Vitamin K Oxidoreductase: function and physiology

描述（由申请人提供）：维生素K氧化还原酶（VKOR）在止血中起关键作用，因为它提供维生素K依赖性（VKD）止血蛋白羧化所需的辅助因子。饮食中的维生素K在血液中循环，在血液中被组织吸收并输送到内质网，用于羧基化。在羧化过程中，减少的维生素K用于驱动VKD蛋白中Glus簇向羧化Glus的转化，使其具有止血功能。羧基化导致维生素K氧化，而VKOR通过一种很大程度上未知的机制减少氧化维生素K的连续羧基化。VKOR是华法林等抗凝血药物的靶标，在维生素K还原过程中失活，因此也需要持续的再循环，这是由一种尚未确定的氧化还原蛋白完成的。因此，VKOR与多个组分一起参与羧基化过程；然而，它如何与这些蛋白质相互作用以完成有效的羧化是未知的，一些蛋白质的身份也是未知的。我们已经开发了表达重组羧基化成分的哺乳动物细胞作为模型系统来理解这一过程，该系统为定义VKOR功能提供了一套独特的工具。我们的具体目标是：1)确定硫氧还蛋白还原酶异构体对VKOR活性和VKD蛋白羧化的重要性。我们发现，维生素K的可用性限制了细胞中的羧基化，而VKOR过表达只会导致羧基化的小幅增加，我们假设这是由于VKOR活性所需的氧化还原蛋白饱和所致。我们确定了硫氧还蛋白还原酶异构体作为羧基化所需的vkor相互作用蛋白，并将通过确定该异构体水平的变化如何改变细胞中的羧基化来验证我们的假设。2)定义细胞质VKOR序列的突变如何赋予华法林耐药性。出乎意料的是，已知引起人类华法林耐药性的VKOR突变在体外分析时显示出对该药物的敏感性。我们假设这种差异是由于vkor相互作用蛋白的影响，如硫氧还蛋白还原酶异构体或脂肪酸结合蛋白，我们也发现并显示对羧化很重要。我们将通过确定这些蛋白对VKOR对华法林敏感性的影响来验证我们的假设。3)确定VKOR如何向羧化酶提供还原维生素K。维生素K在VKOR和羧化酶之间的再循环是非常有效的，这就提出了一个问题，即这两种酶之间的复合体是否促进了再循环。我们将使用我们开发的体外VKD蛋白羧化分析以及细胞中的FRET来测试复合物的存在。这些研究将为我们理解VKD蛋白羧化的VKOR生理学提供基础贡献，这将导致开发优质抗凝血剂和改善治疗用VKD蛋白的生产。

DESCRIPTION (provided by applicant): The vitamin K oxidoreductase (VKOR) plays a critical role in hemostasis because it provides the cofactor required for the carboxylation of vitamin K-dependent (VKD) hemostatic proteins. Vitamin K from the diet circulates in blood, where it is taken up by tissues and delivered to the endoplasmic reticulum to be used in carboxylation. During carboxylation, reduced vitamin K is used to drive the conversion of clusters of Glus to carboxylated Glus in VKD proteins, rendering them functional for hemostasis. Carboxylation results in vitamin K oxidation, and VKOR reduces the oxidized vitamin K for continuous carboxylation by a mechanism that is largely unknown. VKOR, which is the target of anticoagulant drugs like warfarin, becomes inactivated during vitamin K reduction and therefore also requires continuous recycling, which is accomplished by a redox protein not yet identified. Thus, VKOR participates with multiple components in the process of carboxylation; however, how it interacts with these proteins to accomplish efficient carboxylation is unknown, as is the identity of some of the proteins. We have developed mammalian cells expressing recombinant carboxylation components as a model system to understand this process, and this system provides a unique set of tools for defining VKOR function. Our Specific Aims are to: 1) Determine the importance of a thioredoxin reductase isoform to VKOR activity and VKD protein carboxylation. We found that vitamin K availability limits carboxylation in cells and that VKOR overexpression only caused a small increase in carboxylation, which we hypothesize is due to saturation of the redox protein required for VKOR activity. We identified a thioredoxin reductase isoform as a VKOR-interacting protein that is required for carboxylation, and will test our hypothesis by determining how changes in the level of this isoform alter carboxylation in cells. 2) Define how mutations in cytoplasmic VKOR sequences confer resistance to warfarin. Unexpectedly, VKOR mutations known to cause warfarin resistance in humans show sensitivity to this drug when analyzed in vitro. We hypothesize that the difference is due to the impact of VKOR-interacting proteins like the thioredoxin reductase isoform or a fatty acid binding protein that we also identified and showed is important to carboxylation. We will test our hypothesis by determining the effect of these proteins on VKOR susceptibility to warfarin. 3) Determine how VKOR supplies reduced vitamin K to the carboxylase. Vitamin K recycling between VKOR and the carboxylase is very efficient, raising the question of whether recycling is facilitated by complex formation between the two enzymes. We will test for the existence of a complex using in vitro VKD protein carboxylation assays we developed as well as FRET in cells. These studies will provide fundamental contributions to our understanding of VKOR physiology in VKD protein carboxylation that will lead to the development of superior anticoagulants and improved production of VKD proteins for therapeutic use.