A Novel Substrate of Mammalian Thioredoxin Reductase 1

다국어 초록 (Multilingual Abstract)

Recent evidence suggests that a number of proteins are oxidized at their critical cysteine residues as the result of receptor-mediated H₂O₂ production. At the present time, the reduction of these oxidized proteins is thought to be achieved by general electron suppliers such as Trx, Grx and GSH. Evidence(e.g., Trx reduces oxidized Prx Ⅰ-Ⅴ enzymes but not oxidized Prx Ⅵ; Trx, but not Grx or GSH, reduces oxidized PTP1B), however, suggests that the reduction reaction might be governed by specific interactions between the donor and acceptor molecules. Signal transduction paradigms instruct us that both modification and demodification of proteins are controlled processes. As such, we searched new electron donor molecules and found several small proteins containing a CysXXCys(or CysXXSeCys) motif, which is conserved among the members of the thiol-disulfide oxidoreductase superfamily that includes Trx and Grx. One of these is a 14-kDa protein, which we named Trx-related protein 14(TRP14). Its overall similarity to Trx is low(＜20%). The homology search showed that TRP14 homologs exist in a wide range of organisms from bacteria to mammals. Immunoblot analysis indicates that TRP14 is a ubiquitous protein like Trx. TRP14 was expressed at much lower levels than Trx in every tissue examined except in kidney. The reduction potentials were similar for TRP14(-0.257V) and Trx(-0.274V). Studies with mutant proteins in which the two conserved cysteine residues(Cys43 and Cys46) were altered 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 could be reduced by TrxR1 but not by TrxR2, whereas Trx is an equally good substrate for TrxR1 and TrxR2. For the reduction reaction by TrxR1, TRP14 was a better substrate than Trx. Trx serves as a hydrogen donor for the catalytic function of ribonucleotide reductase, methionine sulfoxide reductase, and peroxiredoxins. TRP14 could not support any of these three reactions. This substrate specificity, although surprising in view of their similar reduction potentials, suggests that specific protein-protein interactions or the pKa values of the conserved cysteine residues, in addition to reduction potential, may govern the electron flows from Trx and Trx-like proteins to acceptor proteins. 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 targets of TRP14 in the reduction cascade remain to be identified.