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.
