Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress
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...
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2025-02-01
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| author | Zongmao He Yi Yan Xijun Guo Tong Wang Xinqiao Liu Ren-Bo Ding Yuanfeng Fu Jiaolin Bao Xingzhu Qi |
| author_facet | Zongmao He Yi Yan Xijun Guo Tong Wang Xinqiao Liu Ren-Bo Ding Yuanfeng Fu Jiaolin Bao Xingzhu Qi |
| author_sort | Zongmao He |
| collection | DOAJ |
| description | 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 H<sub>2</sub>O<sub>2</sub> 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. |
| format | Article |
| id | doaj-art-8c70d91cbafb4e94bba01a5d4472ae45 |
| institution | DOAJ |
| issn | 2076-3921 |
| language | English |
| publishDate | 2025-02-01 |
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| spelling | doaj-art-8c70d91cbafb4e94bba01a5d4472ae452025-08-20T02:42:38ZengMDPI AGAntioxidants2076-39212025-02-0114325710.3390/antiox14030257Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative StressZongmao He0Yi Yan1Xijun Guo2Tong Wang3Xinqiao Liu4Ren-Bo Ding5Yuanfeng Fu6Jiaolin Bao7Xingzhu Qi8Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaKey Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Collaborative Innovation Center of One Health, Hainan University, Haikou 570228, ChinaThioredoxin-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 H<sub>2</sub>O<sub>2</sub> 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.https://www.mdpi.com/2076-3921/14/3/257thioredoxin-1Trp31 residueantioxidant activityredox defensedisulfide bond |
| spellingShingle | Zongmao He Yi Yan Xijun Guo Tong Wang Xinqiao Liu Ren-Bo Ding Yuanfeng Fu Jiaolin Bao Xingzhu Qi Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress Antioxidants thioredoxin-1 Trp31 residue antioxidant activity redox defense disulfide bond |
| title | Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress |
| title_full | Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress |
| title_fullStr | Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress |
| title_full_unstemmed | Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress |
| title_short | Trp31 Residue of Trx-1 Is Essential for Maintaining Antioxidant Activity and Cellular Redox Defense Against Oxidative Stress |
| title_sort | trp31 residue of trx 1 is essential for maintaining antioxidant activity and cellular redox defense against oxidative stress |
| topic | thioredoxin-1 Trp31 residue antioxidant activity redox defense disulfide bond |
| url | https://www.mdpi.com/2076-3921/14/3/257 |
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