New Vistas in Vitamin K Metabolism

描述(由申请人提供)：这项研究的长期目标是确定作为维生素K合成代谢和分解代谢基础的综合代谢事件网络，以了解受试者之间对这一基本膳食营养的反应差异。在这一新的应用中，我们将建立P450酶，CYP4F2和CYP4F11作为关键的维生素K羟基酶，启动这一基本膳食成分的分解代谢。此外，我们还将 确定新发现的人类UBDIA1酶在维生素K1和K2相互转化中的作用。这些努力将建立核心维生素K循环蛋白和辅助酶之间的联系，这些辅助酶调节驱动GLA蛋白生产的氢醌辅助因子的可用性。由于在高度脆弱的缺钾人群(如慢性肾脏疾病患者、癌症患者)中，补充维生素K的反应存在高度的个体间差异，这些研究具有很高的意义。这项研究的目标有三个方面：(I)建立人类细胞色素P4F2和细胞色素P4F11在启动维生素K分解代谢中的相对作用；(Ii)确定UBIAD1在维生素K1向K2(MK-4)转化中的作用和作用的化学机制；(Iii)确定这三个基因中的每个基因的共同遗传变异对人体组织和日益复杂的细胞模型中维生素K稳态的影响，作为未来体内研究的先导。在初步研究中，我们已经完成了三种靶酶的异源表达，以及建立了高灵敏和特异的串联MS分析各种生物基质中维生素K的重要任务。有了这些工具，我们建议在Aim1中评估细胞色素P4F2和细胞色素P4F11在维生素K分解代谢中的作用。在目标2中，我们将表达和纯化人UBIAD1，以明确评估该酶将维生素K1转化为K3的催化能力和化学机制。在目标3中，我们将使用储存的、基因分型的人类肝组织和一个新的工程细胞系统来分析GLA蛋白的产生，以确定这些新描述的维生素K循环相关基因中的共同遗传变异对细胞维生素K动态平衡的影响。

DESCRIPTION (provided by applicant): The long-term aim of this research is to define the network of integrated metabolic events that underlie vitamin K anabolism and catabolism, in order to understand inter-subject variability in response to this essential dietary nutrient. In ths new application, we will establish the P450 enzymes, CYP4F2 and CYP4F11 as the key vitamin K hydroxylases that initiate catabolism of this essential dietary constituent. In addition, we will determine the role of the newly identified human UBDIA1 enzyme in the interconversion of vitamins K1 and K2. These efforts will establish the link between the core vitamin K cycle proteins and ancillary enzymes that modulate the availability of the hydroquinone cofactors that drive Gla protein production. Because there is a high degree of inter-individual variability in response to supplementation with vitamin K in highly vulnerable K-deficient populations (e.g. those suffering from chronic kidney disease, cancer patients), these studies are of high significance. The goals of this research proposal are three-fold, (i) to establish the relative roes of human CYP4F2 and CYP4F11 in the initiation of vitamin K catabolism, (ii) to establish the role and chemical mechanism of action of UBIAD1 in conversion of vitamin K 1 to K2 (MK-4), and (iii) to ascertain the influence of common genetic variation in each of these three genes on vitamin K homeostatis in human tissues and cellular models of increasing complexity, as a precursor to future in vivo studies. In preliminary studies, we have accomplished the important tasks of heterologous expression of each of the three target enzymes, and development of highly sensitive and specific tandem MS assays for analysis of vitamin K in a variety of biological matrices. With these tools in hand, we propose, in Aim1, to evaluate the roles of CYP4F2 and CYP4F11 in vitamin K catabolism. In Aim 2, we will express and purify human UBIAD1 to enable an unambiguous assessment of the enzyme's catalytic capability and chemical mechanism for conversion of vitamin K1 to K3. In Aim 3, we will use banked, genotyped human liver tissue and a novel engineered cell system for analysis of Gla protein production to determine the effect of common genetic variation in these newly described vitamin K cycle-associated genes on cellular vitamin K homeostasis.