STRUCTURAL BIOLOGY OF THE HUMAN COPPER TRANSPORTER CTR1

说明(申请人提供)：结合金属存在于大约三分之一的蛋白质中，因此，这些微量营养素是所有生物系统的中心成分。铜直接参与人体许多生理过程，保护细胞免受自由基对DNA的潜在致突变损伤。铜缺乏与贫血、心血管异常、神经变性、肿瘤形成、血管生成和中性粒细胞减少症(化疗的常见毒理学后果)有关。完整的膜蛋白Ctrl负责细胞对铜的摄取。出乎意料的是，hCtrl还负责顺铂等铂细胞毒性抗癌药物的摄取，因此为设计具有更好摄取性能的相关药物提供了一个可能的靶点。hCtrl全长190个氨基酸，包含三个预测的跨膜螺旋，最有可能作为三聚体具有生理功能。Ctrl具有一个新的折叠，其功能的分子基础尚不清楚。将人Ctrl(HCtrl)克隆到巴斯德毕赤酵母中，设计和构建了多个表达载体，并进行了表达优化，实现了可重复性的高效表达。一种稳健的双柱纯化方法已被开发出来，该方法能够方便地产生数毫克量的高纯度hCtrl。根据项目公告PA-03-100“NIGMS高影响/高风险研究的探索性研究”提交的这份R21申请的目的是：1)生产多毫克数量的纯化hCtrl，以及2)对hCtrl和hCtrl络合物进行系统的三维结晶实验。这项建议的目标是获得足够的晶体来用X射线结晶学确定hCtrl的结构。这项工作的长期目标是对hCtrl进行详细的结构-功能研究，批判性地评估hCtrl结构在设计新型铂类抗癌药物方面的实用性，并将我们的研究扩展到铜稳态的结构生物学。

DESCRIPTION (provided by applicant): Bound metals are present in approximately one-third of all proteins and, as such, these micronutrients represent a central component in all biological systems. Copper is directly involved in many human physiological processes and protects cells from potentially mutagenic DNA damage by free radicals. Copper deficiency has been implicated in anemia, cardiovascular abnormalities, neurodegeneration, tumorigenesis, angiogenesis, and neutropenia (a common toxicological consequence of chemotherapy). The integral membrane protein Ctrl is responsible for the cellular uptake of copper. Unexpectedly, hCtrl is also responsible for uptake of platinum cytotoxic anticancer drugs such as cisplatin, and thus presents a possible target for the design of related drugs with improved uptake properties, hCtrl is 190 amino acids in length, contains three predicted transmembrane helices, and is most likely physiologically functional as a trimer. Ctrl possesses a novel fold, and its molecular basis of function is not known. Human Ctrl (hCtrl) has been cloned into the methanotropic yeast Pichia pastoris, multiple constructs have been designed and built, expression has been optimized, and reproducible high-level expression has been achieved. A robust two column purification method has been developed that enables the facile generation of multi-milligram quantities of highly-pure hCtrl. The aims of this R21 application, submitted under Program Announcement PA-03-100 'NIGMS Exploratory Studies for High Impact/High Risk Research', are to: 1) produce multimilligram quantities of purified hCtrl, and 2) conduct systematic three-dimensional crystallization experiments with hCtrl and hCtrl complexes. The goal of this proposal is to obtain crystals sufficient for structure determination of hCtrl by x-ray crystallography. The longer-term goals of this effort are to conduct detailed structure-function studies of hCtrl, to critically evaluate the utility of the hCtrl structure for design of novel platinum anticancer drugs, and to extend our research to the structural biology of copper homeostasis.