The Tumor Necrosis Factor-α Converting Enzyme (TACE):  A Unique Metalloproteinase with Highly Defined Substrate Selectivity

Mohan, Mohita J.; Seaton, Theresa; Mitchell, Justin J.; Howe, Anne; Blackburn, Kevin; Burkhart, William; Moyer, Mary Pat; Patel, Indravadan R.; Waitt, Greg; Becherer, J. David; Moss, Marcia L.; Milla, Marcos E.

doi:10.1021/bi033012g

Cited by 62 publications

(83 citation statements)

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“…In particular, although TACE is considered to be the major physiological TNF sheddase, responsible for 90% of its cleavage in vivo, 3 other cell surface sheddases in the metalloprotease family, such as ADAM-10 and matrix metalloprotease (MMP)-7, have been shown to possess some, albeit small, TNF-cleaving activity. 6,21,22 However, we found that specificity constant (k cat /K m ) for the cleavage of FAM-TAMRA TNF peptide by rADAM-10 was eight-fold lower than that by rTACE, consistent with previous published data by Moss et al 6 Mohan et al 22 reported that the k cat /K m for the cleavage of a similar peptide with a TACE-sensitive TNF sequence was 200-fold lower with rMMP-7 than rTACE. These in vitro experiments using recombinant enzymes suggest that if hydrolysis of the FAM-TAMRA peptide in cell-based assays is metalloprotease-dependent, then the observed enzyme activity should be attributable mainly to TACE, rather than ADAM-10 or MMP-7.…”

Section: Discussionsupporting

confidence: 90%

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Alvarez-Iglesias

Wayne

O’Dea

et al. 2005

Laboratory Investigation

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Tumor necrosis factor-a (TNF) converting enzyme (TACE) is responsible for shedding of various membrane proteins including proinflammatory cytokine TNF. In vivo regulation of TACE is poorly understood mainly due to lack of reliable methodology to measure TACE activity in cell-based assays. Here we report a novel enzyme assay that enables continuous real-time measurement of TACE activity on the surface of live cells. Cells were incubated with a new fluorescent resonance energy transfer peptide consisting of a TACE-sensitive TNF sequence and fluorescein-tetramethylrhodamine (FAM-TAMRA), and enzyme activity was monitored by the rate of increase in fluorescent signal due to peptide cleavage. Validation studies using resting as well as stimulated monocytic cells indicated that the assay was sensitive, reproducible and quantitative. Pharmacological studies with various inhibitors indicated that the observed enzyme activity could largely be ascribed to TACE. Thus, the FAM-TAMRA peptide provides a powerful tool for measurement of constitutive and inducible cellular TACE activity. The principles developed may be applied to analyses of enzyme activity of various sheddases on live cells. Keywords: ADAM; ectodomain shedding; enzyme activity; FRET; protease; TACE; TNF Ectodomain shedding of membrane proteins is recognized as a potent mechanism for downregulation of their cell-associated activity while at the same time enabling their function as soluble mediators. 1,2 Tumor necrosis factor-a (TNF) converting enzyme (TACE), a member of the ADAM (a disintegrin and metalloprotease) family of proteases (ADAM-17), is the first discovered mammalian sheddase, responsible for cleavage of a variety of membrane proteins, including the proinflammatory cytokine TNF, transforming growth factor-a, p75 TNF receptor and L-selectin. 3-8 As a TNF sheddase, TACE regulates the in vivo cleavage of membranebound proTNF to release soluble TNF, 3,4 which has been shown to play a crucial role in acute and chronic inflammation. Thus, analysis of TACE activity should provide important insights into the pathophysiology of inflammatory diseases and potentially help the future development of TACEtargeted therapies. However, the mechanism of in vivo regulation of TACE activity is not well understood, 7-9 mainly due to lack of accurate methods for measuring TACE activity on viable cells. Fluorescence resonance energy transfer (FRET) peptides provide useful tools for kinetic studies of metalloproteases in solution. 10,11 These substrates consist of a donor fluorophore and a light-absorbing acceptor, attached to the terminal residues of a peptide susceptible to cleavage by a protease. Once the substrate is cleaved, increased fluorescence is observed due to loss of internal quenching, allowing quantitative measurement of real-time enzyme activity. The common donor/acceptor pairs are 4-(dimethylaminoazo) benzene-4-carboxyl (Dabcyl) and 5-(2-aminoethylamino)-1-naphthalenesulfonic (Edans), and (7-methoxycoumarin-4-yl) acetyl (Mca) and 2,4-dinitrophenyl (Dnp) or 3-...

