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Wan Jin Jahng, O'Donnell Sylvester, Trevor Moser, Folami Lamoke, Paul Bernstein, Manuela Bartoli, Srinivas Sripathi; Prohibitin Depletion Leads to RPE Apoptosis in Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):695.
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Understanding the molecular mechanisms that mediate oxidative stress-induced proteome changes may provide insight into pathogenesis of retinal degeneration. Our proteomic study demonstrated that prohibitin is involved in oxidative stress signaling in the retina and the RPE. The current study was designed to examine the altered interactions of prohibitin-PIP3/cardiolipin, prohibitin-binding proteins under oxidative stress and how these interactions affect macular degeneration.
Immunoprecipitation and immunocytochemistry were performed to identify protein interactions and visualize protein localization and expressions from ARPE-19 cells. Two dimensional SDS-PAGE and Western blotting were used to identify proteins in AMD eyes. Proteomic data from aged and diabetic mouse/rat model were examined to identify prohibitin downstream signaling. AMD proteome were analyzed by, 2D SDS-PAGE, MALDI-TOF and tandem ESI/MS/MS. Protein-lipid interactions were analyzed to examine protein binding to cardiolipin, cholesterol, and phosphatidyl inositides.
Our study demonstrated that prohibitin-lipid binding switch mediated by cardiolipin and phosphatidylinositol may control dynamic translocalization of prohibitin between mitochondria and the nucleus. Decreased levels of prohibitin in the RPE from AMD eyes, compared to age-matching control, suggest that it may have an anti-apoptosis function in RPE cells, especially under chronic oxidative stress conditions. Prohibitin expression in both macular and peripheral region in the retina increased in AMD, however, prohibitin is down-regulated in the RPE. The lipid interaction assay demonstrated that mitochondrial prohibitin has a strong affinity at low cardiolipin concentrations (6−10 nmol).
We investigated oxidative stress-induced proteome changes in RPE cells using the differential gel electrophoresis (DIGE) technique to identify early biomarkers in AMD. Under oxidative stress, the level of cardiolipin decreased and the level of cardiolipin-prohibitin interactions were altered in RPE cells. Decreased levels of prohibitin in RPE cells of AMD eyes imply that RPE cell death by mitochondrial dysfunction may happen prior to apoptosis of the retina. Anti-apoptotic and transcriptional regulator function of prohibitin might be the crucial step for controlling apoptotic signaling as a cell death determinant from uncontrolled proliferation.
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