Vitamin K Oxidoreductase: Function and Physiology

维生素 K 氧化还原酶：功能和生理学

• 批准号: 8676994
• 负责人: KATHLEEN Lucile BERKNER
• 金额: $ 39.63万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-20 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8676994
• 关键词: Alleles; Anticoagulants; Apoptosis; Atrial Fibrillation; Binding; Biological; Blood coagulation; Calcium; Cells; Complex; Conflict (Psychology); Cytoplasm; Defect; Development; Diet; Dose; Drug Targeting; Electron Transport; Electrons; Endoplasmic Reticulum; Enzymes; Funding; Goals; Growth; Health; Hemorrhage; Hemostatic function; Homeostasis; Homo; Human; Hydroquinones; In Vitro; Integral Membrane Protein; Knockout Mice; Location; Membrane; Mus; Mutate; Mutation; Natural regeneration; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Patients; Pharmaceutical Preparations; Physiology; Play; Positioning Attribute; Predisposition; Process; Production; Proteins; Quinones; Rattus; Reaction; Reporting; Resistance; Role; Signal Transduction; Source; Structure; Sulfhydryl Compounds; Testing; Variant; Vitamin K; Warfarin; calcification; carboxylate; carboxylation; clinical application; dimer; disulfide bond; hydroquinone; improved; in vivo; innovation; insight; mouse model; mutant; novel strategies; oxidation; protein activation; protein function; public health relevance; reduced vitamin K; therapy development; thioredoxin reductase; vitamin K1 oxide

DESCRIPTION (provided by applicant): The vitamin K oxidoreductase (VKORC1) is required for the function of vitamin K-dependent (VKD) proteins, as it supplies the reduced vitamin K used in the carboxylation and consequent activation of these proteins. VKORC1 has broad biological impact, as VKD proteins function in hemostasis, calcium homeostasis, apoptosis, growth control, and signal transduction. VKORC1 is the target of the drug warfarin used to control hemostasis and mutations in VKORC1 cause severe bleeding, indicating the critical role of this enzyme in hemostasis. Understanding VKORC1 mechanism is therefore important, but at present is poorly defined. VKORC1 is inactivated during vitamin K reduction and requires reactivation by a redox protein. We developed an innovative approach for studying VKORC1, which led to the identification of an electron relay pathway in VKORC1 activation. VKORC1 is an integral membrane protein, and we showed that VKORC1 residues in a loop that resides outside of the membrane transfer electrons from a redox protein to membrane-embedded residues that reduce vitamin K. Revealing the importance of the extramembrane loop is significant, because the loop contains a large number of residues whose mutations cause warfarin resistance. We showed that VKORC1 has a dimeric structure, so these mutations likely exist as a heterodimer with wild type VKORC1 in warfarin resistant patients. We found that the dimeric structure allows VKORC1 to reduce vitamin K epoxide to quinone and then to the hydroquinone form used in VKD protein carboxylation. Interestingly, we found that warfarin resistant mutations are impaired in supporting VKD protein carboxylation because they cannot perform both reaction steps efficiently. Thus, warfarin inhibition is more complex than previously appreciated. Aim 1 proposes an innovative hypothesis that residues associated with warfarin resistance normally facilitate activity, and that mutations alter functions that may indirectly blok warfarin access. We will test this hypothesis by determining the activity and warfarin sensitivity of homo- and heterodimers of VKORC1. Aim 2 will generate a mouse model of warfarin resistance and test the hypothesis that a warfarin resistant mutant lowers VKD protein carboxylation in vivo, even in the absence of warfarin. Aim 3 will identify the redox protein that activates VKORC1. The proposed studies will be significant for understanding warfarin therapy and improving the production of VKD proteins for clinical applications.

描述（由申请人提供）：维生素 K 氧化还原酶 (VKORC1) 是维生素 K 依赖性 (VKD) 蛋白质功能所必需的，因为它提供还原的维生素 K，用于这些蛋白质的羧化和随后的激活。 VKORC1 具有广泛的生物学影响，因为 VKD 蛋白在止血、钙稳态、细胞凋亡、生长控制和信号转导中发挥作用。 VKORC1是用于控制止血的药物华法林的靶标，VKORC1的突变会导致严重出血，表明这种酶在止血中的关键作用。因此，了解 VKORC1 机制很重要，但目前定义不明确。 VKORC1 在维生素 K 还原过程中失活，需要通过氧化还原蛋白重新激活。我们开发了一种研究 VKORC1 的创新方法，从而确定了 VKORC1 激活中的电子中继途径。 VKORC1 是一种完整的膜蛋白，我们发现位于膜外的环中的 VKORC1 残基将电子从氧化还原蛋白转移到膜嵌入的残基，从而减少维生素 K。揭示膜外环的重要性意义重大，因为该环包含大量残基，这些残基的突变会导致华法林耐药。我们发现 VKORC1 具有二聚体结构，因此这些突变可能以与野生型 VKORC1 异二聚体的形式存在于华法林耐药患者中。我们发现二聚体结构允许 VKORC1 将维生素 K 环氧化物还原为醌，然后还原为用于 VKD 蛋白羧化的氢醌形式。有趣的是，我们发现华法林抗性突变在支持 VKD 蛋白羧化方面受到损害，因为它们无法有效地执行这两个反应步骤。因此，华法林的抑制作用比之前认识的更为复杂。目标 1 提出了一个创新假设，即与华法林耐药性相关的残基通常会促进活性，而突变会改变可能间接阻止华法林进入的功能。我们将通过测定 VKORC1 同二聚体和异二聚体的活性和华法林敏感性来检验这一假设。目标 2 将生成华法林抗性小鼠模型，并测试华法林抗性突变体即使在没有华法林的情况下也会降低体内 VKD 蛋白羧化的假设。目标 3 将识别激活 VKORC1 的氧化还原蛋白。拟议的研究对于了解华法林疗法和改善临床应用 VKD 蛋白的生产具有重要意义。