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：
9281897
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/9281897
关键词：
Active Sites Affect Alpha Cell 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 Epoxy Compounds 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 Oxides 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 protein function public health relevance reduced vitamin K screening tool vitamin K epoxide reductase vitamin K1 oxide vitamin therapy

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.

Description (Provied by Applicant): Vitamin K-Dependent (VKD) CARBOXYLATION Glutamyl Carboxylase (GGCX), is Required for the The Biological Functioning of ProtEINS THAT CAGULATION, VASCULAR CALCITION Abolism , And Other Important Physiological Processes. Concomitant with Carboxylation, Reduced Vitamin K （KH2）被氧化为维生素K环氧化物。假设维生素K还原酶（VKR）。因素缺乏（VKCFD）与pseudoxanthome弹性（PXE）类似综合征（2）VKR酶的身份是什么？维生素K的生理型成分下的VKOR循环目前的建议旨在识别和表征维生素K周期的未知成分，以了解各种成分在细胞环境中的VKD羧化如何促进这些目标。，我们提出了以下特定目的：使用CRISPR研究GGCX功能（群集经常间隔短的腔植物重复序列）-CAS9介导的GGCX敲除细胞X突变与VKCFD和PXE样综合症有关。在我们的HEK293记者细胞系中，使用基因组尺度的CRISPR-CAS9敲除功能筛选。复杂的机制改善了维生素K相关疾病的疗法。