We have previously shown that Thioredoxin-1 (Trx-1) binds to the cytoplasmic domain of ADAM17 (ADAM17cyto), A Disintegrin And Metalloprotease 17, and that the destabilization in their interface of interaction favors the Trx-1 dimeric and inactive state. These studies opened the question of whether ADAM17 plays a role in the modulation of Trx-1 conformation and activity. Here, we demonstrate that site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) also disrupts the interacting interface with Trx-1, resulting in a decrease of Trx-1 reductive capacity and activity. One of the mechanisms that explain this effect might be that ADAM17cyto favors Trx-1 monomerization state - the active state - by forming a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site. Both the free Trx-1 Cys73 peptide and the disulfide bond Trx-1 Cys73-ADAM17 Cys824 peptide were measured by SRM.

To evaluate the effect of ADAM17cytoWT and ADAM17cytoF730A on Trx-1 Cys73 reduced state, the Trx-1 peptide (73CMPTFQFFK81), which contains the cysteine residue responsible for Trx-1 homodimerization, was submitted to relative quantification using SRM as previously described in (Granato et al., 2018. Antioxid. Redox Signal. 29, 717–734), with modifications. After the proteins´ incubations (Trx-1+ ADAM17cytoWT and Trx-1+ ADAM17cytoF730A, under ADAM17cyto concentrations of 3, 6, 12 and 24µM) during 15 min at 25oC, all reactions were stopped by denaturing condition using 4M urea, in non-reducing conditions. The mixture of proteins was alkylated and submitted to trypsin digestion (1:50). The experiment was performed in three technical replicates.

Proteotypic peptides for Trx-1 and ADAM17cyto proteins were selected based on our DDA analysis and following the rules as previously described (29, 30). Briefly, a total of 3 proteotypic peptides were selected to be monitored label-free, including the disulfide linked peptide at position Cys73, and a total of 2 proteotypic peptides were selected to be monitored label-based, including the carbamidomethylated peptide at position Cys73 with and without methionine oxidation. At least five transitions were monitored for the light and heavy peptides, with a total of 87 transitions. A mixture of 2pmol of the heavy isotopic labeled peptide was added to the samples prior to desalinization performed using Sep-pak C18 cartridge (Waters, Milford, MA). Internal retention time standards (iRT, Pierce Peptide Retention Time Calibration Mixture, Thermo Fisher Scientific, Watlham, MA) were spiked in all samples at 50 fmol/µl, prior to sample injection and four peptides with 12 transitions were monitored. Also, two trypsin auto-lysis peptides were monitored to evaluate the trypsin digestion efficiency.