Characterization of Vitamin K Epoxide Reductase

描述(申请人提供)：该项目的长期目标是不仅要了解维生素K环氧化物还原酶(VKOR)的结构和功能，而且要了解整个维生素K循环。维生素K依赖的羧化是蛋白质翻译后的重要修饰，在多种生理功能中起重要作用，包括凝血和骨代谢。VKOR是华法林的靶标，华法林是治疗血栓栓塞症的最广泛处方的抗凝剂。虽然据估计，华法林每次诱发出血可以预防20次中风，但由于担心出血，华法林仍未得到充分利用。人们希望，由这笔赠款资助的研究将导致改进的抗血栓疗法，从而避免与华法林治疗相关的一些问题。具体地说，我们建议在这笔赠款的期限内完成以下工作：(1)使用标准的纯化和酶分析方法、RNAi技术和改进的表达克隆，我们将研究维生素K循环，以确定生产维生素K氢醌所必需的维生素K环氧化物还原酶(VKOR)以外的成分；(2)利用电子结晶学、生化和分子生物学技术，进行研究，以阐明VKOR的三维结构；以及(3)使用体内和体外方法，研究VKOR所需的结构特征。

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.