The ADAM10 prodomain is a specific inhibitor of ADAM10 proteolytic activity and inhibits cellular shedding events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.     

Catalytic properties of ADAM12 and its domain deletion mutants

Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.     

Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity of the protease domain. In addition to its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of metalloprotease-1, -2, and -3 were not found to block proteolytic activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease in the extracellular environment remains to be identified.     

Catalytic activity of human ADAM33

ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426-430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively (204)EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from beta-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His(14)/Gln(15), not at the alpha-secretase site and was inefficient (k(cat)/K(m) (1.6 +/- 0.3) x 10(2) m(-1) s(-1)). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3' residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, K(i) = 23 +/- 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33.     

The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion

ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.     

Identification of key sequence determinants for the inhibitory function of the prodomain of TACE

The TNFalpha converting enzyme (TACE) is a zinc metalloproteinase that mediates shedding of multiple cell surface proteins. Regulation of TACE enzymatic activity is ultimately mediated via proteolytic removal of its inhibitory prodomain. Sequence determinants for TACE prodomain inhibition of the catalytic domain are yet to be identified. Surprisingly, although TACE and ADAM 10 (closest homologue) share only 23% sequence identity at their prodomains, the latter in isolation inhibits TACE with the same potency as TACE own prodomain. In contrast, the prodomain of ADAM 9 inhibited TACE only weakly. Detailed analysis of ADAM prodomains revealed two short regions for which TACE and ADAM 10 depart dramatically from all other family members. We prepared TACE prodomain variants containing full or partial switches to ADAM 9 residues at those two regions and examined their functional properties. Variants containing ADAM 9 substitutions including amino acid residues 72-82 and 126-137 were fully inactive for TACE inhibition. A third variant comprising residues 114-125 was active but at lower potency relative to wild type. All inactive variants appeared to be correctly folded. Finally, the amino acid residue Phe72 and the motif Asp-Asp-Val-Ile137 were identified within those regions as key determinants for TACE prodomain inhibitory function. We conclude that TACE and ADAM 10 prodomains are functionally equivalent in a way that separates them from the rest of the ADAM family.     

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.     

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.     

ADAM9 inhibition increases membrane activity of ADAM10 and controls α-secretase processing of amyloid precursor protein

Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24-204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nM and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease.     

The C-terminal domains of TACE weaken the inhibitory action of N-TIMP-3

Tumor necrosis factor-alpha converting enzyme (TACE) is an ADAM (a disintegrin and metalloproteinases) that comprises an active catalytic domain and several C-terminal domains. We compare the binding affinity and association rate constants of the N-terminal domain form of wild-type tissue inhibitor of metalloproteinase (TIMP-3; N-TIMP-3) and its mutants against full-length recombinant TACE and the truncated form of its catalytic domain. We show that the C-terminal domains of TACE substantially weaken the inhibitory action of N-TIMP-3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C-terminal domains of ADAM proteinases in influencing TIMP interactions.     

Identification and characterization of human endometase (Matrix metalloproteinase-26) from endometrial tumor

We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase 26 (MMP-26) (matrixin) gene, endometase, an endometrial tumor-derived metalloproteinase. Among more than three million expressed sequence tags sequenced, the endometase gene was only obtained from human endometrial tumor cDNA library. Endometase mRNA was expressed specifically in human uterus, not in other tissues/cells tested, e.g. testis, heart, brain, lungs, liver, thymus, and melanoma G361. Endometase protein has a signal peptide, a propeptide domain, and a catalytic domain with a unique "cysteine switch" propeptide sequence, PHCGVPDGSD, and a zinc-binding motif, VATHEIGHSLGLQH. Endometase is 43, 41, 41, and 39% identical to human metalloelastase, stromelysin, collagenase-3, and matrilysin, respectively. The zymogen was expressed and isolated from Escherichia coli as inclusion bodies with a molecular mass of 28 kDa. The identity and homogeneity of the recombinant protein was confirmed by protein N-terminal sequencing, silver stain, and immunoblot analyses. The pro-enzyme was partially activated during the folding process. Endometase selectively cleaved type I gelatin and alpha(1)-proteinase inhibitor; however, it did not digest collagens, laminin, elastin, beta-casein, plasminogen, soybean trypsin inhibitor, or Bowman-Birk inhibitor. It hydrolyzed peptide substrates of matrixins and tumor necrosis factor-alpha converting enzyme. Endometase may selectively cleave extracellular matrix proteins, inactivate serpins, and process cytokines.     

