title: Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch.
abstract: TNF-alpha-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18-54 (N-TACE(18-54)) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE(18-54) attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE(18-54) inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30-48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.
authors.affiliation: Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Inst., National Institutes of Health, Bldg. 10, Rm. 6D03, MSC 1590, Bethesda, MD 20892-1590, USA.
authors.forename: Caitriona A
authors.initials: CA
authors.lastname: Buckley
authors.forename: Farshid N
authors.initials: FN
authors.lastname: Rouhani
authors.forename: Maryann
authors.initials: M
authors.lastname: Kaler
authors.forename: Barbara
authors.initials: B
authors.lastname: Adamik
authors.forename: Feras I
authors.initials: FI
authors.lastname: Hawari
authors.forename: Stewart J
authors.initials: SJ
authors.lastname: Levine
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000494:pharmacology*; D009000:Monocytes / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D047889:Receptors, Tumor Necrosis Factor, Type II; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D015032:Zinc; D003545:Cysteine