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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要饮食中的维生素K几乎在所有组织中都用于将glus簇转换为γ-羧化GLU（GLAS）在维生素K依赖性（VKD）蛋白中。羧化通过产生钙结合来激活VKD蛋白其功能所需的模块。鉴定出的第一个VKD蛋白是凝血因素，也有影响其他生理的信号传导作用（例如炎症）。肝外VKD蛋白也有其他调节钙化，生长控制，凋亡和信号转导。因此，定义GLA形成是了解VKD蛋白对人类健康和疾病的影响至关重要。一个伽玛 - 谷氨酸羧化酶通过氧化维生素K氢喹酮（KH2）产生GLA至环氧化物（KO）。 ko是然后由维生素K氧化还原酶（VKORC1）以两个步骤进行回收：从环氧化物到维生素K喹酮，然后然后喹酮到氢验。我们表明VKORC1形成了一个二聚体，对于完成这两个反应。 Vkorc1是华法林的目标，华法林是全球数百万人使用的药物血液凝结，例如用机械心脏瓣膜。我们做出了令人惊讶的发现，华法林 Uncouples正常KO降低，需要在治疗期间进行第二次还原酶以生成VKD的KH2 蛋白羧化。结果非常重要，因为肝外VKD蛋白可能差羧化和功能失调，如果第二个还原酶不像VKORC1一样普遍表达。我们证明了VKORC1二聚体对于KO回收至KH2很重要，并且我们最近的初步数据很重要建议VKORC1和羧化酶形成复合物。我们假设通过这些蛋白质蛋白相互作用促进有效的维生素K回收。一些VKORC1突变导致华法林抵抗，即高华法林剂量管理止血的要求，我们假设这些突变破坏了二聚体的完整性。天然发生的羧化酶突变会引起严重的出血，一些突变体在VKORC1-羧化酶相互作用中似乎有缺陷。此应用程序中的目标将定义使VKD蛋白羧化的蛋白质 - 蛋白质相互作用如此有效以及它们发挥的作用在华法林抑制中。 AIM 1将测试维生素K隔离是否通过识别VKORC1二聚化域并在CRISPR/CAS9中测试其功能已删除的细胞系用于内源性VKORC1。 AIM 2将测试VKORC1-羧化酶在维生素K中的重要性通过确定引起严重出血的人类羧化酶突变是否破坏正常维生素K回收，并通过研究羧化酶突变体模型中维生素K回收的效率。 AIM 3将检验以下假设：奎因酮还原酶与VKORC1不同支持VKD羧化在华法林治疗期间，通过测试候选还原酶，我们已经在细胞系模型中鉴定出来。成功的这些目标的完成将揭示有效的VKD蛋白羧化的机制，并提供华法林治疗的重要新信息。