HUMAN FIBRINOGEN: MECHANISMS 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。它是两个半分子的二聚体 由三个不同的链(ayfield、BBeta和Gamma)组成。这个 二聚体的半分子被对称的二硫化物结合在一起。 两个ayfield链和两个Gamma链之间的键。此外, 纤维蛋白原含有许多链间和链间二硫键。 我们的目标是阐明这种多链蛋白是如何组装和 并了解这一过程是如何被监管的。三条链条 是由单独的mRNA合成的，并组装成二聚体 粗面内质网中的纤维蛋白原。一群伽马链和 在肝细胞和纤维蛋白原中检测到一种阿菲尔德-伽马复合体 装配被认为是由预制件的独立附着开始的 Ayfield和Gamma链到新生的、多聚体结合的BBeta链。一个数字 已确定的前体中间体形式包括 BBeta-ayfield、BBeta-Gamma和半分子(ayfield-BBeta-Gamma)。至 研究这些中间体是如何形成的，并学习导致 为了形成功能性纤维蛋白原，我们将：1)分离纤维蛋白原 链和测量不同In条件下链间二硫键的形成 体外条件下存在蛋白质二硫键异构酶。在……里面 另外，分离的纤维蛋白原链将被引入肝脏和 将测量非肝脏微粒子和链间相互作用。2) 每条链的信使核糖核酸将与重组的 含有内源蛋白质二硫键异构酶和 能够进行蛋白质的翻译、转位和加工。在… 不同时期纤维蛋白原前体将被分离、鉴定和 与以前从完整的肝细胞中获得的相比。3)学习 纤维蛋白原和其他物质的细胞内转运和分泌 纤维蛋白原相关前体、替代分泌细胞 每条链上都有基因的转基因。不同的细胞内 形式，它们的运输和最终的命运将被决定。4)在 研究的最后阶段，纤维蛋白原将在关键时刻进行修改 涉及半分子连接处的点(半胱氨酸在Gamma8， Gamma9和ayfield 28)通过相应的 这些基因将被导入宿主分泌细胞，并 修饰的纤维蛋白原的组装和分泌将被确定。

Human fibriogen is a plasma glycoprotein which plays major roles in hemostasis and thrombotic disorders. It is a dimer with each half-molecule composed of three non-identical chains (Ayield, BBeta and Gamma). The half-molecules of the dimer are held together by symmetrical disulfide bonds between two Ayield chains and two Gamma chains. In addition, fibrinogen contains a number of inter- and interchain disulfide linkages. It is our aim to elucidate how this multichain protein is assembled and secreted and to understand how the process is regulated. The three chains are synthesized by separate mRNA's and are assembled into dimeric fibrinogen in the rough endoplasmic reticulum. A pool of Gamma chains and of an Ayield-Gamma complex have been detected in hepatocytes and fibrinogen assembly is thought to begin by the independent attachment of preformed Ayield and Gamma chains to nascent, polysome-bound BBeta chains. A number of precursor intermediate forms have been identified including BBeta-Ayield, BBeta-Gamma, and the half-molecule (Ayield-BBeta-Gamma). To study how these intermediates are formed and to learn the steps which lead to the formation of functional fibrinogen we will: 1) Isolate fibrinogen chains and measure interchain disulfide bond formation under various in vitro conditions in the presence of protein disulfide isomerase. In addition separated fibrinogen chains will be introduced into hepatic and non-hepatic microsomes and interchain interactions will be measured. 2) mRNA's for each of the chains will be incubated with a reconstituted microsomal system which contains endogenous protein disulfide isomerase and is capable of protein translation, translocation and processing. At various times the fibrinogen procursors will be isolated, characterized and compared to those obtained previously from intact hepatocytes. 3) To study intracellular transport and secretion of fibrinogen and other fibrinogen-related precursors, surrogate secretory cells will be transfected with genes for each of the chains. The various intracellular forms, their transport and eventual fate will be determined. 4) In the final phase of the study, the fibrinogen will be modified at critical points involved in the junction of the half-molecules (cysteines at Gamma8, Gamma9 and Ayield 28) by site specific mutagenesis of the corresponding cDNA's. These genes will be introduced into host secretory cells and the assembly and secretion of modified fibrinogen will be determined.