Copper Transport Mechanism of Menkes disease protein and Wilson disease protein

说明(申请人提供)：铜是人体正常新陈代谢所必需的。体内分布铜的蛋白质的先天功能缺陷会导致严重的代谢紊乱，如门克斯病和威尔逊病，其特征是神经和肝脏异常，肾脏和血管损伤。铜转运ATP7A(Menkes病蛋白)和ATP7B(Wilson病蛋白)被认为是人类细胞中铜浓度的关键调节因子。近年来，有关这些转运蛋白的生化和细胞内特性的信息迅速产生。然而，人们对ATP7A和ATP7B的不同生理作用知之甚少。我们的长期目标是确定ATP7A和ATP7B在铜稳态中的特定作用，作为开发和改进这些疾病治疗方法的先决条件。这项提案的主要目标是了解ATP7A和ATP7B在铜运输中的具体作用。通过重组蛋白构建、定点突变、片段交换实验和铜结合亲和力分析，我们将确定一个独特的序列插入在ATP7A和ATP7B的功能和运输中的作用。此外，我们还将通过检测肽基-a-单加氧酶过表达或下调后ATP7A的铜转运速率来研究受体蛋白在铜释放中的作用。最后，我们将研究 ATP7B的活性依赖于CIC-4，CIC-4是一种哺乳动物内体的CI-/H+交换器，但在细胞生理学中的功能尚不清楚。拟议的研究结果将为理解和更好地治疗门克斯病和威尔逊病提供必要的信息。 公共卫生相关性：门克斯病和威尔逊病的特征是神经系统和肝脏异常，肾脏和血管损伤，分别由ATP7A和ATP7B酶功能障碍引起。我们的目标是确定ATP7A和ATP7B的具体作用，作为开发和改进这些疾病治疗方法的先决条件。

DESCRIPTION (provided by applicant): Copper is essential for normal human metabolism. Inborn defects in function of proteins distributing copper within the body lead to severe metabolic disorders, such as Menkes disease and Wilson disease, which are characterized by neurological and hepatic abnormalities, renal and vascular damage. The copper transporting ATPases, ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), have been identified as key regulators of copper concentration in human cells. In recent years, information on biochemical and intracellular properties of these transporters has been rapidly generated. However, little is known about the distinct physiological roles of ATP7A and ATP7B. Our long term goal is to characterize specific roles of ATP7A and ATP7B in copper homeostasis as a prerequisite to the development and improvement of therapeutic treatments for these disorders. The major goal of this proposal is to understand specific roles of ATP7A and ATP7B in copper transport. Using recombinant protein construction, site directed mutagenesis, segment exchanging experiments, and copper binding affinity assays; we will determine the role of a unique sequence insert in the function and trafficking of ATP7A and ATP7B. Also, we will investigate the role of acceptor proteins on copper release by measuring the rate of copper transport of ATP7A after overexpression or knock-down of peptidyl-a-monooxygenase. Finally, we will examine the dependence of ATP7B activity on CIC-4, a mammalian endosomal CI-/H+ exchanger with poorly understood function in cell physiology. The result of the proposed research will yield information essential for understanding and better treatment of Menkes disease and Wilson disease. PUBLIC HEALTH RELEVANCE: Menkes disease and Wilson disease are characterized by neurological and hepatic abnormalities, renal and vascular damage, and are caused by a disruption in function of the enzymes ATP7A and ATP7B, respectively. Our goal is to characterize the specific roles of ATP7A and ATP7B as a prerequisite to the development and improvement of therapeutic treatments for these disorders.