Application of structural dynamic approaches provide novel insights into the enzymatic mechanism of the tumor necrosis factor-alpha-converting enzyme.
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ABSTRACT:
SUBMITTER: Sagi I
PROVIDER: S-EPMC2254313 | biostudies-literature | 2008 Jan
REPOSITORIES: biostudies-literature
Publications
Application of structural dynamic approaches provide novel insights into the enzymatic mechanism of the tumor necrosis factor-alpha-converting enzyme.
Analytical biochemistry 20070926 1
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Project description:Peripheral serotonin (5-hydroxytryptamine, 5-HT) is transported by platelets and released upon stimulation. In the platelet cytoplasm, 5-HT is transamidated to small GTPases, promoting alpha-granule release and primary hemostasis.We hypothesized that 5-HT could also stimulate platelet receptor shedding after binding to the membrane 5-HT receptor (5-HT2AR).Western blot and flow cytometry were used to determine levels of the adhesion receptor glycoprotein (GP)Ibalpha on platelets or its shed fragment glycocalicin in plasma and serum from wild-type mice, Tph1(-/-) mice lacking peripheral 5-HT, and mice lacking functional tumor necrosis factor-alpha-converting enzyme (TACE, ADAM17). Flow chamber experiments and intravital microscopy were used to examine the adhesive properties of platelets after stimulation of 5-HT2AR.Glycocalicin was significantly reduced in Tph1(-/-) plasma and serum. In isolated platelets, 5-HT induced shedding of GPIbalpha, which was increased to 60% when 5-HT uptake was inhibited by the selective serotonin reuptake inhibitor fluoxetine. Specific 5-HT2AR agonism and antagonism suggested activation of this receptor. The shedding could not be induced in TACE(DeltaZn/DeltaZn) platelets, suggesting that activated TACE mediated the shedding of GPIbalpha. Intracellular signaling involved phosphorylation of p38 mitogen-activated protein kinase rather than G-protein signaling. 5-HT2AR stimulation decreased platelet adhesion to collagen-bound von Willebrand factor under arterial shear (1500 s(-1)) and incorporation into FeCl3-induced thrombi in mesenteric arterioles.Stimulation of 5-HT2AR on platelets induces TACE-mediated shedding of GPIbalpha, the key adhesion molecule under high shear conditions. Our observations demonstrate a new pathway through which 5-HT could modulate cardiovascular disease.
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Project description:TNF-α (tumor necrosis factor-α) is initially synthesized as a transmembrane protein that is cleaved by TACE (TNF-α-converting enzyme) to release soluble TNF-α. The elevated level of TNF-α in the brain and circulation in heart failure (HF) suggests an increase in the TACE-mediated ectodomain shedding process. The present study sought to determine whether TACE is upregulated in cardiovascular/autonomic brain regions like subfornical organ and hypothalamic paraventricular nucleus in rats with ischemia-induced HF and whether TACE plays a role in TNF-α-driven sympathetic excitation. We found that TACE was expressed throughout the subfornical organ and paraventricular nucleus, with significantly higher levels in HF than in sham-operated (Sham) rats. Intracerebroventricular injection of recombinant TACE induced a mild increase in blood pressure, heart rate, and renal sympathetic nerve activity that peaked at 15 to 20 minutes in both Sham and HF rats. HF rats had a secondary prolonged increase in these variables that was prevented by the TNF-α inhibitor SPD304. Intracerebroventricular administration of the TACE inhibitor TNF-alpha protease inhibitor 1 decreased blood pressure, heart rate, and renal sympathetic nerve activity in Sham and HF rats, with an exaggerated reduction in heart rate and renal sympathetic nerve activity in the HF rats. Direct microinjection of TACE or TNF-alpha protease inhibitor 1 into paraventricular nucleus or subfornical organ of Sham and HF rats elicited blood pressure, heart rate, and renal sympathetic nerve activity responses similar to intracerebroventricular TACE or TNF-alpha protease inhibitor 1. Intracerebroventricular infusion of Ang II (angiotensin II) and IL (interleukin)-1β increased TACE expression in subfornical organ and paraventricular nucleus of normal rats. These data suggest that a TACE-mediated increase in soluble TNF-α in the brain contributes to sympathetic excitation in HF.
