STRUCTURE/FUNCTION ANALYSIS OF THE GAMMA-CARBOXYLASE

描述(改编自调查人员摘要)：关键片段之一 对止血的调节机制缺乏了解。 其中维生素K依赖(VKD)凝血因子是羧化的。 羧化是一种翻译后修饰，蛋白质喜欢 凝血酶原、凝血因子IX和凝血因子VII需要活性。它涉及到 谷氨酰基团簇转化为γ-羧基谷氨酰基 需要维生素K对苯二酚并被抑制的反应中的残留物 由维生素K拮抗剂华法林抗凝。羧基酶活性 存在于大多数组织中的一种完整的膜蛋白中。 羧化的VKD蛋白也从骨骼和平滑中分离出来 肌肉。不同的VKD是否存在多种羧基酶 蛋白质或只有一种酶仍然是一个悬而未决的问题。这个 对VKD蛋白的羧化作用知之甚少。大多数研究都是 已经使用粗制微粒体制剂对小型合成 基于来自VKD蛋白的序列的肽类似物。这个 已分离出羧基酶cdna，但其编码的蛋白质尚未分离。 与其他已知的蛋白质有同源性，而且cdna几乎没有提供 关于羧基酶结构或功能的提示性信息。Dr。 伯克纳的实验室已经开发出一种新的模型系统来分析 重组VKD与羧基酶的胞内相互作用 哺乳动物细胞中的蛋白质。该系统允许隔离 用于体外分析的羧基酶-VKD蛋白酶底物复合体。Dr。 伯克纳还构建了过表达羧基酶的细胞系 现在可以分离出大量纯净的、有活性的人类羧基酶。 长期目标是了解羧化的机制和 羧基酶在多种生物系统中所起的作用。特定的 本授权期的目标是：1)分析 2)分析VKD蛋白的羧化机理。 确定羧化蛋白质分泌的限制步骤；3) 测试是否有多个羧基酶，或者是否有一个羧基酶修饰 所有VKD蛋白。这些实验的数据将解释为什么 VKD蛋白的羧化在体内是有限的，即使当羧基酶 是过度表达的，以及羧基酶是如何执行这种不寻常的，以及 关键的，反应。这些实验还将提供方法，用于 VKD蛋白的未来分离及其为设计VKD提供的信息 专门改变止血的抗凝血剂。

DESCRIPTION (Adapted from investigator's abstract): One of the key pieces missing in understanding the regulation of hemostasis is the mechanism by which the vitamin K-dependent (VKD) clotting factors are carboxylated. Carboxylation is a post-translational modification that proteins like prothrombin, factor IX and factor VII require for activity. It involves the conversion of clusters of glutamyl residues to gamma carboxyglutamyl residues in a reaction that requires vitamin K hydroquinone and is inhibited by the anticoagulant Warfarin, a vitamin K antagonist. Carboxylase activity resides in an integral membrane protein present in most tissues. Carboxylated VKD proteins have also been isolated from bone and smooth muscle. Whether there are multiple carboxylases for the different VKD proteins or only a single enzyme remains an open question. The carboxylation of VKD proteins is poorly understood. Most of the studies have been performed using crude microsomal preparations on small synthetic peptide analogs based on sequences derived from the VKD proteins. The carboxylase cDNA has been isolated, but the protein encoded by it does not share homology with other known proteins, and the cDNA provides little suggestive information about carboxylase structure or function. Dr. Berkner's lab has developed a novel, model system for analyzing the intracellular interaction of the carboxylase with individual recombinant VKD proteins in mammalian cells. This system allows for isolation of the carboxylase-VKD protein enzyme-substrate complex for in vitro analysis. Dr. Berkner has also constructed cell lines that over-express the carboxylase and can now isolate large amounts of pure, active human carboxylase. Long-term goals are to understand the mechanism of carboxylation and the role that the carboxylase plays in multiple biological systems. Specific aims for this grant period are to: 1) analyze the domain organization of the carboxylase; 2) analyze the mechanism of VKD protein carboxylation by identifying the limiting step in the secretion of carboxylated proteins; 3) test whether there are multiple carboxylases or if one carboxylase modifies all VKD proteins. Data from these experiments will elucidate why the carboxylation of VKD proteins is limited in vivo even when the carboxylase is overexpressed and how the carboxylase carries out this unusual, and critical, reaction. These experiments will also provide methods for the future isolation of VKD proteins and information for the design of anticoagulants that specifically alter hemostasis.