Proteome and Secretome Dynamics of Stem Cell-Derived Retinal Pigmented Epithelium in Response to Acute and Chronic ROS

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, and is characterized by slow retinal degeneration linked to chronic oxidative stress in the retinal pigmented epithelium (RPE). The exact molecular mechanisms that lead to RPE death and dysfunction in response to chronic reactive oxygen species (ROS) are still unclear. In this work, human stem cell-derived RPE samples were treated with a low dose of paraquat (PQ) for 1 week or 3 weeks to induce chronic reactive oxygen species (ROS) stress. Cells were then harvested and both the intracellular and secreted RPE proteomes were quantified by mass spectrometry. Inside the RPE, chronic ROS caused concerted increase of glycolytic proteins but decreased mitochondrial proteins, as well as decreased extracellular matrix proteins and membrane proteins required for endocytosis. From the secreted proteins, we found that stressed RPE secrete over 1,000 detectable proteins, and the composition of the proteins secreted from RPE changes due to chronic ROS. Notably, secreted APOE is decreased 4-fold due to 3 weeks of chronic ROS stress, and urotensin-II, the strongest known vasoconstrictor, doubles. Further, secreted TGF-beta is increased, and its cognate signaler BMP1 decreased in the secretome. Together, these alterations of the RPE proteome and protein secretome paint a detailed molecular picture of the retinal stress response in space and time.

Proteomic and phosphoproteomic analyses identify liver-related signaling in retinal pigment epithelial cells during EMT

“…Comparisons between our data and several published related datasets show closer links to hepatocellular carcinoma and TGF-b-treated keratinocytes than to reactive oxygen species (ROS)-treated hESC-RPE cells We sought to relate our findings to previously observed changes in proteome and phosphoproteome studies of related cell types and/or biological processes. To do so, we selected several previously published datasets encompassing human stem-cell derived RPE cells treated with ROS (Meyer et al, 2019), early-stage hepatocellular carcinoma (Jiang et al, 2019), Twist-induced EMT in human mammary epithelial (HMLE) cells (Uretmen Kagiali et al, 2019), and early responses of keratinocytes to TGF-b (D'Souza et al, 2014). We crossreferenced proteins and phosphosites that were altered significantly by dissociation and/or TGNF and altered significantly in at least one other dataset, then performed hierarchical clustering of the resulting 272 proteins and 233 phosphosites (Figure 5).…”