Browsing by keyword "metalloproteases"
Now showing items 1-2 of 2
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Degradomic identification of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) as an ADAMTS9 and ADAMTS20 substrate [preprint]The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix (ECM) proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative N-terminomics method, terminal amine isotopic labeling of substrates (TAILS) was used to compare parental and gene-edited cells and their medium to identify ADAMTS9 substrates. Among differentially abundant N-terminally labeled internal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease MT1-MMP, whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge i.e., between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by re-expression of ADAMTS9 and ADAMTS20, and was dependent on hinge O-glycosylation. Since MT1-MMP is a type I transmembrane protein, identification of an N-terminally labeled peptide in the medium suggested additional downstream cleavage sites in its ectodomain. Indeed, a C-terminally semi-tryptic MT1-MMP peptide with greater abundance in wild-type RPE-1 medium identified by a targeted search indicated a cleavage site in the hemopexin domain. Consistent with retention of MT1-MMP catalytic domain on the surface of gene-edited cells, pro-MMP2 activation, which requires cell-surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell-surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell-surface activity of MT1-MMP and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.
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Leptin induces cell migration and invasion in a FAK-Src- dependent manner in breast cancer cellsBreast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src, and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 showed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.
