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)。由于人类不能合成维生素K，KO必须经过两步还原才能完成维生素K循环；这个还原是由维生素K环氧化物还原酶(VKOR)和我们假设的维生素K还原酶(VKR)完成的。尽管对维生素K循环中的酶的了解取得了重大进展，但基本的问题仍然存在：1)为什么GGCX的一些突变会导致称为联合维生素K依赖凝血因子缺乏症(VKCFD)的出血性疾病，而另一些突变与弹性假黄瘤(PXE)样综合征有关？2)VKR酶的身份是什么？3)VKOR活性部位再生的机制是什么？4)VKOR的同源酶VKORC1L1在生理条件下对维生素K循环有贡献吗？目前的建议旨在识别和表征维生素K循环的未知成分，了解这些不同的成分如何在细胞环境中对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。为实现这些目标，我们提出了以下具体目标。目的：利用CRISPR-Cas9介导的GGCX基因敲除细胞研究GGCX的功能，以了解GGCX突变与VKCFD和PXE样综合征的关系。目的：通过对HEK293报告细胞系进行CRISPR-Cas9基因敲除功能缺失筛查，鉴定VKR。目的3：在我们的双基因敲除的HEK293报告细胞中鉴定VKOR、自然发生的VKOR突变体和VKORC1L1。从这些研究中获得的信息将帮助我们了解各种维生素K循环成分如何对这些复杂的机制做出贡献，从而获得控制血栓形成和改进维生素K相关疾病的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 functioning 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). Since humans cannot synthesize vitamin K, KO must be converted back into KH2 in a two-step reduction to complete the vitamin K cycle; this reduction is accomplished by the enzyme vitamin K epoxide reductase (VKOR) and, as we hypothesize, vitamin K reductase (VKR). Despite significant progress in understanding of the enzymes in the vitamin K cycle, fundamental questions remain: 1) Why do some mutations of GGCX result in the bleeding disorder referred to as combined vitamin K-dependent coagulation factors deficiency (VKCFD), while others are linked with Pseudoxanthoma elasticum (PXE)-like syndrome? 2) What are the identities of the VKR enzymes? 3) What is the mechanism for VKOR active site regeneration? 4) Does VKORC1L1, the paralogous enzyme of VKOR, contribute to the vitamin K cycle under physiological conditions? The current proposal aims to identify and characterize the unknown components of the vitamin K cycle, to understand how these various components contribute to VKD carboxylation in the cellular milieu, and to 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 GGCX function using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated GGCX knockout cells, in order to understand how GGCX mutations are related to VKCFD and PXE-like syndromes. Aim 2: To characterize and identify VKR using genome-scale CRISPR-Cas9 knockout loss-of-function screening in our HEK293 reporter cell line. Aim 3: To characterize VKOR, naturally-occurring VKOR mutants, and VKORC1L1 in our double-gene knockout HEK293 reporter cells. 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 improving therapies for vitamin K-related disorders.