COPPER IN PEDIATRIC LIVER DISEASES

铜是一种必需的微量元素，在细胞呼吸、抗氧化防御和铁的动态平衡等生化过程中起着关键作用。肝脏是人体铜稳态的中枢器官，这些研究的长期目标是明确铜在儿童肝病中的作用。最近的研究表明，铜向肝细胞内特定蛋白的转运是由不同的细胞内载体蛋白介导的，称为伴侣蛋白。本研究旨在阐明铜伴侣蛋白HAH1在肝细胞铜转运和代谢中的作用。HAH1的结构/功能研究将利用定点突变和HAH1在酿酒酵母中的表达完成。HAH1在铜转运到分泌途径中的作用将通过与威尔逊病ATPase的蛋白质-蛋白质相互作用研究来检验，这些蛋白质在完整的单细胞中的相互作用将通过绿色荧光融合蛋白和荧光共振能量转移来检测。HAH1基因在哺乳动物细胞中的确切功能将通过分析小鼠肝细胞中铜的运输和代谢来确定，这些小鼠的目的是将小鼠的HAH1基因进行种系缺失。最后，将通过对以铜蓄积和毒性为特征的肝病儿童的基因组DNA中的HAH1序列分析来研究HAH1在儿童肝病中的作用，这些儿童包括被诊断为威尔逊病但没有检测到威尔逊ATPase突变的人。综上所述，这些研究结果将有助于进一步深入了解铜在肝细胞中运输的分子和细胞机制，并可能允许采用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.