DYNAMICS AND NUCLEAR EFFECTS OF NON-POLYQ AGGREGATES

非 PolyQ 骨料的动力学和核效应

• 批准号: 6903757
• 负责人: ELIZABETH S SZTUL
• 金额: $ 16.71万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6903757
• 关键词: cell line; cellular pathology; chemical aggregate; fluorescence recovery after photobleaching; gene expression; genetic regulation; green fluorescent proteins; heat shock proteins; homopeptide; immunofluorescence technique; molecular chaperones; molecular dynamics; neurons; oligonucleotides; p53 gene /protein; proteasome; protein protein interaction; synthetic protein; transcription factor

Protein aggregation and the formation of inclusion bodies are two hallmarks of the cytopathology in neurodegenerative diseases. In diseases such as Parkinson's and Alzheimer's, the deposits are cytoplasmic, while in Huntington's disease and the ataxias, the deposits form predominantly within the nuclei. The inclusions recruit key cellular components, such as chaperones and proteasomes, nuclear matrix components, and specific transcriptional regulators. The aim of this proposal is to explore the hypothesis that the pathology associated with aggregation is related to the sequestration of cellular components involved in critical cellular processes such as protein folding and degradation, and gene expression. To this end, we plan to use a novel model system, based on an artificial protein we call GFP170*. GFP170* lacks polyQ stretches, yet induces cellular responses analogous to those caused by the deposition of polyQ aggregates. Like polyQ proteins, GFP170* forms cytoplasmic and nuclear aggregates, recruits cytoplasmic and nuclear components, alters transcriptional regulation, and kills cells. We propose twin lines of investigation. First, we will examine the physical nature of the sequestered cellular components. GFP-based real-time imaging of live cells will be used to explore the mobility of GFP170* and of cellular components recruited to the aggregates. Such data will inform on the physical stability of the aggregates, and will influence possible models of cytopathology. Second, we will follow the functional consequences of nuclear aggregation on gene expression. In vivo read-out systems and genomic approaches will be used to explore the modulation of transcriptional regulators by aggregating GFP170*. Completion of the proposed studies will establish a firm basis for defining the role of nuclear aggregates in cytopathology.

蛋白质聚集和包涵体的形成是神经退行性疾病细胞病理学的两个特征。在帕金森氏症和阿尔茨海默氏症等疾病中，沉积物是细胞质的，而在亨廷顿病和共济失调中，沉积物主要形成在细胞核内。包涵体招募关键的细胞成分，如伴侣和蛋白酶体，核基质成分，以及特定的转录调节因子。这一建议的目的是探索一种假设，即与聚集相关的病理与参与关键细胞过程(如蛋白质折叠和降解以及基因表达)的细胞成分的隔离有关。 为此，我们计划使用一种新的模型系统，基于一种我们称为GFP 170*的人造蛋白质。GFP 170*缺乏多Q伸展，但诱导的细胞反应类似于多Q聚集体沉积引起的反应。像多聚Q蛋白一样，GFP 170*形成细胞质和核聚集体，招募细胞质和核成分，改变转录调控，并杀死细胞。 我们提出了两条调查路线。首先，我们将检查隔离的细胞组件的物理性质。基于GFP的活细胞实时成像将被用来探索GFP 170*和被招募到聚集体中的细胞成分的流动性。这些数据将提供关于聚集体的物理稳定性的信息，并将影响可能的细胞病理学模型。其次，我们将跟踪核聚集对基因表达的功能后果。体内读出系统和基因组方法将被用来探索通过聚集GFP 170*来调节转录调控。 拟议研究的完成将为确定核聚集体在细胞病理学中的作用奠定坚实的基础。