Molecular Mechanisms of Copper Transporting ATPases

铜转运ATP酶的分子机制

基本信息

批准号：
6898524
负责人：
DAVID L HUFFMAN
金额：
$ 21.9万
依托单位：
WESTERN MICHIGAN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-01 至 2007-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The mechanism of heavy-metal transporting P-type ATPases is crucial to the understanding of copper and iron homeostasis mechanisms. Wilson Disease is a disorder of copper metabolism, leading to a buildup of copper in the brain, liver, and kidneys, followed by subsequent neurological disorders, cirrhosis of the liver, and kidney failure. Wilson Protein and its homologues are essential for incorporation of copper into a multicopper oxidase that is vital for high affinity iron uptake. Thus copper and iron uptake are tightly coupled processes. The relationship between Wilson Disease and the mutations that cause the disease are not well understood. The N-terminal metal-binding domains of the Wilson protein are important for copper acquisition and subsequently delivery of copper into the post-Golgi vesicle that matures copper-containing multicopper oxidases. A number of different mutations give rise to Wilson disease, 3 of which occur in the N-terminal metal-binding domains. The molecular effect of these 3 mutations has not been elucidated. Wilson protein contains 6 N-terminal metal-binding domains. Intramolecular interactions between these domains facilitate the copper acquisition pathway. Characterization of these mutations and intradomain interactions is best accomplished by isolation of these metal-binding domains, followed by biochemical and structural studies. Experiments will be designed to test the transfer of copper between domains. Wild type vs. mutant forms will be compared by stability studies and assessment of the folding kinetics. Finally, it is likely that domains 5 and 6 of the Wilson protein act in concert with each other since no intervening sequence occurs between the putative consensus motifs. Expression of a construct containing domains 5 and 6 together will allow one to determine the role of two closely adjacent domains in homologous P-type ATPases. Finally, we are isolating the metal-binding domains of Arabidopsis thaliana RAN1 and the metallochaperone CCH, as a plant model for human disease.

描述（由申请人提供）：重金属转运 P 型 ATP 酶的机制对于理解铜和铁稳态机制至关重要。威尔逊病是一种铜代谢紊乱，导致铜在大脑、肝脏和肾脏中积聚，随后出现神经系统疾病、肝硬化和肾衰竭。 Wilson 蛋白及其同系物对于将铜掺入多铜氧化酶至关重要，而多铜氧化酶对于高亲和力铁吸收至关重要。因此，铜和铁的吸收是紧密耦合的过程。威尔逊病与导致该疾病的突变之间的关系尚不清楚。 Wilson 蛋白的 N 端金属结合结构域对于铜的获取以及随后将铜递送到使含铜多铜氧化酶成熟的高尔基体后囊泡中非常重要。许多不同的突变都会引起威尔逊病，其中 3 种突变发生在 N 端金属结合域。这 3 个突变的分子效应尚未阐明。 Wilson 蛋白含有 6 个 N 端金属结合结构域。这些结构域之间的分子内相互作用促进了铜的获取途径。这些突变和域内相互作用的表征最好通过分离这些金属结合域，然后进行生化和结构研究来完成。将设计实验来测试铜在域之间的转移。将通过稳定性研究和折叠动力学评估来比较野生型与突变型。最后，Wilson 蛋白的结构域 5 和结构域 6 很可能彼此协同作用，因为在假定的共有基序之间没有出现插入序列。包含结构域 5 和 6 的构建体一起表达将允许人们确定同源 P 型 ATP 酶中两个紧密相邻的结构域的作用。最后，我们分离了拟南芥 RAN1 的金属结合域和金属伴侣 CCH，作为人类疾病的植物模型。