Abstract
Purpose :
Oxidative stress contributes to retinal pigment epithelium (RPE) dysfunction in age-related macular degeneration (AMD), but details on how RPE cells respond/adapt to oxidative stress are not fully understood. The purpose of the present study was to identify and quantitate differentially expressed antioxidant proteins in a human telomerase reverse transcriptase (hTERT)-immortalized RPE cell line exposed to the oxidant, tert-Butyl hydroperoxide (tBHP), using a proteomics approach.
Methods :
hTERT-RPE-1 cells (American Type Culture Collection product number CRL-4000) were exposed to 100μM tBHP for 24 hours to induce oxidative stress. Cells were then processed for tandem mass tag (TMT) labeling, multidimensional liquid chromatography (LC), LC-mass spectrometry data acquisition, database searches and TMT quantitation. Data was collected, reconciled, organized, and Gene Ontology and functional category analyses were carried out.
Results :
Exposure of hTERT-RPE-1 cells to tBHP led to a change in expression of proteins that are part of antioxidant signaling networks, including proteins involved in glutathione metabolism, peroxidases or superoxide dismutases and proteins involved in protection against oxidative stress. Oxidative stress increased expression of four proteins involved in glutathione metabolism by between 15.1- and 26.1-fold and decreased the expression of three such proteins by between 5.2- and 35.3-fold. Exposure to oxidative stress led to the upregulation of six proteins in the peroxidase / superoxide dismutase group by between 8.4- and 34.1-fold. Lastly, oxidative stress upregulated, by between 8.3- and 28.6-fold, the expression levels of three and downregulated, by between 4.7- and 16.3-fold, the expression levels of four proteins involved in downstream cytoprotective responses against oxidative stress.
Conclusions :
The response of hTERT-RPE-1 cells to extracellularly applied oxidative stress involves changes in the expression levels of proteins that are part of complex protein signaling networks. The present study identified proteins that may be critical for understanding the antioxidant response of RPE cells and their susceptibility to oxidative stress in AMD and other diseases that are characterized by RPE dysfunction, thereby providing novel targets for the development of novel therapeutics.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.