COPPER IN PEDIATRIC LIVER DISEASES

铜是一种重要的微量元素，在细胞呼吸、抗氧化防御和铁稳态等生物化学过程中起着重要作用。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童肝脏疾病中的作用。最近的研究表明，铜在肝细胞内传递到特定的蛋白质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HAH1在肝细胞铜转运和代谢中的作用。结构/功能研究将利用酿酒酵母HAH1的定点诱变和表达来完成。将通过威尔森病atp酶的蛋白-蛋白相互作用研究HAH1在铜输送到分泌途径中的作用，并使用绿色荧光融合蛋白和荧光共振能量转移研究这些蛋白在完整单细胞中的相互作用。HAH1基因在哺乳动物细胞中的确切功能将通过对小鼠肝细胞铜转运和代谢的分析来确定，并靶向种系删除小鼠HAH1基因。最后，将通过对以铜积累和毒性为特征的肝脏疾病儿童的基因组DNA进行HAH1序列分析，研究HAH1在儿童肝脏疾病中的作用，包括诊断为Wilson病但未检测到Wilson atp酶突变的个体。综上所述，这些研究的结果将有助于进一步了解肝细胞中铜转运的分子和细胞机制，并可能为预防或改善由金属代谢紊乱引起的儿童肝病提供新的治疗方法。

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.