COPPER IN PEDIATRIC LIVER DISEASES

铜与小儿肝病的关系

• 批准号: 6495379
• 负责人: Jonathan David Gitlin
• 金额: $ 17.56万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6495379
• 关键词: clinical research; congenital disorders; copper; hepatolenticular degeneration; human subject; intracellular transport; laboratory mouse; liver disorder; metal metabolism; molecular chaperones; pathologic process; protein structure function

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.

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发挥着关键作用。肝脏是人体铜稳态的中心器官，这些研究的长期目标是明确铜在儿童肝脏疾病中的作用。最近的研究表明，铜向肝细胞内特定蛋白质的递送是由称为伴侣的不同细胞内载体蛋白介导的。本提案中的研究旨在阐明铜伴侣HAH1在肝细胞铜运输和代谢中的作用。结构/功能研究将利用酿酒酵母中 HAH1 的定点诱变和表达来完成。 HAH1 在铜输送到分泌途径中的作用将通过威尔逊病 ATP 酶的蛋白质-蛋白质相互作用研究来检查，并且使用绿色荧光融合蛋白和荧光共振能量转移检查完整单细胞泔水中这些蛋白质的相互作用。 HAH1 在哺乳动物细胞中的精确功能将通过分析小鼠肝细胞中铜的运输和代谢来确定，其中小鼠 HAH1 基因有针对性的种系缺失。最后，HAH1 在儿科肝病中的作用将通过对以铜积累和毒性为特征的肝病儿童的基因组 DNA 进行 HAH1 序列分析来研究，其中包括诊断为威尔逊病但在威尔逊 ATP 酶中未检测到突变的个体。总而言之，这些研究的结果将有助于进一步了解肝细胞中铜运输的分子和细胞机制，并可能提供新的治疗方法来预防或改善因金属代谢紊乱引起的儿童肝病。