Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity

Antioxid Redox Signal. 2018 Sep 10;29(8):717-734. doi: 10.1089/ars.2017.7297. Epub 2018 Feb 27.

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.

Keywords: ADAM17; mass spectrometry; redox signaling; thioredoxin-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM17 Protein / metabolism*
  • Binding Sites
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Oxidation-Reduction
  • Thioredoxins / analysis
  • Thioredoxins / genetics
  • Thioredoxins / metabolism*
  • Tumor Cells, Cultured

Substances

  • TXN protein, human
  • Thioredoxins
  • ADAM17 Protein
  • ADAM17 protein, human