Erratum: Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells (original) (raw)
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Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells
Laboratory Investigation, 2005
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.
Purification of ADAM 10 from bovine spleen as a TNFα convertase
FEBS Letters, 1997
We have purified a protease with characteristics of TNFa convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFa substrate to mature TNFa, and can cleave a synthetic peptide substrate to yield the mature TNFa amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFa processing in a processing-incompetent cell line.
Biochemical Society Symposia, 2003
Tumour necrosis factor ␣ (TNF␣)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor ␣, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions.
TACE/ADAM-17 enzymatic activity is increased in response to cellular stimulation
Biochemical and Biophysical Research Communications, 2003
Tumor necrosis factor-a 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 TNFa processingsite 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.
Purification of TNF Binding Proteins
Tumor Necrosis Factor, 2004
The finding that the two tumor necrosis factor receptors (TNFR) exist in soluble form in various body fluids not only has substantiated the paradigm of naturally existing soluble cytokine receptors but also has represented a milestone on the road to the biochemical and biological characterization of the two TNFRs. This chapter gives a simple, basic protocol for the purification of the two soluble TNFRs. The protocols found here may be easily adapted for the purification of various other soluble cytokine receptors. The purified proteins may be used in biological experiments or for the generation of specific research tools such as polyclonal or monoclonal antibodies.
TNF-α converting enzyme (TACE) is inhibited by TIMP-3
FEBS Letters, 1998
TNF-K K converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-K K to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-K K secretion is involved.
Journal of Immunological Methods, 2002
In the present study, a new flow cytometric method for the identification of TNF-a-secreting cells based on the use of a TNF-a converting enzyme (TACE) inhibitor compound (BB3103) is described. TNF-a secreting cells were measured in parallel in stimulated peripheral blood samples (n = 4), using the BB3103 TACE inhibitor or brefeldin A as secretion blocking agents. To induce TNF-a production by PB T-cells and monocytes, whole blood samples were stimulated either for 4 h with PMA plus ionomycin or for 6 h with LPS plus IFNg, respectively. Interestingly, slightly higher percentages of TNF-a + CD4 + (65 F 11% versus 49 F 11.4%, p = 0.06) and TNF-a + CD8 + (60 F 9.9% versus 47 F 27.7% p = 0.46) T-cells together with a greater amounts of TNF-a/cell-mean fluorescence intensity (MFI) of 1050 F 230 versus 258 F 112 for CD4 + , p = 0.06 and 424 F 169 versus 266 F 201 for CD8 + , p = 0.27-were found for activated T-lymphocytes cultured with BB3103 as compared to those treated with brefeldin A. Kinetic analysis of surface TNF-a expression under these stimulatory conditions showed detectable surface TNF-a levels on both T-cells and monocytes after 30 min. Thereafter, surface TNF-a expression on both Tcells and monocytes progressively increased for up to 3 and 4 h, respectively. From this time on, a decrease in the membrane levels of TNF-a was observed in the monocytes, presumably due to the occurrence of cell death. In order to show that the BB3103 inhibitor was also active on other TACE-associated molecules, CD62L expression on PMA-stimulated PB lymphocytes, monocytes and neutrophils was analyzed by flow cytometry in the presence and absence of BB3103. The TACE inhibitor proved to be active in stabilizing CD62L expression on PMA-stimulated PB leukocytes. In summary, our results show that stimulation of PB T-cells and monocytes in the presence of the TACE inhibitor BB3103 followed by surface staining for TNF-a provides a new, simple and rapid method for the identification of intact TNF-a producting cells present in a sample without the need for prior cell fixation and permeabilization. In addition, this approach could also be applied in order to stabilize
Improvement of a cytokine (TNF-α) bioassay by serum-free target cell (WEHI 164) cultivation
Cytotechnology, 1999
The elaboration of a sensitive bioassay for assessment of tumour necrosis factor alpha (TNF-α) in a defined medium is described. The assay is based on the cytotoxic effect of TNF-α on a target cell line, the murine fibrosarcoma WEHI 164 clone 13. Cytotoxicity was assessed by detecting the rate of tetrazolium salt reduction employing a spectrophotometer (ELISA-reader). A similar bioassay was used previously to assess TNF-α, though this was dependent on cell growth in a medium containing serum. By employing a synthetic serum replacement, the WEHI cells were adapted to growth in a defined medium which allowed both the propagation of the cell line and the assay to be performed under completely defined conditions. Thus, factors in serum that may influence the TNF-α assessment, such as growth factors, cytokines, soluble cytokine-receptors and macroglobulin, were avoided. The only protein required in this bioassay was insulin, while albumin was added as a carrier protein and to protect the...
The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3
FEBS Letters, 2000
A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described`metallosheddase' cleavage sites of tumour necrosis factor K K, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.