Characterization of nuclear proteome in normal and diseased liver

正常和患病肝脏中核蛋白质组的表征

• 批准号: 7134091
• 负责人: SVETLANA LUTSENKO
• 金额: $ 19.19万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7134091
• 关键词: affinity labeling; atomic absorption spectrometry; binding proteins; chromatography; copper; disease /disorder model; gene expression; genetically modified animals; hepatolenticular degeneration; inborn metal metabolism disorder; laboratory mouse; liver metabolism; mass spectrometry; molecular pathology; nuclear proteins; pathologic process; proteomics; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): Wilson disease (WD) is a severe metabolic disorder associated with copper accumulation in several tissues and marked hepatic, neurological, and/or psychiatric abnormalities. The disease is caused by mutations in the gene ATP7B, which encodes the copper-transporting P-type ATPase, or Wilson disease protein (WNDP). Recent studies have begun to uncover the biochemical and cell biological properties of WNDP; however, the detailed information on molecular mechanisms of copper-induced pathology is still lacking. Our laboratory has recently demonstrated that genetically engineered Atp7b-/- knock-out mice recapitulate many symptoms of WD and represent a valuable animal model for characterization of this human disorder. We have also established that at the early stages of the disease copper accumulates in the nuclei of Atp7b-/- hepatocytes, triggering changes in gene expression and overall nuclear morphology. The molecular mechanism of these important early events is unknown and will be investigated in this study. Currently, no information is available on the role of copper in mammalian nuclei, and proteins that bind nuclear copper or proteins regulated by intra-nuclear copper remain unidentified. This information is central for understanding of the effects of copper on liver nuclear proteome, and it will be obtained in this study using modern biochemical approaches. Three aims are proposed. First, to test the hypothesis that copper accumulation induces specific changes in nuclear proteome using quantitative gel-electrophoresis and mass-spectrometry. Second, to identify nuclear proteins that show change in abundance and posttranslational modification in response to copper accumulation. Third, to identify proteins involved in binding of copper in normal and diseased nuclei. The studies will yield the first quantitative data on the role of copper in mammalian nuclei and yield key information on biochemical steps involved in the early stages of WD pathology. The proteomic information and methodology generated during this study will be applicable for analysis of other hepatic disorders in humans.

描述(申请人提供)：威尔逊病(WD)是一种严重的代谢紊乱，与铜在几个组织中的积聚和明显的肝脏、神经和/或精神异常有关。这种疾病是由编码铜运输P型ATPase或威尔逊病蛋白(WNDP)的基因ATP7B突变引起的。最近的研究已经开始揭示WNDP的生化和细胞生物学特性，但关于铜诱导病理的分子机制仍缺乏详细的信息。我们的实验室最近证明了基因工程ATP7B-/-基因敲除小鼠概括了WD的许多症状，并代表了一种描述这种人类疾病的有价值的动物模型。我们还证实，在疾病的早期阶段，铜在ATP7B-/-肝细胞的细胞核中积累，引发基因表达和整体核形态的变化。这些重要早期事件的分子机制尚不清楚，将在本研究中进行研究。目前，还没有关于铜在哺乳动物细胞核中的作用的信息，结合核内铜的蛋白质或核内铜调控的蛋白质仍未确定。这一信息对于了解铜对肝细胞核蛋白质组的影响至关重要，本研究将利用现代生物化学方法获得这一信息。提出了三个目标。首先，用定量凝胶电泳法和质谱仪验证铜积累引起核蛋白质组特异性改变的假说。第二，确定核蛋白的丰度变化和翻译后修饰，以响应铜的积累。第三，确定正常和病变细胞核中参与铜结合的蛋白质。这些研究将首次提供有关铜在哺乳动物细胞核中的作用的定量数据，并提供有关WD病理早期阶段涉及的生化步骤的关键信息。在这项研究中产生的蛋白质组学信息和方法学将适用于人类其他肝脏疾病的分析。