Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin)

Tissue components hydrolyzing matrix metalloproteinases (MMPs) exhibit a high sequence similarity (56 -64% in catalytic domains) and yet a significant degree of functional specificity. The hexapeptide-binding sites of 24 known human MMPs were compared in terms of their force field interaction energies with five probes that are most frequently encountered in substrates and inhibitors. The probes moved along a grid enclosing partially flexible binding sites in rigid catalytic domains that were represented by published experimental structures and comparative models and new comparative models for nine most recently characterized MMPs. For individual MMPs, representative interaction energies were obtained as averages for all suitable experimental structures. Correlations of the representative energies for all MMP pairs were succinctly catalogued for individual probes, subsites, and correlation levels. Among the probes (neutral sp 3 carbon and sp 3 oxygen, positive sp 3 nitrogen and hydrogen, and negative carbonyl oxygen), the last probe is least distinctive. Similarities of subsites are decreasing as S1 > S2 > S3 > S1 ϳ S3 > S2 . Most interesting, occupancies of subsites in published structures of MMP-inhibitor complexes follow an almost parallel trend, alluding to overall low selectivity of known MMP inhibitors. Flexible subsite S1 that appears as the specificity pocket in rigid x-ray structures is actually very similar among individual MMPs. Several correlations indicated that MMPs 3, 8, and 12 have similar binding sites. Modeling results are corroborated with published experimental data on MMP inhibition and substrate specificities. The results provide numerous clues for development of specific inhibitors and substrates, as well as for selection of MMPs for testing that provides maximum information without redundant experiments.

Section: Resultssupporting

confidence: 71%

Similarity of Binding Sites of Human Matrix Metalloproteinases

Lukáčová

Zhang

Mackov

et al. 2004

“…Substrates in group II contain a Gly-Leu-(Lys/Arg) motif. Interestingly, both groups I and II are related to substrates that have been described previously for other MMPs and are somewhat related to sites of cleavage in collagen (21)(22)(23). Group III substrates, however, appear to represent a novel recognition sequence as it contains the Arg-Arg-Hy-Leu (group IIIA) and Arg-X-Leu (group IIIB) motifs.…”

Section: Resultsmentioning

confidence: 62%

“…The single peptide from group II contained a Gly at P 1 , Leu at P 1Ј , and a Lys at P 2Ј . The motifs of groups I and II, and locations of their scissile bonds, are similar to MMP cleavage sites in collagen and gelatin (21)(22)(23). Interestingly, however, the peptides from group III were all cleaved after an Arg residue, and in several cases an Arg-Arg occupied the P 2 and P 1 positions.…”

Section: Resultsmentioning

confidence: 83%

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Kridel

Chen

Kotra

et al. 2001

173

140

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P 3 through P 2 . This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P 3 and P 1 . This study shows that MMP-9 has a unique preference for Arg at both P 2 and P 1 , and a preference for Ser/Thr at P 2 . Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.Matrix metalloproteinase-9 (MMP-9) 1 is a member of the matrixin family of metallo-endopeptidases (1-3). MMP-9 is historically referred to as gelatinase B because of its ability to cleave gelatin, a denatured form of collagen, in vitro. Along with MMP-2, MMP-9 differs from other MMPs because it contains three fibronectin type II repeats that have high binding affinity for collagen. These repeats are thought to mediate the binding of MMP-2 and -9 to collagen (1, 2). This binding interaction brings the catalytic pocket of the MMP in proximity to collagen, thereby enhancing its rate of hydrolysis. Despite these well characterized biochemical interactions, it is now clear that MMP-9 is also able to cleave a number of other proteins and may have a rather wide range of physiologic substrates (4 -8).Much of our understanding of the biological function of MMP-9 comes from the study of mice lacking this gene. For example, MMP-9-deficient mice have impaired ossification of the skeletal growth plate, a defect that has been partially attributed to poor vascularization of developing bone (9). Studies on these mice also show that MMP-9 is essential for the recruitment of osteoclasts into developing bones (10). Other work indicates that MMP-9-deficient mice are resistant to dermal blistering in a bullous pemphigoid model, an effect that has been attributed to the inability of these mice to cleave the SERPIN ␣1-proteinase inhibitor (5). Finally, recent studies in the RIP1-Tag2 transgenic mouse model of multistage carcinogenesis indicate that MMP-9 is part of the angiogenic "switch" that is essential for tumor growth (11,12). Other reports suggests that MMP-9 may play a role in inflammation in the nervous system. MMP-9 is elevated in enceph...

