Characterization of nuclear proteome in normal and diseased liver

描述（由申请人提供）：威尔逊病（WD）是一种严重的代谢性疾病，与铜在多个组织中的积累和显著的肝脏、神经和/或精神异常有关。这种疾病是由编码铜转运p型atp酶或威尔逊病蛋白（WNDP）的基因ATP7B突变引起的。近年来的研究已经开始揭示WNDP的生化和细胞生物学特性；然而，关于铜诱导病理的分子机制的详细信息仍然缺乏。我们的实验室最近证明，基因工程的Atp7b-/-敲除小鼠再现了WD的许多症状，并代表了表征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-/-肝细胞的核中积累，引发基因表达和整体核形态的变化。这些重要的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，并且结合核铜的蛋白质或由核内铜调节的蛋白质仍未确定。这些信息对于理解铜对肝核蛋白质组的影响至关重要，并且将在本研究中使用现代生化方法获得这些信息。提出了三个目标。首先，利用定量凝胶电泳和质谱技术验证铜积累诱导核蛋白质组特异性变化的假设。其次，鉴定在铜积累过程中表现出丰度变化和翻译后修饰的核蛋白。第三，鉴定正常和病变细胞核中参与铜结合的蛋白。这些研究将产生铜在哺乳动物细胞核中的作用的第一个定量数据，并产生涉及WD病理早期阶段的生化步骤的关键信息。本研究中产生的蛋白质组学信息和方法将适用于人类其他肝脏疾病的分析。

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.