Vitamin K Oxidoreductase: Function and Physiology

描述（由申请人提供）：维生素K氧化还原酶（VKORC 1）是维生素K依赖性（VKD）蛋白功能所必需的，因为它提供了这些蛋白羧化和随后活化中所用的还原维生素K。VKORC 1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起作用。VKORC 1是用于控制止血的药物华法林的靶点，VKORC 1的突变导致严重出血，表明这种酶在止血中的关键作用。因此，了解VKORC 1机制很重要，但目前还没有明确的定义。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VKORC 1的创新方法，从而确定了VKORC 1激活中的电子中继途径。VKORC 1是一种完整的膜蛋白，我们发现VKORC 1残基位于膜外的环中，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，因为该环含有大量突变导致华法林耐药的残基。我们发现VKORC 1具有二聚体结构，因此这些突变可能与华法林耐药患者中的野生型VKORC 1以异源二聚体形式存在。我们发现，二聚体结构允许VKORC 1将维生素K环氧化物还原为醌，然后还原为VKD蛋白羧化中使用的氢醌形式。有趣的是，我们发现华法林抗性突变在支持VKD蛋白羧化方面受损，因为它们不能有效地进行两个反应步骤。因此，华法林抑制比以前认识到的更复杂。目的1提出了一个创新的假设，即与华法林耐药相关的残基通常促进活性，突变改变可能间接阻止华法林进入的功能。我们将通过测定VKORC 1的同源和异源二聚体的活性和华法林敏感性来检验这一假设。目的2将产生华法林抗性的小鼠模型，并测试华法林抗性突变体降低体内VKD蛋白羧化的假设，即使在不存在华法林的情况下。目的3将鉴定激活VKORC 1的氧化还原蛋白。这些研究对了解华法林治疗和提高VKD蛋白的产量以供临床应用具有重要意义。

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.