Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch
Caitriona A. Buckley,
Farshid N. Rouhani,
Maryann Kaler,
Barbara Adamik,
Feras I. Hawari, and
Stewart J. Levine
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Submitted 15 November 2004
; accepted in final form 25 February 2005
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ABSTRACT
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TNF-
-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 1854 (N-TACE1854) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE1854 attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE1854 inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 3048, 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.
tumor necrosis factor-
-converting enzyme; 1,10-phenanthroline; a disintegrin and metalloprotease; 55-kDa type I tumor necrosis factor receptor; 75-kDa type II tumor necrosis factor receptor
TNF-
-CONVERTING ENZYME (TACE) or ADAM17 (a disintegrin and metalloprotease-17), a member of the disintegrin and metalloprotease family of zinc metalloproteases, is an important regulator of inflammation, immune regulation, and cellular proliferation as a consequence of its ability to process cell surface integral membrane proteins to soluble forms (2, 4, 24). TACE was originally identified as the enzyme that cleaves the membrane-bound precursor of tumor necrosis factor-
(TNF-
), as well as the type II 75-kDa TNF receptor (TNFR2, TNFRSF1B, CD120b), transforming growth factor (TGF)-
, and L-selectin (3, 23, 24, 29). Other cell surface proteins that have been identified as substrates for TACE include cytokines, chemokines, growth factors, adhesion molecules, and cytokine and growth factor receptors, as well as the cellular prion protein and the amyloid precursor protein (1, 68, 14, 17, 20, 29, 3133, 37, 42, 43, 47, 48).
TACE plays an important role in both lung development and the pathogenesis of pulmonary disease. TACE is expressed by a variety of cells in the lung, including alveolar macrophages, bronchial epithelial cells, and vascular smooth muscle cells (13). Lungs from embryonic TACE-deficient mice display impaired branching morphogenesis, inhibited epithelial cell proliferation and differentiation, and delayed vasculogenesis, thereby demonstrating a role for TACE in normal lung maturation (49). TACE also regulates mucin production by human airway epithelial cells. Activation of TACE by phorbol ester, Pseudomonas aeruginosa, or lipopolysaccharide catalyzes the cleavage of pro-TGF-
into soluble mature TGF-
, which then binds to and induces the phosphorylation of the epidermal growth factor receptor (EGFR), with resultant MUC5AC expression (39). Cigarette smoke, via a process that may involve oxygen free radicals, also activates TACE with resultant ligand-dependent EGFR phosphorylation and MUC5AC production (38). After activation, TACE undergoes stimulation-dependent internalization, which may downregulate catalytic activity at the plasma membrane (11). This may be relevant to the pathogenesis of community-acquired pneumonia as epithelial lining fluid cells from infected lungs have downregulated cell surface TACE expression compared with cells obtained from uninvolved lungs (16).
ADAM family zinc metalloproteases, including TACE, have a conserved structure that includes, from NH2 to COOH terminus, a signal sequence, prodomain, metalloprotease domain, disintegrin domain, cysteine-rich domain containing an EGF-like repeat, a transmembrane domain, and an intracytoplasmic tail (3, 23, 24, 35, 36). An important function of the prodomain is to retain the proenzyme in an inactive state. 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 via a cysteine-switch mechanism. It was recently reported, however, that the cysteine-switch motif is not required for the inhibitory activity of the prodomain (15). The TACE prodomain may also play an important role in protein folding, as TACE mutants lacking the prodomain are inefficiently synthesized in Sf9 cells and possibly undergo intracellular degradation (21). As found with other ADAM family members, cleavage of the TACE prodomain typically occurs COOH-terminal to a consensus proprotein-convertase sequence [RX(K/R)R] (12). Removal of the TACE prodomain is catalyzed by furin and other proprotein-convertases, such as PC7, in the late Golgi compartment (5, 12, 28, 35).
In the present study, we hypothesized that the amino-terminal region of the TACE prodomain might contribute to the ability of the TACE prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism (15). We synthesized a 37-amino acid peptide that corresponds to TACE amino acids 1854 (N-TACE1854) but does not contain the consensus cysteine-switch motif (PKVCGY186) (21). N-TACE1854 attenuated TACE-catalyzed TNFR2 cleavage in a model assay system and constitutive TNFR2 shedding from the human U-937 monocytic cell line. A 19-amino acid, leucine-rich domain, which corresponds to N-TACE amino acids 3048, possessed partial TACE inhibitory activity. Therefore, we propose that a subdomain within the amino terminus of the TACE prodomain attenuates TACE catalytic activity independently of the cysteine-switch mechanism. This study provides new insight into the ability of the TACE prodomain to regulate TACE enzymatic activity.
