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Large inhibitor of metalloproteinases (LIMP) contains tissue inhibitor of metalloproteinases (TIMP)-2 bound to 72,000-M(r) progelatinase.

ABSTRACT: Connective-tissue cells in culture produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that this inhibitor was solely responsible for the inhibition of these enzymes within connective tissue. However, other inhibitors have recently been described, including large inhibitor of metalloproteinases (LIMP) present in the culture medium of human foetal lung fibroblasts. Here we show that a large proportion of the inhibitory activity of LIMP consists of 72,000-M(r)-progelatinase bound to TIMP-2, a recently discovered low-M(r) metalloproteinase inhibitor closely related to TIMP. The physiological implications of the secretion of a complex of 72,000-M(r) progelatinase and TIMP-2 are discussed, and the separation of the complex in 6 M-urea is described.

SUBMITTER: Curry VA

PROVIDER: S-EPMC1132757 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

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Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

Project description:Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.

| S-EPMC1131427 | biostudies-other

Interaction between tissue inhibitor of metalloproteinases-2 and progelatinase A: immunoreactivity analyses.

Project description:By immunoreactivity analysis using monoclonal antibodies, we showed that the C-terminal domain [R415-631; R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected the immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31-45], confirming that the C-terminal domain ("tail" portion of TIMP-2 participates in the binding with the C-terminal domain of proMMP-2. We also demonstrated that not only the C-terminal domain but also the N-terminal domain (R1-417) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of each individual domain to TIMP-2, however, was weak enough that either domain could be fully replaced by proMMP-2 through the same binding sites as does proMMP-2, and also that the high-order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 complexed with the N-terminal domain of pro-MMP-2 had fully inhibitory activity against the collagenolytic activity of MMP-1. We also demonstrated that either the interstitial collagenase-TIMP-2 complex or the gelatinase B(MMP-9)-TIMP-2 complex was able to form a ternary complex with proMMP-2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 complex, but the binding seemed to be less stable than the binding with TIMP-2 alone. Even in the presence of a 10-fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9--TIMP-2 complex. These clear differences might be ascribed to some significant conformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex formation with MMP-9.

| S-EPMC1216985 | biostudies-other

The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1.

Project description:Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.

| S-EPMC1151465 | biostudies-other

Dissociation of tissue inhibitor of metalloproteinases (TIMP) from enzyme complexes yields fully active inhibitor.

Project description:Recombinant human tissue inhibitor of metalloproteinases (TIMP) forms complexes with high-Mr active recombinant stromelysin that are stable over long periods under physiological conditions. TIMP-stromelysin complexes could be dissociated in the presence of EDTA at pH 3, releasing free TIMP and destroying stromelysin activity. The dissociated TIMP was apparently unmodified, in contrast with other known protein inhibitors of metalloproteinases and many classes of serine-proteinase inhibitor, which are slowly cleaved.

| S-EPMC1138932 | biostudies-other

Sequence motifs of tissue inhibitor of metalloproteinases 2 (TIMP-2) determining progelatinase A (proMMP-2) binding and activation by membrane-type metalloproteinase 1 (MT1-MMP).

