Vitamin K Oxidoreductase: Function and Physiology

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）蛋白功能所必需的，因为它提供用于这些蛋白羧化和随后激活的还原维生素K。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起作用。VKORC1是华法林控制止血的靶点，VKORC1突变可导致严重出血，表明该酶在止血中的关键作用。因此，了解VKORC1机制很重要，但目前还没有明确定义。VKORC1在维生素K还原过程中失活，需要氧化还原蛋白重新激活。我们开发了一种创新的方法来研究VKORC1，从而确定了VKORC1激活的电子中继途径。VKORC1是一种完整的膜蛋白，我们发现位于膜外环中的VKORC1残基将电子从氧化还原蛋白转移到膜内嵌入的残基，从而降低维生素k。揭示膜外环的重要性是显著的，因为该环含有大量突变导致华法林抗性的残基。我们发现VKORC1具有二聚体结构，因此这些突变可能以异源二聚体的形式与野生型VKORC1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蛋白羧化的对苯二酚形式。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执行两个反应步骤。因此，华法林的抑制作用比以前认识到的更为复杂。Aim 1提出了一个创新的假设，即与华法林耐药性相关的残基通常促进活性，而突变改变了可能间接阻断华法林获取的功能。我们将通过测定VKORC1的同型和异型二聚体的活性和华法林敏感性来检验这一假设。Aim 2将建立华法林耐药小鼠模型，并验证华法林耐药突变体在体内即使在没有华法林的情况下也会降低VKD蛋白羧化的假设。目的3将鉴定激活VKORC1的氧化还原蛋白。所提出的研究将对了解华法林治疗和改善临床应用的VKD蛋白的产生具有重要意义。

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.