REGULATION OF EXTRACELLULAR PROTEOLYSIS BY SERPINS

描述：该提案重点关注蛋白酶控制机制 丝氨酸蛋白酶抑制剂（serine protease inhibitors，SERPINS） 两种不同型号的SERPIN将 用于研究SERPIN激活的机制， 肝素，以及SERPIN：蛋白酶复合物结合的机制， 内化并被细胞处理。 第一个长期目标是获得 深入了解抗血栓治疗的分子基础。 凝血酶 ATIII抑制活性被肝素加速1,000 - 10,000倍， 但这一重要临床研究背后的结构和分子基础 肝素和相关化合物的应用仍有待阐明。 这种理解水平是开发替代品的必要条件 肝素治疗 这将通过以下方式来实现： 在确定的肝素结合区域内进行定点诱变， ATIII，然后对 这些变化对工程蛋白中肝素活化的影响。 这些 研究将扩展到表征这些变体的相互作用 与血管内皮细胞，代表的生理来源， 用于ATIII-凝血酶相互作用的肝素。 此外，SERPIN水平是 由SERPIN的内化和加工调节：蛋白酶 由特定的细胞类型组成。 本研究的这一方面将 集中在一个组合的生化和定点诱变分析， PN 1来探测SERPIN的机制：蛋白酶与细胞的结合， 这是一种调节其内在化的机制， 处理. 根据先前获得的数据， PN 1的变体和生物活性合成肽将用于 探测负责Th：PN 1复合物进入的分子 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.