Characterization of a novel substrate of mammalian thioredoxin reductase 1

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We identified a rat brain protein containing cysteine residues that are sensitive to the oxidation by H2O2. This 14-kDa protein, consisted of 123 amino acids, contains a Cys-X-X-Cys motif (Cys-Pro-Asp-Cys), which is conserved among the members of thiol-disulfide oxidoreductase superfamily that include thioredoxin (Trx). Because the newly discovered 14-KDa protein is similar to Trx in size, it was named thioredoxin-related protein (TRP14). The BLAST homology search showed that TRP14 homologues are distributed in wide range of organisms including bacteria, yeast, nematode, insect, plant and mammalian, and their Trp-Cys-Pro-Asp-Cys motifs are completely identical. Immunoblot analysis indicates that TRP14 is a ubiquitous protein like Trx. TRP14 was expressed in much lower level than Trx in every tissue except in kidney and its cell distribution was complimentary to that of Trx.The reduction potentials were similar for TRP14 (-0.257 V) and Trx (-0.274 V). Studies with the mutants of the two conserved cysteine residues (Cys43 and Cys46) and direct analysis of the Cys-containing peptides revealed that as in the case of Trx, the conserved cysteine residues form an intramolecular disulfide linkage upon oxidation by hydrogen peroxide. The resulting disulfide can be reduced by the cytosolic Trx reductase (TrxR1) but not by the mitochondrial Trx reductase (TrxR2), whereas Trx is an equally good substrate for TrxR1 and TrxR2. For the reduction reaction by TrxR1, TRP14 was slightly better substrate than Trx (the Kms for TRP14 and Trx were 8.3 uM and 10.8 uM, respectively) and their Vmaxs were nearly similar (the Vmaxs for TRP14 and Trx were 32.4 uM/min and 30.8 uM/min, respectively). Trx serves as a hydrogen donor for the catalytic function of ribonucleotide reductase, methionine sulfoxide reductase and peroxiredoxins, and as a general protein-disulfide reductase that reduce the disulfide bond of insulin and facilitates refolding of ribonuclease. The ability of TRP14 to support the five reactions in replacement of Trx was tested using purified recombinant TRP14. TRP14 could not support any of the five reactions. This specificity is surprising in view of their similar reduction potentials. As an effort to search for proteins that depend on TRP14 for the supply of reducing equivalents, GST-TRP14 fusion protein was immobilized to Sepharose gels. TrxR1 was found to bind specifically to the TRP14-bound Sepharose column. Our results suggest that TRP14 is a disulfide-oxidoreductase that is capable of receiving electrons from NADPH via TrxR1. The downstream target of TRP14 in the reduction cascade remains to be identified.