Structural and Functional Basis of the Vitamin K Cycle

维生素 K 循环的结构和功能基础

• 批准号: 10400676
• 负责人: Weikai Li
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400676
• 关键词: Affinity; Anticoagulants; Anticoagulation; Basic Science; Behavior; Binding; Biochemical; Biology; Blood Coagulation Factor; Blood Vessels; Blood coagulation; Bone Diseases; Calcium; Catalysis; Chemicals; Clinical; Coagulation Process; Crystallization; Data; Deep Vein Thrombosis; Disease; Distant; Dose; Drug or chemical Tissue Distribution; Electron Transport; Environment; Enzymes; Epoxy Compounds; Foundations; Funding; Glutamic Acid; Glutathione; Goals; Hemorrhage; Homeostasis; Homologous Gene; Human; Human Genome; Hydroquinones; In Vitro; Kinetics; Knowledge; Lead; Ligands; Membrane; Membrane Proteins; Methods; Modification; Molecular; Molecular Conformation; Mutation; Myocardial Infarction; Natural regeneration; Nature; Oral; Osteoporosis; Overdose; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Peripheral; Physiologic calcification; Physiological; Physiological Processes; Play; Process; Proteins; Public Health; Pulmonary Embolism; Reaction; Reducing Agents; Regulation; Reporting; Research; Resistance; Resolution; Risk; Role; Stroke; Structure; Testing; Therapeutic; Thrombosis; Variant; Vascular calcification; Vitamin K; Warfarin; antagonist; bone health; carboxylation; cofactor; experience; improved; inhibitor; innovation; insight; mouse model; novel; paralogous gene; prevent; reduced vitamin K; research study; resistance mutation; response; tool; vitamin K epoxide reductase; vitamin K1 oxide

The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane- embedded enzyme that is difficult to characterize with structural and biochemical studies. Our long-term goal is to elucidate the physiological process of the entire vitamin K cycle and its interaction with VKAs. This cycle begins with γ-carboxylation, a modification required for the activity of vitamin-K-dependent proteins, including several coagulation factors. The carboxylase activity requires the epoxidation of vitamin K hydroquinone. VKOR regenerates this cofactor by reducing the epoxide, and this reductase activity is maintained by electron-transfer pathways. VKOR also has a paralog, VKORL, which has the same activity but is relatively insensitive to warfarin inhibition. Owing to differences in tissue distribution, VKOR primarily supports blood coagulation and VKORL likely supports non-coagulation processes. The objective of this application is to elucidate the mechanisms of VKOR and VKORL catalysis and vitamin K antagonism using our expertise in membrane structure biology. Our hypotheses are: (1) the narrow therapeutic window of warfarin is in part because it is a tight-binding inhibitor whose dose range is limited by VKOR levels; (2) the cellular activity of VKOR is maintained by alternative electron-transfer pathways; and (3) a common structural mechanism governs the warfarin insensitivity of VKORL and warfarin-resistant mutations in VKOR. To support these hypotheses, we have achieved a long-standing goal of determining the crystal structures of human VKOR with several VKAs and with the substrate, vitamin K epoxide, and have determined the structures of a VKORL homolog in its warfarin-bound and ligand-free states. We found distinct groups of warfarin-resistant mutations in VKOR, and identified key residues that control the warfarin sensitivity of VKORL. We also showed that warfarin is a tight-binding inhibitor in vitro. We will test our hypotheses with three specific aims: (1) we will show the tight binding of warfarin in a cellular environment, understand its correlation with VKOR's redox status, and test whether reducing VKOR can release bound warfarin; (2) we will determine how VKAs inhibit VKOR catalysis, elucidate the reduction steps and reaction intermediate of VKOR, and characterize the electron-transfer pathways that maintain VKOR activity; and 3) we will define the structural basis of warfarin resistance and investigate whether VKORL variations lead to osteoporosis. Armed with our recently developed structural tools, we will demonstrate innovative concepts about the inhibition range of VKAs, the catalytic pathway of VKOR, and mutations interfering with coagulation and bone health. Thus, the proposed studies will significantly advance our knowledge of VKOR function and its interaction with VKAs, leading to improved warfarin management.

维生素K循环支持血液凝固、骨矿化和血管钙稳态。一个关键 维生素K环氧化物还原酶（VKOR）是维生素K拮抗剂（VKA）的靶点。尽管 其广泛的临床应用，VKA的剂量（例如，华法林）很难调节，过量可导致致命的 流血了改善剂量调节需要了解VKA如何抑制VKOR，VKOR是一种膜- 嵌入的酶难以用结构和生物化学研究表征。我们的长期目标是 阐明整个维生素K循环的生理过程及其与VKA的相互作用。这个周期 从γ-羧化开始，这是维生素K依赖性蛋白质活性所需的修饰，包括 几种凝血因子。羧化酶活性需要维生素K对苯二酚的环氧化。VKOR 通过还原环氧化物再生该辅因子，并且该还原酶活性通过电子转移维持 途径。VKOR还有一个副药VKORL，具有相同的活性，但对华法林相对不敏感 抑制作用由于组织分布的差异，VKOR主要支持血液凝固，VKORL 可能支持非凝固过程。本申请的目的是阐明 VKOR和VKORL催化和维生素K拮抗利用我们在膜结构生物学的专业知识。我们 假设是：（1）华法林治疗窗狭窄的部分原因是它是一种紧密结合的抑制剂 其剂量范围受到VKOR水平的限制;（2）VKOR的细胞活性通过替代的 电子转移途径;和（3）共同的结构机制控制VKORL的华法林不敏感性 和华法林耐药突变。为了支持这些假设，我们实现了一个长期目标 用几种VKA和底物维生素K环氧化物测定人VKOR的晶体结构， 并已确定了在其华法林结合和无配体状态下的VKORL同系物的结构。我们发现 VKOR中不同的华法林耐药突变组，并确定了控制华法林的关键残基 VKORL的灵敏度。我们还表明，华法林是一种紧密结合的抑制剂在体外。我们将测试我们的假设 有三个具体的目标：（1）我们将显示华法林在细胞环境中的紧密结合，了解其 与VKOR的氧化还原状态的相关性，并测试是否还原VKOR可以释放结合华法林;（2）我们将 确定VKA如何抑制VKOR催化，阐明VKOR的还原步骤和反应中间体， 并表征维持VKOR活性的电子转移途径; 3）我们将定义结构 华法林耐药的基础上，并调查是否VKORL变异导致骨质疏松症。装备我们的 最近开发的结构工具，我们将展示有关VKA抑制范围的创新概念， VKOR的催化途径，以及干扰凝血和骨骼健康的突变。因此，拟议的 这些研究将大大提高我们对VKOR功能及其与VKA相互作用的认识， 改善华法林管理。