A comprehensive approach to identify redox and non-redox targets of Trx-like proteins

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by Lia S. Nakao

Like the old dictum that says “birds of a feather flock together”, understanding the specific partners of a given protein provides an important clue about its function. Thioredoxin 1 (Trx1) is a well-known redox protein that contains a CXXC motif (cysteines residues flanking two aminoacid residues), responsible for its disulfide reductase function. The first (C-terminal) Cys of the motif attacks the disulfide of the target protein, producing a short lived mixed disulfide, which is reduced by the second (N-terminal resolving) Cys, releasing Trx1 and the target, in the oxidized and reduced forms, respectively. If the resolving Cys is replaced by a non-redox residue, such as alanine or serine, the target will be trapped in the mixed disulfide and can be identified [1]. This is the basis of the mechanism-based, trapping mutant method, which has identified several Trx1 targets. A recent work [2] adapted this strategy to identify novel partners of Trx1 and of other CXXC-containing proteins, namely Rdx12, nucleoredoxin 1 (Nrx1) and thioredoxin-like protein 1 (Txnl1) in cell culture model (HEK293T), by immunoaffinity purification and liquid chromatography/mass spectrometry. Some important steps were included to improve the method: (i) cellular overexpression of Trx1 and Trx-like proteins allowed the trapping of the targets in situ, increasing the possibility of trapping physiological partners; (ii) the endogenous expression of the studied proteins was knocked down to minimize the resolving of the mixed disulfide; (iii) the overexpressed proteins carried both a hemagglutinin (HA) tag to facilitate the immunopurification using anti-HA coupled to beads, and a tobacco etch virus (TEV) protease cleavage site to allow the elution of the immunopurified complex in a very mild condition; (iv) the comparison of the MS data of each candidate across the 3 constructs (the CXXC, CXXS and the SXXC, where S represents the serine amino acid), as well as of the control lacking expression construct, allowed the calculation of target enrichment, supporting a detailed and quantitative analyses of the datasets. The method was validated by the confirmation of peroxiredoxins 1, 2, 4 and 5 as Trx1 targets. This strategy led to identification of apoptosis inducing factor 1 (AIF) and Mia40 (a protein involved with the oxidative import of mitochondrial proteins) as novel Trx1 redox targets, and glutathione peroxidase 4 as a novel Rdx12 redox target, as they were significantly enriched in their CXXS mutants. Nrx1 and Txnl1 CXXS mutants, however, showed low enrichment of targets, indicating that these proteins are not cellular reductases of disulfide or oxidized cysteine residues. Thus, this work presents a novel strategy to search for both redox and non-redox targets of CXXC-containing proteins, validates some novel and potentially interesting Trx1 and Rdx12 redox targets, and presents datasets of possible redox and non-redox targets of Trx1, Rdx12, Txnl1 and Nrx1.

  1. L. Verdoucq, F. Vignols, J.–P. Jacquot, Y. Chartier, Y. Meyer.
    In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family.
    The Journal of Biological Chemistry, 274: 19714-22, 1999. | dx.doi.org/10.1074/jbc.274.28.19714
  2. L. S. Nakao, R. A. Everley, S. M. Marino, S. M. Lo, Luiz E. de Souza, S. P. Gigy, V. N. Gladyshev.
    Mechanism-based proteomic screen identifies targets of thioredoxin-like proteins.
    The Journal of Biological Chemistry, 290: 5685-95, 2015. | dx.doi.org/10.1074/jbc.M114.597245

Contributed by: Lia S. Nakao, from the Federal University of Paraná, Curitiba.
Dr. Nakao is a former trainee at Redoxoma member’s labs (Augusto, Laurindo)
and has been a member of our INCT-Redoxoma over the last 10 years.

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