ADAM28 is activated by MMP-7 (matrilysin-1) and cleaves insulin-like growth factor binding protein-3

ADAM28, a member of a disintegrin and metalloproteinase (ADAM) family, has two isoforms, membrane-type form (ADAM28m) and secreted form (ADAM28s). Although ADAM28 is expressed and synthesized in a precursor form (proADAM28) by lymphocytes and some cancer cells, its activation mechanism and substrates remain unclear. Here, we report that proADAM28s of 65kDa is processed with active matrix metalloproteinase-7 (MMP-7) to 42- and 40-kDa forms which corresponds to active ADAM28s without propeptide. Processed ADAM28s digested insulin-like growth factor binding protein-3 (IGFBP-3) in both free and complex forms with IGF-I or IGF-II, and the digestion was prevented with EDTA, 1,10-phenanthroline, KB-R7785, tissue inhibitor of metalloproteinases-3 (TIMP-3), and TIMP-4. These data provide the first evidence that proADAM28s is activated by MMP-7 and ADAM28 digests IGFBP-3.     

Inhibitory potential of prodomain of Plasmodium falciparum protease serine repeat antigen 5 for asexual blood stages of parasite

Plasmodium falciparum serine repeat antigen 5 (SERA5) is a target for both drug and vaccine intervention against malaria. SERA5 is secreted in the parasitophorous vacuole where it is proteolytically processed before schizont rupture. Among the processed products is a 50.8-kDa central domain of the protease, which possesses chymotrypsin-like activity and consists of a 28.9-kDa catalytic domain with a 21.9-kDa N-terminal prodomain, which remain attached together. Because SERA5 has been implicated in merozoite egress from host erythrocytes, the effect of the prodomain and a heptapeptide derived from its C-terminus spanning from D(560) to F(566) (DNSDNMF) on parasite growth was studied. When E. coli-expressed prodomain was incubated with parasite culture, a significant delay in transition from schizont to ring stages was observed up to nanomolar concentrations. The peptide, DNSDNMF also showed similar effects but at nearly 1000-fold higher concentrations. The peptide was also found to interact with the catalytic domain. These data demonstrate the crucial role of SERA5 prodomain for the egress process. Given the inhibitory potential of the prodomain for the parasite, we suggest that peptidomimetic inhibitors based on SERA5 prodomain sequences can be developed as future therapeutics against malaria.     

ADAM12 is a four-leafed clover: the excised prodomain remains bound to the mature enzyme

The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.     

Activation of Arabidopsis vacuolar processing enzyme by self-catalytic removal of an auto-inhibitory domain of the C-terminal propeptide

Vacuolar processing enzyme (VPE) is a cysteine proteinase responsible for the maturation of various vacuolar proteins in higher plants. To clarify the mechanism of maturation and activation of VPE, we expressed the precursors of Arabidopsis gamma VPE in insect cells. The cells accumulated a glycosylated proprotein precursor (pVPE) and an unglycosylated preproprotein precursor (ppVPE) which might be unfolded. The N-terminal sequence of pVPE revealed that ppVPE had a 22-amino-acid signal peptide to be removed co-translationally. Under acidic conditions, the 56-kDa pVPE was self-catalytically converted to a 43-kDa intermediate form (iVPE) and then to the 40-kDa mature form (mVPE). N-terminal sequencing of iVPE and mVPE showed that sequential removal of the C-terminal propeptide and N-terminal propeptide produced mVPE. Both iVPE and mVPE exhibited the activity, while pVPE exhibited no activity. These results imply that the removal of the C-terminal propeptide is essential for activating the enzyme. Further removal of the N-terminal propeptide from iVPE is not required to activate the enzyme. To demonstrate that the C-terminal propeptide functions as an inhibitor of VPE, we expressed the C-terminal propeptide and produced specific antibodies against it. We found that the C-terminal propeptide reduced the activity of VPE and that this inhibitory activity was suppressed by specific antibodies against it. Our findings suggest that the C-terminal propeptide functions as an auto-inhibitory domain that masks the catalytic site. Thus, the removal of the C-terminal propeptide of pVPE might expose the catalytic site of the enzyme.     

