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Redox Biology Apr 2024Thioredoxin reductase (TXNRD) is a selenoprotein that plays a crucial role in cellular antioxidant defense. Previously, a distinctive guiding bar motif was identified in...
Guiding bar motif of thioredoxin reductase 1 modulates enzymatic activity and inhibitor binding by communicating with the co-factor FAD and regulating the flexible C-terminal redox motif.
Thioredoxin reductase (TXNRD) is a selenoprotein that plays a crucial role in cellular antioxidant defense. Previously, a distinctive guiding bar motif was identified in TXNRD1, which influences the transfer of electrons. In this study, utilizing single amino acid substitution and Excitation-Emission Matrix (EEM) fluorescence spectrum analysis, we discovered that the guiding bar communicates with the FAD and modulates the electron flow of the enzyme. Differential Scanning Fluorimetry (DSF) analysis demonstrated that the aromatic amino acid in guiding bar is a stabilizer for TXNRD1. Kinetic analysis revealed that the guiding bar is vital for the disulfide reductase activity but hinders the selenocysteine-independent reduction activity of TXNRD1. Meanwhile, the guiding bar shields the selenocysteine residue of TXNRD1 from the attack of electrophilic reagents. We also found that the inhibition of TXNRD1 by caveolin-1 scaffolding domain (CSD) peptides and compound LCS3 did not bind to the guiding bar motif. In summary, the obtained results highlight new aspects of the guiding bar that restrict the flexibility of the C-terminal redox motif and govern the transition from antioxidant to pro-oxidant.
Topics: Antioxidants; Kinetics; Oxidation-Reduction; Selenocysteine; Thioredoxin Reductase 1; Thioredoxin-Disulfide Reductase; Humans
PubMed: 38277963
DOI: 10.1016/j.redox.2024.103050 -
Frontiers in Molecular Biosciences 2024Mammalian cytosolic thioredoxin reductase (TrxR1) serves as an antioxidant protein by transferring electrons from NADPH to various substrates. The action of TrxR1 is...
The C-terminal selenenylsulfide of extracellular/non-reduced thioredoxin reductase endows this protein with selectivity to small-molecule electrophilic reagents under oxidative conditions.
Mammalian cytosolic thioredoxin reductase (TrxR1) serves as an antioxidant protein by transferring electrons from NADPH to various substrates. The action of TrxR1 is achieved via reversible changes between NADPH-reduced and non-reduced forms, which involves C-terminal selenolthiol/selenenylsulfide exchanges. TrxR1 may be released into extracellular environment, where TrxR1 is present mainly in the non-reduced form with active-site disulfide and selenenylsulfide bonds. The relationships between extracellular TrxR1 and tumor metastasis or cellular signaling have been discovered, but there are few reports on small-molecule compounds in targeted the non-reduced form of TrxR1. Using eight types of small-molecule thiol-reactive reagents as electrophilic models, we report that the selenenylsulfide bond in the non-reduced form of TrxR1 functions as a selector for the thiol-reactive reagents at pH 7.5. The non-reduced form of TrxR1 is resistant to hydrogen peroxide/oxidized glutathione, but is sensitive to certain electrophilic reagents in different ways. With 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and S-nitrosoglutathione (GSNO), the polarized selenenylsulfide bond breaks, and selenolate anion donates electron to the dynamic covalent bond in DTNB or GSNO, forming TNB-S-Se-TrxR1 complex or ON-Se-TrxR1 complex. The both complexes lose the ability to transfer electrons from NADPH to substrate. For diamide, the non-reduced TrxR1 actually prevents irreversible damage by this oxidant. This is consistent with the regained activity of TrxR1 through removal of diamide dialysis. Diamide shows effective in the presence of human cytosolic thioredoxin (hTrx1), Cys residue(s) of which is/are preferentially affected by diamide to yield disulfide, hTrx1 dimer and the mixed disulfide between TrxR1-Cys497/Sec498 and hTrx1-Cys73. In human serum samples, the non-reduced form of TrxR1 exists as dithiothreitol-reducible polymer/complexes, which might protect the non-reduced TrxR1 from inactivation by certain electrophilic reagents under oxidative conditions, because cleavage of these disulfides can lead to regain the activity of TrxR1. The details of the selective response of the selenenylsulfide bond to electrophilic reagents may provide new information for designing novel small-molecule inhibitors (drugs) in targeted extracellular/non-reduced TrxR1.
PubMed: 38523661
DOI: 10.3389/fmolb.2024.1274850 -
International Journal of Molecular... Feb 2024The targeting of human thioredoxin reductase is widely recognized to be crucially involved in the anticancer properties of several metallodrugs, including Au(I)...
The targeting of human thioredoxin reductase is widely recognized to be crucially involved in the anticancer properties of several metallodrugs, including Au(I) complexes. In this study, the mechanism of reaction between a set of five N-heterocyclic carbene Au(I) complexes and models of the active Sec residue in human thioredoxin reductase was investigated by means of density functional theory approaches. The study was specifically addressed to the kinetics and thermodynamics of the tiled process by aiming at elucidating and explaining the differential inhibitory potency in this set of analogous Au(I) bis-carbene complexes. While the calculated free energy profile showed a substantially similar reactivity, we found that the binding of these Au(I) bis-carbene at the active CysSec dyad in the TrxR enzyme could be subjected to steric and orientational restraints, underlining both the approach of the bis-carbene scaffold and the attack of the selenol group at the metal center. A new and detailed mechanistic insight to the anticancer activity of these Au(I) organometallic complexes was thus provided by consolidating the TrxR targeting paradigm.
Topics: Humans; Selenocysteine; Thioredoxin-Disulfide Reductase; Antineoplastic Agents; Gold; Coordination Complexes; Methane
PubMed: 38473872
DOI: 10.3390/ijms25052625