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

• Published in: Antioxidants & redox signaling Vol. 29; no. 8; p. 717
• Main Authors: Granato, Daniela C, E Costa, Rute A P, Kawahara, Rebeca, Yokoo, Sami, Aragão, Annelize Z, Domingues, Romênia R, Pauletti, Bianca A, Honorato, Rodrigo V, Fattori, Juliana, Figueira, Ana Carolina M, Oliveira, Paulo S L, Consonni, Silvio R, Fernandes, Denise, Laurindo, Francisco, Hansen, Hinrich P, Paes Leme, Adriana F
• Format: Journal Article
• Language: English
• Published: United States 10.09.2018
• Subjects: mass spectrometry; ADAM17; redox signaling; thioredoxin-1
• ISSN: 1557-7716
• DOI: 10.1089/ars.2017.7297

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. 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-1 and catalytic site mutant Trx-1 rescued ADAM17 activity, although the interaction with Trx-1 was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1 mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1 mutant showed similar oxidant levels to Trx-1 , 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. 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. This unexpected Trx-1 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.