The transmembrane domain of TACE regulates protein ectodomain shedding

Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.     

Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain

Tumor necrosis factor-alpha-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC(50) of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC(50) greater that 2 microm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys(184) residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.     

Tumor necrosis factor-α converting enzyme (TACE) increases RANKL expression in osteoblasts and serves as a potential biomarker of periodontitis

Periodontitis is a very prevalent disease. Therefore, biomarkers for the early and standard diagnoses of periodontitis are urgently needed. TACE is a membrane-bound metalloprotease. Although a recent study suggested that TACE levels in GCF are elevated during periodontal disease, the levels of TACE in GCF at different stages of chronic periodontitis have not been determined. Here, we analyzed the protein levels of TACE in GCF from periodontal disease subjects and confirmed that the protein levels of TACE were higher in the moderate periodontitis groups. TACE is known to be a NF-κB ligand that stimulates RANKL secretion in osteoblasts. To understand the effects of TACE on RANKL and OPG in osteoblasts, we treated MG63 cells with TACE. We observed an increase in RANKL protein expression but a decrease in OPG protein expression. Our data suggest that TACE can induce RANKL expression and promote osteoclastogenesis, thus worsening the outcome of periodontitis.     

Discovery of novel spirocyclopropyl hydroxamate and carboxylate compounds as TACE inhibitors

We have discovered nanomolar inhibitors of TNF-α convertase (TACE) comprised of a novel spirocyclic scaffold and either a carboxylate or hydroxamate zinc binding moiety. X-ray crystal structures and computer models of selected compounds binding to TACE explain the observed SAR. We report the first TACE X-ray crystal structure for an inhibitor with a carboxylate zinc ligand.     

Reactive oxygen species and p38 mitogen-activated protein kinase mediate tumor necrosis factor α-converting enzyme (TACE/ADAM-17) activation in primary human monocytes

Tumor necrosis factor α-converting enzyme (TACE) is responsible for the shedding of cell surface TNF. Studies suggest that reactive oxygen species (ROS) mediate up-regulation of TACE activity by direct oxidization or modification of the protein. However, these investigations have been largely based upon nonphysiological stimulation of promonocytic cell lines which may respond and process TACE differently from primary cells. Furthermore, investigators have relied upon TACE substrate shedding as a surrogate for activity quantification. We addressed these concerns, employing a direct, cell-based fluorometric assay to investigate the regulation of TACE catalytic activity on freshly isolated primary human monocytes during LPS stimulation. We hypothesized that ROS mediate up-regulation of TACE activity indirectly, by activation of intracellular signaling pathways. LPS up-regulated TACE activity rapidly (within 30 min) without changing cell surface TACE expression. Scavenging of ROS or inhibiting their production by flavoprotein oxidoreductases significantly attenuated LPS-induced TACE activity up-regulation. Exogenous ROS (H(2)O(2)) also up-regulated TACE activity with similar kinetics and magnitude as LPS. H(2)O(2)- and LPS-induced TACE activity up-regulation were effectively abolished by a variety of selective p38 MAPK inhibitors. Activation of p38 was redox-sensitive as H(2)O(2) caused p38 phosphorylation, and ROS scavenging significantly reduced LPS-induced phospho-p38 expression. Inhibition of the p38 substrate, MAPK-activated protein kinase 2, completely attenuated TACE activity up-regulation, whereas inhibition of ERK had little effect. Lastly, inhibition of cell surface oxidoreductases prevented TACE activity up-regulation distal to p38 activation. In conclusion, our data indicate that in primary human monocytes, ROS mediate LPS-induced up-regulation of TACE activity indirectly through activation of the p38 signaling pathway.     

Molecular basis for the mechanism of action of an anti-TACE antibody

Inhibitors of tumor necrosis factor-α converting enzyme (TACE) have potential as therapeutics for various diseases. Many small molecule inhibitors, however, exhibit poor specificity profiles because they target the highly conserved catalytic cleft of TACE. We report for the first time the molecular interaction of a highly specific anti-TACE antagonistic antibody (MEDI3622). We characterized the binding of MEDI3622 using mutagenesis, as well as structural modeling and docking approaches. We show that MEDI3622 recognizes a unique surface loop of sIVa-sIVb β-hairpin on TACE M-domain, but does not interact with the conserved catalytic cleft or its nearby regions. The exquisite specificity of MEDI3622 is mediated by this distinct structural feature on the TACE M-domain. These findings may aid the design of antibody therapies against TACE.     

Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch

TNF-alpha-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18-54 (N-TACE(18-54)) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE(18-54) attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE(18-54) inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30-48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.     

