Characterization of enzymes in the vitamin K cycle

维生素 K 循环中酶的表征

基本信息

批准号：
9078285
负责人：
DARREL W STAFFORD
金额：
$ 47.93万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9078285
关键词：
Active Sites Affect Anticoagulants Back Biological Assay Biological Process Blood Coagulation Disorders Blood coagulation Cell Line Cell surface Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Factor Deficiency Complex Cysteine Data Disease Enzymes Fluorescence-Activated Cell Sorting Funding Generations Genes Goals Hemostatic function Hereditary Disease Human Individual Knock-out Knowledge Lead Libraries Link Mediating Missense Mutation Molecular Monitor Mutation Natural regeneration Pathway interactions Patients Physiological Physiological Processes Post-Translational Protein Processing Production Proteins Pseudoxanthoma Elasticum Reducing Agents Reporter Reporting Resistance Role Signal Transduction Single Nucleotide Polymorphism Structure Structure-Activity Relationship Syndrome Testing Thrombosis Variant Vascular calcification Vitamin K Vitamin K Reductase Warfarin base bone metabolism carboxylate carboxylation cardiovascular risk factor clinical phenotype cofactor design dosage enzyme mechanism extracellular fluindione gamma-glutamyl carboxylase genome-wide improved in vivo insight knockout gene loss of function matrix Gla protein mutant novel therapeutics paralogous gene public health relevance reduced vitamin K screening tool vitamin K epoxide reductase vitamin K1 oxide

项目摘要

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.

描述（由申请人提供）：维生素 K 依赖性 (VKD) 羧化是一种由 γ-谷氨酰羧化酶 (GGCX) 催化的重要翻译后修饰，是控制血液凝固、血管钙化、骨代谢的蛋白质的生物功能所必需的以及其他重要的生理过程，还原的维生素 K (KH2) 被氧化为维生素 K 环氧化物 (KO)。人类无法合成维生素 K，KO 必须通过两步还原转化回 KH2，才能完成维生素 K 循环；这种还原是通过 K 环氧化物维生素还原酶 (VKOR) 和维生素 K 还原酶来完成的（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 的某些突变会导致被称为复合维生素 K 依赖性凝血因子缺乏症的出血性疾病。 (VKCFD)，而其他则与弹性假黄瘤 (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？ VKOR，在生理条件下对维生素 K 循环有贡献吗？目前的提案旨在识别和表征维生素 K 循环的未知成分，以了解这些不同成分如何促进维生素 K 循环。细胞环境中的 VKD 羧化，并确定自然发生的 GGCX 突变如何导致不同的疾病状态。为了实现这些目标，我们提出以下具体目标：使用 CRISPR（成簇规则间隔短回文重复序列）研究 GGCX 功能。 )-Cas9 介导 GGCX 敲除细胞，以了解 GGCX 突变如何与 VKCFD 和 PXE 样综合征相关。目标 2：在我们的 HEK293 报告细胞系中使用基因组规模的 CRISPR-Cas9 敲除功能缺失筛选来表征和鉴定 VKR 目标 3：在我们的双基因敲除 HEK293 报告细胞系中表征 VKOR、天然存在的 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 循环成分如何促成这些复杂的机制，从而获得控制血栓形成的新治疗见解并改进维生素疗法K 相关疾病。