Quantitation of MT1-MMP Activity at the Cell Surface

摘要

MT1-MMP plays critical roles during tumor malignancy and is one of the best-validated proteolytic enzyme targets on cancer cells. It is up-regulated in several tumor types, including breast, cervical, and ovarian cancer and is significantly associated with adverse outcome [1]. In spite of the large proteolytic repertoire and of the robust proteolytic activity of engineered soluble forms of the enzyme, several studies indicate that soluble MT1-MMP mediated proteolysis is not sufficient per se for efficient penetration of ECM barriers. Thus, localization of the enzyme at the cell surface is essential to translate its proteolytic activity into a modification of cell function. Overall, the transmembrane nature of MT1-MMP allows it to influence extracellular remodeling of the matrix surrounding tumor cells as well as intracellular signaling events involved in cell invasion. Like virtually all cell surface proteases, quantitative assessment of MT1-MMP in its native environment has not been achieved. A mechanistic examination of MT1-MMP at the cell surface would unravel the influences of binding partners on activities, and set the stage for the development of unique, non-active site inhibitors. Such inhibitors may act only on selective functions of MT1-MMP, reducing potential toxicities. MMP activity assays are typically based on fluorescence resonance energy transfer utilizing synthetic peptides [2]. Fluorescence can be detected with outstanding sensitivity, and these continuous assays allow for rapid, convenient kinetic evaluation of proteases and thus greatly aid mechanistic studies of these enzymes. In this study, MT1-MMP and several MT1-MMP mutants were stably transfected and a cell-based FRET assay utilized to quantify protease activity. Activity comparisons were made for soluble MT1-MMP and surface-bound MTl-MMPs to evaluate the importance of the cell surface and specific MT1-MMP domains on activity.