STRUCTURAL BIOLOGY OF THE HUMAN COPPER TRANSPORTER CTR1

描述（由申请人提供）：结合金属存在于大约三分之一的蛋白质中，因此，这些微量营养素代表了所有生物系统的核心成分。铜直接参与许多人体生理过程，并保护细胞免受自由基的潜在诱变性DNA损伤。铜缺乏与贫血、心血管异常、神经退行性变、肿瘤发生、血管生成和中性粒细胞减少（化疗的常见毒理学后果）有关。整体膜蛋白Ctrl负责铜的细胞摄取。出乎意料的是，hCtrl还负责铂类细胞毒性抗癌药物（如顺铂）的摄取，因此为设计具有改善摄取特性的相关药物提供了可能的靶点。hCtrl长度为190个氨基酸，包含三个预测的跨膜螺旋，并且最有可能具有三聚体的生理功能。Ctrl具有一种新的折叠，其功能的分子基础尚不清楚。将Human Ctrl （hCtrl）克隆到嗜甲烷酵母毕赤酵母（Pichia pastoris）中，设计构建了多个构建体，并对表达进行了优化，实现了可重复的高水平表达。一个强大的两柱纯化方法已经开发，使容易产生数毫克的高纯度的hCtrl量。根据项目公告PA-03-100“高影响/高风险研究的NIGMS探索性研究”提交的R21申请的目的是：1)生产多毫克量的纯化hCtrl， 2)用hCtrl和hCtrl复合物进行系统的三维结晶实验。本提案的目的是获得足以用x射线晶体学测定hCtrl结构的晶体。这项工作的长期目标是进行详细的hCtrl结构-功能研究，批判性地评估hCtrl结构在新型铂抗癌药物设计中的效用，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.