Structure-Function Analysis of the Gamma-Carboxylase

超出所提供的空间。维生素K依赖/补充剂（VKD）在止血中起关键作用。羧化酶利用维生素K氧化的能量将Glu转化为Gla。肝脏中至少10种不同的VKD蛋白与内质网中的单个羧化酶接合以完全羧化（每分子9-12个Gla）。在正常条件下，完全羧化发生，然而如何调节该过程以实现如此显着的保真度尚不清楚。本研究建立了一种模拟正常生理过程的方法来研究蛋白质羧化，结果表明羧化酶的持续合成能力可以解释体内蛋白质的全面羧化过程，并为蛋白质羧化的机理提供了新的认识。在导致底物调节羧化的假设的研究中鉴定了羧化酶活性位点。当VKD蛋白在哺乳动物细胞中表达时，为什么carb 6xylation饱和的长期存在的问题得到解决，并显示了分泌机制在促进羧化中的重要性。发现了两种修饰，羧化酶羧化和磷酸化。长期目标是了解羧化的机制，包括多种VKD蛋白如何竞争组织中的一种羧化酶以完全羧化。为下一个资助期提出的具体目标是：1）确定活性位点如何促进羧化。如何羧化调节的假设将通过表征新发现的VKD蛋白-羧化酶相互作用的第二位点和确定维生素K如何被氧化以启动羧化来测试。2)明确VKD蛋白羧化的机制，验证维生素K的利用率、竞争性VKD蛋白和羧化酶羧化作用影响羧化酶持续合成能力的假设。3)确定羧化酶羧化和磷酸化如何调节羧化。这些修改调节分泌事件如何促进羧化的假设将通过分析对羧化酶运输，稳定性，伴侣缔合和fiX羧化的影响进行测试。这些研究将为理解羧化机制做出根本性贡献，这对于开发改进的抗凝剂，确定饮食和抗凝的变化如何影响止血以及产生治疗量的VKD蛋白质将是重要的。性能现场=

EXCEED THE SPACE PROVIDED. Vitamin K depe/adent (VKD) pr6teing r_-qtiire c_fb6X_l-ation f_iVity_ th_<Vlgcl)q2m'tyoxylase plays a critical role in hemostasis. The carboxylase converts Glu's to Gla's using the energy of vitamin K oxygenation. At least 10 different VKD proteins in liver engage a single carboxylase in the endoplasmic reticulum to become fully carboxylated (9-12 Gla's per molecule). Under normal conditions full carboxylation occurs, however how the process is regulated to achieve such remarkable fidelity is not known. A method that mimics the normal physiological process was developed to study protein carboxylation, which showed conclusively that carboxylase processivity can account for comprehensive carboxylation in vivo and als Oprovided new insights into the mechanism. The carboxylase active site was identified in studies that led to a hypothesis of substrate regulated carboxylation. _ong standing question of why carb6xylation is saturated when VKD proteins are expressed in mammalian cells was addressed and showed the importance of the secretory machinery in facilitating carboxylation. Two modifications, carboxylase carboxylation and phosphorylation, were discovered. The long term goal is to understand the mechanism of carboxylation, including how multiple VKD proteins compete for one carboxylase in tissue to become fully carboxylated. The specific aims proposed for the next funding period are to: 1) Determine how the active site facilitates carboxylation. The hypotheses for how carboxylation is regulated will be tested by characterizing a newly discovered second site of VKD protein-carboxylase interaction and determining how vitamin K is oxygenated to initiate carboxylation. 2) Define the mechanism of VKD protein carboxylation, The hypothesis that vitamin K availability, competing VKD proteins and carboxylase carboxylation affect carboxylase processivity will be tested. 3) Determine how carboxylase carboxylation and phosphorylation regulate carboxylation. The hypothesis that these modifications regulate how secretory events facilitate carboxylation will be tested by analyzing effects on carboxylase trafficking, stability, chaperone association and fiX carboxylation. These studies will make fundamental contributions towards understanding the mechanism of carboxylation, which will be important for developing improved anticoagulants, determining how changes in diet and anticoagulation affect hemostasis and producing therapeutic amounts of VKD proteins. PERFORMANCE SITE ========================================Section End===========================================