REGULATION OF EXTRACELLULAR PROTEOLYSIS BY SERPINS

描述：该提案的重点是蛋白酶控制的机制。 丝氨酸蛋白酶抑制剂(SERPINS)。两个不同型号的SERPIN将 被用来研究SERPIN激活的机制 肝素，以及SERPIN：蛋白酶复合体的结合机制， 内化，并由细胞处理。第一个远程目标是获得 深入了解抗血栓治疗的分子基础。凝血酶 肝素可使ATIII的抑制活性提高1,000到10,000倍， 但这一重要临床研究背后的结构和分子基础 肝素及相关化合物的应用仍有待阐明。 这一水平的理解是开发替代方案所必需的 去接受肝素治疗。这将通过以下几个方面的组合来实现 沙门氏菌肝素结合区的定点突变 ATIII，然后对其影响进行定量生化评估 这些变化对工程蛋白中肝素的激活有影响。这些 研究将扩展到表征这些变种的相互作用 具有血管内皮细胞，它代表了 ATIII-凝血酶相互作用的肝素。此外，SERPIN级别为 受SERPIN内化和加工调控的蛋白酶 按特定细胞类型分类的复合体。本研究的这一方面将 重点进行生化和定点突变相结合的分析 PN1来探讨SERPIN的机制：与细胞的蛋白酶结合，以及 调解其内化的机制，这是必要的 正在处理。基于之前获得的数据，基因工程 PN1的变种和具有生物活性的合成肽将用于 探测Th：PN1复合体进入细胞内的分子 处理它们的细胞。

DESCRIPTION: The proposal is focused on the mechanisms of protease control by serine protease inhibitors (SERPINS). Two different model SERPINs will be employed to investigate the mechanism underlying SERPIN activation by heparin, and the mechanism by which SERPIN: Protease complexes are bound, internalized, and processed by cells. The first long range goal is to gain insight into the molecular basis of antithrombotic therapy. The thrombin inhibitory activity of ATIII is accelerated 1,000 to 10,000 fold by heparin, but the structural and molecular basis underlying this important clinical application of heparin and related compounds, remains to be elucidated. This level of understanding is requisite to the development of alternatives to heparin therapy. This will be approached by a combination of site-directed mutagenesis within the identified heparin binding region of ATIII, followed by a quantitative biochemical assessment of the effects of these changes on heparin activation in the engineered proteins. These studies will be extended to characterize the interactions of these variants with vascular endothelial cells which represent the physiological source of heparin for ATIII-thrombin interactions. In addition, SERPIN levels are regulated by the internalization and processing of SERPIN: Protease complexes by specific cell types. This aspect of the present studies will focus on a combined biochemical and site directed-mutagenesis analysis of PN1 to probe the mechanism of SERPIN:Protease binding to cells, and the mechanism that mediates their internalization, which is requisite to processing. Based on previously obtained data, genetically engineered variants of PN1 and biologically active synthetic peptides will be used to probe the molecules responsible for the entry of Th:PN1 complexes into the cells that process them.