Vitamin K Oxidoreductase: function and physiology

维生素 K 氧化还原酶：功能和生理学

• 批准号: 7540971
• 负责人: KATHLEEN Lucile BERKNER
• 金额: $ 35.33万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-20 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7540971
• 关键词: Affect; Anticoagulants; Biological Assay; Biological Models; Blood; Cell Line; Cells; Complex; Development; Diet; Endoplasmic Reticulum; Enzymes; Epoxy Compounds; Event; Exhibits; Fluorescence Resonance Energy Transfer; Generations; Goals; Hemostatic Agents; Hemostatic function; Human; Hydroquinones; In Vitro; Lead; Life; Mammalian Cell; Mutation; Natural regeneration; Oxidation-Reduction; Oxidoreductase; Patients; Pattern; Pharmaceutical Preparations; Physiology; Play; Predisposition; Process; Production; Protein Binding; Protein Isoforms; Proteins; Prothrombin; Recombinants; Recycling; Resistance; Role; System; Testing; Therapeutic Uses; Tissues; Vitamin K; Warfarin; carboxylate; carboxylation; cofactor; fatty acid-binding proteins; gamma-glutamyl carboxylase; hydroquinone; improved; in vitro testing; overexpression; oxidation; protein complex; reduced vitamin K; therapeutic protein; thioredoxin reductase; tool; vitamin K epoxide reductase; vitamin K1 oxide

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.

描述（由申请人提供）：维生素 K 氧化还原酶 (VKOR) 在止血中发挥着关键作用，因为它提供维生素 K 依赖性 (VKD) 止血蛋白羧化所需的辅因子。饮食中的维生素 K 在血液中循环，被组织吸收并输送到内质网用于羧化。在羧化过程中，还原的维生素 K 用于驱动 VKD 蛋白中的 Glus 簇转化为羧化 Glus，从而使其发挥止血功能。羧化会导致维生素 K 氧化，而 VKOR 会通过一种很大程度上未知的机制来还原氧化的维生素 K，以实现连续羧化。 VKOR 是华法林等抗凝药物的靶标，在维生素 K 还原过程中会失活，因此也需要持续回收，这是通过尚未鉴定的氧化还原蛋白来完成的。因此，VKOR与多个组分一起参与羧化过程；然而，它如何与这些蛋白质相互作用以实现有效的羧化尚不清楚，一些蛋白质的身份也是如此。我们开发了表达重组羧化成分的哺乳动物细胞作为模型系统来理解这一过程，并且该系统提供了一套独特的工具来定义 VKOR 功能。我们的具体目标是： 1) 确定硫氧还蛋白还原酶亚型对 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化作用，并且 VKOR 过度表达仅导致羧化作用小幅增加，我们假设这是由于 VKOR 活性所需的氧化还原蛋白饱和所致。我们确定了一种硫氧还蛋白还原酶亚型作为羧化所需的 VKOR 相互作用蛋白，并将通过确定该亚型水平的变化如何改变细胞中的羧化来检验我们的假设。 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，已知会导致人类华法林耐药的 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（如硫氧还蛋白还原酶异构体或脂肪酸结合蛋白）的影响所致，我们也鉴定并证明它们对羧化很重要。我们将通过确定这些蛋白质对 VKOR 对华法林敏感性的影响来检验我们的假设。 3) 确定 VKOR 如何向羧化酶提供还原的维生素 K。 VKOR 和羧化酶之间的维生素 K 回收非常有效，这就提出了两种酶之间的复合物形成是否促进回收的问题。我们将使用我们开发的体外 VKD 蛋白羧化测定以及细胞中的 FRET 来测试复合物的存在。这些研究将为我们理解 VKD 蛋白羧化中的 VKOR 生理学提供基础贡献，从而开发出优质的抗凝剂并提高用于治疗用途的 VKD 蛋白的生产。