Journal
FREE RADICAL BIOLOGY AND MEDICINE
Volume 172, Issue -, Pages 482-489Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.freeradbiomed.2021.06.023
Keywords
Thioredoxin reductase; Selenoprotein; Menadione; Superoxide anion radical; NADPH oxidase
Funding
- National Natural Science Foundation of China [31670767]
- Fundamental Research Funds for the Central Universities [DUT17JC36, DUT20LK36, DUT21LK29]
- Research and Development Program of Panjin Institute of Industrial Technology of DUT [PJYJY-002-862011]
- Liaoning Key Laboratory of Chemical Additive Synthesis and Separation [ZJKF2004]
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This study reveals that TrxR1 catalyzes the reduction of menadione in a Sec-independent manner, depending highly on Cys(498) instead. Mutation results showed that the U498C variant supported high-efficiency menadione reduction. The study also reveals that the Sec(498) of TrxR1 is the primary target of menadione.
The selenoprotein thioredoxin reductase 1 (TrxR1; TXNRD1) participates in multiple cellular processes and is regarded as a cellular target in anti-tumor drug discovery and development. TrxR1 has been reported to reduce menadione to menadiol and to produce superoxide anion radicals. However, the details of TrxR1-mediated menadione reduction have rarely been studied. In this study, we found that wild-type TrxR1 could reduce menadione in a less efficient way, but the U498C mutant variant supported high-efficiency menadione reduction in a Sec-independent manner. Meanwhile, the site-directed mutagenesis results showed that Cys(64) mutant increased the K-m values and decreased the catalytic efficiency, which was associated with a charge-transfer complex between FAD-Cys(64). Mass spectrometry (MS) revealed that in NADPH pre-reduced TrxR1 but not oxidized TrxR1, the highly active Sec(498) of wild-type TrxR1 was arylated by menadione and strongly impaired the DTNB reducing activity in a dose-dependent manner. TrxR1 reduced menadione more efficiently than glutathione reductase (GR), and interestingly menadione did not inhibit the GSSG reducing activity of GR. In summary, our results demonstrate that TrxR1 catalyzes the reduction of menadione in a Sec-independent manner, which highly depend on Cys(498) instead of N-terminal redox motif, and the Sec(498) of TrxR1 is the primary target of menadione. The interaction between menadione and TrxR1 revealed in this study may provide a valuable reference for the development of anticancer drugs targeting selenoprotein TrxR1.
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