Characterization of Vitamin K Epoxide Reductase

描述（由申请人提供）：该项目的长期目标是不仅了解维生素K环氧化物还原酶（VKOR）的结构和功能，而且了解整个维生素K循环。维生素K依赖性羧化是蛋白质的一种重要的翻译后修饰，在包括血液凝固和骨代谢在内的多种生理功能中具有重要作用。VKOR是华法林的靶点，华法林是血栓栓塞性疾病最常用的抗凝剂。虽然估计每一次诱发性出血事件可预防20次卒中，但由于担心出血，华法林仍未得到充分利用。希望这项资助的研究能带来抗血栓治疗的改进，从而避免与华法林治疗相关的一些问题。具体而言，我们建议在本赠款的任期内完成以下工作：（1）使用标准纯化和酶测定方法、RNAi技术和修饰的表达克隆，我们将研究维生素K循环以鉴定除了维生素K环氧化物还原酶（VKOR）之外的生产维生素K氢醌所必需的组分，（2）使用电子晶体学，生物化学和分子生物学技术，进行研究以阐明VKOR的三维结构，和（3）使用体内和体外方法研究VKOR所必需的结构特征。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand, not only the structure and function of the vitamin K epoxide reductase (VKOR), but also the vitamin K cycle as a whole. Vitamin K-dependent carboxylation is an essential post-translational modification for proteins important in several physiologic functions, including blood coagulation and bone metabolism. VKOR is the target of warfarin, the most widely prescribed anti-coagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is still under-utilized because of fear of bleeding. It is hoped that research funded by this grant will lead to improved anti- thrombotic therapies that will avoid some of the problems related to warfarin treatment. Specifically, we propose to accomplish the following during the tenure of this grant: (1) using standard purification and enzyme assay methods, RNAi technologies, and modified expression cloning, we will investigate the vitamin K cycle to identify components other than the vitamin K epoxide reductase (VKOR) necessary for the production of vitamin K hydroquinone, (2) employing electron crystallography, biochemical and molecular biological techniques, conduct studies to elucidate the three- dimensional structure of VKOR, and (3) using both in vivo and in vitro methods investigate the structural characteristics necessary for VKOR.