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  Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme

Lee, M. H., Verma, V., Maskos, K., Nath, D., Knäuper, V., Dodds, P., et al. (2002). Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme. Biochemical Journal, 364, 227-234.

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Genre: Zeitschriftenartikel
Alternativer Titel : Biochem. J.

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 Urheber:
Lee, M. H., Autor
Verma, V., Autor
Maskos, K.1, Autor           
Nath, D., Autor
Knäuper, V., Autor
Dodds, P., Autor
Amour, A., Autor
Murphy, G., Autor
Affiliations:
1Fässler, Reinhard / Molecular Medicine, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565147              

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Schlagwörter: binding affinity; gelatinase-A; N-terminal domain of TIMP-3; TACE
 Zusammenfassung: We previously reported that full-length tissue inhibitor of metallo-proteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (K- i(app)) and association rates (k(on)) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60 pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-alpha demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-alpha. Furthermore, the Ser-4Met mutant was also significantly more active (P < 0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF- alpha shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.

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Sprache(n): eng - English
 Datum: 2002-05-15
 Publikationsstatus: Erschienen
 Seiten: -
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: eDoc: 41116
ISI: 000175744200027
 Art des Abschluß: -

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Titel: Biochemical Journal
  Alternativer Titel : Biochem. J.
Genre der Quelle: Zeitschrift
 Urheber:
Affiliations:
Ort, Verlag, Ausgabe: -
Seiten: - Band / Heft: 364 Artikelnummer: - Start- / Endseite: 227 - 234 Identifikator: ISSN: 0264-6021