Cytosolic thioredoxin reductase (TXNRD1) is a key selenoenzyme involved in cellular redox regulation and antioxidant defense. Elucidating the catalytic mechanism of TXNRD1 and its conserved residues or domains is crucial for drug discovery and development. In this study, we investigated the functional roles of several conserved residues in TXNRD1, including Trp 114 , Tyr 116 , and residues in the guiding bar motif, and the catalytic C-terminal domain. Using recombinant TXNRD1 mutants, we found that Trp 114 mutants partially retain TXNRD1 activity in reducing TXN1 or TXNL1 but completely abolish the TRP14 reducing activity. Notably, the function of Trp 114 in TXNRD1 catalysis is independent of the Sec 498 residue and unrelated with the oligomerization status of TXNRD1. AlphaFold structural modeling suggested that Trp 114 may form hydrogen bonds with the TXN-like protein, potentially facilitating the approach of the C-terminal tail of TXNRD1 to the buried disulfide bond (Cys 43 -Cys 46 ) in TRP14. In contrast, the disulfide bonds of TXN1 and TXNL1 are more accessible, highlighting a unique structural requirement of Trp 114 in TRP14 reduction. Furthermore, we demonstrated that both the presence and its precise positioning of Sec 498 in the C-terminal tail are critical for catalytic activity. Additionally, Asp 82 and Arg 416 were found to play important roles in maintaining TXNRD1 thermostability. Together, our findings underscore the unique role of Trp 114 in modulating TXNRD1’s catalytic activity and provide new insights into the regulation of TXNRD1 activity and stability, which deepens our understanding of selenoprotein function and the mechanistic basis of redox regulation.
Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx , was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and β-ME. Furthermore, the FDA-approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin-1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress.
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