Characterization of enzymes in the vitamin K cycle

 描述(由申请人提供)：维生素K依赖(VKD)羧化是由伽马-谷氨酰羧基酶(GGCX)催化的一种基本的翻译后修饰，是控制凝血、血管钙化、骨骼代谢和其他重要生理过程的蛋白质的生物学功能所必需的。伴随着羧化，还原的维生素K(Kh2)被氧化成维生素K环氧化物(KO)。KO必须通过维生素K环氧化物还原酶(VKOR)和维生素K还原酶(VKR)在称为维生素K循环的途径中循环回到Kh2。华法林是治疗血栓栓塞性疾病的最广泛的抗凝血剂，它通过抑制VKOR来损害功能性凝血因子的生物合成。尽管这一领域取得了重大进展，但基本的问题仍然存在：1)VKOR活性部位再生和华法林抑制的机制是什么？-2)VKR酶的特性是什么？-3)为什么GGCX的一些突变会导致出血性疾病，称为联合维生素K依赖凝血因子缺乏症(VKCFD)，而另一些突变与弹性假黄瘤(PXE)样综合征有关？当前提案的目标是识别和表征维生素K循环的组成部分，了解这些不同的组成部分如何在其自然环境中对VKD羧化起作用，并确定自然发生的GGCX突变如何对不同的疾病状态做出贡献。为了实现这些目标，我们提出了以下具体目标：目的1)利用最近建立的基于细胞的分析方法来研究VKOR及其辅酶VKORC1L1的结构与功能的关系；目的2)在我们的AV12和HEK293报告细胞系中，使用基因组编辑技术TALENS(转录激活器样效应核酸酶)和CRISPR(簇状规则间隔短回文重复序列)-Cas9来表征和鉴定VKR；目的3)建立一种基于细胞的分析方法，使用TALENS-冥想中敲除的我们HEK293报告细胞中的内源性GGCX基因来研究GGCX突变与VKCFD和PXE样综合征的关系。从这些研究中获得的信息将帮助我们了解不同的维生素K循环成分如何对这些复杂的机制做出贡献；从而获得对血栓形成控制和改进华法林治疗方法的新的治疗见解。

 DESCRIPTION (provided by applicant): Vitamin K-dependent (VKD) carboxylation, an essential post-translational modification catalyzed by gamma- glutamyl carboxylase (GGCX), is required for the biological function of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase (VKOR) and vitamin K reductase (VKR) in a pathway known as the vitamin K cycle. Warfarin, the most widely prescribed anticoagulant for thromboembolic disorders, works to impair the biosynthesis of functional clotting factors by inhibiting VKOR. Despite significant progress in the field, fundamental questions remain: 1) What are the mechanisms for VKOR active site regeneration and warfarin inhibition? - 2) What are the identities of the VKR enzymes? - 3) Why do some mutations of GGCX result in a bleeding disorder, named as combined vitamin K-dependent coagulation factors deficiency (VKCFD), while others are linked with Pseudoxanthoma elasticum (PXE)-like syndrome? The objectives of the current proposal are to identify and characterize the components of the vitamin K cycle, understand how these various components contribute to VKD carboxylation in their native milieu, and determine how naturally-occurring GGCX mutations contribute to different disease states. To accomplish these goals, we propose the following specific aims: Aim 1) To study the structure-function relationships of VKOR and its paralog enzyme VKORC1L1 using the recently established cell-based assay; Aim 2) To characterize and identify VKR using genome editing techniques TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 in our AV12 and HEK293 reporter cell lines; Aim 3) To establish a cell-based assay for the GGCX function study using the TALENs-meditated gene knockout of the endogenous GGCX gene in our HEK293 reporter cells - allowing us to study how GGCX mutations are related to VKCFD and PXE-like syndromes. Information derived from these studies will help us understand how the various vitamin K cycle components contribute to these complex mechanisms; thereby, gaining new therapeutic insights into the control of thrombosis and by improving warfarin therapy treatments.