Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

The transmembrane metzinkin-proteases of the ADAM (a disintegrin and a metalloproteinase)-family ADAM10 and ADAM 17 are both implicated in the ectodomain shedding of various cell surface molecules including the IL6-receptor and the transmembrane chemokines CX3CL1 and CXCL16. These molecules are constitutively released from cultured cells, a process that can be rapidly enhanced by cell stimulation with phorbol esters such as PMA. Recent research supports the view that the constitutive cleavage predominantly involves ADAM10 while the inducible one is mediated to a large extent by ADAM17. We here describe the discovery of hydroxamate compounds with different potency against ADAM10 and ADAM17 and different ability to block constitutive and inducible cleavage of IL6R, CX3CL1 and CXCL16 by the two proteases. By screening a number of hydroxamate inhibitors for the inhibition of recombinant metalloproteinases, a compound was found inhibiting ADAM10 with more than 100-fold higher potency than ADAM17, which may be explained by an improved fit of the compound to the S1' specificity pocket of ADAM10 as compared to that of ADAM17. In cell-based cleavage experiments this compound (GI254023X) potently blocked the constitutive release of IL6R, CX3CL1 and CXCL16, which was in line with the reported involvement of ADAM10 but not ADAM17 in this process. By contrast, the compound did not affect the PMA-induced shedding, which was only blocked by GW280264X, a potent inhibitor of ADAM17. As expected, GI254023X did not further decrease the residual release of CX3CL1 and CXCL16 in ADAM10-deficient cells verifying that the compound's effect on the constitutive shedding of these molecules was exclusively due to the inhibition of ADAM10. Thus, GI254023X may by of use as a preferential inhibitor of constitutive shedding events without effecting the inducible shedding in response to agonists acting similar to PMA.

The tumor necrosis factor-␣-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-␣ secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that fulllength TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE. TNF␣1 is a potent cytokine that is secreted by activated monocytes and macrophages in a tightly regulated manner (1). Upon release, TNF␣ mediates the recruitment and activation of inflammatory cells to injured or infected tissues (2). Elevated levels of circulating TNF␣ have been demonstrated in several acute and chronic pathological states, such as lipopolysaccharide-induced septic shock, arthritis, pleurisy, Crohn's disease, and inflammatory bowel disease (3). TNF␣ is synthesized as a pro, membrane-anchored form facing the lumenal/extracellular side of the secretory pathway. Our group and others have shown that proTNF␣ is released from cells after endoproteolytic cleavage at positions Ala 76 -Val 77 , mediated by a zinc metalloprotease sensitive to hydroxamic acid inhibitors (4 -6). Because neutralization of TNF␣ activity has been demonstrated in the clinic, this enzyme constitutes a potential target for drug discovery.The TNF␣-converting enzyme (TACE) was purified to homogeneity and cloned (7,8). Analysis of its amino acid sequence demonstrates a multidomain protein closely resembling members of the disintegrin family of metalloproteases, also commonly referred to as ADAMs or metalloprotease and disintegrin-containing proteins (9). Starting at the N terminus, TACE exhibits a classical signal peptide followed by a ϳ200-residue pro domain that includes a consensus cysteine switch motif (PKVCGY 186 ), which can act as an inhibitor by ligating the zinc ion in the catalytic site (10, 32). The catalytic domain starts downstream from a consensus furin cleavage site (RVKRR 215 ) and contains a canonical zinc binding site and a MYP loop involved in formation of the P1Ј p...

Tumor necrosis factor-␣-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC 50 of 70 nM. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC 50 greater that 2 M). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys 184 residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.