The molecular regulation of membrane type matrix metalloproteinase 5 (MT5-MMP).

摘要

The matrix metalloproteinases (MMP) belong to an expanding family of enzymes with tightly controlled proteolytic activity against ECM. These proteinases participate in ECM remodeling under both physiological and pathological conditions. The main objective of this study was to determine how MT-MMPs are regulated at the molecular and cellular level using MT5-MMP as a model system.; To further characterize MT5-MMP enzymatic properties, we expressed and purified its soluble form from MDCK cells. A preliminary screen for its potential substrates and assessment of its stability established MT5-MMP as a proteoglycanase with a short half-life at body temperature. The rapid self turnover of MT5-MMP may serve as an off-switch that resides within the molecule.; While characterizing the ectoenzyme of MT5-MMP shed into the extracellular milieu, we identified furin-type convertase as the sheddase. Subsequent mutagenesis studies mapped the cleavage site to the stem region immediately upstream of the transmembrane domain, which contain a cryptic furin recognition site, 545RRKERR. More surface localization and higher proMMP-2 activation for mutants lacking the motif suggest that shedding of MT5-MMP by a furin-type convertase provides a potential mechanism to down-regulate activity of MT-MMPs.; The cell surface expression of MT5-MMP correlates with its activity. In the MT1-MMP model, we identified dynamin-dependent endocytosis as one cellular mechanism regulating MT-MMP activity. Further investigation reveals that internalized MT5-MMP also recycled back to the surface, although at a slower rate than MT1 and MT3. Deficient recycling was observed in truncation mutants of MT1, -3 and -5 lacking the last three residues, suggesting that recycling is mediated by a PDZ motif. The recycling process was significantly delayed in human prostate and breast cancer cell lines, PC3 and MCF7, as compared to normal MDCK cells. Therefore, the recycling of MT-MMP may be modified by tumor cells to utilize the power of MT-MMPs for cancer progression.; In summary, MT5-MMP activity is regulated through auto-proteolysis, shedding cleavage and intracellular trafficking. This study identifies new targets for the development of drugs to fight cancer as well as other diseases with abnormal ECM remodeling.