Aggregation and Clearance of Mutant Huntingtin(RMI)

突变亨廷顿蛋白（RMI）的聚集和清除

• 批准号: 7057708
• 负责人: JAMES ROTHMAN
• 金额: $ 0.48万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-16 至 2006-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057708
• 关键词: Huntington' s disease; cell line; chemical aggregate; confocal scanning microscopy; gene mutation; green fluorescent proteins; high throughput technology; homopeptide; nerve /myelin protein; peptide library; protein degradation; protein protein interaction

DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are a common link across a wide array of neurodegenerative disorders. Recently, an exciting theme has emerged: if mutant protein accumulation is eliminated, symptomatic progression not only halts but also leads to recovery from disease. The first indication that neurodegenerative diseases are reversible comes from an inducible mouse model of the polyglutamine disorder Huntington's disease (HD). In the presence of expanded polyglutamine huntingtin, mice recapitulated HD-like symptoms. When mutant gene expression was abolished, not only did the aggregates disappear, the symptoms regressed. These findings signal that neurodegenerative diseases need no longer be considered a death sentence. Unfortunately, however, the pathway underlying clearance of these mutant proteins are not yet clear. We believe that screening a well-designed cell-based assay with a chemical compound library will allow us to not only further clarify which degradative pathway is important, but may also reveal new means by which these pathways can be activated. We have therefore designed a functional cell-based assay that monitors not only aggregation of mutant huntingtin protein, but also its clearance. To do so, we created a stable cell line that conditionally expresses the N-terminus of huntingtin protein with polyQ proteins of different polyQ lengths fused to variants of GFP. These cell lines permit high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells. We hypothesize that by eliminating the accumulated protein we will bring about recovery of the neurodegenerative process, as shown in several animal models of the disease.

描述（由申请人提供）：突变蛋白的积累和聚集是一系列神经退行性疾病的共同联系。最近，一个令人兴奋的主题出现了：如果突变蛋白积累被消除，症状进展不仅停止，而且导致从疾病中恢复。神经退行性疾病是可逆的第一个迹象来自多谷氨酰胺障碍亨廷顿病（HD）的诱导小鼠模型。当存在扩增的聚谷氨酰胺亨廷顿蛋白时，小鼠重现了hd样症状。当消除突变基因表达时，不仅聚集物消失，症状也有所缓解。这些发现表明，不再需要将神经退行性疾病视为死刑判决。然而，不幸的是，这些突变蛋白的清除途径尚不清楚。我们相信，通过化学化合物文库筛选设计良好的基于细胞的实验，不仅可以使我们进一步阐明哪些降解途径是重要的，而且还可以揭示激活这些途径的新方法。因此，我们设计了一种功能性的基于细胞的检测方法，不仅可以监测突变亨廷顿蛋白的聚集，还可以监测其清除。为此，我们创建了一个稳定的细胞系，该细胞系有条件地表达huntingtin蛋白的n端，并将不同长度的polyQ蛋白融合到GFP变体中。这些细胞系允许高通量共聚焦显微镜检查活细胞中表达的突变蛋白的状态。我们假设，通过消除积累的蛋白质，我们将带来神经退行性过程的恢复，正如几种动物模型所显示的那样。