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1.  Sulfated Glycosaminoglycans Control the Extracellular Trafficking and the Activity of the Metalloprotease Inhibitor TIMP-3 
Chemistry & Biology  2014;21(10):1300-1309.
Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.
Graphical Abstract
•The metalloprotease inhibitor TIMP-3 binds to sulfated extracellular glycans•This inhibits cellular uptake of TIMP-3 by the endocytic receptor LRP-1•Glycans also increase TIMP-3 affinity for selected target proteases•The sulfation of matrix glycans therefore modulates TIMP-3 activity and ECM turnover
Tissue inhibitor of metalloproteinase (TIMP)-3 regulates extracellular matrix turnover. Troeberg et al. show that TIMP-3 levels are regulated by the balance between its binding to matrix glycans and receptor-mediated endocytosis and that glycan sulfation regulates this equilibrium.
PMCID: PMC4210636  PMID: 25176127
2.  Incorporation of Bulky and Cationic Cyclam-Triazole Moieties into Marimastat Can Generate Potent MMP Inhibitory Activity without Inducing Cytotoxicity 
ChemistryOpen  2013;2(3):99-105.
The synthesis and matrix metalloproteinase (MMP) inhibitory activity of a cyclam–marimastat conjugate and its metal complexes are described. The conjugate, synthesized with a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (“click” reaction), contains two zinc-binding groups (ZBGs). The metal complexation behavior with copper(II) and zinc(II) was investigated using UV/Vis spectrophotometry and 1H NMR spectroscopy, respectively, demonstrating that the first equivalent of the metal ion was chelated by the cyclam-triazole moiety rather than the hydroxamic acid site. Thus, the corresponding mononuclear metal–cyclam complexes were successfully prepared with one equivalent of the metal salt. Both the cyclam–marimastat conjugate and its metal complexes exhibited slightly reduced potency against MMP-1, but essentially identical inhibitory activity against MMP-3. The conjugate and its metal complexes displayed little or no cytotoxicity, further supporting their potential suitability for imaging MMP localization and activity. To the best of our knowledge, this is the first report that describes the incorporation of metal complexes into an MMP inhibitor without influencing the preexisting ZBG, and the first report of the evaluation of structures containing more than one ZBG as MMP inhibitors.
PMCID: PMC3703814  PMID: 24551546
biological activity; click chemistry; marimastat; matrix metalloproteinase; metal complexes
3.  Proteases involved in cartilage matrix degradation in osteoarthritis 
Biochimica et biophysica acta  2011;1824(1):133-145.
Osteoarthritis is a common joint disease for which there are currently no disease-modifying drugs available. Degradation of the cartilage extracellular matrix is a central feature of the disease and is widely though to be mediated by proteinases that degrade structural components of the matrix, primarily aggrecan and collagen. Studies on transgenic mice have confirmed the central role of Adamalysin with Thrombospondin Motifs 5 (ADAMTS-5) in aggrecan degradation, and the collagenolytic matrix metalloproteinase MMP-13 in collagen degradation. This review discusses recent advances in current understanding of the mechanisms regulating expression of these key enzymes, as well as reviewing the roles of other proteinases in cartilage destruction.
PMCID: PMC3219800  PMID: 21777704
osteoarthritis; proteinase; cartilage; aggrecanase; collagenase
4.  Defining Requirements for Collagenase Cleavage in Collagen Type III Using a Bacterial Collagen System* 
The Journal of Biological Chemistry  2012;287(27):22988-22997.
Background: Structural requirements of triple-helical collagen for collagenolysis are not fully understood.
Results: Recombinant bacterial collagens with human collagen III sequence insertions defined the minimum sequence for cleavage by human collagenases.
Conclusion: Susceptibility of bacterial-human collagen chimeras to collagenases mimicked that of human collagen III.
Significance: This recombinant system is useful to investigate biological functions of collagen segments in a triple-helical context.
Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11′). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11′) of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.
PMCID: PMC3391134  PMID: 22573319
Collagen; Enzyme Kinetics; Extracellular Matrix; Matrix Metalloproteinase (MMP); Protein Chimeras; Recombinant Protein Expression; Collagenase
5.  Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity 
Matrix Biology  2012;31-20(4):229-233.
Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an ‘aggrecanase’ activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells were shown to express the three most active aggrecanases, namely Adamts1, Adamts4 and Adamts5, suggesting that one or more of these enzymes may be responsible for the microvesicle activity. Microvesicles shed by rheumatoid synovial fibroblasts similarly degraded aggrecan in a TIMP-3-sensitive manner. Our findings raise the novel possibility that microvesicles may assist oligodendroglioma and rheumatoid synovial fibroblasts to invade through aggrecan-rich extracellular matrices.
► Microvesicles are shed by numerous cell types. ► We isolated microvesicles shed by oligrodendroglioma and rheumatoid synovial fibroblasts. ► These microvesicles contained an aggrecan-degrading proteolytic activity. ► Microvesicles may thus assist cells in degrading aggrecan-rich extracellular matrices.
PMCID: PMC3391679  PMID: 22406378
ADAM, adamalysin; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ECM, extracellular matrix; MEF, mouse embryonic fibroblasts; MMP, matrix metalloproteinase; MVs, microvesicles; RA, rheumatoid arthritis; TIMP, tissue inhibitor of metalloproteinase; Membrane vesicles; Aggrecan; Metalloproteinase; ADAMTS
6.  Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex 
Biochemical Journal  2012;443(Pt 1):307-315.
The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.
PMCID: PMC3369482  PMID: 22299597
adamalysin with thrombospondin motifs 5 (ADAMTS-5); aggrecanase; extracellular matrix; glycosaminoglycan; osteoarthritis; tissue inhibitor of metalloproteinases 3 (TIMP-3); ADAMTS, adamalysin with thrombospondin motifs; ATIII, antithrombin III; CysR, cysteine-rich; Dis, disintegrin; DMEM, Dulbecco's modified Eagle's medium; dp, degree of polymerization; HEK, human embryonic kidney; MMP, matrix metalloproteinase; OA, osteoarthritis; PPS, pentosan polysulfate; Bio-PPS, biotinylated PPS; Sp, spacer; TBA, tetrabutyl ammonium; TIMP, tissue inhibitor of metalloproteinases; N-TIMP-3, N-terminal domain of TIMP-3; TS, thrombospondin
7.  MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein 
Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu373–374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser341–342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu2047–2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.
PMCID: PMC3057330  PMID: 21055468
Aggrecan catabolism; MMP; ADAMTS; Interglobular domain; Chondroitin-sulfate-2 region
8.  Localising matrix metalloproteinase activities in the pericellular environment 
The FEBS journal  2010;278(1):2-15.
Matrix metalloproteinases (MMPs) are a group of structurally related proteolytic enzymes containing a zinc ion in the active site. They are secreted from cells or bound to the plasma membrane and hydrolyze extracellular matrix (ECM) and cell surface-bound molecules. They therefore play key roles in morphogenesis, wound healing, tissue repair and remodelling in diseases such as cancer and arthritis. Whilst the cell anchored membrane-type MMPs (MT-MMPs) function pericellularly, the secreted MMPs have been considered to act within the ECM, away from the cells from which they are synthesized. However, recent studies have shown that secreted MMPs bind to specific cell surface receptors, membrane-anchored proteins or cell associated ECM molecules and function pericellularly at focussed locations. This minireview describes examples of cell surface and pericellular partners of MMPs and how they alter enzyme function and cellular behaviour.
PMCID: PMC3004722  PMID: 21087456
integrin; receptor; proteoglycan; collagen; tetraspanin; extracellular matrix
9.  Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications 
The Biochemical journal  2010;431(1):113-122.
