The Role of Copper in Pediatric Liver Disease

描述（由申请人提供）：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具有关键作用。肝脏是人体铜稳态的中心器官，这些研究的长期目标是确定铜在儿科肝病中的作用。虽然先前的研究已经揭示了铜伴侣Atox 1和威尔逊病ATP酶（ATP 7 b）在肝铜代谢中的核心作用，但导致这种金属在小管膜上排泄的肝细胞分泌途径内的机制仍然知之甚少。最近，一个直接的相互作用已被证明之间的ATP 7 b和Murr 1，人类同源的贝德灵顿梗铜中毒基因产物，提供生化证据支持的作用Mutt 1在肝胆病理生理学的铜排泄。本提案的具体目的是阐明Murr 1和ATP 7 b在这一过程中的作用。结构/功能研究将使用定点突变来描述Murr 1与ATP 7 b相互作用的生化细节。Murr 1错义突变患者的肝铜中毒的病理生理学将通过检查这些突变的转基因小鼠以及Murr 1基因有针对性的种系缺失的小鼠中的铜稳态和ATP 7 b功能来阐明。Murr 1在铜排泄中的功能作用将通过分离和表征与ATP 7 b相互作用后形成的Murr 1异源寡聚复合物中存在的特异性相互作用蛋白来确定。最后，将使用体外表达克隆来表征与该结构域相互作用的蛋白质，从而定义ATP 7 b的羧基末端在肝细胞中铜介导的运输和排泄中的作用。总之，这些研究的结果将允许对肝脏内排泄事件的肝胆病理生理学的新见解，并可能允许新的治疗方法来预防或改善许多儿科肝病中的肝损伤。

DESCRIPTION (provided by applicant): 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 humans and the long-term objective of these studies is to define the role of copper in pediatric liver disease. While previous studies have revealed a central role for the copper chaperone Atox1 and the Wilson disease ATPase (ATP7b) in hepatic copper metabolism, the mechanisms within the hepatocyte secretory pathway that lead to excretion of this metal at the canalicular membrane remain poorly understood. Most recently, a direct interaction has been demonstrated between ATP7b and Murr1, the human homologue of the Bedlington terrier copper toxicosis gene product, providing biochemical evidence in support of a role for Mutt1 in the hepatobiliary pathophysiology of copper excretion. The specific aims of this proposal are intended to elucidate the role of Murr1 and ATP7b in this process. Structure/function studies will be accomplished using site-directed mutagenesis to delineate the biochemical details of Murr1 interaction with ATP7b. The pathophysiology of hepatic copper toxicosis in patients with missense mutations in Murr1 will be elucidated by examining copper homeostasis and ATP7b function in mice transgenic for these mutations as well as mice with a targeted germline deletion of the Murr1 gene. The functional role of Murr1 in copper excretion will be determined by isolation and characterization of specific interacting proteins present in Murr1 heterooligomeric complexes formed upon interaction with ATP7b. Finally, the role of the carboxyl terminus of ATP7b in copper-mediated trafficking and excretion in hepatocytes will be defined using in vitro expression cloning to characterize the proteins interacting with this domain. Taken together the results of these studies will permit new insights into the hepatobiliary pathophysiology of excretory events within the liver and may allow for novel therapeutic approaches to prevent or ameliorate hepatic injury in a number of pediatric liver diseases.