CONTROL OF EXTRACELLULAR REGULATORY PROTEASES

这一建议是基于我们最近的研究，这些研究描述了一部小说 一类细胞分泌的蛋白质，称为“蛋白酶连接素”。连接素是 由包括正常人包皮成纤维细胞在内的多种细胞分泌， 牛和人来源的血管内皮细胞和成纤维细胞样细胞 来自啮齿动物胚胎的细胞。Nexine函数用于监视和 通过一种机制调节特定的胞外丝氨酸蛋白酶 包括连接到特定的蛋白酶，然后是细胞结合， 蛋白酶Nexin的内化和细胞内降解： 蛋白酶复合体。这一过程是由细胞特异性受体介导的。 为了Nexin家族。我们已经鉴定了三种不同的蛋白酶连接素，Pn-I (MR 38,000)、PN-II(MR 95,000)和PN-III(MR 31,000)，每一个都显示 与不同丝氨酸蛋白酶连锁的特异性。我的研究计划是 为了将两个连接素(Pn-I和Pn-II)从 人包皮成纤维细胞无血清条件培养液 针对它们的抗体，并为它们开发放射免疫分析。这将是 使我能够对它们的细胞释放进行定量研究 生物合成。我还计划制作肽图，以确定 不同的连接素在结构上是相关的，以及对该地点的研究 连接蛋白与其靶标相互作用时的特定切割 蛋白水解酶，并确定这种裂解在调节 它们的细胞结合。在我学习的最后阶段，我将进行 回答有关Nexin生物合成控制问题的实验 然后释放。由于已知Nexin释放受某些因素的刺激 试剂，包括它们表现出连锁特异性的蛋白酶，即 将试图确定介导这一过程的细胞受体系统 刺激。最后，我将讨论 诱导Nexin释放，即协同或单独，确定 如果这种调节发生在转录、翻译或 一些翻译后的修改。

This proposal is based on our recent studies that have described a novel class of cell-secreted proteins, the "protease nexins.' The nexins are secreted by a variety of cells including normal human foreskin fibroblast, vascular endothelial cells of bovine and human origin, and fibroblast-like cells derived from rodent embryos. The nexine function to monitor and regulate specific extracellular serine proteases through a mechanism that involves linkage to the specific protease, followed by cellular binding, internalization, and intracellular degradation of the protease nexin: protease complex. This process is mediated by cellular receptors specific for the nexins. We have identified three different protease nexins, PN-I (Mr 38,000), PN-II (Mr 95,000), and PN-III (Mr. 31,000), each of which show specificity for linkage to different serine proteases. My research plan is to purify two of the nexins (PN-I and PN-II) to homogeneity from the serum-free conditioned medium of human foreskin fibroblasts, raise antibodies to them, and develop radioimmunoassays for them. This will enable me to perform quantitative studies on their cellular release and biosynthesis. I also plan to generate peptide maps to determine if the different nexins are structurally related, as well as to study the site specific cleavage in the nexins upon interaction with their target proteases, and determine the role of this cleavage in the regulation of their cellular binding. In the final phases of my studies, I will conduct experiments to answer questions regarding the control of nexin biosynthesis and release. Since it is known that nexin release is stimulated by certain agents, including the proteases for which they show linkage specificity, I will try to identify the cellular receptor system that mediates this stimulation. Finally, I will address the question of specificity of the induction of nexin release, i.e., coordinate or individual, the determine if this regulation occurs at the level of transcription, translation, or some post-translational modification.