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: 10163694
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10163694
• 关键词: Affinity; Anticoagulants; Anticoagulation; Basic Science; Behavior; Binding; Biochemical; 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; bone health; carboxylation; cofactor; experience; improved; inhibitor/antagonist; innovation; insight; mouse model; novel; paralogous gene; prevent; reduced vitamin K; research study; resistance mutation; response; structural biology; 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循环支持血液凝血，骨矿化和血管钙稳态。钥匙 在此周期中，维生素K环氧还原（VKOR）是维生素K拮抗剂（VKAS）的靶标。尽管 它们广泛的临床用途，VKAS的剂量（例如Warfarin）很难调节，过量用药会导致致命 流血。改善剂量调节需要了解VKA如何抑制VKOR，这是一种膜 - 结构和生化研究很难表征的嵌入酶。我们的长期目标是 阐明整个维生素K周期的物理过程及其与VKA的相互作用。这个周期 从γ-羧化开始，维生素-K依赖性蛋白的活性所需的修改，包括 几个凝血因素。羧化酶活性需要维生素K氢验酮的环氧化。 VKOR 通过还原环氧化物来再生该辅因子，并且通过电子转移来维持这种还原活性 途径。 VKOR还有一个旁系同源物vkorl，它具有相同的活动，但对华法林不敏感 抑制。由于组织分布的差异，VKOR主要支持血液凝血和VKORL 可能支持非加密过程。该应用的目的是阐明 VKOR和VKORL催化和维生素K拮抗作用使用我们在膜结构生物学方面的专业知识。我们的 假设是：（1）华法林的狭窄治疗窗口部分是因为它是一个紧密的抑制剂 其剂量范围受VKOR水平的限制； （2）VKOR的细胞活性通过替代性维持 电子转移途径； （3）一种常见的结构机制控制Vkorl的华法林不敏感性 和VKOR中的抗华林突变。为了支持这些假设，我们实现了一个长期的目标 用几个VKA和底物，维生素K环氧化物确定人VKOR的晶体结构， 并确定了vkorl同源物在其与配体的状态下的结构。我们发现 VKOR中有不同的华法林的抗性突变，并确定了控制华法林的关键 Vkorl的灵敏度。我们还表明，华法林在体外是一种紧密结合的抑制剂。我们将检验我们的假设 以三个具体的目的：（1）我们将在细胞环境中显示华法林的紧密结合，了解其 与VKOR的氧化还原状态相关，并测试减少VKOR是否可以释放约束华法林； （2）我们会的 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 华法林抵抗的基础，并研究VKORL的变化是否导致骨质疏松症。与我们一起武装 最近开发的结构工具，我们将展示有关VKAS抑制范围的创新概念， VKOR的催化途径以及突变会干扰凝结和骨骼健康。那，提议 研究将大大提高我们对VKOR功能及其与VKA的相互作用的了解，从而导致 改进的华法林管理。