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O'Donnell Sylvester, Srinivas Rao Sripathi, Folami Lamoke, Manuela Bartoli, Paul S Bernstein, Wan Jin Jahng; Region-Specific Phosphorylations in Age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):395.
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The current study explores epigenetic control of gene expression and oxidative responses through modulating phosphoproteome in the retina and RPE cells. We examined a novel role of phosphorylation signaling as an adaptation to extracellular stress and used phosphoproteome alterations to determine early molecular mechanism of retina/RPE cell death and survival. Our hypothesis is that decreased transferrin and increased kinase/phosphatase activity in AMD could be responsible for regulated apoptotic phosphorylation signaling during AMD progression.
Phophoproteomes of peripheral retina and RPE were compared to age-matching control donor eyes to determine senescence-associated molecular events during AMD progression. Phosphoproteins were enriched by charged based spin column chromatography and resolved by 2D gel electrophoresis. Trypsin digested modified peptides were enriched using Ga3+/TiO2 immobilized metal ion chromatography. Eluted phosphopeptides were analyzed using MALDI-TOF and ESI MS/MS. Serine, threonine and tyrosine phosphorylations were confirmed by phospho-western blotting analysis.
Our study demonstrated the down-regulation of transferrin in peripheral retina of AMD donor eyes compared to control. Phospho-Western blotting analysis revealed the phosphorylation of intermediate filament vimentin (Ser), Glial fibrillary acidic like protein and mitochondrial heat shock protein (mtHsp70). Protein database analysis shows the phosphorylation of transferrin in AMD donor eyes. Phosphorylation networks in the macular, peripheral retina, central RPE, and peripheral RPE provide the region-specific phosphorylation reactions involved in the early progression of AMD.
Our study identifies phosphoprotein changes through the activation of receptor tyrosine kinases. Our data suggest that a positive correlation exists between early biomarkers of phosphoproteome under oxidative stress in vitro and retina/RPE proteins from AMD patients. The outcome of our work will be the initial delineation of the underlying physiology of oxidative stress-mediated phosphorylation signaling in the retina/RPE apoptosis. In addition, our study will provide a stimulus for understanding oxidative stress-induced cytoskeletal changes and the aggregate formation mechanism by phosphorylations. As a consequence, an effective therapeutic approach based on the modulation of phosphorylations is expected to result.
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