We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) of aggrecan, gst-IGD-flag, these reactive-site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra alanine residues can be attached to the N-terminus before the Ki (app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [−1] position produced inhibitors as potent as the alanine mutant. The mutants N-TIMP-3(T2G), [–1A]N-TIMP-3 and [–2A]N-TIMP-3 were effective inhibitors of aggrecan degradation, but not of collagen degradation in both IL-1α (interleukin-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [–1A]N-TIMP-3 mutant has additional stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.
PMCID: PMC3003256  PMID: 20645923
a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS); aggrecanase; collagenase; metalloproteinase; osteoarthritis; tissue inhibitor of metalloproteinases 3 (TIMP-3)
10.  Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2) 
The Biochemical journal  2010;430(1):79-86.
The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12–TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor α-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.
PMCID: PMC3025530  PMID: 20533908
a disintegrin and metalloprotease 12 (ADAM12); ectodomain shedding; therapeutic target; tissue inhibitor of metalloproteinase (TIMP); tumour necrosis factor α-converting enzyme (TACE)
11.  Carcinomas contain an MMP-resistant isoform of type I collagen exerting selective support to invasion 
Cancer research  2010;70(11):4366-4374.
Collagen fibers affect metastasis in two opposing ways, by supporting invasive cells but also generating a barrier to invasion. We hypothesized that these functions might be performed by different isoforms of type I collagen. Carcinomas are reported to contain α1(I)3 homotrimers, a type I collagen isoform normally not present in healthy tissues, but the role of the homotrimers in cancer pathophysiology is unclear. In this study, we found that these homotrimers were resistant to all collagenolytic matrix metalloproteinases (MMPs). MMPs are massively produced and utilized by cancer cells and cancer-associated fibroblasts for degrading stromal collagen at the leading edge of tumor invasion. The MMP-resistant homotrimers were produced by all invasive cancer cell lines tested, both in culture and in tumor xenografts, but they were not produced by cancer-associated fibroblasts, thereby comprising a specialized fraction of tumor collagen. We observed the homotrimer fibers to be resistant to pericellular degradation, even upon stimulation of the cells with pro-inflammatory cytokines. Further, we confirmed an enhanced proliferation and migration of invasive cancer cells on the surface of homotrimeric vs. normal (heterotrimeric) type I collagen fibers. In summary, our findings suggest that invasive cancer cells may utilize homotrimers for building MMP-resistant invasion paths, supporting local proliferation and directed migration of the cells while surrounding normal stromal collagen is cleaved. Because the homotrimers are universally secreted by cancer cells and deposited as insoluble, MMP-resistant fibers, they offer an appealing target for cancer diagnostics and therapy.
PMCID: PMC2880213  PMID: 20460529
collagen homotrimers; MMP; collagen degradation; cell-matrix interactions; collagenases
12.  The tissue inhibitors of metalloproteinases (TIMPs): An ancient family with structural and functional diversity 
Biochimica et biophysica acta  2010;1803(1):55-71.
Tissue inhibitors of metalloproteinases (TIMPs) are widely distributed in the animal kingdom and the human genome contains four paralogous genes encoding TIMPs 1 to 4. TIMPs were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has now been found to be broader as it includes the inhibition of several of the disintegrin-metalloproteinases, ADAMs and ADAMTSs. TIMPs are therefore key regulators of the metalloproteinases that degrade the extracellular matrix and shed cell surface molecules. Structural studies of TIMP–MMP complexes have elucidated the inhibition mechanism of TIMPs and the multiple sites through which they interact with target enzymes, allowing the generation of TIMP variants that selectively inhibit different groups of metalloproteinases. Engineering such variants is complicated by the fact that TIMPs can undergo changes in molecular dynamics induced by their interactions with proteases. TIMPs also have biological activities that are independent of metalloproteinases; these include effects on cell growth and differentiation, cell migration, anti-angiogenesis, anti- and pro-apoptosis, and synaptic plasticity. Receptors responsible for some of these activities have been identified and their signaling pathways have been investigated. A series of studies using mice with specific TIMP gene deletions has illuminated the importance of these molecules in biology and pathology.
