Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors.
; ; et al
in Protein Expression & Purification (1999), 16
The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 ... [more ▼]
The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 synthetic inhibitors, we have produced and purified the catalytic domain of ST3, matrilysin, stromelysin-2, and membrane type-1 MMP from inclusion bodies in a bacterial system. Our strategy allowed the purification of MMPs directly in the active form, thereby avoidingin vitroactivation. A total of 140,000 synthetic compounds from the Bristol-Myers Pharmaceutical Research Institute chemical deck were tested, using a substrate-based colorimetric enzymatic assay, in which ST3 activity was evaluated through its ability to cleave and inactivate α-1 proteinase inhibitor. One ST3 inhibitor belonging to the cephalosporin family of antibiotics was thereby identified. [less ▲]Detailed reference viewed: 75 (1 ULg)
Characterisation of structural determinants and molecular mechanisms involved in Stromelysin-3 activation by 4-aminophenylmercuric acetate and furin-type convertases.
; Noël, Agnès ; et al
in Biochemical Journal (1996), 315
Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP) which has been implicated in cancer progression and in a number of conditions involving tissue remodelling. In contrast to other MMPs which are ... [more ▼]
Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP) which has been implicated in cancer progression and in a number of conditions involving tissue remodelling. In contrast to other MMPs which are secreted as zymogens requiring extracellular activation, ST3 is found in the extracellular space as a potentially active mature form, suggesting that the activation of the ST3 proform differs from that of other MMPs. We show in the present study that the ST3 proform is not autocatalytically processed in the presence of 4-aminophenylmercuric acetate (APMA). By using ST3/ST2 chimeras, we demonstrate that resistance to APMA is due to properties associated with both the ST3 pro- and catalytic domains. In agreement with the observation made by Pei and Weiss [Pei and Weiss (1995) Nature (London) 375, 244-247], we find that the requirement for activation of the ST3 proform by the furin convertase is entirely contained within a stretch of 10 amino acids located at the junction between the ST3 pro- and catalytic domains. Furin cleaves human and mouse ST3 equally well. However, PACE-4, a furin-like convertase, is much more efficient on the mouse enzyme, suggesting that ST3 protein determinants other than the conserved Ala-Arg-Asn-Arg-Gln-Lys-Arg sequence preceding the furin cleavage site are implicated in PACE-4 action. Finally, we show that processing of the ST3 proform is inhibited by a furin inhibitor in human MCF7 breast cancer cells stably transfected to constitutively express a full-length human ST3 cDNA. Using brefeldin A, we demonstrate that, in these MCF7 cells, the 56 kDa precursor form of ST3 is post-translationally modified in the cis- or media-Golgi into a 62 kDa proform. Thereafter, its processing into the 47 kDa mature form occurs in the trans-Golgi network and is followed by secretion into the extracellular space. [less ▲]Detailed reference viewed: 8 (1 ULg)
Stromelysin-3 and other stromelysins in breast cancer : importance of epithelial-stromal interactions during tumor progression.
; ; et al
in Lippman, M. E.; Dickinson, R. D. (Eds.) Breast Cancer: Cellular and Molecular Biology (1996)Detailed reference viewed: 5 (0 ULg)
Identification of structural determinants controlling human and mouse stromelysin-3 proteolytic activities
Noël, Agnès ; ; et al
in Journal of Biological Chemistry (1995), 270
Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling ... [more ▼]
Matrix metalloproteinases (matrixins) constitute a group of extracellular proteinases belonging to the metzincin superfamily. They are involved in both physiological and pathological tissue remodeling processes, including those associated with cancer progression. Stromelysin-3, which is expressed in most invasive human carcinomas, is a matrix metalloproteinase with unusual functional properties. In particular, its mature form does not cleave any of the major extracellular matrix components. To define critical structural determinants involved in controlling stromelysin-3 proteolytic activity, we have used site-directed mutagenesis. We show that the deletion of at least 175 C-terminal amino-acids is sufficient to endow mouse stromelysin-3 with activities against casein, laminin, and type IV collagen. In the case of the human enzyme, however, a further and single Ala-235 Pro substitution is necessary to observe similar activities. Ala-235, which characterizes human stromelysin-3 among matrixins, is located immediately after the C terminus of the “Met-turn,” which forms a hydrophobic basis for the catalytic zinc atom in the metzincin family. We conclude that human stromelysin-3 has gained specific functional properties during evolution by amino acid substitution in the catalytic zinc environment, and that it represents an attractive target for specific inhibitors that may be used to prevent cancer progression. [less ▲]Detailed reference viewed: 3 (1 ULg)
Stromelysin-3 - a paradigm of extracellular proteinase expressed in stromal cells of human carcinomas.
; ; et al
in Cell Biology International (1995), 19(3), 242-242Detailed reference viewed: 4 (0 ULg)
Characterization of monoclonal antibodies against stromelysin-3 and their use to evaluate stromelysin-3 levels in breast carcinoma by semi-quantitative immunohistochemistry
; Noël, Agnès ; et al
in International Journal of Cancer = Journal International du Cancer (1995), 64(5), 336-341
Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ... [more ▼]
Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted. The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice. Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts. One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry [less ▲]Detailed reference viewed: 5 (1 ULg)