Structure-Function of the Human Copper Transporter hCTR1

描述（由申请人提供）： 铜是生命所必需的，并用于关键的生物反应，包括呼吸，清除自由基和铁的吸收。铜离子的异常作用与神经退行性门克斯病和阿尔茨海默病有关。尽管铜在生物学中的重要性，但令人惊讶的是，人们对参与细胞最初吸收铜的转运蛋白知之甚少。最近发现的铜转运蛋白CTR 1是人类唯一已知的高亲和力铜摄取转运蛋白。CTR 1在人类中广泛表达，可以在功能上替代酵母铜转运蛋白Ctrlp。CTR 1及其所有同源物在过渡金属转运蛋白中是独特的，每个单体具有最少数量的预测跨膜结构域，这强烈表明功能需要寡聚化。CTR 1也是唯一的，具有细胞外N-末端结构域，可能在运输之前捕获铜离子。人CTR 1（hCTR 1）在酵母遗传学实验中显示为高亲和力铜摄取转运蛋白，但尚未在分子水平上表征。我建议的实验，将证明实际铜运输通过hCTR 1在脂质体和非洲爪蟾卵母细胞，以确定铜运输的亲和力和选择性的hCTR 1。将使用铜结合试验和hCTR 1与相关低亲和力铜转运蛋白的嵌合构建体来确定hCTR 1的N-末端结构域的作用。将采用脂质体中hCTR 1的纯化和重建来确定转运的特异性和动力学。将引发hCTR 1的二维结晶，以允许通过冷冻电子显微镜观察hCTR 1的结构溶液，以观察脂质双层中的孔形成区域、寡聚状态和对称性。这些实验将启动独特的人类CTR 1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.