PubMed · 10506970
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T Porwol, A R Strohmaier, E Spiess. 1999. Cytotomography.. https://doi.org/10.1016/s0076-6879(99)07009-3
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Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here, the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3, and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell-permeable reagents for monitoring legumain activity in complex proteomes.
Cathepsin B and K, two important members in lysosomal proteases, involve in many serious human diseases, such as tumor and osteoporosis. In order to find their novel inhibitors, we performed the inhibition assays of cathepsin B and cathepsin K in vitro, randomly screened compounds from plants, and found six biflavones, named AMF1-5 and HIF, can potently inhibit cathepsin B and cathepsin K, especially AMF4 and HIF with IC(50) of 0.62 and 0.58 microM against cathepsin B. They are novel inhibitors for cathepsin B and K. Inhibition and flexible docking studies indicated that these biflavones are reversible inhibitors of cathepsin B, and their binding patterns and interaction modes with cathepsin B made them more specific to cathepsin B endopeptidase.
Metastasis of malignant tumor cells involves cell-cell and cell-matrix interactions, which regulate the expression and localization of proteolytic enzymes. In the present study, we investigated the expression and localization of the lysosomal cysteine proteinase cathepsin B and its natural inhibitors cystatin A, B and C in high- (MV3), intermediate- (SKmel28) and low-invasive (SKmel23, WM164) human melanoma cell lines grown on plastic or in contact with monomeric or fibrillar collagen type I. Neither the transcript levels of cathepsin B nor those of the natural inhibitors, cystatin B and C, were altered by the interaction of melanoma cells with collagen type I. However, protein expression and cellular localization of cathepsin B and its inhibitors were markedly affected. In contrast to low-invasive cells, high-invasive cells constitutively released procathepsin B when cultured on plastic. In addition, contact of invasive cells with fibrillar collagen type I resulted in the release of both mature forms of the protease. Perturbation studies using inhibitory antibodies against the beta1 subunit of the integrin receptor indicated a role for the beta1 integrin receptor family in the regulation of cathepsin B release. Cystatin B protein expression was much lower in high-invasive cells in both culture conditions, when compared to low-invasive cells. Cystatin C expression was comparable in all cells, but cell contact to fibrillar collagen type I induced its expression. These results strongly implicate a pivotal role of cell-matrix interactions for the regulation of cathepsin B localization and activity in melanoma cells.