Tissue- and cell-specific sites of action of matrix metalloproteinases (MMPs) have recently been identified. However, how MMPs associate with these strategic sites remains unclear, blunting exploitation. MMP-12 was located on the surface of activated macrophages from inflamed lungs and embedded in the elastin fibrils being digested in aneurysms forming in the aorta. MMP-7 was found on the surfaces of tumor cells, intestinal epithelial cells, and between macrophages and elastin fibrils they are digesting. On cells, MMP- 7 associates with cholesterol sulfate and heavily sulfated glycosaminoglycan (GAG) chains of proteoglycans. MT1-MMP is found on the invading front of tumor cells and endothelial cells migrating through the matrix or basement membranes. This project will test new hypotheses of various exosites that support (1) affinity for and activity upon elastin, (2) association with membrane lipids, and (3) association with and activation by sulfated saccharides. NMR structural and dynamics approaches will be applied to representative assemblies in solution. Directed mutagenesis will be used to test functional roles in catalytic efficiency and associations.
Aim 1 will evaluate role and scope of exosites (some remote) and stability-modulating residues in elastin degradation by MMP-12 and -7. Investigation of the elastase activity of MMP-7 will proceed using the molecular recognition and biophysical approaches recently demonstrated with MMP-12 and -3. Probing of MMP-7 recognition of new 1-elastin derivatives will be guided by BINDSIght, which combines bioinformatics and NMR to discover specificity of interactions. The balance that MMP-7 strikes in tradeoffs among activity (upon elastin), folding stability, and millisec dynamics will be compared with its 55% identical MMP-12 and -3 counterparts characterized in the previous period.
In Aim 2, new bioinformatics predictions that MMP catalytic domains associate with lipid membranes will be tested with NMR mapping of MMP-micelle interfaces and NMR structure determination of two micelle complexes with MMPs now known to bind cell surfaces.
Aim 3 will investigate how GAGs accelerate activation of proMMP-7, testing hypotheses of co-localizing trimolecular activation vs. bimolecular "allosteric" activation. Sulfated saccharide competition assays (by surface plasmon resonance) and activation assays will pave the way. Atomic force microscopy and hydrodynamics will clarify the nature of the saccharide complexes with proMMP-7 and MMP-7. A complex will be selected for detailed structural characterization by NMR spectroscopy. The project will profoundly broaden views of molecular recognition by MMPs and how in detail the novel interactions may direct MMP activity to cell surfaces and elastin fibrils in cardiovascular and pulmonary disease and cancer. The binding modes will reveal unique functional epitopes and sites where future MMP-specific antibodies can be targeted to interfere in these associations in diagnostic, research, and therapeutic uses.
Cardiovascular diseases, cancer, lung diseases, and arthritis have a destructive excess of inflammation that is fostered by enzymes called MMPs that cut connective tissue and proteins near cell surfaces. This project will identify working surfaces of MMPs that help them (1) attack the elastic fibrils of arteries, lungs, and skin and (2) associate with cell surface membranes and saccharides to attack proteins strategically from there. These discoveries will suggest where to direct future antibodies to interfere in these processes for clinical insight and possible tools for treatment.
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