硒蛋白 TrxR1 介导甲萘醌还原:催化特性和抑制作用

Thioredoxin reductase (TrxR) is a class of important antioxidant selenoproteins and is involved in regulating the occurrence and development of tumors. Studies have shown that naphthoquinone molecules can target and inhibit the activity of TrxR1 and generate reactive oxygen species mediated by TrxR1, leading to an imbalance in the cellular redox state, making it a potential chemotherapeutic drug for tumors. This article aims to explore the interaction between the selenoprotein TrxR1 and the naphthoquinone compound menadione through biochemical and mass spectrometry analyses, and further reveal the mechanism of TrxR1 catalyzing the reduction of naphthoquinone molecules and the mechanism of naphthoquinone molecules inhibiting the activity of TrxR1. Through site-directed mutagenesis of the catalytic residues of TrxR1 and the recombinant expression of mutants, we determined the steady-state kinetic parameters of menadione reduction mediated by TrxR1 mutants and analyzed the inhibition of TrxR1 activity by menadione. Finally, the interaction between menadione and TrxR1 was identified by mass spectrometry analysis. The results showed that Sec498 of the selenoprotein TrxR1 catalyzes the reduction of menadione, but the U498C mutation makes the reduction of menadione more efficient, indicating that the reduction of menadione is mainly non-selenium-dependent. Mutation experiments found that Cys498 at the C-terminus plays a major catalytic reduction role, while Cys64 at the N-terminus has a slightly stronger influence on the reduction of menadione than Cys59. LC - MS results showed that there is 1 molecule of menadione adduct in TrxR1, and it is speculated that it irreversibly modifies the highly reactive selenocysteine at the C-terminus of the selenoprotein. This study reveals that TrxR1 can catalyze the reduction of menadione in a non-selenium-dependent manner, and its activity is irreversibly inhibited by menadione, providing a useful reference for the research and development of naphthoquinone-based anticancer drugs targeting TrxR1.

硫氧还蛋白还原酶 (Thioredoxin reductase，TrxR) 是一类重要的抗氧化硒蛋白，参与调控肿瘤发生发展。研究表明，萘醌类分子可以靶向抑制TrxR1 活性并经由TrxR1 介导产生活性氧，导致细胞氧化还原失衡，使其成为潜在的肿瘤化疗药物。本文旨在通过生物化学及质谱分析，探究硒蛋白TrxR1 与萘醌化合物甲萘醌的相互作用，进一步揭示TrxR1 催化萘醌分子还原的机理和萘醌分子抑制TrxR1 活性的机制。通过对TrxR1 催化残基的定点突变和突变体的重组表达，我们测定TrxR1 突变体介导甲萘醌还原稳态动力学参数，并分析甲萘醌对TrxR1 活性抑制，最后通过质谱分析鉴定甲萘醌与TrxR1 相互作用。结果表明，硒蛋白TrxR1 的Sec498 催化甲萘醌还原，但是U498C 突变使甲萘醌还原更加高效，表明了甲萘醌还原主要呈现非硒依赖性。突变实验发现C 端Cys498发挥主要催化还原作用，而N 端Cys64 对甲萘醌还原的影响稍强于Cys59。LC-MS 结果发现TrxR1 存在1 分子甲萘醌加合物，推测其不可逆修饰硒蛋白C 末端高反应活性的硒代半胱氨酸。本研究揭示了TrxR1 可以非硒依赖方式催化甲萘醌还原，同时其活性会受到甲萘醌的不可逆抑制，为靶向TrxR1 的萘醌类抗癌药物研发提供有益参考。