Cole Eye Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH 44195, USA.
Int J Mol Sci. 2022 Sep 1;23(17):9938. doi: 10.3390/ijms23179938.
The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.
视网膜和 RPE 细胞经常受到慢性氧化应激的影响，因为它们是代谢需求高且 ROS 生成多的组织。DJ-1 是视网膜和 RPE 中的一种多功能蛋白，已被证明能够在多种细胞类型中强有力地保护细胞免受氧化应激。DJ-1 半胱氨酸（C）残基的氧化对其在氧化条件下的功能很重要。本研究旨在分析 DJ-1 表达变化及其 C 残基氧化对 RPE 功能的影响。使用复制缺陷型腺病毒感染 ARPE-19 细胞系和原代人胎儿 RPE（hfRPE）培养物的单层，以研究这些单层中 DJ-1 水平增加的影响。腺病毒携带全长人 DJ-1 cDNA（hDJ）和 DJ-1 的突变构建体，这些构建体分别将其三个 C 残基全部或逐个突变为丝氨酸（S）。或者，用 shPARK7 慢病毒转染和转导降低内源性 DJ-1 水平。然后在基线和低氧化应激条件下对这些单层进行测定。通过免疫荧光、Western blot、RT-PCR、线粒体膜电位和活力测定分析结果。我们确定内源性 DJ-1 水平降低会导致 ROS 水平升高。此外，我们观察到用所有 DJ-1 构建体转导的所有 RPE 单层中，线粒体结构发生形态变化。过表达所有 DJ-1 构建体的 ARPE-19 单层的线粒体膜电位显示出显著降低，而 hfRPE 单层仅在过表达 C2S 突变时其 ΔΨm 才显示出显著降低。转导 C53S 构建体的 ARPE-19 细胞的活力显著降低。我们的数据表明，C53 的氧化对于调节 RPE 细胞中内源性 ROS 和活力水平至关重要。
