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）在止血中起关键作用，因为它提供了维生素K依赖性（VKD）止血蛋白的羧化所需的辅助因子。饮食中的维生素K在血液中循环，在该血液中被组织吸收并输送到内质网中以用于羧化。在羧化过程中，使用还原的维生素K用于驱动glus的glus簇转化为VKD蛋白中的羧化GLU，从而使它们可用于止血。羧化导致维生素K氧化，VKOR通过一种未知的机制降低了氧化的维生素K，以连续羧化。 VKOR是Warfarin等抗凝药物的靶标，在减少维生素K时会灭活，因此还需要连续回收，这是由尚未确定的氧化还原蛋白来完成的。因此，VKOR参与了羧化过程中的多个组件。但是，它如何与这些蛋白质相互作用以实现有效的羧化是未知的，某些蛋白质的身份也是未知的。我们已经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了解此过程，并且该系统提供了一组独特的工具来定义VKOR功能。我们的具体目的是：1）确定硫氧还蛋白还原酶同工型对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，而VKOR过表达仅导致羧化的增加，我们认为这是由于VKOR活性所需的氧化还原蛋白饱和所致。我们将硫氧还蛋白还原酶同工型鉴定为羧化所需的VKOR相互作用蛋白，并将通过确定该同工型的水平改变细胞中的羧化来检验我们的假设。 2）定义细胞质VKOR序列中如何赋予华法林的耐药性。出乎意料的是，在体外分析时，已知会引起人类抗性的VKOR突变对这种药物表示敏感性。我们假设差异是由于VKOR相互作用的蛋白（如硫氧还蛋白还原酶同工型）的影响或我们也鉴定出和显示的脂肪酸结合蛋白对羧化很重要。我们将通过确定这些蛋白质对VKOR对华法林的敏感性的影响来检验我们的假设。 3）确定VKOR如何供应减少维生素K为羧化酶。 VKOR和羧化酶之间的维生素K回收非常有效，这提出了一个问题，即是否通过两种酶之间的复杂形成来促进回收利用。我们将使用我们开发的体外VKD蛋白羧化测定法以及细胞中的FRET测试复合物的存在。这些研究将为我们对VKD蛋白羧化的VKOR生理学的理解提供基本贡献，这将导致出色的抗凝剂的开发并改善了VKD蛋白用于治疗用途。