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A.L. Hitt, B.S. Phinney, J.R. Reddan, R. Estrada, L.W. Schovan, D.C. Dziedzic, D. Borchman, M.C. Yappert; Proteomic Analysis of Mitochondrial Proteins Associated With a Lipid Raft Fraction in Cultured Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1868.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mitochondrial dysfunction has been implicated as major contributing factor in age–related disorders, including cataract formation. This involves changes in ATP production, leakage of reactive oxygen species, leakage of cytochrome C, and subsequent interactions with caspases which can induce apoptosis. Recently, mitochondrial DNA deletions have been implicated in disease states of muscle, brain and lens. We identified numerous mitochondrial proteins associated with lipid rafts from cultured human lens epithelial cells (HLECs). Methods: Lipid raft fractions were prepared from the HLEC line, FHL124. Two triton–insoluble floating fractions (TIFF) were collected; a top and a bottom band at the 20–28% and 28–38 % sucrose interfaces. Cholesterol and phospholipids were analyzed by matrix–assisted laser desorption/ionization mass spectrometry (MALDI–MS) using 2,5 dihydroxybenzoic acid as the matrix. Proteins were fractionated by SDS–PAGE and proteolyzed in situ prior to liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Results were evaluated using the DAVID suite of tools and EASE program at NIAID. Results: Proteomic analysis of lipid raft fractions from HLECs identified >1000 associated proteins. Lipid rafts were rich in cholesterol and sphingomyelins. This fraction was highly enriched (Fisher Exact score = 1.5 x 10e–35) in mitochondrial proteins. Of the lipid raft associated mitochondrial proteins, 71 were unique to the bottom fraction. Proteins identified included: several with chaperone activity, e.g., AFG3A, NADH dehydrogenase assembly factor 1, paraplegin, numerous oxidative enzymes e.g., cytochrome c oxidase, ABC transporters e.g., ABCB7 and ABCB10, and proteins involved in apoptosis e.g., Grim19, Bcl2–like 13, Bcl2 antagonist/killer 1, programmed cell death 8. To refine our understanding of the role mitocondrial lipid raft associated proteins play in the cell, they were assigned to the three gene ontology groups: cellular component, molecular function and biological process. Conclusions: This work provides a global overview of mitochondrial proteins associated with lipid rafts from HLECs and may provide new information on target proteins involved in pathogenesis.
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