Structure and Function of the Gamma Carboxylase

描述(申请人提供)：γ-谷氨酰羧基酶在维生素K依赖(VKD)蛋白的GLA结构域中产生羧化谷氨酸(GLAS)，使其在包括止血、钙化、细胞凋亡和生长控制在内的多种功能中活跃。一个单一的羧基酶修饰所有的VKD蛋白，这种广泛的生理影响使得理解催化和调节机制变得至关重要。自然产生的羧化酶突变会导致维生素K依赖凝血因子缺乏症(VKCFD)，这与严重出血有关。最近，突变也被证明会导致第二种疾病，弹性假黄色瘤(PXE)，出血轻微，但钙化有缺陷。为什么羧酶突变导致第二种疾病尚不清楚，我们对导致PXE的羧基酶突变的研究表明，完全修改VKD蛋白的GLA结构域存在缺陷。羧化酶突变导致PXE的观察突显了了解羧化如何充分影响人类健康的重要性。羧化在止血中的作用已经得到了很好的证实；然而，几乎每个组织都表达了羧化酶，而且对其非止血功能的了解很少。在接受华法林治疗的许多患者中，这些功能可能会受到影响，华法林可抑制羧化。 我们在下一个资金阶段的目标是了解VKD蛋白羧化的机制，确定羧基酶突变如何导致两种不同的疾病，并开始确定羧化在不同组织中的作用。在目标1中，我们将测试一个新的假设，即羧基酶是如何启动催化的，并将确定两个自然发生的羧基酶突变如何导致VKCFD。目的2将使用我们开发的一种新的方法来分析VKD蛋白的质谱学，以确定低羧化对PXE的影响。Aim 2还将使用一种创新的小鼠模型来确定羧基酶是否在软组织钙化中发挥作用。羧基酶含有功能未知的GLAS，我们现在已经确定了GLA结构域。在目标3中，我们将使用突变方法来验证GLA结构域调节VKD蛋白羧化的假设，并将使用FRET来确定GLA结构域是否促进磷脂相互作用。VKD蛋白是作为影响羧化的分泌过程的一部分而被羧化的，目标4将测试细胞的作用。 在调节VKD蛋白运输中的组成部分。这些研究将为基于机制的抑制剂的开发提供重要的新的基础信息，从而获得更好的抗凝血剂，也将对生产用于血友病、败血症和组织胶的治疗性VKD蛋白具有重要意义。

DESCRIPTION (provided by applicant): The gamma-glutamyl carboxylase generates carboxylated Glus (Glas) in the Gla domain of vitamin K- dependent (VKD) proteins to render them active in a diverse range of functions that includes hemostasis, calcification, apoptosis and growth control. A single carboxylase modifies all VKD proteins, and this broad physiological impact makes it critical to understand the mechanisms of catalysis and regulation. Naturally occurring carboxylase mutations cause vitamin K-dependent clotting factor deficiency (VKCFD), which is associated with severe bleeding. Mutations have recently also been shown to cause a second disease, pseudoxanthoma elasticum (PXE), where bleeding is mild but calcification is defective. Why carboxylase mutations cause this second disease was unknown, and our studies on a carboxylase mutation that causes PXE indicate a defect in fully modifying the Gla domain of VKD proteins. The observation that carboxylase mutations cause PXE highlights the importance of understanding how carboxylation fully impacts human health. The role of carboxylation in hemostasis is well established; however, the carboxylase is expressed in virtually every tissue and nonhemostatic functions are very poorly understood. These functions are likely to be affected in the many patients treated with Warfarin, which inhibits carboxylation. Our goals in the next funding period are to understand the mechanism of VKD protein carboxylation, determine how carboxylase mutations cause two different diseases, and begin defining the role of carboxylation in different tissues. In Aim 1, we will test a novel hypothesis or how the carboxylase initiates catalysis, and will also determine how two naturally occurring carboxylase mutations cause VKCFD. Aim 2 will determine how undercarboxylation impacts PXE using a novel method we developed to analyze VKD proteins by mass spectrometry. Aim 2 will also use an innovative mouse model to determine whether the carboxylase plays a role in calcification in soft tissue. The carboxylase contains Glas, of unknown function, and we have now identified the Gla domain. In Aim 3, we will use a mutagenesis approach to test the hypothesis that the Gla domain regulates VKD protein carboxylation, and will use FRET to determine if the Gla domain facilitates phospholipid interaction. VKD proteins are carboxylated as part of a secretory process that impacts carboxylation, and Aim 4 will test the role of cellular components in mediating VKD protein trafficking. These studies will provide new fundamental information important for the development of mechanism-based inhibitors that lead to superior anticoagulants, and will also be significant for the production of therapeutic VKD proteins for applications in hemophilia, sepsis and tissue glues.