硒蛋白TrxR1介导甲萘醌还原：催化特性和抑制作用

Selenoprotein thioredoxin reductase mediated menadione reduction: catalytic properties & inhibition effects

摘要:

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。

Abstract:

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec498 is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys498 is mainly responsible for menadione catalysis in comparison to Cys497, while the N-terminal Cys64 is slightly stronger than Cys59 regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.