CONTROL OF EXTRACELLULAR REGULATORY PROTEASES

这个提议是基于我们最近的研究，这些研究描述了一种新的 蛋白酶是一类细胞分泌的蛋白质，称为“蛋白酶连接蛋白”。"连接蛋白是 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，以及成纤维细胞样 来自啮齿动物胚胎的细胞。 监控的核心功能， 通过一种机制调节特定的细胞外丝氨酸蛋白酶， 涉及与特定蛋白酶的连接，随后是细胞结合， 蛋白酶连接蛋白的内化和细胞内降解： 蛋白酶复合物 这一过程是由细胞特异性受体介导的 连接蛋白 我们已经确定了三种不同的蛋白酶连接蛋白，PN-I (Mr PN-II（Mr 95，000）和PN-III（Mr 31，000），每种都显示 与不同丝氨酸蛋白酶连接的特异性。 我的研究计划是 为了将两种连接蛋白（PN-I和PN-II）从所述细胞中纯化至均一， 人包皮成纤维细胞无血清条件培养液 抗体，并为他们开发放射免疫测定。 这将 使我能够对其细胞释放进行定量研究， 生物合成 我还计划生成肽图谱，以确定 不同的连接蛋白在结构上是相关的， 在与它们的靶相互作用时在连接蛋白中的特异性切割 蛋白酶，并确定这种切割在调节 它们的细胞结合。 在我学习的最后阶段，我将进行 回答关于连接蛋白生物合成控制问题的实验 然后释放 由于已知连接蛋白的释放是由某些刺激引起的， 试剂，包括它们对其显示连接特异性的蛋白酶， 将尝试识别介导这一过程的细胞受体系统 刺激. 最后，我将讨论的具体问题， 诱导连接蛋白释放，即，坐标或个人，确定 如果这种调节发生在转录、翻译或 一些翻译后修饰。

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.