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：
10621711
负责人：
DARREL W STAFFORD
金额：
$ 50.57万
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 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10621711
关键词：
Acids Active Sites Address Affect Anticoagulants Anticoagulation Applied Genetics Binding Biological Assay Biological Process Blood Coagulation Disorders Blood Coagulation Factor Blood coagulation Bone Development Cell Line Cell Proliferation Cells 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 Structure-Activity Relationship Symptoms Technology Testing Therapeutic United States National Institutes of Health Vascular calcification Vitamin K Vitamin K Reductase Warfarin carboxylate carboxylation cell growth chemical synthesis clinical phenotype cofactor cost crosslink design enzyme mechanism gamma-glutamyl carboxylase genome-wide glucose metabolism high throughput screening improved in vivo inhibitor 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.

项目摘要/摘要维生素K循环酶：γ-谷氨酰基羧化酶（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进入其生物活性形式。羧化主要与血液凝血有关，为四个凝血因子（因子II，VII，IX和X）和三种抗凝蛋白（蛋白C，S和Z）需要羧化的功能。随着新的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，葡萄糖代谢和细胞增殖。 GGCX是将VKD蛋白直接修改为功能的酶表格。在维生素K相关疾病的患者中，已经发现了GGCX的遗传变异。但是，它尚不清楚为什么某些GGCX突变会引起出血性疾病，而其他突变导致非出血症状。另外，一个GGCX突变有时会引起两种不同的临床表型。但是，它尚不清楚为什么维生素K的给药可以减轻一种症状，而不能减轻另一种症状。 VKOR 是维生素K周期的调节酶，也是口服抗凝剂华法林的靶标。机制 VKOR的活跃现场再生仍然难以捉摸。最近的研究表明，超级战争中毒（一种比华法林更强大的抗凝剂）是日益增长的公共健康问题，维生素K疗法因为这是昂贵且效率低下的。将维生素K降低至其氢喹酮形式的酶以支持VKD 羧化为VKR。尽管努力了数十年，但VKR的身份仍然未知。我们的长期研究目标是详细了解其本地所有维生素K周期酶的结构和功能关系改善凝血和其他相关生理过程的环境。当前的提议旨在解决该领域中剩下的主要问题（如上所述）。为了实现这些目标，我们建议以下具体目标：特定目标1-我们将使用我们最近建立的CRISPR（定期聚类间隔短的短文重复序列）-CAS9敲除记者细胞系，以研究所有当前的效果鉴定出天然存在的GGCX突变，这些突变是在与不同的VKD蛋白的羧化上独特的临床表型；特定目标2-我们将应用遗传代码扩展技术来阐明VKOR 活跃的现场再生和设计并合成新型维生素K衍生物以挽救超级沃尔法林中毒；和特定的目标3-我们将使用全基因组CRISPR-CAS9敲除库筛选未知酶，VKR。我们希望从这些研究中得出的信息将有助于我们了解三种维生素K循环酶有助于羧化的复杂机制，从而获得了新的对控制血液凝结和血管钙化的控制的治疗见解，并改善了治疗疗法维生素K相关的疾病。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Molecular basis of the first reported clinical case of congenital combined deficiency of coagulation factors.

DOI：
10.1182/blood-2017-05-782367
发表时间：
2017-08
期刊：
Blood
影响因子：
20.3
作者：
D. Jin;Brian O Ingram;D. Stafford;J. Tie
通讯作者：
D. Jin;Brian O Ingram;D. Stafford;J. Tie

A genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase.

DOI：
10.1038/s41467-023-36446-8
发表时间：
2023-02-14
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Jin, Da-Yun;Chen, Xuejie;Liu, Yizhou;Williams, Craig M.;Pedersen, Lars C.;Stafford, Darrel W.;Tie, Jian-Ke
通讯作者：
Tie, Jian-Ke

Vitamin K epoxide reductase and its paralogous enzyme have different structures and functions.