ADAM17 cytoplasmic domain modulates Thioredoxin-1
E Costa RAP, Granato DC, Trino LD, Yokoo S, Carnielli CM, Kawahara R, Domingues RR, Pauletti BA, Neves LX, Santana AG, Paulo JA, Aragão AZB, Heleno Batista FA, Migliorini Figueira AC, Laurindo FRM, Fernandes D, Hansen HP, Squina F, Gygi SP, Paes Leme AF. ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity. Redox Biol. 2020 Oct;37:101735. doi: 10.1016/j.redox.2020.101735. Epub 2020 Sep 24. PMID: 33011677; PMCID: PMC7513893.
- Organism: Homo sapiens
- Instrument: Xevo TQ-XS
Trx-1 ADAM17 disulfide bond shuffle
Lab head: Adriana Paes Leme
Submitter: Adriana Paes Leme
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.
Created on 6/25/20, 6:13 AM