PMCID: PMC2853873  PMID: 20080133
Matrix metalloproteinase; Extracellular matrix; Evolution; Multifunctional protein; Cell growth; Sorsby fundus dystrophy
13.  The Dimer Interface of the Membrane Type 1 Matrix Metalloproteinase Hemopexin Domain 
The Journal of Biological Chemistry  2010;286(9):7587-7600.
Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.
PMCID: PMC3045013  PMID: 21193411
Cell Migration; Cell Surface Enzymes; Extracellular Matrix; Matrix Metalloproteinase; Protease
14.  The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3 
We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better Ki value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.
PMCID: PMC2835468  PMID: 19643179
aggrecanase; inhibition kinetics; MMPs
15.  Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications 
Biochemical Journal  2010;431(Pt 1):113-122.
We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) of aggrecan, gst-IGD-flag, these reactive-site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra alanine residues can be attached to the N-terminus before the Ki (app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [−1] position produced inhibitors as potent as the alanine mutant. The mutants N-TIMP-3(T2G), [−1A]N-TIMP-3 and [−2A]N-TIMP-3 were effective inhibitors of aggrecan degradation, but not of collagen degradation in both IL-1α (interleukin-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [−1A]N-TIMP-3 mutant has additional stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.
PMCID: PMC3003256  PMID: 20645923
a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS); aggrecanase; collagenase; metalloproteinase; osteoarthritis; tissue inhibitor of metalloproteinases 3 (TIMP-3); ADAM, a disintegrin and metalloproteinase; ADAMTS, ADAM with thrombospondin motifs; CBB, Coomassie Brilliant Blue R-250; DMBA, dimethylaminobenzaldehyde; DMEM, Dulbecco's modified Eagle's medium; DMMB, Dimethylmethylene Blue; Dpa, N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl; GAG, glycosaminoglycan; GST, glutathione transferase; IGD, interglobular domain; IL-1α, interleukin-1α; Mca, (7-methoxycoumarin-4-yl)acetyl; MMP, matrix metalloproteinase; N-TIMP, N-terminal domain of tissue inhibitor of metalloproteinases; OA, osteoarthritis; OSM, oncostatin M; TIMP, tissue inhibitor of metalloproteinases
16.  Molecular Mechanism of Type I Collagen Homotrimer Resistance to Mammalian Collagenases* 
The Journal of Biological Chemistry  2010;285(29):22276-22281.
Type I collagen cleavage is crucial for tissue remodeling, but its homotrimeric isoform is resistant to all collagenases. The homotrimers occur in fetal tissues, fibrosis, and cancer, where their collagenase resistance may play an important physiological role. To understand the mechanism of this resistance, we studied interactions of α1(I)3 homotrimers and normal α1(I)2α2(I) heterotrimers with fibroblast collagenase (MMP-1). Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound α1(I) and α2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site. The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms. Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.
PMCID: PMC2903388  PMID: 20463013
Collagen; Enzyme Kinetics; Extracellular Matrix; Metalloprotease; Protein Degradation; Collagen Homotrimer; Matrix Metalloproteinase; Tissue Remodeling
17.  Role of the Netrin-like Domain of Procollagen C-Proteinase Enhancer-1 in the Control of Metalloproteinase Activity* 
The Journal of Biological Chemistry  2010;285(21):15950-15959.
The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.
PMCID: PMC2871463  PMID: 20207734
ADAM; ADAMTS; Collagen; Extracellular Matrix; Heparin; Matrix Metalloproteinase; Tolloid Proteinase
18.  MT1-MMP is a crucial promotor of synovial invasion in human rheumatoid arthritis 
Arthritis and rheumatism  2009;60(3):686.
