Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.     

Gelatinases and metalloproteinase inhibitor secreted by murine colonic carcinoma cells with differing metastatic potential

Gelatinase activity and inhibitory activity against collagenase were measured in serum-free medium conditioned by murine colonic carcinoma cells with different spontaneous metastatic potentials to the lung. The medium conditioned with poorly metastatic NM11 cells gave higher inhibitory activity than that conditioned with highly metastatic LuM1 cells, while the level of secreted gelatinases in the same medium was lower in NM11 medium than in LuM1 case. Northern analysis showed the higher gene expression of both tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in NM11 cells than in LuM1 cells, suggesting that both TIMPs are responsible for the increase of inhibitory activity in NM11 conditioned medium. Examination of the balance of gelatinases and inhibitor revealed that the amount of inhibitor exceeded that of gelatinases in the medium conditioned with NM11 cells. In contrast, the medium conditioned with LuM1 cells contained excess amounts of gelatinases. The results indicated a close correlation between the balance of gelatinases and inhibitors and the metastatic behavior of murine tumor cells.     

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

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.     

Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs), enzymes that play central roles in the degradation of extracellular matrix components. The balance between MMPs and TIMPs is important in the maintenance of tissues, and its disruption affects tissue homoeostasis. Four related TIMPs (TIMP-1 to TIMP-4) can each form a complex with MMPs in a 1:1 stoichiometry with high affinity, but their inhibitory activities towards different MMPs are not particularly selective. The three-dimensional structures of TIMP-MMP complexes reveal that TIMPs have an extended ridge structure that slots into the active site of MMPs. Mutation of three separate residues in the ridge, at positions 2, 4 and 68 in the amino acid sequence of the N-terminal inhibitory domain of TIMP-1 (N-TIMP-1), separately and in combination has produced N-TIMP-1 variants with higher binding affinity and specificity for individual MMPs. TIMP-3 is unique in that it inhibits not only MMPs, but also several ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin motifs) metalloproteinases. Inhibition of the latter groups of metalloproteinases, as exemplified with ADAMTS-4 (aggrecanase 1), requires additional structural elements in TIMP-3 that have not yet been identified. Knowledge of the structural basis of the inhibitory action of TIMPs will facilitate the design of selective TIMP variants for investigating the biological roles of specific MMPs and for developing therapeutic interventions for MMP-associated diseases.     

Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3

The four tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of matrix metalloproteinases (MMPs) and certain disintegrin and metalloproteinase (ADAM) family proteases in mammals. The protease inhibitory activity is present in the N-terminal domains of TIMPs (N-TIMPs). In this work, the N-terminal inhibitory domain of the only TIMP produced by Drosophila (dN-TIMP) was expressed in Escherichia coli and folded in vitro. The purified recombinant protein is a potent inhibitor of human MMPs, including membrane-type 1-MMP, although it lacks a disulfide bond that is conserved in all other known N-TIMPs. Titration with the catalytic domain of human MMP-3 [MMP-3(DeltaC)] showed that dN-TIMP prepared by this method is correctly folded and fully active. dN-TIMP also inhibits, in vitro, the activity of the only two MMPs of Drosophila, dm1- and dm2-MMPs, indicating that the Drosophila TIMP is an endogenous inhibitor of the Drosophila MMPs. dN-TIMP resembles mammalian N-TIMP-3 in strongly inhibiting human tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) but is a weak inhibitor of human ADAM10. Models of the structures of dN-TIMP and N-TIMP-3 are strikingly similar in surface charge distribution, which may explain their functional similarity. Although the gene duplication events that led to the evolutionary development of the four mammalian TIMPs might be expected to be associated with functional specialization, Timp-3 appears to have conserved most of the functions of the ancestral TIMP gene.     

PEGylation Extends Circulation Half-Life While Preserving In Vitro and In Vivo Activity of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG_20K-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG_20K-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.     

X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. comparison with the latent and active forms of the enzyme

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases. It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.     

Gelatinase-mediated migration and invasion of cancer cells

The matrix metalloproteinases(MMP)-2 and -9, also known as the gelatinases have been long recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. In the recent years, a plethora of non-matrix proteins have also been identified as gelatinase substrates thus significantly broadening our understanding of these enzymes as proteolytic executors and regulators in various physiological and pathological states including embryonic growth and development, angiogenesis and tumor progression, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. Although the effect of broad-spectrum inhibitors of MMPs in the treatment of cancer has been disappointing in clinical trials, novel mechanisms of gelatinase inhibition have been now identified. Inhibition of the association of the gelatinases with cell-surface integrins appears to offer highly specific means to target these enzymes without inhibiting their catalytic activity in multiple cell types including endothelial cells, tumor cells and leukocytes. Here, we review the multiple functions of the gelatinases in cancer, and especially their role in the tumor cell migration and invasion.     

The N-terminus of collagenase MMP-8 determines superactivity and inhibition: a relation of structure and function analyzed by biomolecular interaction analysis.

