Modulation of peritumoral fibroblasts with a membrane-tethered tissue inhibitor of metalloproteinase (TIMP) for the elimination of cancer cells.

BACKGROUND: Peritumoral fibroblasts are key components of the tumor microenvironment. Through remodeling of the extracellular matrix (ECM) and secretion of pro-tumorigenic cytokines, peritumoral fibroblasts foster an immunosuppressive milieu conducive to tumor cell proliferation. In this study, we investigated if peritumoral fibroblasts could be therapeutically engineered to elicit an anti-cancer response by abolishing the proteolytic activities of membrane-bound metalloproteinases involved in ECM modulation. METHODS: A high affinity, glycosylphosphatidylinositol (GPI)-anchored Tissue Inhibitor of Metalloproteinase (TIMP) named "T1PrαTACE" was created for dual inhibition of MT1-MMP and TACE. T1PrαTACE was expressed in fibroblasts and its effects on cancer cell proliferation investigated in 3D co-culture models. RESULTS: T1PrαTACE abrogated the activities of MT1-MMP and TACE in host fibroblasts. As a GPI protein, T1PrαTACE could spontaneously detach from the plasma membrane of the fibroblast to co-localize with MT1-MMP and TACE on neighboring cancer cells. In a 3D co-culture model, T1PrαTACE promoted adherence between the cancer cells and surrounding fibroblasts, which led to an attenuation in tumor development. CONCLUSION: Peritumoral fibroblasts can be modulated with the TIMP for the elimination of cancer cells. As a novel anti-tumor strategy, our approach could potentially be used in combination with conventional chemo- and immunotherapies for a more effective cancer therapy.

A Novel 3D Model for Visualization and Tracking of Fibroblast-Guided Directional Cancer Cell Migration.

Stromal fibroblasts surrounding cancer cells are a major and important constituent of the tumor microenvironment not least because they contain cancer-associated fibroblasts, a unique fibroblastic cell type that promotes tumorigenicity through extracellular matrix remodeling and secretion of soluble factors that stimulate cell differentiation and invasion. Despite much progress made in understanding the molecular mechanisms that underpin fibroblast-tumor cross-talk, relatively little is known about the way the two cell types interact from a physical contact perspective. In this study, we report a novel three-dimensional dumbbell model that would allow the physical interaction between the fibroblasts and cancer cells to be visualized and monitored by microscopy. To achieve the effect, the fibroblasts and cancer cells in 50% Matrigel suspension were seeded as independent droplets in separation from each other. To allow for cell migration and interaction, a narrow passage of Matrigel causeway was constructed in between the droplets, effectively molding the gel into the shape of a dumbbell. Under time-lapse microscopy, we were able to visualize and image the entire process of fibroblast-guided cancer cell migration event, from initial vessel-like structure formation by the fibroblasts to their subsequent invasion across the causeway, attracting and trapping the cancer cells in the process. Upon prolonged culture, the entire population of fibroblasts eventually infiltrated across the passage and condensed into a spheroid-like cell mass, encapsulating the bulk of the cancer cell population within. Suitable for almost every cell type, our model has the potential for a wider application as it can be adapted for use in drug screening and the study of cellular factors involved in cell-cell attraction.

Translocating a High-Affinity Designer TIMP-1 to the Cell Membrane for Total Renal Carcinoma Inhibition: Putting the Prion Protein to Good Use.

Membrane type 1-matrix metalloproteinase (MT1-MMP) and tumor necrosis factor α (TNF-α)-converting enzyme (TACE) are prominent membrane-anchored metalloproteinases that regulate the turnover of extracellular matrix (ECM) components and bioactive molecules required for cancer proliferation. In this study, we describe a novel approach that would allow tissue inhibitor of metalloproteinase 1 (TIMP-1), the endogenous inhibitor of the matrix metalloproteinases (MMPs), to be translocated to the cell membrane for simultaneous MT1-MMP/TACE inhibition. We achieve this by fusing T1TACE, a designer TIMP-1 with superb affinities for MT1-MMP and TACE, to the glycosyl-phosphatidyl inositol anchor of prions to create a membrane-tethered, broad-spectrum inhibitor, named T1Pr αTACE, that colocalizes with MT1-MMP and TACE on the cell surface. Transduction of T1Pr αTACE in human fibrosarcoma cells results not only in a substantial reduction in gelatinolytic and TNF-α/heparin binding epithelial growth factor shedding activities but also in a loss of tubulogenic capability in three-dimensional matrices. In renal carcinoma, T1Pr αTACE triggers cellular senescence and disrupts MMP-mediated proteolysis of ECM components such as fibronectin and collagen I, leading to an impairment in cell motility and survival under both in vitro and in vivo conditions. Taken together, our findings may provide a new perspective in TIMP targeting that could be exploited to halt metastatic renal carcinoma progression.