A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage and this step requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane-type 1 MMP (MT1-MMP), in synovial pannus invasiveness.
Expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemistry of RA joints specimens. The functional role of MT1-MMP was analyzed by 3D collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, or GM6001. The effect of adenoviral expression of a dominant negative MT1-MMP construct lacking a catalytic domain was also examined.
MT1-MMP was highly expressed at the pannus-cartilage junction of RA joints. Freshly isolated rheumatoid synovial tissues and isolated RA synovial fibroblasts invaded into a 3D collagen matrix in an MT1-MMP-dependent manner. Invasion was blocked by TIMP-2 and GM6001, but not by TIMP-1. It was also inhibited by the over-expression of a dominant negative MT1-MMP which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP-dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.
MT1-MMP is an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP-dependent invasion may form a novel therapeutic strategy for RA.
PMCID: PMC2819053  PMID: 19248098
MT1-MMP; synovial pannus; rheumatoid arthritis
19.  Progress in matrix metalloproteinase research 
Molecular aspects of medicine  2008;29(5):290-308.
Matrix metalloproteinases (MMPs) are now acknowledged as key players in the regulation of both cell–cell and cell–extracellular matrix interactions. They are involved in modifying matrix structure, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis. They play central roles in morphogenesis, wound healing, tissue repair and remodelling in response to injury and in the progression of diseases such as arthritis, cancer and cardiovascular disease. Because of their wide spectrum of activities and expression sites, the elucidation of their potential as drug targets in disease or as important features of the repair process will be dependent upon careful analysis of their role in different cellular locations and at different disease stages. Novel approaches to the specific regulation of individual MMPs in different contexts are also being developed.
PMCID: PMC2810947  PMID: 18619669
Matrixin; Tissue inhibitors of metalloproteinases; Cancer; Cardiovascular disease; Arthritis
20.  Screening of potential ADAMTS-4 inhibitors utilizing a collagen-model FRET substrate 
Analytical biochemistry  2007;373(1):43-51.
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, while the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen, hence aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase 2 (ADAMTS-5) is desirable. We have previously described collagen-model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multi-well formats. In the present study, a collagen-model FRET substrate has been examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n = 960) with known pharmacologic activity. Five compounds were identified that inhibited ADAMTS-4 >60% at a concentration of 1 μM. A secondary screen using RP-HPLC was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC50 value of 1 μM. Because the collagen-model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), non-active site binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow for a more definitive evaluation of this protease in osteoarthritis, as well as representing a potential next generation in metalloproteinase therapeutics.
PMCID: PMC2245870  PMID: 17949675
21.  Cell Surface Collagenolysis Requires Homodimerization of the Membrane-bound Collagenase MT1-MMP 
Molecular Biology of the Cell  2006;17(12):5390-5399.
Pericellular degradation of interstitial collagens is a crucial event for cells to migrate through the dense connective tissue matrices, where collagens exist as insoluble fibers. A key proteinase that participates in this process is considered to be membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP-14), but little is known about the mechanism by which it cleaves the insoluble collagen. Here we report that homodimerization of MT1-MMP through its hemopexin (Hpx) domain is essential for cleaving type I collagen fibers at the cell surface. When dimerization was blocked by coexpressing either a membrane-bound or a soluble form of the Hpx domain, cell surface collagenolytic activity was inhibited in a dose-dependent manner. When MMP-13, a soluble collagenase active as a monomer in solution, was expressed as a membrane-anchored form on the cell surface, homodimerization was also required to cleave collagen. Our results introduce a new concept in that pericellular collagenolysis is regulated by correct molecular assembly of the membrane-anchored collagenase, thereby governing the directionality of the cell to migrate in tissue.
PMCID: PMC1679699  PMID: 17050733
22.  Crystal Structure of an Active Form of Human MMP-1 
Journal of Molecular Biology  2006;362(1):78-88.
