FIBRINOGEN--MECHANISM OF ASSEMBLY

描述：本续约建议书的目标为1)至 确定组成成分链的细胞内机制 纤维蛋白原被组装成具有功能的生物活性 蛋白质和2)定位在纤维蛋白中起重要作用的结构域 利用重组工程技术进行聚合和交联 纤维蛋白原。为了实现这些目标，申请者将1) 用重组内质网囊泡系统表征内质网因子 参与纤维蛋白原组装，并确定与 纤维蛋白原前体与这些因素，2)表达缺失和 COS细胞中各纤维蛋白原链的替代突变体 绘制组装和分泌所需的关键结构域，3) 在HepG2细胞中进行脉冲追逐研究，以确定序列 纤维蛋白原组装的步骤，4)诱变分泌因子 酵母表达系统研究纤维蛋白原的组装和分泌，以及 5)在假定的交联中诱变重组纤维蛋白原和 聚合位点来定义结构-功能关系。

DESCRIPTION: The objectives of this renewal proposal are 1) to determine the intracellular mechanisms by which the component chains of fibrinogen are assembled into a functional biologically active protein and 2) to map domains which play important roles in fibrin polymerization and crosslinking using recombinant engineered fibrinogen. To accomplish these objectives, the Applicant will 1) use a reconstituted ER vesicle system to characterize ER factors involved in fibrinogen assembly and to determine the association of fibrinogen precursors with such factors, 2) express deletion and substitution mutants of each of the fibrinogen chains in COS cells to map the critical domains necessary for assembly and secretion, 3) perform pulse-chase studies in HepG2 cells to define the sequential steps in fibrinogen assembly, 4) mutagenize secretion factors in a yeast expression system to study fibrinogen assembly and secretion, and 5) mutagenize recombinant fibrinogen in putative crosslinking and polymerization sites to define structure-function relationships.