Structural features and biochemical properties of TNF-α converting enzyme (TACE)
Introduction
Two years ago, we discovered that a hydroxamic acid, GW 9471 (Fig. 1), inhibited TNF secretion from a monocytic cell line, THP-1(McGeehan et al., 1994). The inhibitor is a British Biotech compound that was previously demonstrated to be a broad spectrum inhibitor of the matrix metalloproteases (MMPs) (Campion et al., 1990). The MMPs are a family of enzymes that can be divided into three domains: pro, catalytic, and hemopexin (Fig. 2). Membrane bound MMPs (MT-MMPs), have a transmembrane region in addition to the above domains. The pro region contains a cysteine switch, a feature that maintains the enzyme in an inactive state. The pro domain can be autocatalytically removed to generate an active MMP. The catalytic domain contains a standard zinc ligand binding motif and the hemopexin domain is important for recognition of natural substrates of the enzymes such as collagen and aggrecan.
The MMPs are involved in normal processes such as tissue remodeling, wound repair and endometrial cycling (Talhouk et al., 1992; Jeffrey, 1991; Salamonsen, 1994). However, the MMPs are associated with disease states as well, such as cancer metastasis, multiple sclerosis, arthritis and restenosis (Stetler-Stevenson et al., 1993; Gijbels et al., 1994; Matrisian, 1990; Galis et al., 1995).
TNF-α has also been implicated in diverse pathologies such as arthritis and multiple sclerosis. For example, Centecor has demonstrated that in clinical trials, antibody treatment of patients with rheumatoid arthritis results in improvement in several clinical scores such as joint pain and tenderness and grip strength (Elliot et al., 1994).
In addition, neutralizing antibodies to TNF-α inhibit the development of experimental auto-immune encephalomyelitis (EAE) in rodent models (Selmaj et al., 1991). Other neuroimmunological conditions where the presence of TNF-α is well documented and may play a role in the etiology of the diseases are Alzheimers and stroke (Cochran and Vitek, 1996; Reiss et al., 1993).
Section snippets
Identification of TNF-α converting enzyme as a metalloprotease
Unlike the substrates for the MMPs, TNF-α exists as a membrane bound precursor form that is processed to yield a 17 kDa secreted form (Fig. 3). Processing of precursor TNF-α is required for TNF secretion (Perez et al., 1990; Utsumi et al., 1993). Therefore, we thought that the target of GW 9471, was the processing metalloprotease for precursor TNF-α (TNF-α converting enzyme or TACE). Attempts at identifying a convertase activity were initiated using a macrophage like cell line called Mono-Mac
Purification of TACE
Purification of TACE was accomplished from Mono-Mac 6 cell membrane fractions but there was not enough protein for sequence analysis. Therefore, we switched to a tissue source for purification of the enzyme. Pig spleen was chosen because the cleavage sequence of porcine is similar to the human cleavage sequence and spleen contains a variety of immune competent cell types. Purification was accomplished by the scheme described in Fig. 4. Briefly, a particulate fraction was washed with 0.05% NP-40
Complementary cDNA of TACE
Sequence analysis was performed on the Lys-C digested enzyme. A 41 amino acid residue was identified. Data base searches indicated that the sequence was unique. Therefore, the porcine enzyme was partially cloned from a cDNA library made from spleen mRNA. Results from the cloning indicated the presence of a zinc ligand binding motif. Therefore we had cloned a metalloprotease. Unlike the matrix metalloproteases, there was a cytoplasmic tail that followed the transmembrane region and a cysteine
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2019, Journal of Molecular StructureCitation Excerpt :Black et al. [8] and Moss et al. [9] had identified ADAM17/TACE as the enzyme responsible for inflammatory conditions by shedding of TNF-α causing internalization of sTNF-α (soluble TNF- α) from membrane-bound TNF-α (Fig. 1). The contribution of ADAM17/TACE in a number of pathophysiological conditions have been observed through proteolysis of different cytokines like HB-EGF, ALCAM, carbonic anhydrases (CAs), interferon-γ (INF-γ), amyloid precursor protein (APP), Interleukins (ILs), etc [9,10]. ADAM17/TACE contributes a major role in different life-threatening diseases such as cancer, inflammation, Alzheimer's disease (AD), lymphoma, rheumatoid arthritis (RA), etc [9–11].
TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death
2016, Biochemical PharmacologyCitation Excerpt :Tumor Necrosis Factor (TNF) is a potent pro-inflammatory cytokine produced by many cell types including activated macrophages, monocytes, T and B lymphocytes, keratinocytes, endothelial cells, osteoclasts, neutrophils and fibroblasts [1–3]. TNF is expressed as a 26 kDa transmembrane protein that is further processed into a secreted 17 kDa soluble product [4]. TNF elicits a broad spectrum of responses, both at the cellular and at the systemic level, including lymphocyte and leukocyte activation and migration, fever, inflammatory responses, cell proliferation, differentiation and cell death [2].
The cytoplasmic domain of a disintegrin and metalloproteinase 10 (ADAM10) regulates its constitutive activity but is dispensable for stimulated ADAM10-dependent shedding
2015, Journal of Biological ChemistryCitation Excerpt :Because the transmembrane domain of ADAM17 is required for its response to a variety of stimuli (11), we tested whether the transmembrane domain of ADAM10 is important for its activation by IO. For this purpose, we generated chimeric constructs containing the extracellular domain of ADAM10 fused to the transmembrane and cytoplasmic domains of ADAM17 or only the transmembrane domain of ADAM17, without its cytoplasmic domain (KKTT or KKT, where K stands for Kuzbanian, an alternative name for ADAM10 (6), and T stands for TACE, or TNFα convertase (21), an alternative name for ADAM17; see Table 1 and the diagram in Fig. 5A for details). We observed slightly increased shedding of BTC from Adam10/17−/− mEFs co-transfected with KKTT or KKT compared with ADAM10 WT under unstimulated conditions.
TNF and MAP kinase signalling pathways
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- 1
Present address: Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305, USA.
- 2
Present address: RPR, 600 Arcola Rd., Collegville, PA 19426, USA.
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Present address: Dow Corning Corporation, Midland, MI 48686-0994, USA.