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

Abstract

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-...

Reactive Oxygen Species and p38 Mitogen-activated Protein Kinase Mediate Tumor Necrosis Factor α-Converting Enzyme (TACE/ADAM-17) Activation in Primary Human Monocytes

“…Surface TACE Activity-TACE activity was quantified by monitoring the rate of increase in fluorescent signal due to cleavage of the FAM-TAMRA peptide with a TACE-specific TNF sequence (27). Although previously described in cell lines, we confirmed in primary human monocytes that the rate of peptide cleavage was stable and proportional to cell number, indicating that the TACE activity assay was quantitative (Fig.…”

“…It has been firmly established that PMA stimulation of monocytic cell lines up-regulates TACE activity (14,15,27,36). In contrast, there are few data concerning the more physiologically relevant LPS stimulation of primary cells.…”

“…Well volumes totaled 100 l with 5 M FAM-TAMRA TNF peptide and an appropriate volume of assay buffer. TACE enzymatic activity was quantified by continuous measurement of fluorescence intensity in a microplate fluorometer (Flx-800; Bio-tek Instruments Inc., Bedfordshire, UK) at 37 ºC and at ex 485 nm and em 535 nm in accordance with the protocol described previously (27). Data were acquired with KC4 data analysis software (Bio-tek Instruments Inc.).…”

“…EO 1428 was purchased from Tocris Bioscience (Bristol, UK). Biomol International LP (Exeter, UK) supplied the fluorescein-tetramethylrhodamine (FAM-TAMRA) fluorescence resonance energy transfer (FRET) peptide that consists of a TACE-sensitive TNF sequence (FAM-SPLAQAVRSSSRK-TAMRA) (27).…”

“…We previously developed a highly sensitive fluorometric assay to directly quantify cell surface TACE activity (27). We employed this assay to investigate the regulation of TACE activity on primary human monocytes in response to LPS stimulation.…”