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. 2015 Feb 27;290(9):5685-95.
doi: 10.1074/jbc.M114.597245. Epub 2015 Jan 5.

Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins

Lia S Nakao  1 , Robert A Everley  2 , Stefano M Marino  3 , Sze M Lo  4 , Luiz E de Souza  4 , Steven P Gygi  2 , Vadim N Gladyshev  5
Affiliations

Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins

Lia S Nakao et al. J Biol Chem. .

Abstract

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.

Keywords: Mammal; Oxidation-reduction (Redox); Proteomics; Redox Regulation; Selenocysteine; Thiol; Thioredoxin; Thioredoxin Reductase.

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Figures

FIGURE 1.
FIGURE 1.
Mechanism-based proteomic screening. A, the basis of the trapping mutant approach (left panel). Replacement of the resolving cysteine of the CXXC motif of the Trx-like protein by a redox-inactive amino acid, such as serine or alanine, renders the mixed disulfide stable (right panel), amenable to identification. B, general flow chart of the experimental strategy to identify targets of Trx-like proteins. C, representative scheme of constructs employed for cell transfection. HA, hemagglutin tag.
FIGURE 2.
FIGURE 2.
Knockdown of the endogenous protein and overexpression of Trx-like proteins by transient transfection. HEK 293T cells were knocked down with either the specific or scrambled On-targetTM siRNA (day 1). The next day, the cell cultures were expanded (day 2), and 48 h after siRNA transfection, they were transfected with the construct for protein overexpression (day 3). Forty-eight h after DNA transfection (day 5), cells were harvested, lysed, and analyzed by immunoblotting with the indicated antibody. Cells incubated only with Dharmafect 1 and the calcium transfection reagents were used as controls. In the case of Rdx12, cells were not knocked down, because the endogenous Rdx12 was not detected. The overexpressed (arrow) and endogenous (*) proteins are indicated.
FIGURE 3.
FIGURE 3.
Immunoaffinity purification of Trx-like proteins with their targets. HEK 293T cells were transfected with siRNA targeting the untranslated regions of the Trx-like protein genes, followed by overexpression of the corresponding Trx-like proteins (with CXXC, CXXS, and SXXC constructs or nothing = mock). Cells were lysed and the HA-tagged proteins were immunopurified with agarose beads coupled to anti-HA. The complexes were eluted with TEV protease, and resolved in reducing SDS-PAGE. Bands were detected with silver staining. The overexpressed proteins (arrow) and TEV protease (*) are indicated.
FIGURE 4.
FIGURE 4.
Validation of the proteomic approach by immunoblotting of the immunoaffinity purified targets of Trx1 under basal and oxidative conditions. HEK 293T cells were transfected with siRNA targeting the untranslated regions of Trx1 gene, followed by transfection with Trx1 constructs (with CXXC, CXXS, and SXXC constructs or nothing = mock). Two days after transfection, cells were treated or not with 0.1 mm H2O2 in PBS for 3 min before lysis and immunoaffinity purification with agarose beads coupled to anti-HA. The complexes were eluted with TEV protease, and resolved in reducing SDS-PAGE. Resolved proteins were immobilized in PVDF membranes and probed with the indicated antibodies. A whole cell lysate was used as a positive control of the immunoblotting. IP, immunoprecipitation.
FIGURE 5.
FIGURE 5.
Immunoaffinity purification of Trx1 with their targets under basal and oxidative conditions. HEK 293T cells were transfected with siRNA targeting the untranslated regions of the Trx1 gene, followed by transfection with Trx1 constructs (with CXXC, CXXS, and SXXC constructs or nothing = mock). Two days after the transfection, cells were treated or not with 0.1 mm H2O2 in PBS for 3 min before lysis and immunoaffinity purification with agarose beads coupled to anti-HA. The complexes were eluted with TEV protease, and resolved in reducing SDS-PAGE. Bands were detected with silver staining. The overexpressed Trx1 (arrow) and TEV protease (*) are indicated.
FIGURE 6.
FIGURE 6.
Validation of the interaction between Trx1 and AIF or Mia40. A, immunoaffinity purified eluates from HEK 293T transfected with the Trx1 constructs or mock were resolved in reducing SDS-PAGE, blotted, and probed with anti-AIF. A whole cell lysate was used as a positive control of the immunoblotting. B, direct interaction between reduced Trx1 and AIF(Δ1–120). Reduced CXXC or CXXS Trx1 (10 μm) was incubated with 2.5 μm AIF(Δ1–120), AIF(Δ1–120) in the presence of 0.5 mm H2O2 or with pre-oxidized AIF(Δ1–120). Reactions were performed at 37 °C for 15 min. Samples were resolved in 4–12% SDS-PAGE under non-reducing conditions and probed with anti-Trx1 and anti-AIF. Migration of Trx1-AIF(Δ1–120)-mixed disulfide (arrow) and non-reacted Trx1 and AIF(Δ1–120) (*) is indicated. C, direct interaction between reduced Trx1 and Mia40. Reduced CXXC or CXXS Trx1 (10 μm) was incubated with 10 μm Mia40, or Mia40 preincubated with H2O2 or diamide (DA). Reactions were performed at 37 °C for 15 min. Samples were resolved by 10% SDS-PAGE under non-reducing conditions and probed with anti-Trx1 antibody. Migration of Trx1-Mia40-mixed disulfide (arrow) and non-reacted Trx1 (*) is indicated. IP, immunoprecipitation; IB, immunoblotting.
FIGURE 7.
FIGURE 7.
Validation of the interaction between Rdx12 and GPx4 and between Nrx1 and PDI. Immunoaffinity purified eluates from HEK 293T cells overexpressing CXXC, CXXS, and SXXC constructs of Rdx12 (A) and Nrx1 (B) were resolved by reducing SDS-PAGE, blotted, and probed with anti-GPx4 and anti-PDI, respectively. Mock represents eluates from control cells. A whole cell lysate was used as a positive control. In B, the SXXC lysate sample was developed with West Pico Supersignal, whereas the eluates were developed with Femto Pico Supersignal chemiluminescence kit. IP, immunoprecipitation.
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