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）：重金属运输P型ATPases的机制对于理解铜和铁稳态机制至关重要。威尔逊病是一种铜的代谢疾病，导致大脑，肝脏和肾脏的铜积聚，随后发生了神经系统疾病，肝脏肝硬化和肾衰竭。 Wilson蛋白及其同源物对于将铜掺入多功能氧化酶至关重要，该氧化酶对高亲和力铁的摄取至关重要。因此，铜和铁吸收是紧密耦合的过程。威尔逊疾病与引起疾病的突变之间的关系尚不清楚。威尔逊蛋白的N末端金属结合结构域对于铜采集和随后将铜递送到后高尔基球囊泡中，该囊泡适时含有含铜的多型氧化酶。许多不同的突变引起了威尔逊疾病，其中3种发生在N末端金属结合结构域中。这三个突变的分子效应尚未阐明。威尔逊蛋白包含6个N末端金属结合结构域。这些结构域之间的分子内相互作用有助于铜采集途径。通过分离这些金属结合结构域，随后进行生化和结构研究，可以最好地完成这些突变和域内相互作用的表征。实验将设计用于测试域之间铜的传递。野生型与突变体形式将通过稳定性研究和折叠动力学的评估进行比较。最后，威尔逊蛋白的域5和6可能相互起作用，因为假定的共识基序之间没有中间序列发生。包含域5和6的构建体的表达将允许一个人确定两个紧密相邻域在同源P型ATPase中的作用。最后，我们将拟南芥RAN1的金属结合结构域和金属伴侣CCH分离为人类疾病的植物模型。