Thioredoxin-1 negatively modulates ADAM17 activity through direct binding and indirect reductive activity

Daniela C. Granato, Rute A. P. e Costa, Rebeca Kawahara, Sami Yokoo, Annelize Z. Aragão, Romênia R. Domingues, Bianca A. Pauletti, Rodrigo V. Honorato, Juliana Fattori, Ana Carolina M. Figueira, Paulo S. L. Oliveira, Silvio R. Consonni, Denise Fernandes, Francisco Laurindo, Hinrich P. Hansen, Adriana F. Paes Leme

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Aims: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17.

Results: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity.

Innovation: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases.

Conclusion: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717–734.
Original languageEnglish
Pages (from-to)717-734
Number of pages18
JournalAntioxidants and Redox Signaling
Volume29
Issue number8
Early online date27 Feb 2018
DOIs
Publication statusPublished - 10 Sep 2018
Externally publishedYes

Keywords

  • ADAM17
  • thioredoxin-1
  • mass spectrometry
  • redox signaling

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