Early biosignature of oxidative stress in the retinal pigment epithelium

Abstract

The retinal pigment epithelium (RPE) is essential for retinoid recycling and phagocytosis of photoreceptors. Understanding of proteome changes that mediate oxidative stress-induced degeneration of RPE cells may provide further insight into the molecular mechanisms of retinal diseases. In the current study, comparative proteomics has been applied to investigate global changes of RPE proteins under oxidative stress. Proteomic techniques, including 2D SDS-PAGE, differential gel electrophoresis (DIGE), and tandem time-of-flight (TOF-TOF) mass spectrometry, were used to identify early protein markers of oxidative stress in the RPE. Two biological models of RPE cells revealed several differentially expressed proteins that are involved in key cellular processes such as energy metabolism, protein folding, redox homeostasis, cell differentiation, and retinoid metabolism. Our results provide a new perspective on early signaling molecules of redox imbalance in the RPE and putative therapeutic target proteins of RPE diseases caused by oxidative stress.

Figure 1. Comparative proteome analysis of bovine RPE cells and human RPE D407 cells under oxidative stress

(A) 2D-SDS-PAGE analysis of bovine RPE proteins. RPE proteins (100 μg) were separated by electrophoresis and visualized by Coomassie blue staining. X axis represents isoelectric point (3-10) and Y axis shows molecular weight in kDa. Arrows indicate protein spots that were up-or down-regulated proteins under oxidative stress. (B) 2D-SDS-PAGE of control RPE cells with treated with PBS. (C) 2D-DIGE protein analysis of human RPE D407. Five μg of total proteins were prelabeled with Cy3 (untreated control, green, emission wavelength 580nm) and with Cy5 (oxidative stress, red, emission wavelength 670nm). Red spots indicate that proteins were up-regulated and green spots were down-regulated in H₂O₂ treated cells. (D) Coomassie blue stained gel (100 μg) from H₂O₂ treated RPE D407 cells. Arrows indicate protein spots that were up-regulated by the treatment. (E) Coomassie blue stained gel of control D407 cells.

Figure 2. Quantitative analysis of 2D-PAGE

(A) Magnified protein spots in oxidative stress are shown with the control. (B) Each protein spots were analyzed based on volume and intensity. (C) Western blot analysis of 14-3-3 and neurofilament in control (lane 1) and H₂O₂ treated (lane 2) bovine RPE cells. (D) Quantification of western blot shows that 14-3-3 and neurofilament in oxidative stress were up-regulated by 1.8 fold and 2.8 fold compared to control.