There are evidence that both a disintegrin and metalloproteinase 17 (ADAM17) and calpain are involved in platelet glycoprotein (GP)Ibα ectodomain cleavage. However, the relationship between the two enzymes in the shedding process remains unclear. Here we show that calcium ionophore A23187- and α-thrombin-induced GPIbα shedding is completely inhibited by the metalloproteinase inhibitor GM6001, whereas it is only partially inhibited by calpain inhibitors. Calpain activator dibucaine-induced GPIbα shedding was completely inhibited by both metalloproteinase and calpain inhibitors. On the other hand, calpain inhibitors did not inhibit GPIbα shedding induced by the reagents that specifically activate ADAM17. Furthermore, A23187-induced GPIbα shedding in Chinese hamster ovary cells expressing wild-type or mutant GPIb-IX was also partially inhibited by calpain inhibitors and almost completely inhibited by GM6001. Therefore, these data indicate that calpain plays an important role in the regulation of ADAM17-dependent GPIbα ectodomain shedding in both platelets and nucleated cells. 相似文献
Tumor necrosis factor-α (TNF-α) is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-α-converting enzyme (TACE/ADAM17) is the major sheddase for ectodomain shedding of TNF-α. At present, however, it is poorly understood how its catalytic activity is regulated. Here, we show that nardilysin (N-arginine dibasic convertase; NRDc) enhanced TNF-α shedding. In a cell-based shedding assay, expression of NRDc synergistically enhanced TACE-induced TNF-α shedding. A peptide cleavage assay in vitro showed that recombinant NRDc enhances the cleavage of TNF-α induced by TACE. Notably, co-incubation of NRDc completely reversed the inhibitory effect of a physiological concentration of salt on TACE’s activity in vitro. Overexpression of NRDc in TACE-deficient fibroblasts resulted in an increase in the amount of TNF-α released. Co-expression of NRDc with ADAM10 promoted the release compared with the sole expression of ADAM10. These results suggested that NRDc enhances TNF-α shedding through activation of not only TACE but ADAM10. Our results indicate the involvement of NRDc in ectodomain shedding of TNF-α, which may be a novel target for anti-inflammatory therapies. 相似文献
ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala76:Val77 within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity. 相似文献
The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer’s disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases. 相似文献
Very low-density lipoprotein receptor (VLDLR) is a multifunctional transmembrane protein. Beyond the function of the full-length VLDLR in lipid transport, the soluble ectodomain of VLDLR (sVLDLR) confers anti-inflammatory and antiangiogenic roles in ocular tissues through inhibition of canonical Wnt signaling. However, it remains unknown how sVLDLR is shed into the extracellular space. In this study, we present the first evidence that a disintegrin and metalloprotease 17 (ADAM17) is responsible for sVLDLR shedding in human retinal pigment epithelium cells using pharmacological and genetic approaches. Among selected proteinase inhibitors, an ADAM17 inhibitor demonstrated the most potent inhibitory effect on sVLDLR shedding. siRNA-mediated knockdown or CRISPR/Cas9-mediated KO of ADAM17 diminished, whereas plasmid-mediated overexpression of ADAM17 promoted sVLDLR shedding. The amount of shed sVLDLR correlated with an inhibitory effect on the Wnt signaling pathway. Consistent with these in vitro findings, intravitreal injection of an ADAM17 inhibitor reduced sVLDLR levels in the extracellular matrix in the mouse retina. In addition, our results demonstrated that ADAM17 cleaved VLDLR only in cells coexpressing these proteins, suggesting that shedding occurs in a cis manner. Moreover, our study demonstrated that aberrant activation of Wnt signaling was associated with decreased sVLDLR levels, along with downregulation of ADAM17 in ocular tissues of an age-related macular degeneration model. Taken together, our observations reveal the mechanism underlying VLDLR cleavage and identify a potential therapeutic target for the treatment of disorders associated with dysregulation of Wnt signaling. 相似文献
Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease disintegrin that cleaves a variety of membrane proteins, releasing ("shedding") their extracellular domains from cells. Most TACE-mediated shedding events occur at low basal rates that are enhanced by treatment of cells with a variety of stimuli. To study the mechanism of induced shedding, we developed a peptide-cleavage assay that measures the cellular TACE activity. In unstimulated cells, cleavage of a TNFalpha processing-site peptide was mediated mainly by enzymes other than TACE. However, stimulation of cells with phorbol-12-myristate-13-acetate (PMA) increased peptide cleavage in a TACE-dependent manner. PMA treatment did not increase the amount of TACE on the cell surface. Moreover, the cytoplasmic domain of TACE was not required for the induced activity. Based on these observations, induction of TACE-mediated shedding events occurs at least in part via an increase in the enzymatic activity of cellular TACE, independent of its cytoplasmic domain. 相似文献
Meltrin beta (Mel beta, also called ADAM19) is a member of the ADAM (adisintegrin and metalloprotease) family, which are membrane-anchored glycoproteins that play crucial roles in various biological processes. Many intercellular signaling molecules are membrane-anchored proteins, which are proteolytically processed after becoming membrane-bound, to liberate their extracellular domains (ectodomain shedding). Genetic and biochemical studies have shown that some ADAMs participate in these events. We found previously that Mel beta can cleave the extracellular region of the membrane-anchored beta-exon-containing Neuregulin-1 (NRG beta1) protein, which is one of the main ligands for the neural ErbB receptor. Mel beta-deficient mice showed developmental defects in the nervous system. These observations raise the possibility that the NRG ectodomain shedding mediated by Mel beta is closely related to the neural development. Here we show that Mel beta-mediated ectodomain shedding of NRG beta1 takes place in the lipid rafts of neurons. The lipid rafts localization of Mel beta requires its membrane-anchoring region, and NRG beta1 ectodomain shedding is not enhanced if Mel beta cannot reach the lipid rafts. These results indicate that localization of Mel beta in lipid rafts is critical for its ectodomain shedding. 相似文献
The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC 3.4.24.86) continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized. 相似文献
All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are made as transmembrane precursors. Proteolytic processing by ADAMs (a disintegrin and metalloprotease) regulates the bioavailability of several EGFR-ligands, yet little is known about the enzyme responsible for processing the recently identified EGFR ligand, epigen. Here we show that ectodomain shedding of epigen requires ADAM17, which can be stimulated by phorbol esters, phosphatase inhibitors and calcium influx. These results suggest that ADAM17 might be a good target to block the release of bioactive epigen, a highly mitogenic ligand of the EGFR which has been implicated in wound healing and cancer. 相似文献
All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors. 相似文献
Defects in heart development are the most common congenital abnormalities in humans, providing a strong incentive to learn more about the underlying causes. Previous studies have implicated the metalloprotease-disintegrins ADAMs (a disintegrin and metalloprotease) 17 and 19 as well as heparin binding EGF-like growth factor (HB-EGF) and neuregulins in heart development in mice. Here, we show that mice lacking both ADAMs 17 and 19 have exacerbated defects in heart development compared to mice lacking either ADAM, providing the first evidence for redundant or compensatory functions of ADAMs in development. Moreover, we identified additional compensatory or redundant roles of ADAMs 9 and 19 in morphogenesis of the mitral valve and cardiac outflow tract. Cell biological studies designed to address the functions of these ADAMs in shedding of HB-EGF uncovered a contribution of ADAM19 to this process, but this was only evident in the absence of the major HB-EGF sheddase, ADAM17. In addition, ADAM17 emerged as the major sheddase for neuregulins beta1 and beta2 in mouse embryonic fibroblasts. These results raise the possibility that ADAMs 9, 17, and 19 contribute to heart development in humans and have implications for understanding the mechanisms underlying congenital heart disease. 相似文献