Expression and function of tumour necrosis factor-alpha-converting enzyme in the central nervous system

Tumour necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE/ADAM17) is a membrane protein belonging to the ADAM (a disintegrin and a metalloprotease) family able to cleave various membrane proteins, including the transmembrane form of TNF-alpha at its physiological processing site. Being an ADAM, TACE may mediate not only proteolysis but also adhesive interactions; however, the role of the disintegrin domain of TACE has not been studied. In the central nervous system (CNS), little is known about the physiological role of TACE, but some important pathophysiological functions have been reported recently, with both neurotoxic and neuroprotective repercussions. This article discusses and reviews the main contributions to this field of investigation addressing the expression and function of TACE in the CNS.     

Hydantoins, triazolones, and imidazolones as selective non-hydroxamate inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

We have discovered selective and potent inhibitors of TACE that replace the common hydroxamate zinc binding group with a hydantoin, triazolone, and imidazolone heterocycle. These novel heterocyclic inhibitors of a zinc metalloprotease were designed using a pharmacophore model that we previously described while developing hydantoin and pyrimidinetrione (barbiturate) inhibitors of TACE. The potency and binding orientation of these inhibitors is discussed and they are modeled into the X-ray crystal structure of TACE and compared to hydroxamate and earlier hydantoin TACE inhibitors which share the same 4-[(2-methyl-4-quinolinyl)methoxy]benzoyl P1' group.     

Current perspective of TACE inhibitors: A review

Rheumatoid Arthritis (RA) is one of the most common autoimmune inflammatory conditions, affecting approximately 1% of the adult population worldwide. TNF-α is a pleitropic, pro-inflammatory cytokine which plays a pivotal role in the origin and progression of RA and other immune mediated disorders. The success of anti-TNF-α biological agents proved that inhibition of TNF-α could result in effective control of RA. Since the discovery of anti-TNF-α biologicals, much efforts have gone into developing an orally bioavailable small size TNF-α antagonist. One of the ways to block TNF-α in biological fluids is to inhibit TNF-α converting enzyme (TACE). This target has been validated in preclinical trials using TACE inhibitors. But, even after more than a decade no single TACE inhibitor has passed the Phase II clinical trials. Very recently, it has been shown that TACE inhibitors could also be used for inhibition of pathogenic EGFR signaling in cancer. Hence, TACE inhibitors could perform a dual role, in curing not only RA but also certain cancerous conditions. Developments in the field have prompted us to review the research work on TACE inhibitors, especially their structure activity relationships and molecular modeling studies.     

Mammalian iRhoms have distinct physiological functions including an essential role in TACE regulation

Loss of iRhom2, a catalytically inactive rhomboid‐like protein, blocks maturation of TACE/ADAM17 in macrophages, resulting in defective shedding of the cytokine tumor necrosis factor. Apart from the resulting inflammatory defects, iRhom2‐null mice appear normal: they do not show the several defects seen in TACE knockouts, suggesting that TACE maturation is independent of iRhom2 in cells other than macrophages. Here we show that the physiological role of iRhoms is much broader. iRhom1 knockout mice die within 6 weeks of birth. They show a severe phenotype, with defects in several tissues including highly penetrant brain haemorrhages. The non‐overlapping phenotypes imply that iRhom 1 and 2 have distinct physiological roles, although at a cellular level both promote the maturation of TACE (but not other ADAM proteases). Both iRhoms are co‐expressed in many contexts where TACE acts. We conclude that all TACE activity, constitutive and regulated, requires iRhom function. iRhoms are therefore essential and specific regulators of TACE activity, but our evidence also implies that they must have additional physiologically important clients.     

The discovery of novel tartrate-based TNF-α converting enzyme (TACE) inhibitors

A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP’s. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.     

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.     

Patterns of sorafenib and TACE treatment of unresectable hepatocellular carcinoma in a Chinese population: subgroup analysis of the GIDEON study

