Flexibility and variability of TIMP binding: X-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.

MMP-TIMP interaction depends on residue 2 in TIMP-4.

Extracellular matrix remodeling and degradation are of great importance in both physiological and pathological situations. Matrix metalloproteinases (MMPs) and their natural occurring inhibitors - tissue inhibitors of metalloproteinases (TIMPs) - are involved in matrix turnover. Among the TIMPs there is only little specificity for inhibiting individual MMPs. In this report we describe the mutational analysis of the interaction of human TIMP-4 with several MMPs. The effects of different substitutions of residue 2 (Ser(2)) in the inhibitory domain of TIMP-4 were determined by kinetic measurements. Size, charge and polarity of residue 2 in the TIMP structure are key factors in MMP inhibition.

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure.

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.

Structure-based design and synthesis of potent matrix metalloproteinase inhibitors derived from a 6H-1,3,4-thiadiazine scaffold.

We describe a new generation of heterocyclic nonpeptide matrix metalloproteinase (MMP) inhibitors derived from a 6H-1,3,4-thiadiazine scaffold. A screening effort was utilized to identify some chiral 6-methyl-1,3,4-thiadiazines that are weak inhibitors of the catalytic domain of human neutrophil collagenase (cdMMP-8). Further optimization of the lead compounds revealed general design principles that involve the placement of a phenyl or thienyl group at position 5 of the thiadiazine ring, to improve unprimed side affinity; the incorporation of an amino group at position 2 of the thiadiazine ring as the chelating agent for the catalytic zinc; the placement of a N-sulfonamide-substituted amino acid residue at the amino group, to improve primed side affinity; and the attachment of diverse functional groups at position 4 or 5 of the phenyl or thienyl group at the unprimed side, to improve selectivity. The new compounds were assayed against eight different matrix metalloproteinases, MMP-1, cdMMP-2, cdMMP-8, MMP-9, cdMMP-12, cdMMP-13, cdMMP-14, and the ectodomain of MMP-14, respectively. A unique combination of the above-described modifications produced the selective inhibitor (2R)-N-[5-(4-bromophenyl)-6H-1,3,4-thiadiazin-2-yl]-2-[(phenylsulfonyl)amino]propanamide with high affinity for MMP-9 (K(i) = 40 nM). X-ray crystallographic data obtained for cdMMP-8 cocrystallized with N-allyl-5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-amine hydrobromide gave detailed design information on binding interactions for thiadiazine-based MMP inhibitors.

Characterization of C-terminally truncated human tissue inhibitor of metalloproteinases-4 expressed in Pichia pastoris.

The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover. In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris. The inhibition of MMP-8, -9, -12, -13 and -14 by the N-terminal domain of TIMP-4 was analysed. Using a fluorescent MCA-peptide, Ki values for each subclass of MMPs were determined. With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.

Novel heterocyclic inhibitors of matrix metalloproteinases: three 6H-1,3,4-thiadiazines.

The title compounds, (2S)-N-[5-(4-chlorophenyl)-2,3-dihydro-6H-1,3,4-thiadiazin-2-ylidene]-2-[(phenylsulfonyl)amino]propanamide, C18H17ClN4O3S2, (I), (2R)-N-[5-(4-fluorophenyl)-6H-1,3,4-thiadiazin-2-yl]-2-[(phenylsulfonyl)amino]propanamide, C18H17FN4O3S2, (II), and (2S)-N-[5-(5-chloro-2-thienyl)-6H-1,3,4-thiadiazin-2-yl]-2-[(phenylsulfonyl)amino]propanamide, C16H15ClN4O3S3, (III), are potent inhibitors of matrix metalloproteinases. In all three compounds, the thiadiazine ring adopts a screw-boat conformation. The molecules of compound (I) show a short intramolecular N(Ala)-H...N(exo) hydrogen bond [N...N 2.661 (3) A] and are linked into a chain along the c axis by N(endo)-H...S(endo) and N(endo)-H...O(Ala) hydrogen bonds [N...S 3.236 (3) and N...O 3.375 (3) A] between neighbouring molecules. In compound (II), the molecules are connected antiparallel into a chain along the a axis by N(exo)-H...O(Ala) and N(Ala)-H...N(endo) hydrogen bonds [N...O 2.907 (6) and N...N 2.911 (6) A]. The molecules of compound (III) are dimerized antiparallel through N(exo)-H...N(endo) hydrogen bonds [N...N 2.956 (7) and 2.983 (7) A]. The different hydrogen-bonding patterns can be explained by an amido-imino tautomerism (prototropic shift) shown by different bond lengths within the 6H-1,3,4-thiadiazine moiety.

The human neutrophil lipocalin supports the allosteric activation of matrix metalloproteinases.