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METHODS
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Characterization of N-TACE1854 inhibitory activity.
A model assay system was developed to assess the ability of N-TACE1854 to attenuate TACE enzymatic activity. Recombinant human TACE (rhTACE), as well as the recombinant human 55-kDa, type I TNF receptor and TNFR2 fusion proteins (rhTNFR1:Fc and rhTNFR2:Fc, respectively) were purchased from R&D Systems (Minneapolis, MN). Both TNFR1:Fc and TNFR2:Fc are recombinant human chimeric proteins that encode the extracellular receptor domain, fused to a carboxy-terminal 6x-histidine-tagged Fc region of human IgG1 via a linker peptide (IEGRMD). rhTACE corresponds to the mature form after removal of the prodomain and has an apparent molecular size of 70 kDa. N-TACE1854 was synthesized by Sigma-Genosys (The Woodlands, TX). N-TACE1854 truncation mutants were also synthesized: an amino-terminal mutant corresponding to N-TACE amino acids 1829, a middomain mutant corresponding to N-TACE amino acids 3042, a carboxy-terminal mutant corresponding to amino acids 4354, and an extended middomain mutant corresponding to N-TACE amino acids 3048 (N-TACE3048). A scrambled peptide containing the N-TACE1854 amino acids in a random order was also synthesized by Sigma-Genosys. Chou-Fasman analysis was performed using MacVector (Accelrys, Burlington, MA). Vasoactive intestinal peptide (VIP) and
-defensin were purchased from Bachem (Torrance, CA). TNF-
proteinase inhibitor (TAPI-2) was purchased from Peptides International (Louisville, KY). 1,10-Phenanthroline monohydrate and zinc chloride (ZnCl2) were purchased from Sigma-Aldrich (St. Louis, MO).
Assays (50 µl) were performed in 50 mM Tris·HCl and 25 mM NaCl, pH 8.0, and incubated at 30°C for 30 min. Proteins were separated by SDS-PAGE using 412% Bis-Tris NUPAGE gels (Invitrogen, Carlsbad, CA) and visualized with the SilverQuest Silver Staining kit (Invitrogen). For Western blot analysis, proteins were separated via SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated overnight (4°C) with a murine IgG1 monoclonal antibody (200 ng/ml) directed against the 6x-histidine tag (Tetra-His; Qiagen, Valencia, CA), which reacts with the COOH terminus of the rhTNFR2:Fc fusion protein. A rabbit polyclonal antibody generated against N-TACE amino acids 1854 (Sigma-Genosys) was utilized for Western blotting at a 1:1,000 dilution. Detection was by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies.
Quantification of TNFR2 shedding.
U-937 cells purchased from ATCC (Manassas, VA) were maintained in RPMI 1640 medium with 10% fetal bovine serum. U-937 cells were plated in six-well plates at a density of 2 x 106 cells/well in 1 ml of media. Release of TNFR2 into U-937 cell culture medium during a 24-h period was quantified by a sandwich ELISA (R&D Systems). Cellular apoptosis and necrosis were measured using the TACS Annexin V-FITC Apoptosis Detection kit (R&D Systems) and an XL-MCL flow cytometer (Beckman-Coulter, Miami, FL). Statistical analysis was performed by a paired Student's t-test with a Bonferroni correction for multiple comparisons and by single-factor ANOVA. Differences were considered significant at a P value
0.05.
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RESULTS
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N-TACE1854 attenuates TACE-mediated TNFR2 cleavage.
Experiments were conducted to assess whether the amino terminus of the TACE prodomain can regulate TACE catalytic activity independently of the cysteine-switch mechanism. A peptide corresponding to amino acids 1854 of the TACE coding sequence (N-TACE1854), which lacks the hydrophobic signal peptide sequence and the cysteine-switch consensus motif, was synthesized (Fig. 1). The N-TACE1854 amino acid sequence was deduced from a RT-PCR product generated from NCI-H292 human pulmonary epithelial cell line total RNA and primers spanning the full-length TACE coding region. N-TACE1854 has a predicted molecular mass of 4,186 Da and isoelectric point of 4.61.

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Fig. 1. Characterization of a 37-amino acid peptide that corresponds to TNF- -converting enzyme amino acids 1854 (N-TACE1854). A: TACE protein structure. The TACE prodomain comprises TACE amino acids 18214 and encodes a consensus cysteine-switch motif. B: N-TACE1854 amino acid sequence. The synthesized N-TACE1854 peptide corresponds to TACE amino acids 1854. The signal peptide (underlined), encoded by TACE amino acids 117, is not included in the N-TACE1854 sequence. The 19-amino acid, leucine-rich inhibitory domain is denoted by the double underline.
