GELATINASE A/MT MMP SYSTEM IN CELL ADHESION AND MOTILITY

描述：(改编自调查员摘要)矩阵 真核细胞分泌的金属蛋白酶(MMPs)启动组织 通过降解现有的细胞外基质大分子如胶原蛋白进行重塑 和蛋白多糖。恶性细胞利用这些蛋白酶促进肿瘤生长 侵袭和转移。调查者对生物学的研究 MMPs的功能建立在空间调控假设的基础上 胞外蛋白分解是通过将细胞内的 酶与细胞表面分子结构的相互作用 和/或细胞外基质的纤维，其中酶原的生理活性 发生。因此，对这种相互作用的机理研究是 调查员请注意。在这方面，细胞表面的发现 明胶酶A的激活机制及其膜的分离 激活剂是近年来发现的一种完整的膜金属蛋白酶MT-MMPs 突破性进展。Gela的调节性C末端结构域(GelACTD)发挥着 在细胞表面激活机制中的关键作用，相互作用 酶与整合素、抑制剂TIMP-2的结合和底物识别。 这位研究人员最近报道了这个结构域的晶体结构。 现在基于这个结构，调查者已经创建并描述了 60个跨越溶剂的单一氨基酸替代突变体文库 该结构域暴露的残基。使用这些突变体，调查员 测定了GelACTD的TIMP-2结合面。这些突变体将会是 有助于进一步研究激活机制。这个 研究人员还发现，C-末端结构域直接与 催化结构域，这种相互作用严重影响了 酶到底物。根据这一观察结果，调查者开发了 “反式”互补试验，这种酶是由两个 独立表达的域，因此这种交互现在可以 学习。最后，调查人员发现，GeIA膜激活剂 MT-MMPs可被募集到局灶性粘连中。因此MT-MMPs的调控 跨内吞细胞室和细胞表面之间的运输可能会 在细胞黏附和运动中起关键作用。这项建议结合了 设计了生化、生物物理、细胞和分子生物学方法 利用这些结果来促进对 明胶酶和整体膜降解胞外蛋白的机制 在分子和细胞水平上的蛋白酶。详细了解 催化、细胞表面活化的机制及其作用 Gela/MT-MMP/TIMP-2系统在细胞侵袭性表型中的演变 这些研究的结果。

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.