Structural and Functional Basis of the Vitamin K Cycle

维生素 K 循环的结构和功能基础

基本信息

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
项目类别：
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

项目摘要

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。该循环中的酶维生素 K 环氧化物还原酶 (VKOR) 是维生素 K 拮抗剂 (VKA) 的靶标。由于临床用途广泛，VKA（例如华法林）的剂量很难调节，过量服用会导致致命改善剂量调节需要了解 VKA 如何抑制 VKOR，VKOR 是一种膜- 我们的长期目标是难以通过结构和生化研究来表征的嵌入酶。阐明整个维生素 K 循环的生理过程及其与 VKA 的相互作用。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，包括多种凝血因子的羧化酶活性需要维生素 K 对苯二酚的环氧化。通过还原环氧化物来再生该辅因子，并且该还原酶活性通过电子转移来维持 VKOR 也有一个旁系同源物 VKORL，它具有相同的活性，但对华法林相对不敏感。由于组织分布的差异，VKOR 主要支持血液凝固，而 VKORL 则主要支持凝血。可能支持非凝血过程。该应用的目的是阐明其机制。利用我们在膜结构生物学方面的专业知识进行 VKOR 和 VKORL 催化和维生素 K 拮抗作用。假设是：(1) 华法林的治疗窗狭窄部分是因为它是一种紧密结合抑制剂 (2) VKOR的细胞活性由替代物维持电子转移途径；(3) 共同的结构机制控制 VKORL 的华法林不敏感性为了支持这些假设，我们已经实现了一个长期目标。确定人 VKOR 与几种 VKA 和底物维生素 K 环氧化物的晶体结构，并确定了 VKORL 同系物在华法林结合状态和无配体状态下的结构。 VKOR 中不同组的华法林耐药突变，并确定了控制华法林的关键残基我们还证明了华法林是一种体外紧密结合抑制剂。我们将测试我们的假设。具有三个具体目标：（1）我们将展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，并测试降低VKOR是否可以释放结合的华法林；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；华法林耐药性的基础并研究 VKORL 变异是否会导致骨质疏松症。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。研究将显着增进我们对 VKOR 功能及其与 VKA 相互作用的了解，从而改善华法林管理。