Characterization of enzymes in the vitamin K cycle

项目摘要/摘要 维生素K循环酶：γ-谷氨酰羧基酶(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(VKD)的翻译后羧化。 蛋白质转化为具有生物活性的形式。羧化作用主要与凝血有关，如四种 凝血因子(因子II、VII、IX和X)和三种抗凝蛋白(蛋白质C、S和Z)需要 为它们的功能进行了羧化。随着新的VKD蛋白及其新的生物学功能的发现， 羧化的重要性已经扩展到血管钙化、骨骼发育、葡萄糖 新陈代谢和细胞增殖。GGCX是一种直接将VKD蛋白修饰成其功能的酶 表格。已在维生素K相关疾病患者中发现了GGCX基因变异。然而，它 目前尚不清楚为什么一些GGCX突变会导致出血性疾病，而其他突变会导致非出血性疾病 症状。此外，一个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，而不能改善另一种症状。VKOR 是维生素K循环的一种调节酶，也是口服抗凝剂华法林的靶标。这一机制 VKOR活跃的站点再生仍然难以捉摸。最近的研究表明，超级华法林中毒 (一种比华法林更有效的抗凝剂)是一个日益严重的公共卫生问题，而维生素K疗法 因为这既昂贵又低效。一种将维生素K还原为对苯二酚形式以支持VKD的酶 羧化反应是VKR。尽管经过几十年的努力，VKR的身份仍然不为人知。我们的长期研究目标 是详细了解其天然体内所有维生素K循环酶的结构和功能关系 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 以下具体目标：具体目标1--我们将使用我们最近建立的CRISPR(定期分组 间隔短回文重复序列)-Cas9基因敲除报告细胞系研究目前ALL 不同VKD蛋白的羧化相关的自然发生的GGCX突变 独特的临床表型；特定目标2-我们将应用遗传密码扩展技术来阐明VKOR 抗超级华法林活性部位再生及其新型维生素K衍生物的设计合成 和特定目标3-我们将使用全基因组的CRISPR-Cas9基因敲除文库来筛选 未知的酶，VKR。我们预计，从这些研究中获得的信息将帮助我们了解 三种维生素K循环酶参与了复杂的羧化机制，从而获得了新的 对控制凝血和血管钙化的治疗见解和改进治疗 维生素K相关疾病。

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.