Targeting a Designer TIMP-1 to the Cell Surface for Effective MT1-MMP Inhibition: A Potential Role for the Prion Protein in Renal Carcinoma Therapy.

Renal carcinoma cells express Membrane Type 1-Matrix Metalloproteinase (MT1-MMP, MMP-14) to degrade extracellular matrix components and a range of bioactive molecules to allow metastasis and cell proliferation. The activity of MT1-MMP is modulated by the endogenous inhibitors, Tissue Inhibitor of Metalloproteinases (TIMPs). In this study, we describe a novel strategy that would enable a "designer" TIMP-1 tailored specifically for MT1-MMP inhibition (V4A/P6V/T98L; Kiapp 1.66 nM) to be targeted to the plasma membrane for more effective MT1-MMP inhibition. To achieve this, we fuse the designer TIMP-1 to the glycosyl-phosphatidyl inositol (GPI) anchor of the prion protein to create a membrane-tethered, high-affinity TIMP variant named "T1Pr αMT1" that is predominantly located on the cell surface and co-localised with MT1-MMP. Confocal microscopy shows that T1Pr αMT1 is found throughout the cell surface in particular the membrane ruffles where MT1-MMP is most abundant. Expression of T1Pr αMT1 brings about a complete abrogation of the gelatinolytic activity of cellular MT1-MMP in HT1080 fibrosarcoma cells whilst in renal carcinoma cells CaKi-1, the GPI-TIMP causes a disruption in MMP-mediated proteolysis of ECM components such as fibronectin, collagen I and laminin that consequently triggers a downstream senescence response. Moreover, the transduced cells also suffer from an impairment in proliferation and survival in vitro as well as in NOD/SCID mouse xenograft. Taken together, our findings demonstrate that the GPI anchor of prion could be exploited as a targeting device in TIMP engineering for MT1-MMP inhibition with a potential in renal carcinoma therapy.

Ensnaring membrane type 1-matrix metalloproteinase (MT1-MMP) with tissue inhibitor of metalloproteinase (TIMP)-2 using the haemopexin domain of the protease as a carrier: a targeted approach in cancer inhibition.

Metastatic cancer cells express Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) to degrade the extracellular matrix in order to facilitate migration and proliferation. Tissue Inhibitor of Metalloproteinase (TIMP)-2 is the endogenous inhibitor of the MMP. Here, we describe a novel and highly effective fusion strategy to enhance the delivery of TIMP-2 to MT1-MMP. We can reveal that TIMP-2 fused to the haemopexin +/- transmembrane domains of MT1-MMP (two chimeras named T2PEX+TM and T2PEX) are able to interact with MT1-MMP on the cell surface as well as intracellularly. In the case of T2PEX+TM, there is even a clear sign of MT1-MMP:T2PEX+TM aggregation by the side of the nucleus to form aggresomes. In vitro, T2PEX+TM and T2PEX suppress the gelatinolytic and invasive abilities of cervical carcinoma (HeLa) and HT1080 fibrosarcoma cancer cells significantly better than wild type TIMP-2. In mouse xenograft, we further demonstrate that T2PEX diminishes cervical carcinoma growth by 85% relative to the control. Collectively, our findings indicate the effectiveness of the fusion strategy as a potential targeted approach in cancer inhibition.

ADAMTS13 and 15 are not regulated by the full length and N-terminal domain forms of TIMP-1, -2, -3 and -4.

