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Srinivas Sripathi, O'Donnell Sylvester, Trevor Moser, Paul Bernstein, Folami Lamoke, Manuela Bartoli, Wan Jin Jahng; Phosphorylation Networks in Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5002.
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Age-related macular degeneration (AMD) results from the chronic atrophy of retina and retinal pigment epithelial (RPE) cells (dry AMD) or choroidal neovascularization (wet AMD). However, the molecular mechanism underlying the progression of AMD is not well defined yet. Light-induced oxidative stress, lipid oxidation, accumulation of soft drusen, and crystalline aggregation may contribute to AMD pathogenesis. The aim of our phosphoproteome study is to identify the early biochemical events and specific phosphorylated sites in the macular, peripheral retina and RPE. Our data demonstrated that specific kinase/phosphatase regulated apoptotic phosphorylation signaling in AMD progression.
The senescence-associated molecular events during AMD progression were identified by comparing the phosphoproteome of macular and peripheral retina, RPE from different stages of AMD compared to age-matching control donor eyes. Phosphoproteins were enriched by charge-based spin column chromatography and resolved by 2D gel electrophoresis. Tryptic peptides were enriched using Ga3+/TiO2 immobilized metal ion chromatography. Eluted phosphopeptides were analyzed and confirmed by MALDI-TOF or ESI/MS/MS mass spectrometry and phospho-Western blot respectively.
Our preliminary data shows tyrosine phosphorylation of beta crystallin A3 and A4 increased but serine phosphoryaltion of beta crystallin decreased in RPE under stress. Our phospho-Western analysis reveals phosphorylation of intermediate filament vimentin (Ser) and mitochondrial heat shock protein mt Hsp70. Serine/threonine phosphatase PP2A-Cα/β (Tyr) and tubulin α1B/β2 were dephosphorylated in the RPE. Further, our data shows that prohibitin is upregulated in the retina, but down regulated in the RPE from AMD donor eyes. Our study suggests that altered lipid composition may determine phosphorylation-dependent interactions of target proteins that contain PH, PX, and SH domains.
The region specific proteome changes in central/peripheral retina and RPE from AMD donor eyes and age-matching controls were analyzed. Phosphorylation changes and downstream activation may leads to the early biochemical events in AMD. Our phosphoproetomic strategy provides the detailed phosphorylation signaling,phospholipid changes, altered protein aggregation,binding affinity and expression that include depleted levels of anti-apoptotic, mitochondrial chaperone prohibitin in the RPE of AMD eyes.
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