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The inhibitory activity of N-TACE1854 was assessed in an in vitro assay system utilizing rhTACE and TNF chimeric receptor fusion proteins as model substrates. Because rhTNFR1:Fc and rhTNFR2:Fc each contain the entire extracellular domain of the receptor, we reasoned that they might be susceptible to cleavage by rhTACE. As shown in Fig. 2A, rhTACE cleaved the rhTNFR2:Fc model substrate, generating two predominant cleavage products, which were detected by silver staining. Furthermore, the TACE-catalyzed cleavage of the TNFR2:Fc model substrate was attenuated by 80 µM N-TACE1854. In contrast, rhTACE did not cleave the rhTNFR1:Fc model substrate. Because both rhTNFR1:Fc and rhTNFR2:Fc encode the same linker and Fc region of IgG1, we conclude that rhTACE cleaves TNFR2, but not TNFR1 or the IgG1 chimera. Therefore, in subsequent experiments, rhTNFR2:Fc was used as a substrate to assess the ability of N-TACE1854 to attenuate rhTACE activity. The ability of rhTACE to cleave the rhTNFR2:Fc model substrate was also shown to be zinc dependent, which is consistent with the classification of TACE as a member of the ADAM family of zinc metalloproteases. As shown in Fig. 2B, incubation with the predominantly zinc-specific chelator 1,10-phenanthroline significantly attenuated rhTACE-mediated TNFR2:Fc cleavage, which was partially restored by the addition of 25100 µM ZnCl2. As has been described for other zinc metalloproteases, a further increase in ZnCl2 concentration resulted in a decline in enzyme activity (10).

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Fig. 2. A: recombinant human (rh) TACE catalyzes the cleavage of recombinant human type II TNF receptor (rhTNFR2), but not rhTNFR1. rhTACE (0.5 µM) was incubated with 0.95 µM rhTNFR1:Fc (left) or TNFR2:Fc (right) alone or in combination with 80 µM N-TACE1854 for 30 min at 30°C. Samples were separated by SDS-PAGE, and proteins were visualized by silver staining. Positions of protein standards (kDa) are on the left. Diagrams of the rhTNFR:Fc chimeric substrates are at the bottom. The entire extracellular domain of human TNFR1 (Met 1-Thr211) (34) or TNFR2 (Met 1-Asp257) (19, 40) was fused to the Fc region of human IgG1 (Pro 100-Lys330) via a linker peptide (IEGRMD). Both chimeric fusion proteins contain a 6x-histidine tag (6x HIS) at the carboxy terminus. B: cleavage of TNFR2:Fc by rhTACE is zinc dependent. rhTACE (0.5 µM) was incubated with 1,10-phenanthroline (1,10-Phe) for 30 min before the addition of the indicated concentration of zinc chloride (ZnCl2) for 30 min at 24°C. The rhTNFR2:Fc substrate (0.95 µM) was added for an additional 30 min at 30°C. Proteins were separated by SDS-PAGE, and rhTNFR2:Fc cleavage products were identified by Western blotting utilizing the antibody against the carboxy-terminal 6x-histidine tag of TNFR2:Fc. Positions of protein standards (kDa) are on the right.
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As shown by SDS-PAGE and silver staining (Fig. 3A), N-TACE1854 attenuated the proteolytic cleavage of 0.95 µM rhTNFR2:Fc by 0.5 µM rhTACE in a concentration-dependent fashion between 20 and 160 µM. The identity of rhTNFR2:Fc and its cleavage products was confirmed by immunoblotting utilizing an anti-6x-histidine antibody, which reacts with the COOH-terminal 6x-histidine tag of the rhTNFR2:Fc chimeric protein (Fig. 3B). Together, these experiments demonstrate that N-TACE1854 can attenuate TACE proteolytic activity toward TNFR2.

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Fig. 3. N-TACE1854 inhibits cleavage of TNFR2 by TACE. rhTACE (0.5 µM) was incubated with 0.95 µM rhTNFR2:Fc alone or with increasing concentrations of N-TACE1854 (20160 µM) for 30 min at 30°C. Proteins were separated by SDS-PAGE before silver staining (A) or immunoblotting with an antibody against the carboxy-terminal 6x-histidine tag of TNFR2:Fc (B). Positions of protein standards (kDa) are on the left.
