Structure-Function Analysis of the Gamma-Carboxylase

超出提供的空间。维生素K依赖性(VKD)在止血中起重要作用。羧基酶利用维生素K氧化的能量将谷氨酸转化为谷氨酸。肝脏中至少有10种不同的VKD蛋白与内质网中的一种单一的羧化酶结合，使其完全羧化(每个分子9-12个GLA)。在正常情况下，会发生完全的羧化作用，然而，这个过程是如何调节的，以达到如此显著的保真度，目前尚不清楚。建立了一种模拟正常生理过程的方法来研究蛋白质的羧化作用，结果表明，羧化酶的活性可以解释体内的全面羧化作用，ALS O为研究其作用机制提供了新的见解。在导致底物调节的羧化假说的研究中，确定了羧基酶的活性部位。解决了为什么当VKD蛋白在哺乳动物细胞中表达时，C6x化是饱和的这一长期存在的问题，并表明了分泌机制在促进羧化方面的重要性。发现了两种修饰，即羧化酶羧化和磷酸化。长期目标是了解羧化的机制，包括多个VKD蛋白如何竞争组织中的一个羧基酶使其完全羧化。为下一个资金阶段提出的具体目标是：1)确定活性部位如何促进羧化。通过鉴定新发现的VKD蛋白-羧基酶相互作用的第二个位点，并确定维生素K是如何被氧化来启动羧化的，将检验如何调节羧化的假说。2)明确VKD蛋白的羧化机制，即维生素K的可获得性、竞争性的VKD蛋白和羧基酶的羧化作用影响羧基化酶活性的假说将得到检验。3)确定羧化酶羧化和磷酸化是如何调节羧化的。这些修饰调节分泌事件如何促进羧化的假设将通过分析对羧基酶运输、稳定性、伴侣结合和固定羧化的影响来检验。这些研究将有助于了解羧化作用的机制，这将对开发改进的抗凝剂、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蛋白具有重要意义。表演网站========================================Section End===========================================

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===========================================