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。 公共卫生相关性：除了开发控制血栓形成的新方法外，我们对维生素K循环的研究所获得的知识应该可以深入了解维生素在骨形成中的明显主要作用。虽然对骨代谢的贡献是明确的，但维生素K是否可用于治疗或减缓骨质疏松症中骨密度损失的进展仍存在争议。与公共卫生相关的另一个发展中领域是凝血/血栓形成在癌症中的作用，以及维生素K及其拮抗剂是否在癌症治疗中占有一席之地。

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. PUBLIC HEALTH RELEVANCE: In addition to development of new ways of controlling thromboses, knowledge gained from our research on the vitamin K cycle should give insight into the vitamin's apparently major role in bone formation. While the contribution to bone metabolism is clear, it is still controversial whether vitamin K is useful in treating or slowing progression of bone density loss in osteoporosis. Another developing area relevant to public health is the role of coagulation/thrombosis in cancer, and in turn whether vitamin K and its antagonists have a place in cancer treatment.