A disintegrin and metalloproteinase with thombospondin motifs (ADAMTS) 13 and 15 are secreted zinc proteinases involved in the turnover of von Willebrand factor and cancer suppression. In the present study, ADAMTS13 and 15 were subjected to inhibition studies with the full-length and N-terminal domain forms of tissue inhibitor of metalloproteinases (TIMPs)-1 to -4. TIMPs have no ability to inhibit the ADAMTS proteinases in the full-length or N-terminal domain form. While ADAMTS13 is also not sensitive to the hydroxamate inhibitors, batimastat and ilomastat, ADAMTS15 can be effectively inhibited by batimastat (Kiapp 299 nM). In conclusion, the present results indicate that TIMPs are not the regulators of these two ADAMTS proteinases.

Expanding the Activity of Tissue Inhibitors of Metalloproteinase (TIMP)-1 against Surface-Anchored Metalloproteinases by the Replacement of Its C-Terminal Domain: Implications for Anti-Cancer Effects.

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, we replaced the C-terminal domain of TIMP-1 with those of TIMP-2, -3 and -4 to create a series of "T1:TX" chimeras. The affinity of the chimeras against ADAM10, ADAM17, MMP14 and MMP19 was investigated. We can show that replacement of the C-terminal domain by those of other TIMPs dramatically increased the affinity of TIMP-1 for some MPs. Furthermore, the chimeras were able to suppress TNF-α and HB-EGF shedding in cell-based setting. Unlike TIMP-1, T1:TX chimeras had no growth-promoting activity. Instead, the chimeras were able to inhibit cell migration and development in several cancer cell lines. Our findings have broadened the prospect of TIMPs as cancer therapeutics. The approach could form the basis of a new strategy for future TIMP engineering.

Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex.

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.

Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2).

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 alpha-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.

The intrinsic protein flexibility of endogenous protease inhibitor TIMP-1 controls its binding interface and affects its function.

Protein flexibility is thought to play key roles in numerous biological processes, including antibody affinity maturation, signal transduction, and enzyme catalysis, yet only limited information is available regarding the molecular details linking protein dynamics with function. A single point mutation at the distal site of the endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1) enables this clinical target protein to tightly bind and inhibit membrane type 1 matrix metalloproteinase (MT1-MMP) by increasing only the association constant. The high-resolution X-ray structure of this complex determined at 2 A could not explain the mechanism of enhanced binding and pointed to a role for protein conformational dynamics. Molecular dynamics (MD) simulations reveal that the high-affinity TIMP-1 mutants exhibit significantly reduced binding interface flexibility and more stable hydrogen bond networks. This was accompanied by a redistribution of the ensemble of substrates to favorable binding conformations that fit the enzyme catalytic site. Apparently, the decrease in backbone flexibility led to a lower entropy cost upon formation of the complex. This work quantifies the effect of a single point mutation on the protein conformational dynamics and function of TIMP-1. Here we argue that controlling the intrinsic protein dynamics of MMP endogenous inhibitors may be utilized for rationalizing the design of selective novel protein inhibitors for this class of enzymes.

The activity of a designer tissue inhibitor of metalloproteinases (TIMP)-1 against native membrane type 1 matrix metalloproteinase (MT1-MMP) in a cell-based environment.

The surface-anchored membrane type 1 matrix metalloproteinase (MT1-MMP) degrades a wide range of extracellular matrix components that includes collagens, laminins, fibronectin and the structural proteoglycan aggrecan. The enzyme modulates cell motility and plays an important role in tumour invasion and proliferation. We have previously designed a variant of tissue inhibitor of metalloproteinase (TIMP)-1 bearing a triple mutation (V4A+P6V+T98L, or N-TIMP-1(mt1)) that forms tight binary complex with the soluble catalytic domain of MT1-MMP [M.H. Lee, M. Rapti, G. Murphy, J. Biol. Chem. 278 (2003) 40224-40230]. Here, we report our latest findings on the cellular potency of this mutant against native MT1-MMP in cell-based environment. We show that N-TIMP-1(mt1) is a highly potent inhibitor against the ectodomain form of MT1-MMP (K(i) 9.53nM) with potential for further development as a therapeutic agent. The mutant is devoid of pro-MMP-2-activating capability but is highly effective in blocking MT1-MMP-mediated FITC-labelled collagen and gelatin film degradation in HTC75 fibrosarcoma and MCF7 breast cancer models. Most encouragingly, N-TIMP-1(mt1) is also effective against CD44 shedding in HTC75 cells and able to prevent tubule formation in human umbilical vascular endothelial cells (HUVEC) in a 3D fibrin gel model. We are interested in the development of the TIMPs as therapeutic agents against MT1-MMP related disorders such as cancers. Our findings here indicate the potential for the design of selective TIMPs with refined specificity and possibility for future therapeutic application.