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Project description:Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands [e.g., transforming growth factor type alpha (TGF-alpha)] and EGFR phosphorylation are implicated in mucin production in airway epithelial cells. Tumor necrosis factor alpha-converting enzyme (TACE) is reported to cleave precursor of TGF-alpha, with release of soluble mature TGF-alpha in various epithelial tissues. We hypothesized that TACE increases the shedding of TGF-alpha, resulting in EGFR phosphorylation and inducing mucin production in human airway epithelial (NCI-H292) cells. To examine this hypothesis, we stimulated NCI-H292 cells with phorbol 12-myristate 13-acetate (PMA, an activator of TACE) and pathophysiologic stimuli [lipopolysaccharide (LPS) and supernatant from the Gram-negative bacterium Pseudomonas aeruginosa (PA sup)]. PMA, PA sup, and LPS increased MUC5AC gene expression and mucin protein production, effects that were prevented by pretreatment with AG1478, a selective inhibitor of EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF-alpha-neutralizing Ab, implicating ligand (TGF-alpha)-dependent EGFR phosphorylation in mucin production. These stimuli induced release of soluble TGF-alpha, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor-alpha protease inhibitor-1 and tissue inhibitor of metalloprotease-3. We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE. Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF-alpha shedding, EGFR phosphorylation, and mucin production. From these results, we conclude that TACE plays a critical role in mucin production by airway epithelial cells by means of a TACE ligand-EGFR cascade in response to various stimuli.
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Project description:Transforming growth factor-alpha (TGF-alpha) is abnormally expressed in autosomal recessive polycystic kidney disease (ARPKD). Tumor necrosis factor-alpha converting enzyme (TACE), a metalloproteinase, mediates TGF-alpha processing. In this study, we sought to determine whether TGF-alpha was an absolute requirement for renal cystogenesis and whether its absence would modulate disease severity or related growth factors/receptors expression. Bpk heterozygotes were bred with TGF-alpha null mice to produce cystic and noncystic offspring with or without TGF-alpha. Assessments included kidney weight (KW), body weight (BW), blood urea nitrogen (BUN), and kidney and liver immunohistology. Western analysis assessed kidney expression of amphiregulin (AR), epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), and their receptors, EGFR and ErbB4. A PCR-based methodology for genotyping bpk mice was also developed. No significant differences in KW, BW, KW/BW%, or BUN were seen in cystic mice with versus without TGF-alpha. Cystic kidney disease and liver disease histology were similar. AR, EGF, HB-EGF, EGFR, and ErbB4 were abnormally expressed to an equal degree in kidneys of mice with versus without TGF-alpha. Although previous data suggest a critical role of TGF-alpha in murine PKD, these data show that TGF-alpha is not required for renal cyst formation or kidney or liver disease progression. We speculate that the therapeutic effect of WTACE2 could have been due to effects on several TACE targets, including TGF-alpha, AR, and ErbB4, as well as metalloproteinases other than TACE.
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Project description:Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 +/- 2007 TACE/ADAM17(+) MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304(TNF)), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304(TNF) cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17(+) MPs could regulate the inflammatory balance in the culprit lesion.
| S-EPMC2043531 | biostudies-literature
Project description:MMPs and TACE (ADAM-17) assume independent, parallel, or opposite pathological roles in cancer, arthritis, and several other diseases. For therapeutic purposes, selective inhibition of individual MMPs and TACE is required in most cases due to distinct roles in diseases and the need to preserve activities in normal states. Toward this goal, we compared force-field interaction energies of five ubiquitous inhibitor atoms with flexible binding sites of 24 known human MMPs and TACE. The results indicate that MMPs 1-3, 10, 11, 13, 16, and 17 have at least one subsite very similar to TACE. S3 subsite is the best target for development of specific TACE inhibitors. Specific binding to TACE compared to most MMPs is promoted by placing a negatively charged ligand part at the bottom of S2 subsite, at the entrance of S1' subsite, or the part of S3' subsite that is close to catalytic zinc. Numerous other clues, consistent with available experimental data, are provided for design of selective inhibitors.
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Project description:Tumor necrosis factor alpha converting enzyme (TACE) is a sheddase overexpressed in cancers that generates cancer cell growth and survival factors, and is implicated in carcinogenesis and tumor growth. This indicates that TACE could be a potentially important cancer biomarker. Unexpectedly, TACE expression in cancer tissues does not correlate with cancer stage or invasiveness. Although TACE sheddase activity is a more direct and potentially better indicator of TACE biology and might be a better cancer biomarker than TACE expression, it has not been studied in cancer tissues. In the present study, we developed a reliable specific assay for quantification of TACE sheddase activity, investigated TACE activity and TACE protein expression in head and neck cancer (HNC) tissues, and examined the correlation of the results with HNC clinical stages and likelihood to recur. We found that HNC cell lines and tissues contained remarkably higher quantities of TACE activity and TACE protein than normal keratinocytes or oral mucosa. siRNA silencing of TACE resulted in the inhibition of release of the tumorogenic factors amphiregulin and transforming growth factor alpha, and tumor protective factors tumor necrosis factor receptors from HNC cells. Importantly, TACE activity, but not TACE protein expression, was significantly higher in large, T3/T4, primary tumors relative to small, T1/T2, primary tumors, and especially in primary tumors likely to recur relative to those unlikely to recur. These data show that increased TACE activity in cancer is biologically and clinically relevant, and indicate that TACE activity could be a significant biomarker of cancer prognosis.
