Metalloproteases (MMPs) play a pivotal role in tissue remodeling during morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Malignant cells exploit MMPs to promote tumor invasion and metastasis. The long-term goals of this laboratory are (1) to understand the molecular mechanisms of spatial regulation of extracellular proteolysis catalyzed by MMPs and (2) to elucidate the specific role of MMPs in the physiology and pathology of the ECM. The mechanisms of spatial control of enzymatic activity in tissues involve MMP binding to the surface of resident cells, in situ activation, and a unique mode of interaction with the underling ECM substrata. Our most recent results represent a significant progress in these areas, i. Our experiments firmly establish that MMPs utilize a remarkable surface diffusion mechanism for substrate interaction. MMP-1, -2 and 9 can diffuse laterally on the substrate surface without noticeable dissociation. The lateral diffusion of MMP-2 requires the hemopexin C-terminal domain. Complex formation of pro-MMP-2 and -9 with inhibitors 1TMP-2 and -1 respectively does not interfere with the diffusion process, ii. Most interestingly, we have shown that activated MMP-1 is a novel type of diffusion-based, ATP-independent motor enzyme that is driven by proteolysis of its substrate, collagen, iii. we demonstrate that pro- MMP-1, but not activated MMP-1, specifically binds to the cell surface via the pro-peptide domain causing activation in situ;iv. Binding of pro-MMP-9 to gelatin or type IV collagen substrates is sufficient for the enzyme to acquire partial activity with its pro-peptide still intact. These findings have profound implications for mechanistic understanding of the role of MMPs in controlled cell spreading and motility. We propose a flexible, mobile cell surface-substrata interface that allows for controlled ECM remodeling by the moving cells. We have already identified several of the key interactions in this interface. Here we propose to examine the mechanisms of lateral diffusion and in situ activation, and the possible linkage between the two. These studies will form the basis for elucidating the functional components of the cell surface-substrata interface and for biophysical studies on workings of the MMP-1 molecular motor.
Specific Aim 1 proposes to extend the MMP-1 cell surface binding and activation studies by biochemically defining the cell surface interaction.
Specific Aims 2 and 3 propose to start with the gelatinase model to define biochemical interactions of gelatinases with substrate and to examine how these can enable processive diffusion and enzyme activation.
