HUMAN FIBRINOGEN: MECHANISMS OF ASSEMBLY

人纤维蛋白原是一种血浆糖蛋白，其在人纤维蛋白原的形成中起主要作用。 止血和血栓形成疾病。 它是一个二聚体，每个半分子 由三个不同的链（Abyield、BBeta和Gamma）组成。 的 二聚体的半分子通过对称的二硫化物结合在一起 两个Abyield链和两个Gamma链之间的键。 此外，本发明还提供了一种方法， 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的， 并了解该过程是如何调节的。 三条锁链 由单独的mRNA合成，并组装成二聚体 纤维蛋白原在粗面内质网中。 一堆伽马链， 在肝细胞和纤维蛋白原中检测到Ayield-Gamma复合物 组装被认为是由预制的独立连接开始的， 将A和γ链转化为新生的、多核糖体结合的B β链。 一些 已确定的前体中间体形式包括 BBeta-Abyield、BBeta-Gamma和半分子（Ayield-BBeta-Gamma）。 到 研究这些中间体是如何形成的，并了解导致 为了形成功能性纤维蛋白原，我们将：1）分离纤维蛋白原 链和测量链间二硫键的形成下， 在蛋白质二硫键异构酶存在下的体外条件。 在 另外，分离的纤维蛋白原链将被引入肝脏， 将测量非肝微粒体和链间相互作用。 （二） 每条链的mRNA将与重构的 含有内源性蛋白质二硫键异构酶的微粒体系统， 能够进行蛋白质翻译、移位和加工。 在 将纤维蛋白原前体分离、表征并 与以前从完整肝细胞获得的那些相比。 3)研究 纤维蛋白原和其他纤维蛋白原细胞内转运和分泌 纤维蛋白原相关的前体，替代分泌细胞将是 每一条链的基因都被转染了。 各种细胞内 形式，他们的运输和最终的命运将被确定。 4)在 在研究的最后阶段，纤维蛋白原将在关键时进行修改。 参与半分子连接的点（γ 8处的半胱氨酸， Gamma 9和Abyield 28）通过对相应的 cDNA的。 这些基因将被引入宿主分泌细胞， 将测定修饰的纤维蛋白原的组装和分泌。

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.