Project description:Fundamental cellular processes including angiogenesis and cell migration require a proteolytic cascade driven by interactions of membrane-type matrix metalloproteinase 1 (MT1-MMP) and progelatinase A (proMMP-2) that are dependent on the presence of tissue inhibitor of metalloproteinases 2 (TIMP-2). There are unique interactions between TIMP-2 and MT1-MMP, which we have previously defined, and here we identify TIMP-2 sequence motifs specific for proMMP-2 binding in the context of its activation by MT1-MMP. A TIMP-2 mutant encoding the C-terminal domain of TIMP-4 showed loss of proMMP-2 activation, indicating that the C-terminal domain of TIMP-2 is important in establishing the trimolecular complex between MT1-MMP, TIMP-2 and proMMP-2. This was confirmed by analysis of a TIMP-4 mutant encoding the C-terminal domain of TIMP-2, which formed a trimolecular complex and promoted proMMP-2 processing to the intermediate form. Mutants encoding TIMP-4 from Cys(1) to Leu(185) and partial tail sequence of TIMP-2 showed some gain of activating capability relative to TIMP-4. The identified residues were subsequently mutated in TIMP-2 (E(192)-D(193) to I(192)-Q(193)) and this inhibitor showed a significantly reduced ability to facilitate proMMP-2 processing by MT1-MMP. Furthermore, the tail-deletion mutant Delta(186-194)TIMP-2 was completely incapable of promoting proMMP-2 activation by MT1-MMP. Thus the C-terminal tail residues of TIMP-2 are important determinants for stable trimolecular complex formation between TIMP-2, proMMP-2 and MT1-MMP and play an important role in MT1-MMP-mediated processing to the intermediate and final active forms of MMP-2 at the cell surface.

| S-EPMC1223438 | biostudies-other

Steps involved in activation of the complex of pro-matrix metalloproteinase 2 (progelatinase A) and tissue inhibitor of metalloproteinases (TIMP)-2 by 4-aminophenylmercuric acetate.

Project description:Tissue inhibitor of metalloproteinases (TIMP)-2 forms a noncovalent complex with the precursor of matrix metalloproteinase 2 (proMMP-2, progelatinase A) through interaction of the C-terminal domain of each molecule. We have isolated the proMMP-2-TIMP-2 complex from the medium of human uterine cervical fibroblasts and investigated the processes involved in its activation by 4-aminophenylmercuric acetate (APMA). The treatment of the complex with APMA-activated proMMP-2 by disrupting the Cys73-Zn2+ interaction of the zymogen. This is triggered by perturbation of the proMMP-2 molecule, but not by the reaction of the SH group of Cys73 with APMA. The 'activated' proMMP-2 (proMMP-2*) formed a new complex with TIMP-2 by binding to the N-terminal inhibitory domain of the inhibitor without processing the propeptide. Thus the APMA-treated proMMP-2*-TIMP-2 complex exhibited no gelatinolytic activity. In the presence of a small amount of free MMP-2, however, proMMP-2* in the complex was converted into the 65 kDa MMP-2 by proteolytic attack of MMP-2, but the complex did not exhibit gelatinolytic activity. The gelatinolytic activity detected after APMA treatment was solely derived from the activation of free proMMP-2. The removal of the propeptide of the proMMP-2* bound to TIMP-2 was also observed by MMP-3 (stromelysin 1), but not by MMP-1 (interstitial collagenase). MMP-3 cleaved the Asn80-Tyr81 bond of proMMP-2*. On the other hand, when MMP-3 was incubated with the proMMP-2-TIMP-2 complex, it bound to TIMP-2 and rendered proMMP-2 readily activatable by APMA. These results indicate that the blockage of TIMP-2 of the complex with an active MMP is essential for the activation of proMMP-2 when it is complexed with TIMP-2.

| S-EPMC1136975 | biostudies-other

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering.

Project description:Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.

| S-EPMC2373703 | biostudies-literature

Characterization of the functional domain of tissue inhibitor of metalloproteinases-2 (TIMP-2).

Project description:Analysis of the functional domain of tissue inhibitor of metallo-proteinases-2 (TIMP-2) was performed using limited proteolytic degradation with trypsin. This treatment generated a 13.5 kDa fragment which was purified and shown to consist of an uncleaved N-terminal region extending from residue 1 to residue 132. The fragment retains the ability to inhibit activated interstitial collagenase and to block the autocatalytic activation of procollagenase.

| S-EPMC1132131 | biostudies-other

Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Project description:Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain-associated protein kinase-70 (ZAP-70) signaling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1-CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1-CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide-abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1-CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

| S-EPMC8429975 | biostudies-literature

Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10.

Project description:Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.

| S-EPMC8902619 | biostudies-literature

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