The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP-9). Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases. We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly. A significant enhancement of the activity could be demonstrated for the HgCl2- and the plasma kallikrein-induced activation of all three secretory forms of proMMP-9 and of proMMP-8. The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli. This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL. Activation and activity enhancement are obviously mediated by entrapping the remaining N-terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL. In conclusion these results document that HNL can exert an enzyme-activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.

Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin.

ADAM 9 is a member of the cellular metalloprotease/disintegrin/cysteine-rich (MDC) gene family, related to soluble snake venom metalloproteases (SVMP). ADAMs may play important roles in cell-cell fusion, cell-matrix interaction, and other cellular functions. To investigate catalytic activity of human ADAM 9 we have cloned and expressed the metalloprotease domain of human ADAM 9 in Pichia pastoris. The recombinant protein was purified in a three-step purification procedure and activity was detected against gelatin, beta-casein, and fibronectin. In addition we identified five normal and cancer cell lines expressing mRNA of human ADAM 9.

Recombinant cryptic human fibronectinase cleaves actin and myosin: substrate specificity and possible role in muscular dystrophy.

The N-terminal heparin/fibrin binding domain of human plasma fibronectin (pFN) contains a cryptic proteinase. The enzyme could be generated and activated in the presence of Ca2+ from the purified 70 kDa pFN fragment produced by cathepsin D digestion of pFN. In this work we cloned and expressed the serine proteinase, designated fibronectinase (Fnase), in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting fibronectin. Cleavage of pFN and the synthetic peptides Ac-I-E-G-K-pNA and Bz-I-E-G-R-pNA demonstrated identical specificity of the recombinant and the isolated fibronectinase. Further investigations of the substrate specificity revealed for the first time the muscle proteins actin and myosin as being substrates of fibronectinase. The enzyme can be inhibited by alpha1-proteinase inhibitor. In the context of induced cathepsin D release, e. g. from granulocytes under inflammatory conditions, these results indicate an increase in specific proteolytic potential against muscular proteins in dystrophic diseases by the release of cryptic fibronectinase.

Pregnancy-associated plasma protein-E (PAPP-E).

A full-length cDNA encoding a novel human protein was cloned from placenta cDNA. The corresponding 1542 amino acid protein sequence was termed 'pregnancy-associated plasma protein-E' (PAPP-E) as it shows a 62% homology to the human pregnancy-associated plasma protein-A (PAPP-A) that is a diagnostic marker for trisomies, especially Down syndrome. The conserved domain structure contains five motifs related to the short consensus repeats of complement proteins and selectins, three motifs related to the lin-notch motifs of proteins regulating early tissue differentiation, and a putative zinc-binding motif and active site of the metzincin-superfamily of metalloproteases. The PAPP-E gene was localized to chromosome 1q23-25. Northern blot analysis showed that PAPP-E is predominantly expressed in placenta.

Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (Membrane type 1)-MMP-dependent activation of pro-MMP-2.

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.

Two crystal structures of human neutrophil collagenase, one complexed with a primed- and the other with an unprimed-side inhibitor: implications for drug design.

Two crystal structures of human neutrophil collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-L-Trp(P)(OH)(2) occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S(1) subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3, 4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained D-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S(1)' subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation-aromatic interactions can stabilize the formation of both complexes, and the beneficial effect of aromatic substituents in proximity of the catalytic zinc ion is discussed. The phosphonate group bound to either a primed- or unprimed-side inhibitor maintains the same relative position with respect to the catalytic zinc ion, suggesting that this binding function can be exploited for the design of combined inhibitors assembled to interact with both primed and unprimed regions of the active cleft.

Matrix metalloproteinases collagenase-2, macrophage elastase, collagenase-3, and membrane type 1-matrix metalloproteinase impair clotting by degradation of fibrinogen and factor XII.

The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity.

The expression of angiogenin in tissue samples of different brain tumours and cultured glioma cells.

BACKGROUND: As a potential angiogenetic factor the 14.1 kDa polypeptide angiogenin induces neovascularisation. MATERIALS AND METHODS: We investigated the angiogenin expression by immunoblotting and an ELISA in 60 tissue specimens (40 gliomas, 20 other intracranial tumours), in 22 glioma cell cultures and in 4 supernantants of cultivated glioblastoma cells. RESULTS: We could show that angiogenin is detectable in different kinds of intracranial tumours with the highest amount in meningiomas and the lowest amount in low grade astrocytomas. In tissue specimens, a significantly higher angiogenin expression was measured in meningiomas compared to gliomas and metastases. Angiogenin could be detected in primary cultivated glioma cells, but not in the permanent cell lines. There was a significant correlation to the malignancy within the gliomas with an increase of angiogenin concentration according to the higher grade of malignancy. CONCLUSIONS: Our data suggest that angiogenin may contribute to the malignant transformation of gliomas and could perhaps advise that the physiological role of angiogenin is not restricted exclusively to angiogenesis. Based on these findings the clinical importance of angiogenin for therapeutic decisions in malignant brain tumours remains unclear and further analyses on m-RNA-levels are required.

