Structure-Function of the Human Copper Transporter hCTR1

描述(由申请人提供)： 铜是生命所必需的，并被用于关键的生物反应，包括呼吸、清除自由基和铁摄取。铜离子的异常作用与神经退行性变的孟克斯和阿尔茨海默病有关。尽管铜在生物学上很重要，但令人惊讶的是，人们对细胞最初吸收铜所涉及的转运蛋白知之甚少。最近发现的铜转运蛋白CTR1是人类唯一已知的高亲和力铜摄取转运蛋白。CTR1在人类中广泛表达，可以在功能上替代酵母铜转运蛋白Ctrlp。CTR1及其所有同系物在过渡金属转运蛋白中是独一无二的，因为每个单体具有最少的预测跨膜结构域，这强烈表明功能需要齐聚。CTR1也是独一无二的，因为它有一个胞外N-末端结构域，可能在运输之前捕获铜离子。人CTR1(HCTR1)在酵母遗传学实验中被证明是一种高亲和力的铜摄取转运体，但尚未在分子水平上进行表征。我建议进行实验，通过脂质体和非洲爪哇卵母细胞中的hCTR1进行铜的实际转运，以确定hCTR1对铜的亲和力和选择性。HCTR1的N-末端结构域的作用将通过铜结合分析和hCTR1与相关的低亲和力铜转运蛋白的嵌合构建来确定。HCTR1在脂质体中的纯化和重组将被用来确定转运的特异性和动力学。HCTR1的二维结晶将被启动，以允许hCTR1的结构溶液通过低温电子显微镜来显示孔形成区域、低聚状态和在脂质双层中的对称性。这些实验将启动独特的人类CTR1高亲和力铜转运蛋白的分子表征，并确定该蛋白通过脂质双层转运铜的机制。

DESCRIPTION (provided by applicant): Copper is essential for life and is utilized in critical biological reactions including respiration, clearance of free radicals and iron uptake. Abnormal action of copper ions has been implicated in the neurodegenerative Menkes and Alzheimer's diseases. Despite the importance of copper in biology, surprisingly little is known about the transporters involved in the initial uptake of copper into cells. The recently discovered copper transporter CTR1 is the only known high-affinity copper uptake transporter in humans. CTR1 is ubiquitously expressed in humans and can functionally substitute for the yeast copper transporter Ctrlp. CTR1 and all its homologs are unique among transition metal transporters in having the least number of predicted transmembrane domains per monomer strongly suggesting that oligomerization is required for function. CTR1 is also unique in having an extracellular N-terminal domain that is likely to capture copper ions prior to transport. Human CTR1 (hCTR1) was shown to be a high affinity copper uptake transporter in yeast genetics experiments but has not been characterized at the molecular level. I propose experiments that will demonstrate actual copper transport through hCTR1 in liposomes and Xenopus oocytes to determine the affinity and selectivity of hCTR1 for copper transport. The role of the N-terminal domain of hCTR1 will be determined using copper binding assays and chimeric constructs of hCTR1 with a related low-affinity copper transporter. Purification and reconstitution of hCTR1 in liposomes will be employed to determined the specificity and kinetics of transport. Two-dimensional crystallization of hCTR1 will be initiated to allow structural solution of hCTR1 by cryoelectron microscopy for visualization of pore forming regions, oligomeric state and symmetry in the lipid bilayer. These experiments will initiate the molecular characterization of the unique human CTR1 high-affinity copper transporter and determine the mechanisms by which this protein transports copper through the lipid bilayer.