Characterization of enzymes in the vitamin K cycle

 描述（由申请方提供）：维生素K依赖性（VKD）羧化是一种由γ-谷氨酰羧化酶（GGCX）催化的必需翻译后修饰，是控制凝血、血管钙化、骨代谢和其他重要生理过程的蛋白质的生物功能所必需的。伴随着羧化，还原的维生素K（KH 2）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必须在两步还原中转化回KH 2以完成维生素K循环;这种还原是通过酶维生素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循环中的酶的理解取得了重大进展，但基本问题仍然存在：1）为什么GGCX的一些突变会导致称为联合维生素K依赖性凝血因子缺乏症（VKCFD）的出血性疾病，而其他突变则与弹性假黄瘤（PXE）样综合征有关？2)VKR酶的特性是什么？3)VKOR活性位点再生的机制是什么？4)VKORC 1 L1是VKOR的旁系同源酶，在生理条件下有助于维生素K循环吗？目前的提案旨在鉴定和表征维生素K循环的未知组分，了解这些不同组分如何促进细胞环境中的VKD羧化，并确定天然存在的GGCX突变如何促进不同的疾病状态。为实现这些目标，我们提出以下具体目标。目标1：使用CRISPR（重复的规则间隔短回文重复序列）-Cas9介导的GGCX敲除细胞研究GGCX功能，以了解GGCX突变如何与VKCFD和PXE样综合征相关。目的2：在我们的HEK 293报告细胞系中使用基因组规模的CRISPR-Cas9敲除功能丧失筛选来表征和鉴定VKR。目的3：在我们的双基因敲除HEK 293报告细胞中表征VKOR、天然存在的VKOR突变体和VKORC 1 L1。从这些研究中获得的信息将帮助我们了解各种维生素K循环成分如何促进这些复杂的机制，从而获得控制血栓形成和改善维生素K相关疾病治疗的新见解。

 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.