“…The enzymes were incubated with various concentrations of TIMP-4 at 25°C for 30 min before adding the substrate to start the kinetic assay. The assays were carried out at 25°C using a Perkin Elmer LS-5 fluorescence spectrometer (40).…”

Section: Methodsmentioning

confidence: 99%

Preparation and Characterization of Recombinant Tissue Inhibitor of Metalloproteinase 4 (TIMP-4)

Liu¹,

Wang²,

Greene³

et al. 1997

129

MMPs 1 and their inhibitors TIMPs play a critical role in ECM homeostasis. Controlled remodeling of the ECM is an essential aspect of normal development, and deregulated remodeling has been indicated to have a role in the etiology of diseases such as arthritis, periodontal disease, and cancer metastasis (1-5). Four mammalian TIMPs have been identified so far: TIMP-1 (6), TIMP-2 (7), TIMP-3 (8 -11), and the recently cloned TIMP-4 (12, 41). The proteins are classified based on structural similarity to each other, as well as their ability to inhibit metalloproteinases.TIMPs are secreted multifunctional proteins that have anti-MMP activity as well as erythroid-potentiating and cell growth-promoting activities. The stimulating effect on cell growth was initially recognized when TIMP-1 and TIMP-2 were identified as having erythroid-potentiating activities (14,15). It is now clear through several recent reports that TIMP-1 and TIMP-2 are mitogenic for non-erythroid cells, including normal keratinocytes (16), fibroblasts (17), lung adenocarcinoma cells (18), and melanoma cells (18). The involvement of TIMPs in the activation of pro-MMP has also been demonstrated (19). In addition, the recent evidence indicates that the TIMP family may be involved in steroidogenesis of rat testis and ovary indicating the potential role of TIMP in the reproduction (20).The most widely appreciated biological function of the TIMPs is their role in the inhibition of cell invasions in vitro (21-24) and tumorigenesis (25-29) and metastasis in vivo (25-31). Since the net MMP activity is the result of the balance between activated enzyme levels and TIMP levels, an increase in the amount of TIMPs relative to MMPs could function to block tumor cell invasion and metastasis. The tumor-suppressing activity of TIMP on primary tumor growth may be in part due to its anti-angiogenic activity. In fact, both TIMP-1 (32) and TIMP-2 (33, 34) have been demonstrated to have an antiangiogenic activity, and such inhibition of angiogenesis is mediated by inhibition of both endothelial cell proliferation (34) and migration (32). The underlying molecular mechanism for the tumor suppressing activities of TIMPs, nevertheless, is thought to depend on their anti-MMP activities.We had recently cloned and characterized a human TIMP-4 (12). Transfection of TIMP-4 into human breast cancer cells inhibited the invasion potential of the cells in the in vitro invasion assay (13). When injected orthotopically into nude mice, TIMP-4 transfectants were significantly inhibited in their tumor growth and axillary lymph node and lung metastasis as compared with controls (13). These results suggest the therapeutic potential of TIMP-4 in treating cancer malignant progression. These results suggest an important role of TIMP-4 in inhibiting primary tumor growth and progression leading to invasion and metastasis. In the present study, we have produced and purified rTIMP4p from baculovirus infected cells. rTIMP4p was shown to inhibit MMP activity and tumor cell invasion across reconstitute...