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We next performed experiments utilizing irrelevant peptides,
-defensin and VIP, to assess the specificity of N-TACE1854 attenuation of TACE-catalyzed TNFR2 cleavage. As shown in Fig. 4, neither
-defensin nor VIP affected the ability of TACE to proteolytically cleave rhTNFR2:Fc. In contrast, TACE-catalyzed rhTNFR2 cleavage was partially inhibited by 80 µM N-TACE1854 and completely inhibited by 25 µM TAPI-2, a hydroxamic acid-based zinc metalloprotease inhibitor. These experiments are consistent with the conclusion that the inhibitory activity of N-TACE1854 is not a nonspecific peptide effect. We also assessed whether N-TACE1854 is a substrate for TACE catalytic activity. There was no decrease in the quantity of N-TACE1854 by immunoblotting after incubation with rhTACE for 4 h (data not shown), suggesting that N-TACE1854 is not a substrate for TACE.
Characterization of N-TACE1854 inhibitory activity.
Experiments were next performed to characterize the N-TACE domains that mediate its inhibitory activity. Truncation mutants were synthesized corresponding to the amino-terminal, middle, and carboxy-terminal domains of N-TACE, not including the signal peptide. The amino-terminal mutant corresponded to TACE amino acids 1829, the middomain mutant corresponded to TACE amino acids 3042, and the carboxy-terminal mutant corresponded to amino acids 4354. As shown in Fig. 5, none of these truncation mutants (80 µM) attenuated the ability of TACE to proteolytically cleave rhTNFR2:Fc. This demonstrates that these N-TACE1854 truncation mutants do not possess TACE inhibitory activity. Furthermore, since these truncation mutants were synthesized in a fashion identical to N-TACE1854, this experiment also demonstrates that the ability of N-TACE1854 to function as a TACE inhibitor is not an artifact related to its synthesis and/or purification.

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Fig. 5. Effect of N-TACE truncation mutants on TACE-catalyzed TNFR2 cleavage. rhTACE (0.5 µM) was incubated with 0.95 µM TNFR2:Fc alone or in combination with 80 µM N-TACE or proteins corresponding to the amino terminus (N: N-TACE amino acids 1829), middle (M: N-TACE amino acids 3042), or carboxy terminus (C: amino acids 4354) of N-TACE1854 for 30 min at 30°C. Proteins were separated by SDS-PAGE before silver staining. Positions of protein standards (kDa) are on the left.
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To characterize further the structural requirements for inhibitory activity, another truncation mutant corresponding to N-TACE amino acids 3048 (N-TACE3048) was synthesized. N-TACE3048 is predicted to have a helical structure by Chou-Fasman analysis and is leucine rich, which may be important for its ability to attenuate TACE activity. As shown in Fig. 6, both N-TACE1854 and N-TACE3048 attenuated the TACE-catalyzed proteolytic cleavage of rhTNFR2:Fc. The inhibitory activity of N-TACE3048, however, was less than that of N-TACE1854. These experiments demonstrate that the domain corresponding to amino acids 3048 of N-TACE partially mediates the TACE inhibitory activity of N-TACE1854.

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Fig. 6. Effect of N-TACE3048 on TACE-catalyzed TNFR2 cleavage. rhTACE (0.5 µM) was incubated with 0.95 µM TNFR2:Fc alone or in combination with 80 µM N-TACE1854 or N-TACE3048 for 30 min at 30°C. Proteins were separated by SDS-PAGE before silver staining. Positions of protein standards (kDa) are on the left.
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N-TACE1854 attenuates constitutive TNFR2 shedding from U-937 cells.
We next assessed whether N-TACE1854 was capable of attenuating TNFR2 shedding in a cell-based system. The U-937 monocytic cell line was incubated with either N-TACE1854 (0.0440 µM) or the amino-terminal mutant, corresponding to N-TACE amino acids 1829 (40 µM), for 24 h. As shown in Fig. 7, the quantity of sTNFR2 present in medium from cells treated with N-TACE1854 was significantly reduced in a concentration-dependent fashion. Furthermore, 40 µM N-TACE1854 significantly attenuated TNFR2 shedding by 42% compared with cells treated with media alone (107.3 ± 3.3 vs. 184.6 ± 2.2 pg/ml, n = 6, P
108). In contrast, the amino-terminal mutant had no effect on TNFR2 shedding compared with cells treated with medium alone (186.5 ± 1.8 vs. 184.6 ± 2.2 pg/ml, n = 6, P = not significant). The ability of N-TACE1854 to decrease TNFR2 shedding was not a consequence of either apoptosis or necrosis, as assessed by annexin V binding and propidium iodide uptake (data not shown). Additional experiments were performed utilizing a scrambled 37-amino acid peptide that contained the N-TACE1854 amino acids in a random order to confirm that the inhibition of TNFR2 shedding byN-TACE1854 is dependent upon its amino acid sequence. The scrambled peptide did not attenuate TNFR2 shedding but instead was associated with a 4% increase in constitutive TNFR2 shedding compared with cells treated with medium alone (115.7 ± 0.6 vs. 111.4 ± 1.7 pg/ml, n = 6, P = 0.038). These data demonstrate that N-TACE1854 significantly attenuates constitutive TNFR2 shedding from U-937 cells.