The extracellular matrix is a dynamic environment that constantly undergoes remodelling and degradation during vital physiological processes such as angiogenesis, wound healing, and development. Unbalanced extracellular matrix breakdown is associated with many diseases such as arthritis, cancer and fibrosis. Interstitial collagen is degraded by matrix metalloproteinases with collagenolytic activity by MMP-1, MMP-8 and MMP-13, collectively known as the collagenases. Matrix metalloproteinase 1 (MMP-1) plays a pivotal role in degradation of interstitial collagen types I, II, and III. Here, we report the crystal structure of the active form of human MMP-1 at 2.67 Å resolution. This is the first MMP-1 structure that is free of inhibitor and a water molecule essential for peptide hydrolysis is observed coordinated with the active site zinc. Comparing this structure with the human proMMP-1 shows significant structural differences, mainly in the relative orientation of the hemopexin domain, between the pro form and active form of the human enzyme.
PMCID: PMC1885970  PMID: 16890240
MMP, matrix metalloproteinase; ECM, extracellular matrix; matrix metalloproteinases; fibroblast collagenase; collagen; X-ray crystallography; inhibitor-free
23.  Development of a Solid-Phase Assay for Analysis of Matrix Metalloproteinase Activity 
Proteases play fundamentally important roles in normal physiology and disease pathology. Methods for detection of active proteolysis may greatly aid in the diagnosis of disease progression, and suggest modes of therapeutic intervention. Most assays for proteolytic potential are limited by a lack of specificity and/or quantification. We have developed a solid-phase activity assay for members of the matrix metalloproteinase (MMP) family that is specific and can be used to quantify active enzyme concentration. The assay has two principal components: a capture antibody that immobilizes the MMP without perturbing the enzyme active site, and a fluorescence resonance energy transfer substrate for monitoring proteolysis at low enzyme concentrations. The assay was standardized for MMP-1, MMP-3, MMP-13, and MMP-14. The efficiency of the assay was found to be critically dependent upon the quality of the antibodies, the use of substrates exhibiting high specific activities for the enzymes, and enzyme samples that are fresh. The assay was applied to studies of constitutive and induced MMP activity in human melanoma cells. Analysis of several melanoma cell lines, and comparison with prior studies, correlated higher constitutive MMP-13 activity with higher levels of the cell surface receptor CD44. Ligands to two different melanoma cell surface receptors (the α2β1 integrin or CD44) were found to induce different proteolytic profiles, suggesting that the extracellular matrix can modulate melanoma invasion. Overall, the solid-phase MMP activity assay was found to be valuable for analysis of protease activity in cellular environments. The solid-phase assay is suitably flexible to allow studies of virtually any proteolytic enzyme for which appropriate substrates and antibodies are available.
PMCID: PMC2291699  PMID: 15585827
matrix metalloproteinase; fluorogenic substrate; melanoma; solid-phase assay; ELISA
25.  N-Cadherin cleavage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1 
Comparative Hepatology  2004;3(Suppl 1):S8.
Apoptosis of hepatic stellate cells (HSC) has previously been shown to occur during spontaneous resolution of experimental liver fibrosis. TIMP-1 has also been shown to have a key role because of its ability to inhibit apoptosis of HSC via matrix metalloproteinase (MMP) inhibition. This has led to further study of novel substrates for MMPs that might impact on HSC survival. N-Cadherin is known to mediate cell-cell contacts in fibroblasts. In this study we demonstrate that N-Cadherin is expressed by activated rat HSC. Furthermore, during apoptosis of HSC, the N-Cadherin is cleaved into smaller fragments. Apoptosis of HSC may be inhibited by TIMP-1. This is associated with reduced fragmentation of N-Cadherin. N-Cadherin may have an important role in supporting HSC survival while N-Cadherin cleavage may play a part in promoting HSC apoptosis in recovery from liver fibrosis.
PMCID: PMC2410231  PMID: 14960160

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