GELATINASE A/MT MMP SYSTEM IN CELL ADHESION AND MOTILITY

明胶酶 A/MT MMP 系统在细胞粘附和运动中的作用

• 批准号: 6511707
• 负责人: GREGORY I GOLDBERG
• 金额: $ 31.2万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511707
• 关键词: affinity labeling; catalyst; collagenase; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; extracellular matrix; gene mutation; integrins; intermolecular interaction; laboratory rabbit; metalloendopeptidases; molecular site; protein localization; proteolysis; site directed mutagenesis; synthetic peptide; transfection

DESCRIPTION: (adapted from the investigator's abstract) Matrix Metalloproteases (MMPs) secreted by eucaryotic cells initiate tissue remodeling by degradation of existing ECM macromolecules such as collagens and proteoglycans. Malignant cells exploit these proteases to promote tumor invasion and metastasis. The investigator's research into biological function of MMPs is based on the hypothesis that spatially regulated extracellular proteolysis is accomplished by compartmentalization of the enzymes via their interaction with molecular structures on the cell surfaces and/or fibrils of the ECM where the physiological activation of proenzymes occurs. Thus mechanistic study of such interactions is a major focus of the investigator's attention. In this respect the discovery of a cell surface activation mechanism of Gelatinase A (GeIA) and isolation of its membrane activator, an integral membrane metalloprotease MT-MMP, were recent breakthroughs. The regulatory C-terminal domain of GelA (GelACTD) plays a pivotal role in the cell surface activation mechanism, interaction of the enzyme with integrin, binding of inhibitor TIMP-2 and substrate recognition. The investigator has recently reported the crystal structure of this domain. Now based on this structure, the investigator has created and characterized a library of 60 single amino-acid substitution mutants that span solvent exposed residues of this domain. Using these mutants the investigator determined TIMP-2 binding surface of GelACTD. These mutants will be instrumental in further investigation of the activation mechanism. The investigator also discovered that C-terminal domain directly interacts with catalytic domain and this interaction critically affects the binding of the enzyme to substrate. Based on this observation the investigator developed "in trans" complementation test where the enzyme is assembled from two independently expressed domains, so that this interaction can be now studied. Finally, the investigator discovered that GeIA membrane activator MT-MMP can be recruited into focal adhesions. Thus regulation of MT-MMP trafficking between transendocytic compartment and cell surface is likely to play a critical role in cell adhesion and motility. This proposal combining biochemical, biophysical, cell and molecular biology approaches is designed to capitalize on these results to advance the understanding of the mechanisms of extracellular proteolysis by gelatinases and integral membrane proteases on a molecular and cellular level. Detailed understanding of mechanisms of catalysis, cell surface activation and the role of GelA/MT-MMP/TIMP-2 system in invasive phenotype of cells will evolve as a result of these studies.

描述：（改编自研究者的摘要）矩阵 真核细胞分泌的金属蛋白酶（MMP）启动组织 通过降解现有的 ECM 大分子（例如胶原蛋白）进行重塑 和蛋白多糖。 恶性肿瘤细胞利用这些蛋白酶促进肿瘤生长 侵袭和转移。 研究者对生物学的研究 MMP 的功能基于空间调节的假设 细胞外蛋白水解是通过区室化来完成的 酶通过与细胞表面分子结构的相互作用 和/或 ECM 的原纤维，其中酶原的生理激活 发生。 因此，这种相互作用的机制研究是该领域的一个主要焦点。 调查员的注意。 在这方面，细胞表面的发现 明胶酶A(GeIA)的激活机制及其膜的分离 激活剂，一种整合膜金属蛋白酶 MT-MMP，是最近出现的 突破。 GelA (GelACTD) 的 C 端调节域发挥着 在细胞表面活化机制、相互作用中发挥着关键作用 酶与整合素、抑制剂 TIMP-2 的结合和底物识别。 研究人员最近报道了该区域的晶体结构。 现在，基于这个结构，研究人员创建并表征了 包含 60 个跨溶剂的单氨基酸取代突变体的文库 该结构域的暴露残基。 研究者利用这些突变体 确定了 GelACTD 的 TIMP-2 结合表面。 这些突变体将是 有助于进一步研究激活机制。 这 研究人员还发现 C 端结构域直接与 催化结构域和这种相互作用严重影响 酶到底物。 根据这一观察，研究人员开发了 “反式”互补测试，其中酶由两个组装而成 独立表达的域，因此现在可以进行这种相互作用 研究过。 最后，研究者发现GeIA膜激活剂 MT-MMP 可以募集到粘着斑中。 因此MT-MMP的调节 跨内吞区室和细胞表面之间的运输可能 在细胞粘附和运动中发挥关键作用。 该提案结合 设计了生物化学、生物物理、细胞和分子生物学方法 利用这些结果来加深对 明胶酶和整合膜的细胞外蛋白水解机制 分子和细胞水平上的蛋白酶。 详细了解 催化机制、细胞表面活化及其作用 细胞侵袭表型中的 GelA/MT-MMP/TIMP-2 系统将进化为 这些研究的结果。