Mechanisms controlling the efficiency of hemostatic vitamin K-dependent protein activation

控制止血维生素 K 依赖性蛋白激活效率的机制

• 批准号: 10594567
• 负责人: KATHLEEN Lucile BERKNER
• 金额: $ 54.93万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594567
• 关键词: Affinity Chromatography; Anticoagulants; Apoptosis; Artificial Heart; Binding; Biological; Biological Assay; Blood Coagulation Factor; Blood coagulation; Bone Development; CRISPR/Cas technology; Calcium Binding; Cell Line; Cells; Complex; Data; Defect; Diet; Dimerization; Disease; Dose; Drug usage; Embryonic Development; Epoxy Compounds; Excretory function; Extrahepatic; Functional disorder; Growth; Growth and Development function; Health; Heart Valves; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; Hydroquinones; Inflammation; Ingestion; Mammalian Cell; Mediating; Metabolism; Modeling; Monitor; Mus; Mutation; Oxidoreductase; Persons; Pharmaceutical Preparations; Physiology; Platelet Activation; Play; Production; Proteins; Quinone Reductases; Quinones; Reaction; Recycling; Regulation; Resistance; Role; Signal Transduction; Testing; Therapeutic; Tissues; Ubiquitin; Vitamin K; Vitamin K 2; Warfarin; Yeasts; calcification; carboxylate; carboxylation; cell growth; dietary; dimer; gamma-glutamyl carboxylase; in vivo; mutant; mutant mouse model; oxidation; paralogous gene; protein activation; protein complex; protein protein interaction; reduced vitamin K; virtual; vitamin K1 oxide

Project Summary Dietary vitamin K is used in virtually all tissues to convert clusters of Glus to gamma-carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins. Carboxylation activates VKD proteins by generating a calcium-binding module required for their function. The first VKD proteins identified were coagulation factors, which also have signaling roles that impact other physiologies (e.g. inflammation). Additional extrahepatic VKD proteins also regulate calcification, growth control, apoptosis and signal transduction. Defining Gla formation is therefore essential for understanding the impact of VKD proteins on human health and disease. A single gamma- glutamyl carboxylase generates Gla by oxygenating vitamin K hydroquinone (KH2) to an epoxide (KO). KO is then recycled by the vitamin K oxidoreductase (VKORC1) in two steps: from epoxide to vitamin K quinone, and then quinone to hydroquinone. We showed that VKORC1 forms a dimer that is important in accomplishing these two reactions. VKORC1 is the target of warfarin, a drug used by millions of people worldwide to control blood clotting, for example with mechanical heart valves. We made the surprising discovery that warfarin uncouples normal KO reduction, necessitating a second reductase during therapy to generate KH2 for VKD protein carboxylation. The results are highly significant because extrahepatic VKD proteins may be poorly carboxylated and dysfunctional if the second reductase is not ubiquitously expressed like VKORC1. We showed that a VKORC1 dimer is important to KO recycling to KH2, and our recent preliminary data suggest that VKORC1 and the carboxylase form a complex. We hypothesize that vitamin K sequestration by these protein-protein interactions promotes efficient vitamin K recycling. Some VKORC1 mutations cause warfarin resistance, i.e. the requirement for higher warfarin doses to manage hemostasis, and we hypothesize that these mutations disrupt dimer integrity. Naturally occurring carboxylase mutations cause severe bleeding, and some mutants appear to be defective in VKORC1-carboxylase interaction. The aims in this application will define the protein-protein interactions that make VKD protein carboxylation so efficient and what role they play in warfarin inhibition. Aim 1 will test whether vitamin K sequestration mediates VKORC1 reduction by identifying VKORC1 dimerization domains and testing their function in CRISPR/Cas9 edited cell lines deleted for endogenous VKORC1. Aim 2 will test the importance of VKORC1-carboxylase association in vitamin K recycling by determining whether human carboxylase mutations that cause severe bleeding disrupt normal vitamin K recycling, and by studying the efficiency of vitamin K recycling in a carboxylase mutant mouse model. Aim 3 will test the hypothesis that a quinone reductase distinct from VKORC1 supports VKD carboxylation during warfarin therapy by testing candidate reductases we have identified in cell line models. Successful completion of these aims will reveal the mechanisms of efficient VKD protein carboxylation, and provide important new information for warfarin therapy.

项目概要 膳食维生素 K 几乎用于所有组织，将 Glus 簇转化为 γ-羧化 Glus (Glas) 维生素 K 依赖性 (VKD) 蛋白质。羧化通过产生钙结合激活 VKD 蛋白 其功能所需的模块。第一个发现的 VKD 蛋白是凝血因子，它也具有 影响其他生理学（例如炎症）的信号传导作用。其他肝外 VKD 蛋白也 调节钙化、生长控制、细胞凋亡和信号转导。因此，Gla 形成的定义是 对于了解 VKD 蛋白对人类健康和疾病的影响至关重要。单个伽马- 谷氨酰羧化酶通过将维生素 K 对苯二酚 (KH2) 氧化成环氧化物 (KO) 来生成 Gla。 KO 是 然后由维生素 K 氧化还原酶 (VKORC1) 分两步回收：从环氧化物到维生素 K 醌，以及 然后将醌转化为对苯二酚。我们证明了 VKORC1 形成的二聚体对于完成 这两个反应。 VKORC1 是华法林的靶点，华法林是一种全世界数百万人用来控制病情的药物。 血液凝固，例如机械心脏瓣膜。我们惊奇地发现华法林 解开正常的 KO 还原，在治疗期间需要第二种还原酶来生成 VKD 的 KH2 蛋白质羧化。结果非常重要，因为肝外 VKD 蛋白可能很难 如果第二种还原酶不像 VKORC1 那样普遍表达，则会发生羧基化和功能障碍。 我们表明 VKORC1 二聚体对于 KO 回收至 KH2 很重要，我们最近的初步数据 表明 VKORC1 和羧化酶形成复合物。我们假设维生素 K 的螯合是通过 这些蛋白质与蛋白质之间的相互作用促进了维生素 K 的有效回收。一些 VKORC1 突变导致 华法林耐药，即需要更高的华法林剂量来控制止血，我们假设 这些突变破坏了二聚体的完整性。自然发生的羧化酶突变会导致严重出血， 一些突变体似乎在 VKORC1-羧化酶相互作用方面存在缺陷。本申请的目标是 定义使 VKD 蛋白质羧化如此有效的蛋白质-蛋白质相互作用以及它们发挥的作用 在华法林抑制作用中。目标 1 将测试维生素 K 隔离是否介导 VKORC1 还原 识别 VKORC1 二聚化结构域并测试其在 CRISPR/Cas9 编辑删除的细胞系中的功能 对于内源性 VKORC1。目标 2 将测试 VKORC1-羧化酶关联在维生素 K 中的重要性 通过确定导致严重出血的人类羧化酶突变是否扰乱正常情况来回收 维生素 K 回收，并通过研究羧化酶突变小鼠模型中维生素 K 回收的效率。 目标 3 将检验以下假设：不同于 VKORC1 的醌还原酶支持 VKD 羧化 在华法林治疗期间，通过测试我们在细胞系模型中鉴定的候选还原酶。成功的 完成这些目标将揭示有效 VKD 蛋白羧化的机制，并提供 华法林治疗的重要新信息。