UBC Theses and Dissertations
Matrix metalloproteinase substrate recognition, characterization and proteomic discovery Tam, Eric M.
The matrix metalloproteinase (MMP) family of secreted and cell membrane proteases are known to cleave all extracellular matrix proteins and a large number of bioactive molecules. However, the complete role of MMPs in vivo is not fully known. Thus, it is pertinent to identify all substrates of MMPs and to also characterize their interactions with substrates in order to comprehend the molecular mechanisms that lead to proteolysis. Membrane type l-MMP (MT1- MMP, MMP-14) and MMP-2 (gelatinase A) are cooperative dynamic components of a cell surface proteolysis apparatus involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Here, we have investigated the structural elements of MT1- MMP and MMP-2 involved in the recognition of type I collagen, in order to dissect the enigmatic mechanism of native (triple helical) collagen cleavage by MMPs. Both MT1-MMP and MMP-2 consist of a catalytic domain tethered to a C domain by a linker peptide. MMP-2 contains an additional insert of three fibronectin type II modules within the catalytic domain. We demonstrate that recombinant proteins representing the C domain of MT1-MMP and the fibronectin domain of MMP-2 act in a dominant negative manner to block MT1-MMP and MMP-2 cleavage of type I collagen. These domains alone are also capable of perturbing the native structure of collagen. Together, these results reveal an important divergence in the mode of collagen cleavage between these two enzymes. To probe the in vivo role of MT1-MMP, we developed a rapid proteomic screen for identifying new MT1-MMP substrates in cells using isotope coded affinity tag (ICAT) method of protein quantification. The abundance levels of several extracellular proteins were altered upon overexpression of MT1-MMP, which we took as evidence of proteolytic modification. These potential substrates were confirmed biochemically confirmed and here we report that interleukin (IL)-8, secretory leukocyte protease inhibitor (SLPI), death receptor-6, and connective tissue growth factor (CTGF) are previously undescribed substrates of MT1-MMP. Moreover, the utility and quantitative nature of ICAT as a proteomic screen for protease substrate discovery should make it adaptable for studying the proteolytic function of other protease classes in complex and dynamic biological contexts.
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