Interleukin-1 stimulates ADAM17 through a mechanism independent of its cytoplasmic domain or phosphorylation at threonine 735

ADAM17 (a disintegrin and metalloproteinase) is a membrane-anchored metalloproteinase that regulates the release of EGFR-ligands, TNFα and other membrane proteins from cells. ADAM17 can be rapidly activated by a variety of signaling pathways, yet little is known about the underlying mechanism. Several studies have demonstrated that the cytoplasmic domain of ADAM17 is not required for its rapid activation by a variety of stimuli, including phorbol esters, tyrosine kinases and some G-protein coupled receptors. However, phosphorylation of cytoplasmic residue T735 was recently reported as a crucial step for activation of ADAM17 by IL-1β and by the p38 MAP-kinase pathway. One possible mechanism to reconcile these results would be that T735 has an inhibitory role and that it must be phosphorylated as a pre-requisite for the activation of ADAM17, which would then proceed via a mechanism that is independent of its cytoplasmic domain. To test this hypothesis, we performed rescue experiments of Adam17-/- cells with wild type and mutant forms of ADAM17. However, these experiments showed that an inactivating mutation (T735A) or an activating mutation (T735D) of cytoplasmic residue T735 or the removal of the cytoplasmic domain of ADAM17 did not significantly affect the stimulation of ADAM17 by IL-1β or by activation of MAP-kinase with anisomycin. Moreover, we found that the MAP-kinase inhibitor SB203580 blocked activation of cytoplasmic tail-deficient ADAM17 and of the T735A mutant by IL-1β or by anisomycin, providing further support for a model in which the activation mechanism of ADAM17 does not rely on its cytoplasmic domain or phosphorylation of T735.     

N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

Flt is one of the cell surface VEGF receptors which can be cleaved to release an N-terminal extracellular fragment which, like alternately transcribed soluble Flt1 (sFlt1), can antagonize the effects of VEGF. In HUVEC and in HEK293 cells where Flt1 was expressed, metalloprotease inhibitors reduced Flt1 N-terminal cleavage. Overexpression of ADAM10 and ADAM17 increased cleavage while knockdown of ADAM10 and ADAM17 reduced N-terminal cleavage suggesting that these metalloproteases were responsible for Flt1 cleavage. Protein kinase C (PKC) activation increased the abundance and the cleavage of Flt1 but this did not require any residues within the intracellular portion of Flt1. ALLN, a proteasomal inhibitor, increased the abundance of Flt1 which was additive to the effect of PKC. Removal of the entire cytosolic region of Flt1 appeared to stimulate cleavage of Flt1 and Flt1 was no longer sensitive to ALLN suggesting that the cytosolic region contained a degradation domain. Knock down of c-CBL, a ring finger ubiquitin ligase, in HEK293 cells increased the expression of Flt1 although it did not appear to require a previously published tyrosine residue (1333Y) in the C-terminus of Flt1. Increasing VEGFR2 expression increased VEGF-stimulated sFlt1 expression and progressively reduced the cleavage of Flt1 with Flt1 staying bound to VEGFR2 as a heterodimer. Our results imply that secreted sFlt1 and cleaved Flt1 will tend to have local effects as a VEGF antagonist when released from cells expressing VEGFR2 and more distant effects when released from cells lacking VEGFR2.     

Heparan sulfate regulates ADAM12 through a molecular switch mechanism

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.     

Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme.

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.