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：
9885011
负责人：
DARREL W STAFFORD
金额：
$ 54.3万
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 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9885011
关键词：
Acids Active Sites Address Affect Anticoagulants Anticoagulation Binding Biological Biological Assay Biological Process Blood Coagulation Disorders Blood Coagulation Factor Blood coagulation Bone Development CRISPR/Cas technology Cell Line Cell Proliferation Cells Chemicals Clinical Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Process Collection Complement Complex Cysteine Data Disease Disease susceptibility Disputes Dose Enzymes Funding Generations Genes Genetic Code Genetic Variation Glutamic Acid Goals Hemorrhage Human Hydroquinones Knock-out Knowledge Libraries Mediating Modification Molecular Mutation Natural regeneration Noise Oral Osteocalcin Patients Physiological Physiological Processes Play Poisoning Protein C Proteins Prothrombin Public Health Reaction Recovery Reducing Agents Reporter Research Role Signal Transduction Structure-Activity Relationship Symptoms Technology Testing Therapeutic United States National Institutes of Health Vascular calcification Vitamin K Vitamin K Reductase Warfarin base carboxylation cell growth clinical phenotype cost crosslink design enzyme mechanism gamma-glutamyl carboxylase genome-wide glucose metabolism high throughput screening improved in vivo inhibitor/antagonist insight loss of function matrix Gla protein nanoluciferase novel novel therapeutics public health relevance reduced vitamin K screening unnatural amino acids vitamin K epoxide reductase vitamin therapy

项目摘要

Project Summary/Abstract The vitamin K cycle enzymes: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and vitamin K reductase (VKR) are responsible for the post-translational carboxylation of vitamin K-dependent (VKD) proteins into their biologically active forms. Carboxylation is mainly associated with blood coagulation, as four coagulation factors (factors II, VII, IX, and X) and three anticoagulant proteins (proteins C, S, and Z) require carboxylation for their function. With the discovery of new VKD proteins and their new biological functions, the importance of carboxylation has been expanded to vascular calcification, bone development, glucose metabolism, and cell proliferation. GGCX is the enzyme that directly modifies VKD proteins to their functional forms. Genetic variations in GGCX have been identified in patients with vitamin K-related disorders. However, it is not clear why some GGCX mutations cause bleeding disorders while other mutations result in non-bleeding symptoms. Additionally, one GGCX mutation can sometimes cause two distinct clinical phenotypes. However, it remains unclear as to why the administration of vitamin K can ameliorate one symptom but not the other. VKOR is a regulatory enzyme of the vitamin K cycle and the target of the oral anticoagulant, warfarin. The mechanism of VKOR's active site regeneration remains elusive. Recent studies have shown that superwarfarin poisonings (an anticoagulant more powerful than warfarin) are a growing public health concern and that vitamin K therapy for that is costly and inefficient. The enzyme that reduces vitamin K to its hydroquinone form to support VKD carboxylation is VKR. Despite decades of effort, the identity of VKR is still unknown. Our long-term research goal is to understand in detail the structure and function relationship of all vitamin K cycle enzymes within their native milieu for better control coagulation and other related physiological processes. The current proposal aims to solve the main questions remaining in the field (as mentioned above). To accomplish these goals, we propose the following specific aims: Specific Aim 1 - we will use our recently established CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 knockout reporter cell lines to study the effect of all currently identified naturally occurring GGCX mutations on the carboxylation of different VKD proteins associated with distinct clinical phenotypes; Specific Aim 2 - we will apply genetic code expansion technology to clarify VKOR's active site regeneration and design and synthesize novel vitamin K derivatives to rescue superwarfarin poisonings; and Specific Aim 3 - we will use the genome-wide CRISPR-Cas9 knockout library to screen for the unknown enzyme, VKR. We expect that information derived from these studies will help us understand how the three vitamin K cycle enzymes contribute to the complex mechanisms of carboxylation, thereby gaining new therapeutic insights into the control of blood coagulation and vascular calcification and improving therapies for vitamin K-related disorders.

项目概要/摘要维生素 K 循环酶：γ-谷氨酰羧化酶 (GGCX)、维生素 K 环氧化物还原酶 (VKOR) 和维生素 K 还原酶 (VKR) 负责维生素 K 依赖性 (VKD) 的翻译后羧化蛋白质转化为生物活性形式。羧化主要与血液凝固有关，如四种凝血因子（因子 II、VII、IX 和 X）和三种抗凝蛋白（蛋白 C、S 和 Z）需要羧化以发挥其功能。随着新 VKD 蛋白及其新生物学功能的发现，羧化的重要性已扩展到血管钙化、骨骼发育、葡萄糖新陈代谢和细胞增殖。 GGCX 是直接修饰 VKD 蛋白使其功能性的酶形式。已在患有维生素 K 相关疾病的患者中发现了 GGCX 的遗传变异。然而，它尚不清楚为什么某些 GGCX 突变会导致出血性疾病，而其他突变则导致不出血症状。此外，一种 GGCX 突变有时会导致两种不同的临床表型。然而，它目前尚不清楚为什么服用维生素 K 可以改善一种症状，但不能改善另一种症状。维科是维生素 K 循环的调节酶，也是口服抗凝剂华法林的靶标。机制 VKOR 的活性位点再生仍然难以捉摸。最近的研究表明，超级华法林中毒（一种比华法林更强大的抗凝剂）是一个日益严重的公共卫生问题，维生素 K 疗法因为这样做成本高昂且效率低下。将维生素 K 还原为对苯二酚形式以支持 VKD 的酶羧化是VKR。尽管经过数十年的努力，VKR 的身份仍然未知。我们的长期研究目标是详细了解所有维生素K循环酶在其天然体内的结构和功能关系更好地控制凝血和其他相关生理过程的环境。目前的提案旨在解决该领域遗留的主要问题（如上所述）。为了实现这些目标，我们建议具体目标如下：具体目标 1 - 我们将使用我们最近建立的 CRISPR（定期聚类） Interspaced Short Palindromic Repeats)-Cas9敲除报告细胞系研究目前所有的效果鉴定出自然发生的 GGCX 突变与与相关的不同 VKD 蛋白的羧化有关不同的临床表型；具体目标2——我们将应用遗传密码扩展技术来阐明VKOR的活性位点再生以及设计和合成新型维生素K衍生物以拯救超级华法林中毒；具体目标 3 - 我们将使用全基因组 CRISPR-Cas9 敲除文库来筛选未知酶，VKR。我们希望从这些研究中获得的信息将帮助我们了解如何三种维生素 K 循环酶有助于复杂的羧化机制，从而获得新的对控制血液凝固和血管钙化的治疗见解以及改进治疗维生素 K 相关疾病。