REGULATION OF COPPER EXPORT FROM HUMAN CELLS

人体细胞铜输出的监管

• 批准号: 7690600
• 负责人: SVETLANA LUTSENKO
• 金额: $ 35.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7690600
• 关键词: ATP phosphohydrolase; Address; Appearance; Binding; Biochemical; Biological; Biological Assay; Buffers; Cell physiology; Cells; Characteristics; Collaborations; Copper; Data; Dependence; Disease; Down-Regulation; Equilibrium; Event; Funding; Goals; Health; Hepatic; Hepatocyte; Hepatolenticular Degeneration; Homeostasis; Human; In Vitro; Ions; Kidney; Laboratories; Length; Link; Measures; Membrane; Menkes Kinky Hair Syndrome; Messenger RNA; Metabolic Diseases; Metabolism; Metal Binding Site; Metals; Methods; Molecular Weight; Mus; Mutation; Organelles; Pathway interactions; Play; Procedures; Process; Property; Proteins; Proximal Kidney Tubules; Recombinants; Regulation; Role; Serum; Signal Transduction; Site; Small Interfering RNA; Structure; Testing; Time; Urine; Variant; Vesicle; Wilson disease protein; age related; apical membrane; copper-transporting ATPase; disease phenotype; kidney cell; liver function; novel; orexin A receptor; research study; response; trafficking; uptake

The major goal of this project is to generate a comprehensive picture of cellular mechanisms regulating intracellular copper levels through copper delivery to the secretory pathway and copper export from the cell. The project focuses on the analysis of copper-transporting ATPases ATP7A and ATP7B, which play an essential role in this regulation. In humans, mutations in these transporters cause severe metabolic disorders, Menkes disease and Wilson's disease, respectively. Specific Aim 1 outlines experiments dissecting the mechanism of copper release from Cu-ATPases. The hypothesis will be tested that the first luminal loop of the Cu-ATPases stimulates copper release from the transporters by sequestering copper at the previously unidentified metalbinding sites. The metal-binding properties of the loop region will be characterized, its structure will be determined by NMR, and the effect of mutations within the first loop on the function of Cu-ATPases will be examined. Specific Aim 2 will determine the role of luminal copper acceptor proteins in transport activity and trafficking of Cu-ATPases from the secretory pathway. The experiments under Specific Aim 3 will investigate specific functions of ATP7A and ATP7B in balancing copper concentration in kidney. Both Wilson's disease and Menkes disease are associated with copper accumulation in renal proximal tubules suggesting distinct and nonoverlapping roles of ATP7A and ATP7B in renal copper homeostasis. This hypothesis will be tested by determining the effect of Cu-ATPases inactivation on total cellular copper content and on copper efflux from the basolateral and apical membranes of polarized renal cells. The effect of small molecular weight compound, which is highly elevated in the serum and urine of Atp7b-/- mice, on copper uptake and localization of ATP7A will be determined. The studies will produce novel mechanistic information necessary for better understanding of human copper homeostasis and better treatment of disorders associated with copper misbalance.

该项目的主要目标是生成调节细胞机制的全面图片 细胞内铜水平通过铜递送到分泌途径和从细胞出口的铜出口。 该项目着重于对铜运输ATPases ATP7A和ATP7B的分析，这些ATP7A和ATP7B起着重要的 在此法规中的作用。在人类中，这些转运蛋白的突变会引起严重的代谢性疾病，menkes 疾病和威尔逊病。特定目标1大纲实验剖析了机制 从Cu-atpase释放铜。将测试该假设是Cu-atpases的第一个腔循环 通过在先前未识别的金属束缚处隔离铜来刺激铜从转运蛋白释放 站点。环路区域的金属结合特性将被表征，其结构将为 由NMR确定，以及第一个循环中突变对Cu-atpases功能的影响 检查。具体目标2将确定腔铜受体蛋白在运输活性和 从分泌途径中贩运cu-atpase。特定目标下的实验将调查 ATP7A和ATP7B在肾脏中平衡铜浓度的特定功能。威尔逊氏病和 Menkes疾病与肾脏近端小管中的铜积聚有关，表明明显和非重叠 ATP7A和ATP7B在肾铜稳态中的角色。该假设将通过 确定Cu-atpases失活对总细胞铜含量和对铜外排的影响 极化肾细胞的基底外侧和顶膜。小分子量化合物的作用，该化合物的作用 在ATP7B - / - 小鼠的血清和尿液中高度升高，铜摄取和ATP7A的定位将是 决定。研究将产生必要的新机械信息，以更好地理解 人类的铜稳态和更好地治疗与铜失衡有关的疾病。