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Project description:Glial erbB1 receptors play a significant role in the hypothalamic control of female puberty. Activation of these receptors by transforming growth factor alpha (TGFalpha) results in production of prostaglandin E2, which then stimulates luteinizing hormone releasing hormone (LHRH) neurons to secrete LHRH, the neuropeptide controlling sexual development. Glutamatergic neurons set in motion this glia-to-neuron signaling pathway by transactivating erbB1 receptors via coactivation of AMPA receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Because the metalloproteinase tumor necrosis factor alpha converting enzyme (TACE) releases TGFalpha from its transmembrane precursor before TGFalpha can bind to erbB1 receptors, we sought to determine whether TACE is required for excitatory amino acids to activate the TGFalpha-erbB1 signaling module in hypothalamic astrocytes, and thus facilitate the advent of puberty. Coactivation of astrocytic AMPARs and mGluRs caused extracellular Ca2+ influx, a Ca2+/protein kinase C-dependent increase in TACE-like activity, and enhanced release of TGFalpha. Within the hypothalamus, TACE is most abundantly expressed in astrocytes of the median eminence (ME), and its enzymatic activity increases selectively in this region at the time of the first preovulatory surge of gonadotropins. ME explants respond to stimulation of AMPARs and mGluRs with LHRH release, and this response is prevented by blocking TACE activity. In vivo inhibition of TACE activity targeted to the ME delayed the age at first ovulation, indicating that ME-specific changes in TACE activity are required for the normal timing of puberty. These results suggest that TACE is a component of the neuron-to-glia signaling process used by glutamatergic neurons to control female sexual development.
| S-EPMC6674310 | biostudies-literature
Project description:Tumor necrosis factor (TNF)-α is a major pro-inflammatory cytokine produced in response to toll-like receptor stimulation. TNF-α release is controlled by the activity of TNF-α converting enzyme (TACE) that cut membrane-bound TNF-α to shed its ectodomain as a soluble cytokine. The purinergic receptor P2X ligand-gated ion channel 7 (P2X7) is activated in response to elevated concentrations of extracellular ATP and induces different pro-inflammatory pathways in macrophages to establish an inflammatory response. P2X7 receptor promotes the activation of the inflammasome and the release of interleukin-1β, the production of inflammatory lipids, and the generation of reactive oxygen species. In this study, we analyzed the mechanism of P2X7 receptor responsible of TNF-α release after priming macrophages with LPS doses ≤100 ng/ml. We found that P2X7 receptor increases the extracellular activity of TACE through the release of the mature form of TACE in exosomes. This effect was blocked using P2X7 receptor inhibitors or in macrophages obtained from P2X7 receptor-deficient mice. Elevation of intracellular Ca2+ and p38 mitogen-activated protein kinase after P2X7 receptor activation were involved in the release of TACE, which was able to process TNF-α on nearby expressing cells. Finally, we observed an increase of TNF-α in the peritoneal lavage of mice treated with LPS and ATP. In conclusion, P2X7 receptor induces the release of TACE in exosomes to the extracellular compartment that could amplify the pro-inflammatory signal associated to this receptor. These results are important for the development of therapeutics targeting P2X7 receptor.
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Project description:Several transmembrane molecules are cleaved at juxtamembrane extracellular sites leading to shedding of ectodomains. We analysed shedding of members of the Vps10p-D (Vps10p domain; where Vps is vacuolar protein sorting) family of neuronal type-I receptors with partially overlapping functions, and additional proteolytic events initiated by the shedding. When transfected into CHO (Chinese-hamster ovary) cells (CHO-K1), sorCS1a-sorCS1c isoforms were shed at high rates (approximately 0.61% x min(-1)) that were increased approx. 3-fold upon stimulation with phorbol ester. sorCS1c identified in the cultured neuroblastoma cell line SH-SY5Y was shed similarly. In CHO-K1 transfectants, constitutive and stimulated shedding of sorCS3 also occurred at high rates (0.29% and 1.03% x min(-1)). By comparison, constitutive and stimulated shedding of sorLA occurred at somewhat lower rates (0.07% and 0.48% x min(-1)), whereas sorCS2 and sortilin were shed at very low rates even when stimulated (approximately 0.01% x min(-1)). Except for sorCS2, shedding of the receptors was dramatically reduced in mutant CHO cells (CHO-M2) devoid of active TACE (tumour necrosis factor alpha-converting enzyme), demonstrating that this enzyme accounts for most sheddase activity. The release of sorCS1 and sorLA ectodomains initiated rapid cleavage of the membrane-tethered C-terminal stubs that accumulated only in the presence of gamma-secretase inhibitors. Purified shed sorLA bound several ligands similarly to the entire luminal domain of the receptor, including PDGF-BB (platelet-derived growth factor-BB) and amyloid-beta precursor protein. In addition, PDGF-BB also bound to the luminal domains of sorCS1 and sorCS3. The results suggest that ectodomains shed from a subset of Vps10p-D receptors can function as carrier proteins.
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