Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain

Tumor necrosis factor-alpha-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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-07, Vol.279 (30), p.31638-31645
Main Authors: Gonzales, Patricia E, Solomon, Ariel, Miller, Ann B, Leesnitzer, M Anthony, Sagi, Irit, Milla, Marcos E
Format: Article
Language:English
Subjects:
ADAM Proteins
ADAM17 Protein
Amino Acid Sequence
Animals
Catalytic Domain - genetics
Cysteine - chemistry
In Vitro Techniques
Metalloendopeptidases - antagonists & inhibitors
Metalloendopeptidases - chemistry
Metalloendopeptidases - genetics
Metalloendopeptidases - metabolism
Mutagenesis, Site-Directed
Protein Folding
Protein Structure, Tertiary
Recombinant Proteins - antagonists & inhibitors
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Tumor Necrosis Factor-alpha - metabolism
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Summary:Tumor necrosis factor-alpha-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 microm). 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M401311200