To analyze safety and efficacy of patterns of sorafenib and TACE therapy under real-life clinical practice conditions. A total of 338 Chinese patients with unresectable hepatocellular carcinoma (HCC) from the international database of the GIDEON non-interventional trial were included in this analysis. Endpoints were overall survival (OS), progression-free survival (PFS), time to progression (TTP) and safety. Two major patterns in the use of sorafenib observed in current Chinese clinical practice were: sorafenib administration subsequent to transarterial chemoembolization (TACE) treatment (n?=?226, 66.9%) and sorafenib administration concomitant to TACE (n?=?80, 35.4%). Patients receiving TACE prior to sorafenib had worse liver function (43.8% BCLC stage Cat diagnosis and 62.1% BCLC stage C at study entry) than those receiving TACE concomitant to sorefenib (35.0% BCLC stage C at diagnosis and 51.3% BCLC stage three at study entry). For patients undergoing prior TACE and concomitant TACE treatment, median OS time was 354 days vs. 608 days, PFS time was 168 days vs. 201 days, and TTP was 214 days vs. 205 days; and the percentage of patients who experienced drug-related adverse effects after sorafenib therapy in these two groups were 33.3 and 50.0%, respectively. Sorafenib treatment is usually administered in cases of tumor progression or poor liver function status after TACE treatment in China. Under such conditions, patients still gained a relatively satisfactory survival outcome. In addition, the present study suggests that concomitant sorafenib and TACE treatments may lead to a better prognosis, although differences in baseline characteristics may have contributed in part to the better outcomes.     

Impaired trafficking and activation of tumor necrosis factor-alpha-converting enzyme in cell mutants defective in protein ectodomain shedding

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.     

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion

When studying how HIV‐1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor‐α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase‐regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)‐dependent but was not associated with ER stress. These data indicate that Hck‐activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom‐dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.      

Chloroquine Promotes the Anticancer Effect of TACE in a Rabbit VX2 Liver Tumor Model

Background: To investigate the efficacy of TACE combined with CQ, an autophagic inhibitor, in a rabbit VX2 liver tumor model.Methods: Tumor size was measured. And tumor growth rate was calculated to examine the effect of the combined treatment. Apoptosis was detected by TUNEL assay. Meanwhile, autophagic activity was detected by immunohistochemistry and Western blotting to investigate the mechanism underlying. Liver function was also examined to assess feasibility and safety of the combined therapy.Results: Tumors in the control grew more than 4 times bigger after 14 days, while that in the group of TACE alone just showed mild growth. But a slight shrinkage was shown after the treatment of CQ+TACE. Growth ratio of TACE alone was 96.45% ± 28.958% while that of CQ+TACE was -28.73% ± 12.265%. Compared with TACE alone, necrosis in CQ+TACE showed no significant difference, however, the apoptosis was much higher. There were only 14.8±3.11% apoptotic cells in TACE, but 33±4.18% in CQ+TACE, which suggests the increased apoptosis in CQ+TACE contributed to the decrease of tumor volume. In terms of autophagic activity, the result is negative when we immunostained sections of the control with LC3 antibody, but positive in TACE alone and CQ+TACE. And the result of Western blot showed that there was just a low level of LC3Ⅱexpressed in the control and CQ alone, but higher in TACE, and much higher in CQ+TACE because CQ inhibited its degradation in autophagy. Compared with control, p62 decreased in TACE, but the decrease was partially reversed in CQ+TACE. In addition, toxicity of CQ+TACE was assessed not higher than TACE alone, which supports the safety of CQ+TACE.Conclusion: CQ+TACE works better than TACE alone in rabbit VX2 liver tumor model because CQ inhibits autophagy induced by TACE. The inhibited autophagy loses its resistance to apoptosis that apoptosis increased, which contributes to the inhibition of tumor growth. This study indicates CQ may be a promising adjuvant to promote the effect of TACE.     

Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme

The extracellular domains of many proteins, including growth factors, cytokines, receptors, and adhesion molecules, are proteolytically released from cells, a process termed "shedding." Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease-disintegrin that sheds tumor necrosis factor-alpha and other proteins. To study the regulation of TACE-mediated shedding, we examined the effects of stimulation of cells on TACE localization and expression. Immunofluorescence microscopy revealed a punctate distribution of TACE on the surface of untreated cells, and stimulation of monocytic cells with lipopolysaccharide did not affect TACE staining. Phorbol 12-myristate 13-acetate (PMA), a potent inducer of shedding, decreased cell-surface staining for TACE. Surface biotinylation experiments confirmed and extended this observation; PMA decreased the half-life of surface-biotinylated TACE without increasing the turnover of total cell-surface proteins. Soluble fragments of TACE were not detected in the medium of cells that had down-regulated TACE, and TACE was not down-regulated when endocytosis was inhibited. Antibody uptake experiments suggested that cell-surface TACE was internalized in response to PMA. Surprisingly, a metalloprotease inhibitor prevented the PMA-induced turnover of TACE. Thus, PMA activates shedding and causes the down-regulation of a major "sheddase," suggesting that induced shedding may be regulated by a mechanism that decreases the amount of active TACE on the cell surface.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.