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 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...

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 ؋ 10 8 independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1, P2, and P3 subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2,4-dinitrophenyl-GPLGM-RGL-NH 2 (CP), has a k cat /K m value of 4.22 ؋ 10 6 M ؊1 s ؊1 for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens. Human collagenase 3 (MMP-13)1 is a member of the collagenase subfamily of matrix metalloproteinases (MMPs) (1). Several potential roles for this enzyme have been proposed, including aggrecan degradation associated with osteoarthritis (2), cleavage of type II collagen in osteoarthritic cartilage explants (3), and in tumor progression and metastasis (1). Its expression is also detected in human T lymphocytes (4). Human collagenase 3 degrades type II collagen, the primary collagen in articular cartilage, more than 10 times as efficiently as collagenase 1, another MMP known to be up-regulated in arthritic diseases (5). In addition, collagenase 3 cleaves soluble type II collagen 5 to 6 times more efficiently than type I or type III collagen (6). Furthermore a preferential collagenase 3 inhibitor significantly reduces collagen degradation from unstimulated osteoarthritic cartilage (3). These cumulative data suggest that collagenase 3 may play a significant role in the cleavage of type II collagen in arthritic cartilage.The specificity of this enzyme for aggrecan and other collagenous substrates has also been investigated. Collagenase 3 cleaves aggrecan at the same major and minor sites identified for other members of the MMP family as well as at a novel site (7). In addition, collagenase 3 can degrade fibronectin and type IV,...

TNF alpha converting enzyme (TACE) processes precursor TNF alpha between Ala76 and Val77, yielding a correctly processed bioactive 17 kDa protein. Genetic evidence indicates that TACE may also be involved in the shedding of other ectodomains. Here we show that native and recombinant forms of TACE efficiently processed a synthetic substrate corresponding to the TNF alpha cleavage site only. For all other substrates, conversion occurred only at high enzyme concentrations and prolonged reaction times. Often, cleavage under those conditions was accompanied by nonspecific reactions. We also compared TNF alpha cleavage by TACE to cleavage by those members of the matrix metalloproteinase (MMP) family previously implied in TNF alpha release. The specificity constants for TNF alpha cleavage by the MMPs were approximately 100-1000-fold slower relative to TACE. MMP 7 also processed precursor TNF alpha at the correct cleavage site but did so with a 30-fold lower specificity constant relative to TACE. In contrast, MMP 1 processed precursor TNF alpha between Ala74 and Gln75, in addition to between Ala76 and Val77, while MMP 9 cleaved this natural substrate solely between Ala74 and Gln75. Additionally, the MMP substrate Dnp-PChaGC(Me)HK(NMA)-NH(2) was not cleaved at all by TACE, while collagenase (MMP 1), gelatinase (MMP 9), stromelysin 1 (MMP 3), and matrilysin (MMP 7) all processed this substrate efficiently. All of these results indicate that TACE is unique in terms of its specificity requirements for substrate cleavage.

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Obesity is associated with multiple comorbidities such as cardiovascular disease, type 2 diabetes, and cancer ultimately resulting in increased mortality. Because of this, many individuals attempt to lose weight, but weight loss is often not maintained leading to the practice of weight cycling. Since human studies evaluating the effects of weight cycling on mortality are in disagreement with one another, and no animal studies had been performed on this topic, our laboratory conducted the first animal study to determine if stable obesity or weight cycling is more detrimental to lifespan. Our results showed that weight cycled mice significantly outlived their obese counterparts suggesting that weight cycling is healthier than remaining obese. However, weight cycling is a complex behavior with an infinite number of parameters. Thus, in the current study, we sought to expand our weight cycling model by investigating the effect of cycle duration on lifespan. Specifically, we evaluated the effect of doubling our previous four week cycle to eight weeks and measured the effect of this change on lifespan. One hundred male C57BL/6J mice were placed on one of four lifelong diets: a high fat diet (HF; n=30), a low fat diet (LF; n=30), a cycled diet alternating between 4 weeks on a low fat diet followed by 4 weeks on a high fat diet (4wk; n=20) or a cycled diet alternating between 8 weeks on a low fat diet and 8 weeks on a high fat diet (8wk; n=20). Caloric consumption, body weight, body composition, blood glucose, and lifespan were assessed. As expected caloric consumption, body weight, fat mass, and blood glucose levels were significantly higher in HF fed mice than LF controls and these values fluctuated significantly between cycles in weight cycled mice (increasing while on the HF diet and decreasing while on the LF diet). The mean lifespans were the following for each group: LF: 896.7 days, HF: 638.7 days, 4wk: 724.9 days, and 8wk: 749.7 days. Log-rank (Mantel-cox) analyses reveal that HF fed mice were significantly shorter lived than the other three groups while LF fed mice were significantly longer lived than the other three groups. Both weight cycled groups were shorter lived than the LF fed group and longer lived than the HF fed group. Finally, lifespan did not differ between 4wk and 8wk weight cycled groups. In conclusion, these findings demonstrate that altering cycle duration in weight cycled mice from 4 to 8 weeks does not alter lifespan. Importantly, both 4wk and 8wk weight cycled groups outlived stable obese controls suggesting that lifelong weight cycling is not more deleterious to health than remaining obese.

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