The transmembrane and multidomain neural cell adhesion molecule (NCAM) plays important functional roles in the developing and adult nervous system. NCAM is proteolytically processed and appears in soluble forms in the cerebrospinal fluid and in serum under normal and pathological conditions. In this report, we present evidence that the metalloprotease a disintegrin and a metalloprotease (ADAM)17/tumour necrosis factor alpha converting enzyme (TACE) cleaves the polysialylated as well as the non-polysialylated transmembrane isoforms of NCAM, whereas the glycophosphatidylinositol-linked isoform of NCAM is not proteolytically cleaved. A truncated, enzymatically inactive mutant of TACE did not result in release of the NCAM110 cleavage product. Proteolytic cleavage was enhanced by a calmodulin-specific inhibitor and the actin-destabilizing agents cytochalasin D and latrunculin B. In contrast, the microtubule-stabilizing agent colchicine or microtubule-destabilizing agent paclitaxel did not affect the release of the 110-kDa fragment of NCAM. Neurite outgrowth from cerebellar microexplants was inhibited in the presence of the metalloprotease inhibitor GM 6001 on substrate-coated NCAM, but not on poly-l-lysine. Upon transfection of hippocampal neurones with an enzymatically inactive mutant of TACE, NCAM-stimulated neurite outgrowth was inhibited without affecting neurite outgrowth on poly-l-lysine, showing that proteolytic processing of NCAM by the metalloprotease TACE is involved in NCAM-mediated neurite outgrowth. 相似文献
Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation. 相似文献
The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts. 相似文献
Epithin/PRSS14, a type II transmembrane serine protease, plays critical roles in cancer metastasis. Previously, we have reported that epithin/PRSS14 undergoes ectodomain shedding in response to phorbol myristate acetate (PMA) stimulation. In this study, we show that transforming growth factor-β (TGF-β) induces rapid epithin/PRSS14 shedding through receptor mediated pathway in 427.1.86 thymoma cells. Tumor necrosis factor-α converting enzyme (TACE) is responsible for this shedding. Amino acid sequence encompassing the putative shedding cleavage site of epithin/PRSS14 exhibit strong homology to the cleavage site of l-selectin, a known TACE substrate. TACE inhibitor, TAPI-0 and TACE siRNA greatly reduced TGF-β-induced epithin/PRSS14 shedding. TGF-β treatment induces translocation of intracellular pool of TACE to the membrane where epithin/PRSS14 resides. These findings suggest that TGF-β induces epithin/PRSS14 shedding by mediating translocation of epithin/PRSS14 sheddase, TACE, to the membrane. 相似文献
Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory process, in which the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α is considered to play a role. In the present study the putative involvement of TNF-α gene polymorphisms in pathogenesis of COPD was studied by analysis of four TNF-α gene polymorphisms in a Caucasian COPD population. 相似文献
EphrinA/EphA‐dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid‐NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase‐inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E‐F loop of the second fibronectin type III domain (Leu671‐Lys672/Ser673‐Leu674) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3‐induced shedding of NCAM in HEK293T cells. EphrinA5‐induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia.
Tumor necrosis factor-alpha (TNF-α) is a pleiotropic inflammatory cytokine. Tumor necrosis factor-alpha was evaluated in the serum samples of patients with idiopathic retinal periphlebitis in young adults (Eales’ disease). Retinal periphlebitis was graded according to a new grading system based on severity of inflammation (grade 1–4). Quantification of the TNF-α levels was carried out using ELISA kit in the serum samples of young adults with idiopathic retinal periphlebitis (n = 17) and healthy controls (n = 17) of similar age. Tumor necrosis factor-α level was found to be significantly raised in cases with retinal periphlebitis as compared with controls (p < 0.001). Higher levels of TNF-α were found to be associated with increased severity of retinal periphlebitis. Tumor necrosis factor-α represents a novel target for controlling inflammatory activity in idiopathic retinal periphlebitis. Higher levels of TNF-α, in association with the increased severity of retinal periphlebitis, have implications for early anti-TNF-α therapy. 相似文献