REGULATION & BIOSYNTHESIS OF VITAMIN K-DEPENDENT FACTORS

这个项目的总体目标是加深对 转录和翻译后加工中涉及的元素 依赖维生素K的凝血因子。使用人类因素X作为 研究范式，这里提出的实验将(1)确定顺式和 因子X表达所需的反式调节元件。使用 足迹分析、凝胶移位和基因突变，然后再转染法 用报告基因检测功能转录因子的位置 将确定F.X启动子内的(Tf)结合位点。装订 将寻找几种肝脏特异性转铁蛋白(HNF-3和C/EBP)的位点，并 共转染实验用来确定这些转铁蛋白对细胞的影响 FX发起人。这些发现将被用来探索另外两个现象， 血友病B-Leyden的病理生理及凝血模式 因子在HepG2细胞中的表达。(2)我们会探讨 这些蛋白质中的信号序列。分泌物的信号序列 蛋白质的功能是将这些蛋白质定位于内质网。 我们已经描述了一个因子X变体(因子X Santo Domingo)，其中 信号序列的突变会导致严重的凝血因子X缺乏。我们 建议进行进一步的实验来确定细胞内的 这种突变蛋白质的命运。在描述这种变种的过程中， 我们首次建立了信号肽(SP)的裂解 在F.X。我们建议确定因子的SP裂解位点 VII和II也是如此。因此，这将定义以下项目的起点 翻译后羧化所需的前肽 依赖维生素K的蛋白质。(3)我们将确定 通过对自然产生的X因子中特异性GLA残基的研究 以及在GLA结构域发生突变的重组X因子变异体。vbl.使用 定点突变，GLA(16)对Asp和GLA(20)对Asp变异体将 是被建造的。这些，连同对天冬氨酸自然产生的GLA(26) 变种，将通过物理化学手段和凝血来表征 检测，以确定特定残留物在维持 构象及其对磷脂结合的影响。

The overall aim of this project is to achieve an enhanced understanding of the elements involved in transcription and post-translational processing of the vitamin K-dependent clotting factors. Using human Factor X as a paradigm for study, experiments proposed here will (1) determine cis and trans regulatory elements required for the expression of Factor X. Using footprinting assays, gel shift, and mutagenesis followed by transfection assays with reporter genes, the location of functional transcription factor (TF) binding sites within the F.X promoter will be determined. Binding sites for several liver-specific TF's (HNF-3 and C/EBP) will be sought and co-transfection assays used to determine the effects of these TF's on the F.X promoter. These findings will be used to explore two other phenomena, the pathophysiology of hemophilia B Leyden, and the pattern of clotting factor expression in HepG2 cells. (2) We shall explore the function of the signal sequences in these proteins. The signal sequences of secreted proteins function to target these proteins to the endoplasmic reticulum. We have described a Factor X variant (Factor X Santo Domingo) in which a mutation in the signal sequence results in severe Factor X deficiency. We propose to carry out further experiments to determine the intracellular fate of this mutant protein. In the course of characterizing this variant, we have established for the first time the signal peptidase (SP) cleavage site in F.X. We propose to determine the SP cleavage sites for Factors VII and II as well. This will, as a consequence, define the start sites of the propeptides, which are required for the posttranslational carboxylation of the vitamin K-dependent proteins. (3) We shall determine the role of specific Gla residues in Factor X through the study of naturally occurring and recombinant Factor X variants with mutations in the Gla domain. Using site directed mutagenesis, Gla(16) to Asp and Gla(20) to Asp variants will be constructed. These, along with a naturally occurring Gla(26) to Asp variant, will be characterized by physicochemical means and by coagulation assays, to define the roles of specific residues in maintaining conformation and effecting phospholipid binding.