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Ji-Yeon Um, Srinivas Sripathi, O'Donnell Sylvester, Madu Joshua, Folami Lamoke, So Ra Kim, Paul S Bernstein, Manuela Bartoli, Wan Jin Jahng; AMD BIOMARKER MAPPING GUIDED BY TISSUE-SPECIFIC PHOSPHORYLATIONS. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):818.
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Reversible protein phosphorylation in the retinal pigment epithelium (RPE) is a central signaling response as a survival tool under oxidative environment. Previously, our unbiased proteomic approach demonstrated that the early apoptotic network and post-translational mechanisms including the proteins, erythropoietin, prohibitin, phosphatase, and vimentin, exist to dertermine cell survivor or death in RPE cells under stress conditions. We hypothesized that region-specific (macular vs peripheral retina) and tissue-specific (retina vs. RPE) phosphorylations may lead to 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. Trypsin digested phosphorylated peptides were enriched using Ga3+/TiO2 immobilized metal ion chromatography. Ser, Thr and Tyr phosphorylations were confirmed by phospho-Western blotting.
Our study demonstrated that region- and tissue-specific phosphorylations exist as a protection mechanism in the retina and the RPE. Specific biomarkers in pheripheral region of the retina suggest that the altered energy metabolism (pyruvate kinase, creatine kinase, enolase), iron metabolism (transferrin), extracellular ligand binding (receptor tyrosine kinase), and cytoskeletal reorganization (GFAP) exist in AMD eyes. Western blotting analysis revealed the phosphorylations of intermediate filament vimentin (Ser), GFAP, and mtHsp70. Vimentin might be critically involved in the process of light-induced damage in the retina. Melatonin reduces light-induced post-translational modification of vimentin, assisting in maintaining the proper filament network in Muller cells.
The region-specific phosphoproteome changes were analyzed in macular, peripheral retina, central RPE, and peripheral RPE from AMD donor eyes. Our data suggest that a positive correlation exists between early biomarkers of phosphoproteome under oxidative stress in vitro and retina/RPE proteins from AMD eyes. Phosphorylations of creatine kinase, pyruvate kinase, tyrosine kinase, PP2A, transferrin, GFAP and vimentin may contribute to AMD progression. Specific lipid accumulation and mitochondrial dysfunction by prohibitin depletion may initiate peripheral RPE apoptosis in AMD pathway.
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