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Fig. 7. Effect of N-TACE1854 on TNFR2 shedding from U-937 cells. U-937 cells were incubated for 24 h with 0.0440 µM N-TACE1854 or 40 µM of the truncation mutant corresponding to the amino terminus of TACE (N). Soluble (s) TNFR2 in cell culture medium was quantified by ELISA. N-TACE1854 inhibited TNFR2 shedding in a concentration-dependent fashion (n = 6, P < 0.05 compared with control, single-factor ANOVA). *P < 0.05 vs. untreated cells (Control).
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Experiments were also performed to assess whether N-TACE3048 inhibits TNFR2 shedding from U-937 cells. Treatment with 40 µM N-TACE3048 inhibited TNFR2 shedding by 16% compared with cells treated with media alone (160.8 ± 3.4 vs. 190.4 ± 2.9 pg/ml, n = 6, P
104). This suggests that although N-TACE3048 partially attenuates TNFR2 shedding, N-TACE amino acids 1854 are required for a maximal effect in this cell-based system.
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DISCUSSION
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TACE can regulate inflammatory responses via the proteolytic cleavage and shedding of TNFR2 to function as a soluble TNF binding protein (29). The important role that soluble TNFR2 (sTNFR2) plays in regulating TNF bioactivity is exemplified by virally encoded soluble TNF binding proteins that function as highly effective modulators of innate immune responses (9). For example, both the Shope fibroma and myxoma viruses express T2 proteins, which are structurally similar to TNFR2 and are secreted by infected cells to subvert TNF-dependent host defenses (40, 46). Similarly, a soluble human TNFR2-Ig fusion protein is utilized clinically to attenuate TNF bioactivity and disease severity in patients with inflammatory arthritides and psoriasis (26). Furthermore, sTNFR2 may modulate pulmonary inflammatory responses in the acute respiratory distress syndrome, asthma, sarcoidosis, bacterial pneumonia, and tuberculosis (18, 25, 27, 30, 44, 45).
Regulation of TACE enzymatic activity is important to prevent excessive or unanticipated cleavage of target proteins. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although this interaction was thought to be mediated via a cysteine-switch mechanism, it has recently been shown that the prodomain cysteine-switch motif is not required for this inhibitory activity (15). This is based upon the finding that a TACE prodomain variant containing a cysteine-to-alanine substitution at position 184 showed the same inhibitory activity toward a recombinant TACE catalytic-domain construct as the wild-type TACE prodomain (15).
Here we report that the amino terminus of the TACE prodomain also possesses TACE inhibitory activity that is independent of the cysteine-switch mechanism. We synthesized a 37-amino acid peptide that corresponded to the amino terminus of the TACE prodomain but did not include the consensus cysteine-switch motif. This peptide, termed N-TACE1854, comprises amino acids 1854 of the TACE protein and was demonstrated to attenuate TACE-catalyzed cleavage of TNFR2 in vitro. This inhibitory activity appeared to be specific, as neither truncation mutants corresponding to the amino-terminal, middle, and carboxy-terminal domains of N-TACE nor irrelevant small proteins (VIP and
-defensin) possessed TACE inhibitory activity. N-TACE1854 also attenuated by 42% constitutive TNFR2 shedding from the U-937 monocytic cell line, which suggests that N-TACE may partially attenuate the activity of native, cell-associated TACE. This is consistent with a role for TACE in constitutive TNFR2 shedding, as was described in HEK293 cells expressing a dominant negative TACE (41). Neither the amino-terminal truncation mutant nor a scrambled peptide inhibited constitutive U-937 cell TNFR2 shedding, which suggests that N-TACE1854 mediates this inhibitory activity in a sequence-specific fashion. Our findings, however, do not establish whether the ability of N-TACE1854 to attenuate constitutive TNFR2 shedding in intact cells is specific for TACE alone, as N-TACE1854 could conceivably inhibit other enzymes that also function as TNFR2 sheddases. We propose that the amino-terminal region of the TACE prodomain can attenuate TACE catalytic activity independently of the cysteine-switch mechanism.
