COPPER IN PEDIATRIC LIVER DISEASES

铜是一种必需的微量元素，在细胞呼吸、抗氧化防御和铁稳态的生物化学中起关键作用。肝脏是人体铜稳态的中心器官，这些研究的长期目标是确定铜在儿科肝病中的作用。最近的研究表明，铜在肝细胞内的特定蛋白质的传递是由不同的细胞内载体蛋白称为伴侣介导的。本提案中的研究旨在阐明铜伴侣HAH 1在肝细胞中铜运输和代谢中的作用。结构/功能研究将利用定点诱变和在酿酒酵母中表达HAH 1来完成。HAH 1在铜传递到分泌途径中的作用将通过与Wilson病ATP酶的蛋白质-蛋白质相互作用研究来检查，并且这些蛋白质在完整的单细胞中的相互作用将使用绿色荧光融合蛋白和荧光共振能量转移来检查。HAH 1在哺乳动物细胞中的精确功能将通过分析具有鼠HAH 1基因的靶向种系缺失的小鼠肝细胞中的铜转运和代谢来确定。最后，HAH 1在儿科肝病中的作用将通过HAH 1序列分析来研究，这些基因组DNA来自患有以铜蓄积和毒性为特征的肝病的儿童，包括被诊断患有威尔逊病但威尔逊ATP酶中没有可检测到的突变的个体。总之，这些研究的结果将允许进一步深入了解铜在肝细胞中运输的分子和细胞机制，并可能允许新的治疗方法来预防或改善由金属代谢紊乱引起的儿童肝脏疾病。

Copper is an essential trace element with a critical role in the biochemistry of cellular respiration, antioxidant defense and iron homeostasis. The liver is the central organ of copper homeostasis in human and the long-term objective of these studies is to define the role of copper in pediatric liver disease. Recent studies have revealed that the delivery of copper to specific proteins within hepatocytes is mediated by distinct intracellular carrier proteins termed chaperones. The studies in this proposal are intended to elucidate the role of the copper chaperone HAH1 in copper trafficking and metabolism in hepatocytes. Structure/function studies will be accomplished utilizing site-directed mutagenesis and expression of HAH1 in Saccharomyces cerevisiae. The role of HAH1 in copper delivery to the secretory pathway will be examined by protein-protein interaction studies with the Wilson disease ATPase and the interaction of these proteins in intact single cell swill be examined using green fluorescent fusion proteins and fluorescence resonance energy transfer. The precise function of HAH1 in mammalian cells will be determined by analysis of copper trafficking and metabolism in the hepatocytes of mice with a targeted germline deletion of the murine HAH1 gene. Finally, the role of HAH1 in pediatric liver disease will be studied by HAH1 sequence analysis in genomic DNA from children with liver disease characterized by copper accumulation and toxicity including individuals diagnosed with Wilson disease but without detectable mutations in the Wilson ATPase. Taken together the results of these studies will permit further insight into the molecular and cellular mechanisms of copper trafficking in hepatocytes and may allow for novel therapeutic approaches to prevent or ameliorate childhood liver disease resulting from perturbations in metal metabolism.