Langlands Hannah D, Shoemark Deborah K, Toye Ashley M
School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK.
Antioxidants (Basel). 2024 Sep 2;13(9):1070. doi: 10.3390/antiox13091070.
The regulation of reactive oxygen species (ROS) in red blood cells (RBCs) is crucial for maintaining functionality and lifespan. Indeed, dysregulated ROS occurs in haematological diseases such as sickle cell disease and β-thalassaemia. In order to combat this, RBCs possess high levels of protective antioxidant enzymes. We aimed to further boost RBC antioxidant capacity by overexpressing peroxiredoxin (Prxs) and glutathione peroxidase (GPxs) enzymes. Multiple antioxidant enzyme cDNAs were individually overexpressed in expanding immortalised erythroblasts using lentivirus, including Prx isoforms 1, 2, and 6 and GPx isoforms 1 and 4. Enhancing Prx protein expression proved straightforward, but GPx overexpression required modifications. For GPx4, these modifications included adding a SECIS element in the 3'UTR, the removal of a mitochondrial-targeting sequence, and removing putative ubiquitination sites. Culture-derived reticulocytes exhibiting enhanced levels of Prx and GPx antioxidant proteins were successfully engineered, demonstrating a novel approach to improve RBC resilience to oxidative stress. Further work is needed to explore the activity of these proteins and their impact on RBC metabolism, but this strategy shows promise for improving RBC function in physiological and pathological contexts and during storage for transfusion. Enhancing the antioxidant capacity of reticulocytes has exciting promise for developing culture-derived RBCs with enhanced resistance to oxidative damage and offers new therapeutic interventions in diseases with elevated oxidative stress.
红细胞（RBC）中活性氧（ROS）的调节对于维持其功能和寿命至关重要。事实上，在镰状细胞病和β地中海贫血等血液系统疾病中会出现ROS调节异常。为了应对这一问题，红细胞拥有高水平的保护性抗氧化酶。我们旨在通过过表达过氧化物还原酶（Prxs）和谷胱甘肽过氧化物酶（GPxs）来进一步提高红细胞的抗氧化能力。使用慢病毒在永生化红细胞祖细胞中分别过表达多种抗氧化酶的cDNA，包括Prx亚型1、2和6以及GPx亚型1和4。提高Prx蛋白表达相对简单，但GPx的过表达需要进行修饰。对于GPx4，这些修饰包括在3'UTR中添加一个硒代半胱氨酸插入序列（SECIS）元件、去除线粒体靶向序列以及去除假定的泛素化位点。成功构建了培养来源的网织红细胞，其Prx和GPx抗氧化蛋白水平升高，这展示了一种提高红细胞对氧化应激恢复力的新方法。需要进一步研究来探索这些蛋白质的活性及其对红细胞代谢的影响，但这种策略有望在生理和病理情况下以及输血储存期间改善红细胞功能。提高网织红细胞的抗氧化能力对于开发具有更强抗氧化损伤能力的培养来源红细胞具有令人兴奋的前景，并为氧化应激升高的疾病提供了新的治疗干预措施。
