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.

这个项目的主要目标是产生一个全面的图片细胞机制调节 细胞内铜水平通过铜递送到分泌途径和铜从细胞输出。 该项目的重点是分析铜转运ATP酶ATP 7A和ATP 7 B，这两种酶在细胞内起着重要的作用。 在这一规定中的作用。在人类中，这些转运蛋白的突变会导致严重的代谢紊乱， 疾病和威尔逊病，分别。具体目标1概述了解剖 从Cu-ATP酶释放铜。将检验Cu-ATP酶的第一个管腔环 通过在以前未识别的金属结合处螯合铜，刺激铜从转运蛋白释放 网站.环区域的金属结合特性将被表征，其结构将被表征。 通过NMR确定，并且将研究第一环内的突变对Cu-ATP酶功能的影响。 考察特异性目标2将确定管腔铜受体蛋白在转运活性中的作用， 从分泌途径运输Cu-ATP酶。具体目标3下的实验将研究 ATP 7A和ATP 7 B在平衡肾脏铜浓度中的特定功能。威尔逊氏病和 Menkes病与肾近端小管铜蓄积相关，提示不同且不重叠 ATP 7A和ATP 7 B在肾铜稳态中的作用。这一假设将由以下人员进行检验： 测定Cu-ATP酶失活对总细胞铜含量和铜流出的影响， 极化肾细胞的基底膜和顶膜。小分子量化合物的作用， 在Atp 7 b-/-小鼠的血清和尿液中高度升高，对铜的摄取和ATP 7A的定位将是 测定这些研究将产生新的机制信息，为更好地理解 人体铜稳态和更好地治疗与铜失衡相关的疾病。