DYNAMICS AND NUCLEAR EFFECTS OF NON-POLYQ AGGREGATES

非 PolyQ 骨料的动力学和核效应

• 批准号: 7014032
• 负责人: ELIZABETH S SZTUL
• 金额: $ 19.58万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014032
• 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形成是神经退行性疾病细胞病理学的两个标志。在诸如帕金森氏症和阿尔茨海默氏症的疾病中，沉积物是细胞质的，而在亨廷顿氏症和共济失调中，沉积物主要在细胞核内形成。包涵体募集关键的细胞组分，如分子伴侣和蛋白酶体、核基质组分和特异性转录调节因子。本提案的目的是探索与聚集相关的病理学与关键细胞过程（如蛋白质折叠和降解以及基因表达）中所涉及的细胞组分的隔离相关的假设。 为此，我们计划使用一种新的模型系统，该系统基于我们称为GFP 170 * 的人工蛋白质。GFP 170 * 缺乏polyQ片段，但诱导的细胞反应类似于由polyQ聚集体沉积引起的细胞反应。与polyQ蛋白一样，GFP 170 * 形成细胞质和细胞核聚集体，募集细胞质和细胞核组分，改变转录调控并杀死细胞。 我们提出两条调查线索。首先，我们将研究被隔离的细胞成分的物理性质。将使用基于GFP的活细胞实时成像来探索GFP 170 * 和募集到聚集体的细胞组分的迁移率。这些数据将为聚集体的物理稳定性提供信息，并将影响可能的细胞病理学模型。其次，我们将遵循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用于探索通过聚集GFP 170 * 调节转录调节因子。 完成拟议的研究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。