REGULATION & BIOSYNTHESIS OF VITAMIN K-DEPENDENT FACTORS

规定

• 批准号: 2224395
• 负责人: Katherine A High
• 金额: $ 26.19万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-17 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224395
• 关键词: DNA footprinting; active sites; binding proteins; blood chemistry; carboxylation; clotting factor; coagulation factor VII; coagulation factor X; endoplasmic reticulum; enzyme biosynthesis; exopeptidase; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genetic regulatory element; genetic transcription; hemophilia As; human subject; liver cells; liver metabolism; posttranslational modifications; protein signal sequence; protein structure function; reporter genes; site directed mutagenesis; tissue /cell culture; transcription factor; transfection; vitamin K

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.

该项目的总体目标是提高对以下方面的认识： 参与转录和翻译后加工的元件 维生素K依赖性凝血因子 使用人类因子X作为 研究范例，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、凝胶迁移和诱变，然后转染 用报告基因的测定，功能性转录因子的定位 (TF)将确定F.X启动子内的结合位点。 结合 将寻找几种肝脏特异性TF（HNF-3和C/EBP）的位点， 共转染测定用于确定这些TF’s对肿瘤细胞的作用。 F.X启动子。 这些发现将用于探索另外两种现象， 血友病B Leyden的病理生理学和凝血模式 在HepG 2细胞中表达。 (2)我们将探讨 这些蛋白质中的信号序列。 分泌的信号序列 蛋白质的功能是将这些蛋白质靶向内质网。 我们已经描述了因子X变体（因子X Santo Domingo），其中 信号序列的突变导致严重的X因子缺乏。 我们 建议进行进一步的实验，以确定细胞内 这种突变蛋白的命运。 在描述该变体的特征的过程中， 我们首次建立了信号肽酶（SP）切割 地点在F. X 我们建议确定因子的SP切割位点 第七和第二。 因此，这将确定 翻译后羧化所需的前肽 维生素K依赖性蛋白质。 (3)我们将确定 因子X中特异性Gla残基的研究 和在Gla结构域中具有突变的重组因子X变体。 使用 定点诱变，Gla（16）至Asp和Gla（20）至Asp变体将 被建造。 这些，沿着天然存在的Gla（26）到Asp 变体，将通过物理化学方法和凝血来表征 测定，以确定特定残基在维持 构象和影响磷脂结合。