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。 公共卫生意义：除了开发控制血栓的新方法外，我们从维生素K循环研究中获得的知识应该会深入了解维生素显然在骨骼形成中的主要作用。虽然维生素K对骨代谢的贡献是明确的，但维生素K在治疗或减缓骨质疏松症骨密度丢失进展方面是否有效仍存在争议。另一个与公共健康相关的发展中领域是凝血/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.