He Zongmao, Yan Yi, Guo Xijun, Wang Tong, Liu Xinqiao, Ding Ren-Bo, Fu Yuanfeng, Bao Jiaolin, Qi Xingzhu
Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, China.
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
Antioxidants (Basel). 2025 Feb 24;14(3):257. doi: 10.3390/antiox14030257.
Thioredoxin-1 (Trx-1) is an important redox protein found in almost all prokaryotic and eukaryotic cells, which has a highly conserved active site sequence: Trp-Cys-Gly-Pro-Cys. To investigate whether the Trp31 residue is essential for the antioxidant activity of human Trx-1 (hTrx-1), we mutated Trx-1 by replacing Trp31 with Ala31 (31Ala) or deleting Trp31 residue (31Del). We introduced 31Ala and 31Del mutations into prokaryotic cells for hTrx-1 protein expression, protein purification and evaluation of antioxidant activity. The results showed that neither the replacing mutation to Ala31 nor the deletion of Trp31 residue affected the efficient expression of hTrx-1 protein in prokaryotic cells, indicating that neither form of Trp31 mutation would disrupt the folded structure of the Trx-1 protein. Comparison of the antioxidant activity of purified hTrx-1 proteins of wild-type, 31Ala and 31Del forms revealed that both mutant forms significantly decreased the antioxidant capacity of hTrx-1. Further investigations on eukaryotic cells showed that HO treatment caused massive cell death in EA.Hy926 human endothelial cells with 31Ala and 31Del mutations compared to wild-type cells, which was associated with increased ROS production and downregulation of antioxidant Nrf2 and HO-1 expression in the mutant cells. These results suggested that mutations in the Trp31 residue of hTrx-1 remarkably disrupted cellular redox defense against oxidative stress. The antioxidant activity of hTrx-1 relies on the thiol-disulfide exchange reaction, in which the content of thiol groups forming disulfide bonds in hTrx-1 is critical. We found that the content of free thiol groups specifically participating in disulfide bond formation was significantly lower in Trp31 mutant hTrx-1 than in wild-type hTrx-1; that was speculated to affect the formation of disulfide bonds between Cys32 and Cys35 by virtual analysis, thus abolishing the antioxidant activity of hTrx-1 in cleaving oxidized groups and defending against oxidative stress. The present study provided valuable insights towards understanding the importance of Trp31 residue of hTrx-1 in maintaining the correct conformation of the Trx fold structure, the antioxidant functionality of hTrx-1 and the cellular redox defense capability against oxidative stress.
硫氧还蛋白-1（Trx-1）是一种在几乎所有原核细胞和真核细胞中都能找到的重要氧化还原蛋白，其具有高度保守的活性位点序列：Trp-Cys-Gly-Pro-Cys。为了研究Trp31残基对于人硫氧还蛋白-1（hTrx-1）的抗氧化活性是否至关重要，我们通过将Trp31替换为Ala31（31Ala）或删除Trp31残基（31Del）对Trx-1进行了突变。我们将31Ala和31Del突变引入原核细胞以进行hTrx-1蛋白表达、蛋白纯化及抗氧化活性评估。结果表明，将Trp31替换为Ala31的突变以及Trp31残基的缺失均未影响hTrx-1蛋白在原核细胞中的高效表达，这表明Trp31的两种突变形式均不会破坏Trx-1蛋白的折叠结构。对野生型、31Ala和31Del形式的纯化hTrx-1蛋白的抗氧化活性进行比较发现，两种突变形式均显著降低了hTrx-1的抗氧化能力。对真核细胞的进一步研究表明，与野生型细胞相比，HO处理导致具有31Ala和31Del突变的EA.Hy926人内皮细胞大量死亡，这与突变细胞中ROS生成增加以及抗氧化剂Nrf2和HO-1表达下调有关。这些结果表明，hTrx-1的Trp31残基突变显著破坏了细胞对氧化应激的氧化还原防御。hTrx-1的抗氧化活性依赖于硫醇-二硫键交换反应，其中hTrx-1中形成二硫键的硫醇基团含量至关重要。我们发现，特异性参与二硫键形成的游离硫醇基团含量在Trp31突变型hTrx-1中显著低于野生型hTrx-1；通过虚拟分析推测这会影响Cys32和Cys35之间二硫键的形成，从而消除hTrx-1在裂解氧化基团和抵御氧化应激方面的抗氧化活性。本研究为理解hTrx-1的Trp31残基在维持Trx折叠结构的正确构象、hTrx-1的抗氧化功能以及细胞对氧化应激的氧化还原防御能力方面的重要性提供了有价值的见解。
