Structure-Function Analysis of the Gamma Carboxylase

描述(申请人提供)：维生素K依赖(VKD)羧基酶对止血至关重要，因为它将VKD蛋白质中的谷氨酸转化为羧化的谷氨酸，使其与止血发生的细胞表面结合。VKD蛋白在分泌过程中在内质网上被羧化，一个单一的羧化酶修饰所有VKD蛋白，其中许多蛋白在组织中共表达。在之前的资助期间，我们开发了一种直接分析哺乳动物细胞内羧化的方法，该方法表明分泌过程影响羧化，细胞内处理并不是所有VKD蛋白都相同的，并且羧化受Glu羧化所需的减少的维生素K辅因子的可用性调节。研究还表明，VKD转化的限速步骤在细胞内与体外反应中不同，这可能是由于羧基酶的翻译后修饰。其他研究表明，引起钩端螺旋体病的细菌病原体钩端螺旋体含有VKD羧基酶的同源基因，这种酶似乎是通过水平基因转移获得的，并已被调整为羧化以外的角色。我们的研究还涉及到新的功能性羧基酶残基，包括启动羧化的催化碱基和替代导致VKD凝血因子缺乏症的残基。我们的长期目标是了解羧化的机制，包括它是如何与分泌机制结合的，以及多种VKD蛋白是如何被一个羧化酶修饰而变得完全羧化和活性的。我们建议：1.确定活性部位如何促进羧化。我们将确定启动羧化的催化碱基，并将确定羧基酶残基的取代如何导致组合VKD因子缺乏症。2.确定VKD蛋白的转换率是否不同，是否受VKD蛋白-羧基酶相互作用的第二个位点的影响。我们将确定VKD蛋白因子X和凝血酶原是否以相同的效率进行羧化，以及VKD蛋白-羧基酶的两个位点相互作用是否会影响效率。3.为了验证我们的假设，即翻译后羧基酶修饰对VKD蛋白周转很重要。将识别和突变羧基酶中的翻译后修饰位点，以确定它们是否影响VKD蛋白的周转。这些研究将对了解羧化作用做出重要贡献，这将对开发优良的抗凝血剂和生产用于治疗血友病和败血症的VKD蛋白具有重要意义。保持抽象。饮食中的维生素K被用来激活一系列对凝血至关重要的因子，因此了解它们是如何被激活的是很重要的。这些研究将影响用于治疗血友病和感染性休克的抗凝血剂的开发和治疗性蛋白的生产。

DESCRIPTION (provided by applicant): The vitamin K-dependent (VKD) carboxylase is critical to hemostasis because it converts Glus to carboxylated Glus in VKD proteins to allow their binding to cell surfaces where hemostasis occurs. VKD proteins are carboxylated in the endoplasmic reticulum during their secretion, and a single carboxylase modifies all VKD proteins, many of which are coexpressed in tissue. In the previous grant period, we developed an approach to directly analyze intracellular carboxylation in mammalian cells, which showed that the secretory process impacts carboxylation, that intracellular processing is not identical for all VKD proteins and that carboxylation is regulated by the availability of the reduced vitamin K cofactor required for Glu carboxylation. The studies also showed that the rate-limiting step in VKD turnover is different in cells than in an in vitro reaction, which may be due to post-translational modifications in the carboxylase. Other studies revealed that Leptospira, the bacterial pathogen that causes leptospirosis, contains an ortholog of the VKD carboxylase, which appears to have been acquired by horizontal gene transfer and to have been adapted for a role other than carboxylation. Our studies also implicated novel functional carboxylase residues, including the catalytic base that initiates carboxylation and residues whose substitution cause combined VKD coagulation factor deficiency. Our long-term goal is to understand the mechanism of carboxylation, including how it interfaces with the secretory machinery and how multiple VKD proteins are modified by one carboxylase to become fully-carboxylated and active. We propose: 1. To determine how the active site facilitates carboxylation. We will identify the catalytic base that initiates carboxylation and will determine how substitutions in carboxylase residues cause combined VKD factor deficiency. 2. To determine if turnovers of VKD proteins differ and are impacted by a second site of VKD protein-carboxylase interaction. We will determine whether the VKD proteins factor X and prothrombin are carboxylated with equal efficiencies, and whether having two sites of VKD protein-carboxylase interaction impacts efficiency. 3. To test our hypothesis that post-translational carboxylase modifications are important to VKD protein turnover. Sites of post-translational modification in the carboxylase will be identified and mutated to determine if they impact VKD protein turnover. These studies will make important contributions to understanding carboxylation, which will be significant for developing superior anticoagulants and for producing VKD proteins for therapies in hemophilia and sepsis. Lay abstract. Vitamin K in the diet is used to activate a set of factors critical to blood clotting, and therefore it is important to understand how they become activated. The studies will impact the development of anticoagulants and the production of therapeutic proteins for treating hemophilia and septic shock.