HUMAN FIBRINOGEN: MECHANISM OF ASSEMBLY

人纤维蛋白原是一种血浆糖蛋白，其在以下方面起主要作用： 止血和血栓形成疾病，并在许多疾病中具有隐含的作用， 其它生物学过程如细胞粘附和聚集、促进剂 细胞生长、血管生成、发育过程和伤口愈合。 纤维蛋白原是一种二聚体，每半分子由3个不同的 链（A α、B β和tau）。 二聚体的半分子是 通过2A α和2A τ之间的对称二硫键结合在一起 店 此外，纤维蛋白原还含有大量的链间和链内蛋白， 二硫键。 我们的目的是了解这种机制， 合成、组装、加工和分泌多链蛋白。 我们 以前的研究详细介绍了 单个链被组装，并表明，在Hep G2细胞中， B β链的合成是生产中的限速因素 纤维蛋白原 用B β cDNA转染Hep G2细胞， 不仅通过特异性升高B， β链的合成，也可以合成其他两种组分 店 我们现在有以下具体目标1）确定如何 纤维蛋白原链之一的合成的增强会影响 另外两条链的表达。 2)研究相互作用和运输 纤维蛋白原链，我们将检测异源分泌细胞与 每个纤维蛋白原链的单独cDNA和与纤维蛋白原链的组合 A α、B β和tau链的cDNA。 产品表现 将被表征，它们的组装、细胞内转运和 分泌测定。 3)体外系统将使用 每个纤维蛋白原链的特异性mRNA和重构的 微粒体系统能够翻译和易位的分泌 proteins. 氧化还原电位将通过添加 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)常驻内质网蛋白的作用 在纤维蛋白原链组装、封存和降解过程中， 测定 5)通过位点特异性诱变修饰纤维蛋白原cDNA， 确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.