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 genetic loci agouti and extension control the relative amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments in mammals: extension encodes the receptor for melanocyte-stimulating hormone (MSH) and agouti encodes a novel 131-amino-acid protein containing a signal sequence. Agouti, which is produced in the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur. Here we use partially purified agouti protein to demonstrate that agouti is a high-affinity antagonist of the MSH receptor and blocks alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. Agouti was also found to be an antagonist of the melanocortin-4 receptor, a related MSH-binding receptor. Consequently, the obesity caused by ectopic expression of agouti in the lethal yellow (Ay) mouse may be due to the inhibition of melanocortin receptor(s) outside the hair follicle.

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...

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