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Srinivas R. Sripathi, Ji-Yeon Um, Rissa McDonough, Sydney Bruestle, Trevor Moser, Jian-xing Ma, Paul S. Bernstein, Wan Jin Jahng; Phosphoproteome and Lipid Network in RPE Apoptosis under Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1597.
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Phosphoproteomic alterations and lipid composition changes during elevated oxidative stress in AMD remain elusive. Previous studies suggest that early events of senescence-associated protein phosphorylation and lipid oxidation are the crucial reactions of RPE apoptosis under oxidative stress. AMD involves progressive cell death of post mitotic RPE and loss of photoreceptors. Oxidative stress-induced cytoskeletal reorganization,disrupted communication between mitochondria-nucleus, and crystalline aggregation may contribute towards the apoptotic process in the RPE. Our quantitative phosphoproteome analysis provides a powerful strategy that may unveil the specific target and the molecular mechanisms of AMD pathogenesis.
Phosphoproteins and lipids from ARPE-19 cells and RPE cells from both normal and AMD eyes were enriched and analyzed by using Gallium chromatography and MALDI-TOF, ESI-MS/MS. Serine, threonine and tyrosine phosphorylations at different disease stages were visualized by phospho-western blot and site-specific phosphorylation analysis.Scanning electron microscopy and immunocytochemistry were performed.
Our preliminary data demonstrates that cytoskeletal phosphorylations, crystalline aggregation, and mitochondrial signaling may contribute to AMD pathogenesis. Altered phosphorylations of mitochondrial heat shock protein mtHsp70, αA/αB crystalline, vimentin, and ATP synthase were observed in RPE cell death under oxidative stress. Previous data demonstrated that Hsp70 (c-Jun N-terminal kinase), crystallins (Akt), and the increased expression of VDAC might be involved in AMD progression. We observed altered lipid compositions that include increased carbon number of fatty acids, double bond saturation, higher cholesterol, and phosphatidylcholine, whereas cardiolipin levels decreased.
Understanding quantitative phosphorylation signaling and lipid composition may describe the biochemical mechanisms that promote the early stages of AMD. Changes in phospholipids, long chain fatty acids at sn-2 in phosphotidylcholine increased from 16 carbons to 24/26 carbons as well as cholesteryl esters were observed under oxidative stress, which may accelerate the soft drusen formation during AMD progression. In addition, oxidized lipoproteins and cholesterol may trigger the complement activation. Our phosphoproteomic and lipidomic approach may suggest new evidence that connects the pathophysiology of RPE cell death and AMD through protein aggregation,lipid oxidation, protein phosphorylation,and inflammation switch mechanism.
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