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

项目摘要 膳食维生素K用于几乎所有组织，将Glus簇转化为γ-羧基Glus（Glas） 维生素K依赖性（VKD）蛋白。羧基化通过产生钙结合蛋白激活VKD蛋白 其功能所需的模块。第一个鉴定的VKD蛋白是凝血因子，其也具有 影响其他生理学（例如炎症）的信号传导作用。另外的肝外VKD蛋白也 调节钙化、生长控制、凋亡和信号转导。因此，定义Gla形成是 这对于了解VKD蛋白对人类健康和疾病的影响至关重要。一个伽玛- 谷氨酰羧化酶通过将维生素K氢醌（KH 2）氧化成环氧化物（KO）来产生Gla。KO是 然后通过维生素K氧化还原酶（VKORC 1）在两个步骤中再循环：从环氧化物到维生素K醌， 然后醌转化为氢醌。我们发现VKORC 1形成二聚体，这在完成 这两种反应。VKORC 1是华法林的靶点，华法林是一种被全球数百万人用于控制 血液凝固，例如用机械心脏瓣膜。我们有了惊人的发现华法林 解偶联正常KO还原，在治疗期间需要第二种还原酶产生VKD的KH 2 蛋白质羧化结果是非常有意义的，因为肝外VKD蛋白可能很差， 如果第二还原酶不像VKORC 1那样普遍表达，则第二还原酶被羧化和功能障碍。 我们发现VKORC 1二聚体对于KO再循环为KH 2是重要的，我们最近的初步数据表明， 表明VKORC 1和羧化酶形成复合物。我们假设，维生素K的螯合作用， 这些蛋白质-蛋白质相互作用促进有效的维生素K再循环。一些VKORC 1突变导致 华法林抵抗，即需要更高的华法林剂量来管理止血，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， 并且一些突变体似乎在VKORC 1-羧化酶相互作用中有缺陷。本申请的目的将 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所起的作用 华法林抑制作用目标1将通过以下方式测试维生素K螯合是否介导VKORC 1减少： 鉴定VKORC 1二聚化结构域并测试它们在CRISPR/Cas9编辑的细胞系中的功能 内源性VKORC 1。目的2将测试VKORC 1-羧化酶联合在维生素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破坏正常的血液循环， 维生素K再循环，并通过研究羧化酶突变小鼠模型中维生素K再循环的效率。 目的3将检验与VKORC 1不同的醌还原酶支持VKD羧化的假设 通过测试我们在细胞系模型中鉴定的候选还原酶，成功 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。

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.