Interestingly, the TACE disintegrin/cysteine-rich domain has been reported to diminish the inhibitory potency of the prodomain for the catalytic domain (15). Although the full-length TACE prodomain was a potent inhibitor of a recombinant TACE catalytic domain construct (IC50 = 70 nM), its inhibitory activity was significantly less against a construct that contained both the TACE catalytic and disintegrin/cysteine-rich domains (IC50 > 2 µM) (15). Furthermore, the disintegrin/cysteine-rich domain appeared to decrease the ability of the prodomain to stably bind the catalytic domain (15). Thus it is possible that in our study, the disintegrin/cysteine-rich domain impaired the ability of N-TACE1854 to inhibit TACE catalytic activity, since rhTACE corresponds to the mature TACE ectodomain. Furthermore, this may in part explain why micromolar concentrations of N-TACE1854 were required to inhibit rhTACE-mediated TNFR2:Fc cleavage, as well as constitutive TNFR2 shedding from U-937 cells.
TNFR1 has been reported to represent a substrate for TACE based upon the demonstration of increased TNFR1 shedding following reconstitution of TACE-deficient cell lines (31). In our model system, rhTNFR2, but not rhTNFR1, served as a substrate for rhTACE enzymatic activity. Similarly, TACE has been reported to have no detectable activity against a TNFR1 model peptide substrate corresponding to the known TNFR1 cleavage site (22). The inability of rhTACE to cleave rhTNFR1:Fc raises the question as to whether TNFR1 serves as a substrate for TACE in vivo or, alternatively, whether there is a requirement for either or both proteins to be membrane anchored or whether additional regulatory proteins are required.
In conclusion, we have identified that a subdomain within the amino terminus of the TACE prodomain attenuates TACE catalytic activity toward TNFR2. We propose that the ability of N-TACE1854 to inhibit TACE activity in vitro, as well as constitutive TNFR2 shedding in a cell-based system, provides a new insight into the mechanism by which the activity of a disintegrin metalloprotease might be attenuated by its prodomain independently of a cysteine-switch mechanism.
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GRANTS
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Research funding was provided by the Division of Intramural Research, National Heart, Lung, and Blood Institute.
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ACKNOWLEDGMENTS
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The authors thank Drs. Martha Vaughan and Joel Moss for helpful advice and critical review of the manuscript.
B. Adamik is on scientific leave from the Department of Anesthesiology and Intensive Therapy, Wroclaw Medical University, Poland.
F. I. Hawari's current address: Division of Pulmonary and Critical Care Medicine, King Hussein Cancer Center, Amman, Jordan.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. J. Levine, Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Inst., National Institutes of Health, Bldg. 10, Rm. 6D03, MSC 1590, Bethesda, MD 20892-1590 (E-mail: levines{at}nhlbi.nih.gov)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
|
---|
- Althoff K, Reddy P, Voltz N, Rose-John S, and Mullberg J. Shedding of interleukin-6 receptor and tumor necrosis factor alpha. Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins. Eur J Biochem 267: 26242631, 2000.[Abstract/Free Full Text]
- Black RA. Tumor necrosis factor-
converting enzyme. Int J Biochem Cell Biol 34: 15, 2002.[CrossRef][ISI][Medline]
- Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, and Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385: 729733, 1997.[CrossRef][ISI][Medline]
- Blobel CP. Metalloprotease-disintegrins: links to cell adhesion and cleavage of TNF alpha and Notch. Cell 90: 589592, 1997.[CrossRef][ISI][Medline]
- Borroto A, Ruiz-Paz S, De La Torre TV, Borrell-Pages M, Merlos-Suarez A, Pandiella A, Blobel CP, Baselga J, and Arribas J. Impaired trafficking and activation of tumor necrosis factor-
-converting enzyme in cell mutants defective in protein ectodomain shedding. J Biol Chem 278: 2593325939, 2003.[Abstract/Free Full Text]
- Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black RA, and Israel A. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5: 207216, 2000.[CrossRef][ISI][Medline]
- Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ, Johnson RS, Castner BJ, Cerretti DP, and Black RA. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 273: 2776527767, 1998.[Abstract/Free Full Text]