The isolated N-terminal domains of TIMP-1 and TIMP-3 are insufficient for ADAM10 inhibition.

ADAM (a disintegrin and metalloproteinase) 10 is a key member of the ADAM family of disintegrin and metalloproteinases which process membrane-associated proteins to soluble forms in a process known as 'shedding'. Among the major targets of ADAM10 are Notch, EphrinA2 and CD44. In many cell-based studies of shedding, the activity of ADAM10 appears to overlap with that of ADAM17, which has a similar active-site topology relative to the other proteolytically active ADAMs. The tissue inhibitors of metalloproteinases, TIMPs, have proved useful in the study of ADAM function, since TIMP-1 inhibits ADAM10, but not ADAM17; however, both enzymes are inhibited by TIMP-3. In the present study, we show that, in comparison with ADAM17 and the MMPs (matrix metalloproteinases), the N-terminal domains of TIMPs alone are insufficient for the inhibition of ADAM10. This knowledge could form the basis for the design of directed inhibitors against different metalloproteinases.

Identification of the extracellular matrix (ECM) binding motifs of tissue inhibitor of metalloproteinases (TIMP)-3 and effective transfer to TIMP-1.

Tissue inhibitor of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent endopeptidases of the matrix metalloproteinase families. There are four mammalian TIMPs (TIMP-1 to -4) but only TIMP-3 is sequestered to the extracellular matrix (ECM). The molecular basis for the TIMP-3:ECM association has never been fully investigated until now. In this report, we identify the unique amino acid configuration that constitutes the basis of the ECM binding motif in TIMP-3. By systematically exchanging the subdomains of the TIMPs and exhaustive mutation of TIMP-3, we have identified the surface residues directly responsible for ECM association. Contrary to the accepted view, we have found that TIMP-3 interacts with the ECM via both its N- and C-terminal domains. The amino acids involved in ECM binding are all basic in nature: Lys-26, Lys-27, Lys-30, Lys-76 of the N-terminal domain and Arg-163, Lys-165 of the C-terminal domain. Replacement of these residues with glutamate (E) and glutamine (Q) (K26/27/30/76E + R163/K165Q) resulted in a soluble TIMP-3 devoid of ECM-adhering ability. Using the ECM binding motif derived from TIMP-3, we have also created a TIMP-1 mutant (K26/27/30 + K76 transplant) capable of ECM association. This is the first instance of TIMPs being intentionally rendered soluble or ECM-bound. The ability to prepare TIMPs in soluble or ECM-bound forms also opens new avenues for future TIMP research.

Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting: fine-tuning TIMP-4 for optimal inhibition of tumor necrosis factor-{alpha}-converting enzyme.

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs (a disintegrin and metalloproteinase) and the ADAM-TS (ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-alpha-converting enzyme (TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 (N-TIMP-4) with TACE and showed that N-TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N-TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N-TIMP-4 resulted in a 10-fold enhancement in binding affinity; the K(i) values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.

Delineating the molecular basis of the inactivity of tissue inhibitor of metalloproteinase-2 against tumor necrosis factor-alpha-converting enzyme.

Tumor necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE, ADAM-17) is a zinc-dependent ADAM (a disintegrin and metalloproteinase) metalloproteinase (MP) of the metzincin superfamily. The enzyme regulates the shedding of a variety of cell surface-anchored molecules such as cytokines, growth factors, and receptors. The activities of the MPs are modulated by the endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Among the four mammalian TIMPs (TIMP-1 to -4), TACE is selectively inhibited by TIMP-3. The rationale for such selectivity is not fully understood. Here, we examine the molecular basis of TIMP-TACE selectivity using TIMP-2 as the scaffold. By systematically replacing the surface epitopes of TIMP-2 with those of TIMP-3 and a TIMP-1 variant V4S/TIMP-3 AB-loop/V69L/T98L, we created a novel TIMP-2 mutant that exhibits inhibitory potency almost equal to that of the TIMP-3. The affinity of the mutant with TACE is 1.49 nm, a marked improvement in comparison to that of the wild-type protein (Ki 893 nM). The inhibitory pattern of the mutant is typical of that of a slow, tight binding inhibitor. We identify phenylalanine 34, a residue unique to the TIMP-3 AB-loop, as a vital element in TACE association. Mutagenesis carried out on leucine 100 also upholds our previous findings that a leucine on the EF-loop is critical for TACE recognition. Replacement of the residue by other amino acids resulted in a dramatic decrease in binding affinity, although isoleucine (L100I) and methionine (L100M) are still capable of producing the slow, tight binding effect. Our findings here represent a significant advance toward designing tailor-made TIMPs for specific MP targeting.

