HUMAN FIBRINOGEN: MECHANISM OF ASSEMBLY

人纤维蛋白原是一种血浆糖蛋白，它在 止血和血栓性疾病，并暗示在一些 其他生物过程，如细胞黏附和聚集、启动子 对细胞生长、血管生成、发育过程和伤口愈合的影响。 纤维蛋白原是一个二聚体，每个半分子由3个不同的分子组成。 链(Aα、Bβ和tau)。二聚体的半分子是 通过2Aα和2tau之间的对称二硫键结合在一起 锁链。此外，纤维蛋白原还含有许多链间和链内蛋白 二硫键。我们的目标是了解这一现象的机制 多链蛋白质的合成、组装、加工和分泌。我们的 以前的研究详细地介绍了这些步骤 单个链被组装并表明，在Hep G2细胞中， Bβ链的合成是生产中的一个限速因素 纤维蛋白原。Bβ基因转染Hep G2细胞的实验研究 通过特别提高纤维蛋白原的产量，而不仅仅是B β链合成，还有另外两个组分的合成 锁链。我们现在有以下具体目标1)决定如何 其中一条纤维蛋白原链的合成增强会影响 其他两条链的表达式。2)研究相互作用和运输 在纤维蛋白原链中，我们将用 每条纤维蛋白原链和与其组合的单个cDNA Aα、Bβ和tau链的cDNA。所表达的产品 将被表征，它们的组装，细胞内运输和 分泌物已确定。3)将开发一个体外系统，使用 每条纤维蛋白原链和重组的 能够翻译和转位分泌物的微体系统 蛋白质。氧化还原电势将通过添加 谷胱甘肽，我们将详细研究纤维蛋白原的早期步骤 链式相互作用。4)驻留内质网蛋白的作用 对纤维蛋白原链的组装、隔离和降解 下定决心。5)通过定点突变修饰纤维蛋白原cDNA以 测定对纤维蛋白原链组装和分泌的影响。

Human fibrinogen is a plasma glycoprotein which plays major roles in hemostasis and thrombotic disorders and has implied roles in a number of other biological processes such as cell adhesion and aggregation, promoter of cell growth, angiogenesis, developmental processes and wound healing. Fibrinogen is a dimer with each half-molecule composed of 3 non-identical chains (A alpha, B beta, and tau). The half-molecules of the dimer are held together by symmetrical disulfide bonds between 2 A alpha and 2 tau chains. In addition fibrinogen contains a number of inter- and intrachain disulfide linkages. Our aim is to understand the mechanism by which this multi-chain protein is synthesized, assembled, processed and secreted. Our previous studies gave a detailed view of the sequential steps by which the individual chains are assembled and indicated that, in Hep G 2 cells, synthesis of the B beta chain is a rate-limiting factor in the production of fibrinogen. Transfection of Hep G2 cells with B beta cDNA caused increased production of fibrinogen by specifically elevating, not only B beta chain synthesis, but also the synthesis of the other two component chains. We now have the following specific aims 1) Determining how enhancement of the synthesis of one of the fibrinogen chains affects the expression of the other two chains. 2) To study interactions and transport of fibrinogen chains, we shall transfect heterologous secretory cells with individual cDNAs for each of the fibrinogen chains and with a combination of cDNAs for the A alpha, B beta and tau chains. The produce expressed will be characterized and their assembly, intracellular transport and secretion determined. 3) An in vitro system will be developed using specific mRNAs for each of the fibrinogen chains and a reconstituted microsomal system capable of translation and translocation of secretory proteins. The redox potential will be varied, by the addition of glutathione, and we will study in detail the early steps in fibrinogen chain interaction. 4) The role of resident endoplasmic reticulum proteins on fibrinogen chain assembly, sequestration and degradation will be determined. 5) Modify fibrinogen cDNAs by site specific mutagenesis to determine the effects on fibrinogen chain assembly and secretion.