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Section: Discussionsupporting

confidence: 90%

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Alvarez-Iglesias

Wayne

O’Dea

et al. 2005

Laboratory Investigation

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“…This may well be initiated by shedding of c-kit ligand. Not only ADAM33 [32] but also ADAM8 [33], ADAM9 [34], ADAM17 [35,36], and ADAM19 [20] are capable of shedding of c-kit ligand, also known as stem cell factor (SCF). SCF plays, apart from its main role as hemopoietic growth factor, a role in recruitment and in maturation of mast cell progenitors [37], sustaining the survival and maintaining phenotypic properties of mast cells in mucosal tissues [38,39].…”

Section: Discussionmentioning

confidence: 99%

Expression of ADAMs (“a disintegrin and metalloprotease”) in the human lung

et al. 2009

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In view of the associations of "a disintegrin and metalloprotease" (ADAM) with respiratory diseases, we assessed the expression of various ADAMs in human lung tissue. Lung tissue was obtained from nine individuals who underwent surgery for lung cancer or underwent lung transplantation for emphysema. Also, 16HBE 14o-(human bronchial epithelial) and A549 (alveolar type II epitheliumlike) cell lines were used. Immunohistochemistry was performed with antibodies recognizing different ADAM domains. The ADAMs were typically distributed over the bronchial epithelium. ADAM8 and ADAM10 were expressed diffusely in all layers of the epithelium. ADAM9, ADAM17, and ADAM19 were predominantly expressed in the apical part of the epithelium, and ADAM33 was predominantly and strongly expressed in basal epithelial cells. In smooth muscle, ADAM19 and ADAM17 were strongly expressed, as was ADAM33, though this expression was weaker. ADAM33 was strongly expressed in vascular endothelium. All ADAMs were generally expressed in inflammatory cells. The typical distribution of ADAMs in the lung, especially in the epithelium, is interesting and suggests a localized function. As most ADAMs are involved in release of (pro-) inflammatory mediators and growth factors, they may play an important role in the first line of defense and in initiation of repair events in the airways.

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“…Once the protein has been translated to its precursor form it is inserted in the cell membrane as a pro-TNF 26 kDa form. Membrane-bound TNF may play some role in cell-cell interactions 20 but full activation requires proteases, including TACE and several MMPs, 21 to cleave the protein. Once released as a bioactive trimer it exerts its function by interaction with its membrane receptors TNFRSF1A (TNFRI) and TNFRSF1B (TNFRII).…”

Section: Introductionmentioning

confidence: 99%

Is there a future for TNF promoter polymorphisms?

2004

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The in vitro study of TNF promoter polymorphism (SNP) function was stimulated by the numerous case-control (association) studies of the polymorphisms in relation to human disease and the appearance of several studies claiming to show a functional role for these SNPs provided a further impetus to researchers interested in the role of TNF in their disease of interest. In this review we consider case-control studies, concentrating on the autoimmune and inflammatory diseases rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, and asthma, and on infectious diseases including malaria, hepatitis B and C infection, leprosy and sepsis/septic shock. We also review the available evidence on the functional role of the various TNF promoter polymorphisms. In general, case-control studies have produced mixed results, with little consensus in most cases on whether any TNF polymorphisms are actually associated with disease, although results have been more consistent in the case of infectious diseases, particularly malaria. Functional studies have also produced mixed results but recent work suggests that the much studied À308G/A polymorphism is not functional, while the function of other TNF polymorphisms remains controversial. Studies of the TNF region are increasingly using extended haplotypes that can better capture the variation of the MHC region.

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The Tumor Necrosis Factor-α Converting Enzyme (TACE): A Unique Metalloproteinase with Highly Defined Substrate Selectivity

Cited by 62 publications

References 0 publications

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Expression of ADAMs (“a disintegrin and metalloprotease”) in the human lung

Is there a future for TNF promoter polymorphisms?

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