Threonine 98, the pivotal residue of tissue inhibitor of metalloproteinases (TIMP)-1 in metalloproteinase recognition.

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous modulators of the zinc-dependent mammalian matrix metalloproteinases (MMPs) and their close associates, proteinases of the ADAM (a disintegrin and metalloproteinase) and ADAM with thrombospondin repeats families. There are four variants of TIMPs, and each has its defined set of metalloproteinase (MP) targets. TIMP-1, in particular, is inactive against several of the membrane-type MMPs (MT-MMPs), MMP-19, and the ADAM proteinase TACE (tumor necrosis factor-alpha-converting enzyme, ADAM-17). The molecular basis for such inactivity is unknown. Previously, we showed that TIMP-1 could be transformed into an active inhibitor against MT1-MMP by the replacement of threonine 98 residue with leucine (T98L). Here, we reveal that the T98L mutation has in fact transformed TIMP-1 into a versatile inhibitor against an array of MPs otherwise insensitive to wild-type TIMP-1; examples include TACE, MMP-19, and MT5-MMP. Using T98L as the scaffold, we created a TIMP-1 variant that is fully active against TACE. The binding affinity of the mutant (V4S/TIMP-3-AB-loop/V69L/T98L) (K (app)(i) 0.14 nm) surpassed that of TIMP-3 (K (app)(i) 0.22 nm), the only natural TIMP inhibitor of the enzyme. The requirement for leucine is absolute for the transformation in inhibitory pattern. On the other hand, the mutation has minimal impact on the MPs already well inhibited by wild-type TIMP-1, such as gelatinase-A and stromelysin-1. Not only have we unlocked the molecular basis for the inactivity of TIMP-1 against several of the MPs, but also our findings fundamentally modify the current beliefs on the molecular mechanism of TIMP-MP recognition and selectivity.

Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail.

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.

Unveiling the surface epitopes that render tissue inhibitor of metalloproteinase-1 inactive against membrane type 1-matrix metalloproteinase.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity.

Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-alpha converting enzyme.

Tumour necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) is a membrane-anchored, multiple-domain zinc metalloproteinase responsible for the release of the potent pro-inflammatory cytokine, TNF-alpha. The extracellular part of the active enzyme is composed of a catalytic domain and several cysteine-rich domains. Previously, we reported that these cysteine-rich domains significantly weakened the inhibitory potency of the N-terminal-domain form of tissue inhibitor of metalloproteinases-3 (N-TIMP-3). In the present paper, we describe a novel strategy developed to overcome this weakening effect. We have engineered a new generation of N-TIMP-3 mutants that are capable of withstanding the repulsion of the cysteine-rich domains by the formation of electrostatic bonds with the catalytic domain of the enzyme. These N-TIMP-3 mutants displayed markedly improved binding affinity with the soluble extracellular domain form of recombinant TACE. With K (i) (app) values of <0.1 nM, these mutants were dramatically better than the wild-type N-TIMP-3 [K (i) (app) 1.7 nM]. We accounted for this by proposing that Glu(31), an acidic residue situated at the base of the AB-loop of N-TIMP-3, is drawn into contact with Lys(315), a prominent basic residue adjacent to the TACE catalytic site. The mutagenesis strategy involved reorientation of the edge of N-TIMP-3; in particular, the beta-strand A where Glu(31) was located. Further expression of one of the mutants, Lys(26/27/30/76)-->Glu, in a mammalian expression system confirmed that TIMP-3 associates with the extracellular matrix via its C-terminal domain.