- Contin C, Pitard V, Itai T, Nagata S, Moreau JF, and Dechanet-Merville J. Membrane-anchored CD40 is processed by the TNF-alpha-converting enzyme: implications for CD40 signaling. J Biol Chem 278: 3280132809, 2003.[Abstract/Free Full Text]
- Cunnion KM. Tumor necrosis factor receptors encoded by poxviruses. Mol Genet Metab 67: 278282, 1999.[CrossRef][ISI][Medline]
- Demaegdt H, Laeremans H, De Backer JP, Mosselmans S, Le MT, Kersemans V, Michotte Y, Vauquelin G, and Vanderheyden PM. Synergistic modulation of cystinyl aminopeptidase by divalent cation chelators. Biochem Pharmacol 68: 893900, 2004.[CrossRef][ISI][Medline]
- Doedens JR and Black RA. Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme. J Biol Chem 275: 1459814607, 2000.[Abstract/Free Full Text]
- Endres K, Anders A, Kojro E, Gilbert S, Fahrenholz F, and Postina R. Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation. Eur J Biochem 270: 23862393, 2003.[Abstract/Free Full Text]
- Ermert M, Pantazis C, Duncker HR, Grimminger F, Seeger W, and Ermert L. In situ localization of TNFalpha/beta, TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue. Cytokine 22: 89100, 2003.[CrossRef][ISI][Medline]
- Garton KJ, Gough PJ, Blobel CP, Murphy G, Greaves DR, Dempsey PJ, and Raines EW. Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). J Biol Chem 276: 3799338001, 2001.[Abstract/Free Full Text]
- Gonzales PE, Solomon A, Miller AB, Leesnitzer MA, Sagi I, and Milla ME. Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain. J Biol Chem 279: 3163831645, 2004.[Abstract/Free Full Text]
- Greene C, Lowe G, Taggart C, Gallagher P, McElvaney N, and O'Neill S. Tumor necrosis factor-alpha-converting enzyme: its role in community-acquired pneumonia. J Infect Dis 186: 17901796, 2002.[CrossRef][ISI][Medline]
- Hansen HP, Dietrich S, Kisseleva T, Mokros T, Mentlein R, Lange HH, Murphy G, and Lemke H. CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-alpha-converting enzyme. J Immunol 165: 67036709, 2000.[Abstract/Free Full Text]
- Hino T, Nakamura H, Shibata Y, Abe S, Kato S, and Tomoike H. Elevated levels of type II soluble tumor necrosis factor receptors in the bronchoalveolar lavage fluids of patients with sarcoidosis. Lung 175: 187193, 1997.[ISI][Medline]
- Kohno T, Brewer MT, Baker SL, Schwartz PE, King MW, Hale KK, Squires CH, Thompson RC, and Vannice JL. A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor. Proc Natl Acad Sci USA 87: 83318335, 1990.[Abstract/Free Full Text]
- Merlos-Suarez A, Ruiz-Paz S, Baselga J, and Arribas J. Metalloprotease-dependent protransforming growth factor-alpha ectodomain shedding in the absence of tumor necrosis factor-alpha-converting enzyme. J Biol Chem 276: 4851048517, 2001.[Abstract/Free Full Text]
- Milla ME, Leesnitzer MA, Moss ML, Clay WC, Carter HL, Miller AB, Su JL, Lambert MH, Willard DH, Sheeley DM, Kost TA, Burkhart W, Moyer M, Blackburn RK, Pahel GL, Mitchell JL, Hoffman CR, and Becherer JD. Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE). J Biol Chem 274: 3056330570, 1999.[Abstract/Free Full Text]
- Mohan MJ, Seaton T, Mitchell J, Howe A, Blackburn K, Burkhart W, Moyer M, Patel I, Waitt GM, Becherer JD, Moss ML, and Milla ME. The tumor necrosis factor-alpha converting enzyme (TACE): a unique metalloproteinase with highly defined substrate selectivity. Biochemistry 41: 94629469, 2002.[CrossRef][ISI][Medline]
- Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Warner J, Willard D, and Becherer JD. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385: 733736, 1997.[CrossRef][ISI][Medline]
- Moss ML, White JM, Lambert MH, and Andrews RC. TACE and other ADAM proteases as targets for drug discovery. Drug Discov Today 6: 417426, 2001.[CrossRef][ISI][Medline]
- O'Grady NP, Preas HL, Pugin J, Fiuza C, Tropea M, Reda D, Banks SM, and Suffredini AF. Local inflammatory responses following bronchial endotoxin instillation in humans. Am J Respir Crit Care Med 163: 15911598, 2001.[Abstract/Free Full Text]
- Olsen NJ and Stein CM. New drugs for rheumatoid arthritis. N Engl J Med 350: 21672179, 2004.[Free Full Text]
- Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F II, Park DR, Pugin J, Skerrett SJ, Hudson LD, and Martin TR. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 164: 18961903, 2001.[Abstract/Free Full Text]
- Peiretti F, Canault M, Deprez-Beauclair P, Berthet V, Bonardo B, Juhan-Vague I, and Nalbone G. Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme. Exp Cell Res 285: 278285, 2003.[CrossRef][ISI][Medline]
- Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, and Black RA. An essential role for ectodomain shedding in mammalian development. Science 282: 12811284, 1998.[Abstract/Free Full Text]
- Petelin M, Naruishi K, Shiomi N, Mineshiba J, Arai H, Nishimura F, Takashiba S, and Murayama Y. Systemic up-regulation of sTNFR2 and IL-6 in Porphyromonas gingivalis pneumonia in mice. Exp Mol Pathol 76: 7681, 2004.[CrossRef][ISI][Medline]
- Reddy P, Slack JL, Davis R, Cerretti DP, Kozlosky CJ, Blanton RA, Shows D, Peschon JJ, and Black RA. Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme. J Biol Chem 275: 1460814614, 2000.[Abstract/Free Full Text]
- Rio C, Buxbaum JD, Peschon JJ, and Corfas G. Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4. J Biol Chem 275: 1037910387, 2000.[Abstract/Free Full Text]
- Rovida E, Paccagnini A, Del Rosso M, Peschon J, and Dello Sbarba P. TNF-alpha-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation. J Immunol 166: 15831589, 2001.[Abstract/Free Full Text]
- Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GHW, Gatanaga T, Granger GA, Lentz R, Raab H, Kohr WJ, and Goeddel DV. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61: 361370, 1990.[CrossRef][ISI][Medline]
- Schlondorff J, Becherer JD, and Blobel CP. Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). Biochem J 347: 131138, 2000.[CrossRef][ISI][Medline]
- Schlondorff J and Blobel CP. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J Cell Sci 112: 36033617, 1999.[Abstract/Free Full Text]
- Schlondorff J, Lum L, and Blobel CP. Biochemical and pharmacological criteria define two shedding activities for TRANCE/OPGL that are distinct from the tumor necrosis factor alpha convertase. J Biol Chem 276: 1466514674, 2001.[Abstract/Free Full Text]
- Shao MX, Nakanaga T, and Nadel JA. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 287: L420L427, 2004.[Abstract/Free Full Text]
- Shao MX, Ueki IF, and Nadel JA. Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells. Proc Natl Acad Sci USA 100: 1161811623, 2003.[Abstract/Free Full Text]
- Smith CA, Davis T, Anderson D, Solam L, Beckman MP, Jerzy R, Dower SK, Cosman D, and Goodwin RG. A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 248: 10191023, 1990.[ISI][Medline]
- Solomon KA, Pesti N, Wu G, and Newton RC. Cutting edge: a dominant negative form of TNF-alpha converting enzyme inhibits proTNF and TNFRII secretion. J Immunol 163: 41054108, 1999.[Abstract/Free Full Text]
- Sunnarborg SW, Hinkle CL, Stevenson M, Russell WE, Raska CS, Peschon JJ, Castner BJ, Gerhart MJ, Paxton RJ, Black RA, and Lee DC. Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J Biol Chem 277: 1283812845, 2002.[Abstract/Free Full Text]
- Thathiah A, Blobel CP, and Carson DD. Tumor necrosis factor-alpha converting enzyme/ADAM 17 mediates MUC1 shedding. J Biol Chem 278: 33863394, 2003.[Abstract/Free Full Text]
- Tillie-Leblond I, Pugin J, Marquette CH, Lamblin C, Saulnier F, Brichet A, Wallaert B, Tonnel AB, and Gosset P. Balance between proinflammatory cytokines and their inhibitors in bronchial lavage from patients with status asthmaticus. Am J Respir Crit Care Med 159: 487494, 1999.[Abstract/Free Full Text]
- Tsao TC, Hong J, Li LF, Hsieh MJ, Liao SK, and Chang KS. Imbalances between tumor necrosis factor-alpha and its soluble receptor forms, and interleukin-1beta and interleukin-1 receptor antagonist in BAL fluid of cavitary pulmonary tuberculosis. Chest 117: 103109, 2000.[Abstract/Free Full Text]
- Upton C, Macen JL, Schreiber M, and McFadden G. Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence. Virology 184: 370382, 1991.[ISI][Medline]
- Vincent B, Paitel E, Saftig P, Frobert Y, Hartmann D, De Strooper B, Grassi J, Lopez-Perez E, and Checler F. The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein. J Biol Chem 276: 3774337746, 2001.[Abstract/Free Full Text]
- Zhang Y, Jiang J, Black RA, Baumann G, and Frank SJ. Tumor necrosis factor-alpha converting enzyme (TACE) is a growth hormone binding protein (GHBP) sheddase: the metalloprotease TACE/ADAM-17 is critical for (PMA-induced) GH receptor proteolysis and GHBP generation. Endocrinology 141: 43424348, 2000.[Abstract/Free Full Text]
- Zhao J, Chen H, Peschon JJ, Shi W, Zhang Y, Frank SJ, and Warburton D. Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis. Dev Biol 232: 204218, 2001.[CrossRef][ISI][Medline]
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