Halvey Patrick J, Hansen Jason M, Johnson Jennifer M, Go Young-Mi, Samali Afshin, Jones Dean P
Division of Pulmonary, Allergy, Cystic Fibrosis and Sleep, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
Antioxid Redox Signal. 2007 Jul;9(7):807-16. doi: 10.1089/ars.2007.1526.
The effects of nuclear-localized oxidative stress on both nuclear antioxidant systems, and the processes that they regulate, are not clearly understood. Here, we targeted a hydrogen peroxide (H(2)O(2))-producing enzyme, D-amino acid oxidase (DAAO), to the nucleus (NLS-DAAO) and used this to generate H(2)O(2) in the nuclei of cells. On addition of N-acetyl-D-alanine (NADA), a substrate of DAAO, to NLS-DAAO-transfected HeLa cells, a twofold increase in ROS production relative to untreated, transfected control was observed. Staining of cellular thiols confirmed that NLS-DAAO-induced ROS selectively modified the nuclear thiol pool, whereas the cytoplasmic pool remained unchanged. Furthermore, NLS-DAAO/NADA-induced ROS caused significant oxidation of the nuclear GSH pool, as measured by nuclear protein S-glutathionylation (Pr-SSG), but under the same conditions, nuclear Trx1 redox state was not altered significantly. NF-kappaB reporter activity was diminished by NLS-DAAO/NADA-stimulated nuclear oxidation. We conclude that nuclear GSH is more susceptible to localized oxidation than is nuclear Trx1. Furthermore, the attenuation of NF-kappaB reporter activity in the absence of nuclear Trx1 oxidation suggests that critical nuclear redox proteins are subject to control by S-glutathionylation during oxidative stress in the nucleus.
核定位氧化应激对核抗氧化系统及其调控过程的影响尚不清楚。在此，我们将一种产生过氧化氢（H₂O₂）的酶——D-氨基酸氧化酶（DAAO）靶向至细胞核（NLS-DAAO），并利用其在细胞核中产生H₂O₂。向转染了NLS-DAAO的HeLa细胞中添加DAAO的底物N-乙酰-D-丙氨酸（NADA）后，相对于未处理的转染对照，观察到ROS生成增加了两倍。细胞硫醇染色证实，NLS-DAAO诱导的ROS选择性地修饰了核硫醇池，而细胞质硫醇池保持不变。此外，通过核蛋白S-谷胱甘肽化（Pr-SSG）测量发现，NLS-DAAO/NADA诱导的ROS导致核GSH池显著氧化，但在相同条件下，核Trx1的氧化还原状态没有明显改变。NLS-DAAO/NADA刺激的核氧化降低了NF-κB报告基因的活性。我们得出结论，核GSH比核Trx1更容易受到局部氧化的影响。此外，在没有核Trx1氧化的情况下NF-κB报告基因活性的减弱表明，在细胞核氧化应激期间，关键的核氧化还原蛋白受S-谷胱甘肽化的调控。
