REGULATION OF COPPER EXPORT FROM HUMAN CELLS

这个项目的主要目标是生成细胞调控机制的全面图景。 细胞内铜水平通过铜向分泌途径输送和铜从细胞输出。 该项目重点分析了铜转运ATP7A和ATP7B，这两个ATP7A和ATP7B对铜的运输起着至关重要的作用 在这项规定中扮演的角色。在人类中，这些转运蛋白的突变会导致严重的代谢紊乱，门克斯说 肝豆状核变性和肝豆状核变性。具体目标1概述了剖析其机制的实验 铜-三磷酸腺苷酶释放铜。这一假设将得到检验，即铜-三磷酸腺苷酶的第一个管腔环 通过将铜隔离在以前未知的金属结合部位来刺激铜从转运体中释放 网站。环区的金属结合特性将被表征，其结构将是 通过核磁共振确定，第一环内的突变对铜-ATPase功能的影响将是 检查过了。具体目标2将确定腔铜受体蛋白在转运活性和 分泌途径中的铜-三磷酸腺苷酶的运输。在特定目标3下的实验将调查 ATP7A和ATP7B在平衡肾脏铜浓度中的特殊作用。肝豆状核变性和 门克斯病与肾脏近端小管铜蓄积有关，提示不同且不重叠。 ATP7A和ATP7B在肾脏铜稳态中的作用这一假设将通过以下方式检验 铜-三磷酸腺苷酶失活对细胞总铜含量和铜外流的影响 极化的肾细胞的基侧膜和顶膜。小分子化合物的作用 在ATP7B-/-小鼠的血清和尿液中高度升高，对铜的摄取和ATP7A的定位将 下定决心。这些研究将产生新的机制信息，这是更好地理解 人类铜的动态平衡和更好地治疗与铜失衡相关的疾病。

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.