Tissue inhibitors of metalloproteinases (TIMPs) are the physiological, specific inhibitors of matrix metalloproteinases (MMPs) forming tight, noncovalent complexes. Therefore they control the proteolytic activity of MMPs toward the extracellular matrix. To analyze the inhibition of the "activated" and "superactivated" variants of human neutrophil collagenase (MMP-8) by TIMP-2, we determined complex dissociation constants using biomolecular interaction analysis (BIA). As it is known that the association rate constants can exceed the limits of the BIA instruments, the biomolecular interaction analysis was used to examine the equlibrium situation. The dissociation constants were determined by fitting the parameters of the mathematical term for the binding of collagenase onto the TIMP-coupled sensor chip surface to the saturation curve derived from individual sensorgrams. The resulting values are in the nanomolar range and correlate with the results of fluorescence kinetics. These data reveal that TIMP-2 (the recombinant inhibitory domain of human TIMP-2 and bovine TIMP-2 isolated from seminal plama) is a better inhibitor of the activated neutrophil collagenase than of the superactivated variant (the recombinant catalytic domain of human MMP-8). It has been demonstrated by X-ray analysis that the N-terminal heptapeptide only of superactivated MMP-8 is attached by a salt bridge and hydrophobic interaction to the C-terminal helix. Because these interactions have to be disrupted in the complex formation with TIMP we assume that the activated variant enables higher flexibility and a tighter induced fit in the complex formation. Therefore superactivation of MMP-8 correlates with weaker inhibition by TIMP-2.     

Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction

Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.     

Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis

Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the breakdown of extracellular matrix components and play an important role in tissue remodelling and growth, in both physiological and pathological conditions. We studied the autoimmune response to TIMPs in patients with rheumatoid arthritis (RA). Eighty-nine paired blood and synovial fluid samples from patients with RA were assessed for their reactivity with recombinant tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 by an ELISA and were compared with blood from 62 healthy controls and 21 synovial fluid samples from patients with degenerative joint diseases. Presence of antibodies was established as the absorbance of the sample more than 2 standard deviations above the mean of the controls. In addition, immunoglobulin G (IgG) from blood samples of RA patients possessing TIMP antibodies was isolated on protein A–sepharose and tested for the in vitro ability to neutralize TIMP-2-dependent effects on metalloproteinase 9 (MMP9). Anti-TIMP antibodies were found in 56% of RA samples but in only 5% of the controls (P < 0.005). RA patients had high frequencies of antibodies against all TIMPs except TIMP-3. TIMP-2 antibodies were most frequently found (33%), being significantly more prevalent (P = 0.024) in patients with nonerosive than erosive RA. TIMP-1 antibodies were significantly more often found in synovial fluid samples than in the matched blood samples (P < 0.025). Importantly, the IgG fraction containing TIMP antibodies down-regulated the TIMP-2 inhibitory effect, thereby supporting MMP9 activity in vitro. In the present study, we show that RA patients frequently develop autoimmune response to TIMPs that may act as a functionally significant regulator of MMP activity and thereby of joint destruction.     

Kinetic analysis of the binding of hemopexin-like domain of gelatinase B cloned and expressed in Pichia pastoris to tissue inhibitor of metalloproteinases-1

The gelatinases are a subgroup of the matrix metalloproteinase family. The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases. To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris. The active refolded domain was purified by ion exchange chromatography and gel filtration. We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR). The dissociation constant Kd was calculated to be 0.86 nM. Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M. W. et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model.     

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.     

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.     

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.     

Activity modulation of microbial enzymes by llama (Lama glama) heavy-chain polyclonal antibodies during in vivo immune responses

Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.     

Structural basis of the matrix metalloproteinases and their physiological inhibitors,the tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases with a metzincin-like catalytic domain, which are involved in extracellular matrix degradation but also in a number of other important biological processes. Under healthy conditions, their proteolytic activity is precisely regulated by their main endogenous protein inhibitors, the tissue inhibitors of metalloproteinases. Disruption of this balance results in pathophysiological processes such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and for rational drug design. Since the first appearance of atomic MMP structures in 1994, a large amount of structural information has become available on the catalytic domains of MMPs and their substrate specificity, interaction with synthetic inhibitors and the TIMPs, the domain organization, and on complex formation with other proteins. This review will outline our current structural knowledge of the MMPs and the TIMPs.     

Unbalanced collagenases/TIMP-1 expression and epithelial apoptosis in experimental lung fibrosis

In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.     

TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2

ADAMTS-2 is an extracellular metalloproteinase responsible for cleaving the N-propeptides of procollagens I-III; an activity necessary for the formation of collagenous ECM (extracellular matrix). The four TIMPs (tissue inhibitors of metalloproteinases) regulate the activities of matrix metalloproteinases, which are involved in degrading ECM components. Here we delineate the abilities of the TIMPs to affect biosynthetic processing of procollagens. TIMP-1, -2 and -4 show no inhibitory activity towards ADAMTS-2, in addition none of the TIMPs showed inhibitory activity towards bone morphogenetic protein 1, which is responsible for cleaving procollagen C-propeptides. In contrast, TIMP-3 is demonstrated to inhibit ADAMTS-2 in vitro with apparent Ki values of 160 and 602 nM, in the presence of heparin or without respectively; and TIMP-3 is shown to inhibit procollagen processing by cells.