细胞分选检测脑源性 CAD5 细胞中的 PrPC 和 PrPSc （流式细胞仪）。我们将使用这些敏感的分选方法来进行 CRISPR（定期聚类）间隔短回文重复序列）/CAD5 细胞中的 Cas9 全基因组文库筛选感染不同的朊病毒株。这些研究将是第一个全面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朊病毒细胞生物学的基础知识。我们的无偏见的筛选还将揭示朊病毒形成和清除的限速步骤，从而确定药物治疗最敏感的靶点。