Human keratinocyte cell lines differ in the expression of the collagenolytic matrix metalloproteinases-1,-8, and -13 and of TIMP-1.

We investigated cells and conditioned media of the three human keratinocyte cell lines HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic) with regard to production and secretion of the collagenases-1 to -3 (MMP-1, MMP-8 and MMP-13) and TIMP-1 using semi-nested RT-PCR, Western blots, ELISA, immunocytochemistry and casein zymography. Transcripts of MMP-1, -8, -13 and TIMP-1 were detected in all cell lines by RT-PCR and the corresponding proteins were found in the cytoplasm of all three cell lines by Western blot analysis and/or immunocytochemistry. The conditioned media of the malignant II-4RT cells contain significantly more MMP-1 and MMP-8 than those of HaCaT or A5 as evidenced by immunoblotting and ELISA. In addition to the presence of latent MMP-1, zymography also detected the active form of this enzyme. TIMP-1 was found only in extracts of all three cell lines, predominantly in A5. This study clearly indicates that the epithelial tumor cells synthesize different collagenases and TIMP-1. The malignant clone secretes increased amounts of distinct collagenases compared to the non-tumorigenic cell line, thereby verifying a correlation between biological behaviour and the amount of collagenases. In addition, we provide clear evidence that MMP-8 is not exclusively found in polymorphonuclear granulocytes, but also in keratinocyte cell lines.

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris.

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).

Neutrophil tissue inhibitor of matrix metalloproteinases-1 occurs in novel vesicles that do not fuse with the phagosome.

The human neutrophil granule location of precursors of matrix metalloproteinases (MMPs), MMP-8 and -9, has been established, but that of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) has not. In this study, labeling for TIMP-1, pro-MMP-8, pro-MMP-9, and established granule marker proteins reveals that TIMP-1 is mainly located in distinct oval, electron translucent organelles, a little larger than azurophil granules. A lack of labeling for the fluid phase endocytic marker, bovine serum albumin-gold, the lysosome-associated membrane protein markers, and for glycosylphosphatidylinositol-linked proteins, which are enriched in secretory vesicles, indicates the non-endosomal, non-lysosomal, and non-secretory nature of this organelle. Density gradient cofractionation with the least dense, secretory population and some pleomorphism of the organelle suggest it is a "vesicle" rather than a "granule" population. Colocalization with pro-MMP-9 or pro-MMP-8, in minor subpopulations, suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and terminal differentiation and before secretory vesicle synthesis. Pulse-chased IgG-coated latex beads and immunolabeling show that specific and azurophil granules fuse with the phagosome whereas TIMP-1 and pro-MMP-9-containing organelles do not. This suggests that these play no role in phagosomal destruction of IgG-opsonized bacteria. Separate localization and colocalization of these proteins may, however, facilitate fine regulation of extracellular proteolysis.

Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases.

BACKGROUND: The general consensus is that interstitial collagens are digested by collagenases and denatured collagen by gelatinases, although processing of fibrillar and acetic-acid-soluble collagen by gelatinase A has also been reported. One of the main difficulties in studying the mechanism of action of these matrix metalloproteinases (MMPs) derives from the physicochemical properties of the natural triple-helical collagen, which makes it difficult to handle. RESULTS: Synthetic heterotrimeric collagenous peptides that contain the collagenase cleavage site of human collagen type I and differ in the thermal stability of the triple-helical fold were used to mimic natural collagen and gelatin, respectively. Results from digestion of these substrates by fibroblast and neutrophil collagenases (MMP-1 and MMP-8), as well as by gelatinase A (MMP-2), confirmed that the two classes of enzymes operate within the context of strong conformational dependency of the substrates. It was also found that gelatinases and collagenases exhibit two distinct proteolytic mechanisms: gelatinase digests the gelatin-like heterotrimer rapidly in individual steps with intermediate releases of partially processed substrate into the medium, whereas collagenases degrade the triple-helical heterotrimer by trapping it until scission through all three alpha chains is achieved. CONCLUSIONS: The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.

The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E. coli expression.

Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain. The enzyme could be generated and activated in the presence of Ca2+ from the purified 70-kDa pFN fragment produced by cathepsin D digestion. In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, alpha- and beta-casein, insulin b-chain, and a synthetic Mca-peptide. In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates. Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.