Structure and Function of the Gamma Carboxylase

描述（由申请人提供）：γ-谷氨酰羧化酶在维生素K依赖性（VKD）蛋白的Gla结构域中产生羧化Glus（Glas），使其在多种功能中具有活性，包括止血、钙化、细胞凋亡和生长控制。一个单一的羧化酶修饰所有的VKD蛋白，这种广泛的生理影响使得理解催化和调节机制至关重要。自然发生的羧化酶突变导致维生素K依赖性凝血因子缺乏症（VKCFD），这与严重出血有关。突变最近也被证明会导致第二种疾病，弹性假黄瘤（PXE），出血轻微，但钙化是有缺陷的。为什么羧化酶突变导致第二种疾病是未知的，我们对导致PXE的羧化酶突变的研究表明，VKD蛋白的Gla结构域的完全修饰存在缺陷。羧化酶突变导致PXE的观察结果突出了理解羧化如何完全影响人类健康的重要性。羧化在止血中的作用已得到充分证实;然而，羧化酶几乎在每个组织中表达，而非止血功能知之甚少。这些功能可能会受到影响，在许多患者治疗warcantine，抑制羧化。 我们在下一个资助期的目标是了解VKD蛋白羧化的机制，确定羧化酶突变如何导致两种不同的疾病，并开始定义羧化在不同组织中的作用。在目标1中，我们将测试一种新的假设或羧化酶如何启动催化，并确定两种天然存在的羧化酶突变如何导致VKCFD。目的2将确定如何欠羧化影响PXE使用一种新的方法，我们开发的质谱分析VKD蛋白。目标2还将使用一种创新的小鼠模型来确定羧化酶是否在软组织钙化中起作用。羧化酶含有Glas，功能未知，我们现在已经确定了Gla结构域。在目标3中，我们将使用诱变方法来测试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蛋白被羧化，作为影响羧化的分泌过程的一部分，Aim 4将测试VKD蛋白在细胞中的作用。 介导VKD蛋白运输的组分。这些研究将提供新的基本信息，重要的机制为基础的抑制剂，导致上级抗凝剂的发展，也将是重要的治疗性VKD蛋白的生产应用于血友病，败血症和组织胶。

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.