Accumulation of large protein fragments in prematurely senescent ARPE-19 cells.

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TitleAccumulation of large protein fragments in prematurely senescent ARPE-19 cells.
Publication TypeJournal Article
Year of Publication2009
AuthorsLiao, W-L, Turko, IV
JournalInvest Ophthalmol Vis Sci
Date Published2009 Oct
KeywordsCell Aging, Cells, Cultured, Chromatography, Liquid, Cytosol, Electrophoresis, Gel, Two-Dimensional, Eye Proteins, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating), Humans, Models, Biological, Oxidative Stress, Peptide Fragments, Phenotype, Pyruvate Kinase, Retinal Pigment Epithelium, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, tert-Butylhydroperoxide, Triose-Phosphate Isomerase

PURPOSE: Senescence of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of age-related macular degeneration (AMD). This study was designed to improve the understanding of proteomic changes that underlie RPE senescence. Specifically, the levels of several protein fragments in prematurely senescent ARPE-19 cells were quantitatively compared with those in control cells.

METHODS: Premature senescence of human ARPE-19 cells was induced by repeated treatments with 6 mM tert-butylhydroperoxide (tert-BHP). Whole senescent cells were then treated with deuterated D(3)-acrylamide, and control cells were treated with normal D(0)-acrylamide. The D(3) and D(0) samples were mixed at a 1:1 ratio, and the proteins were separated by FPLC (fast protein liquid chromatography) and 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis). After in-gel trypsinolysis, the relative quantification of selected proteins and fragments in the senescent cells versus control ARPE-19 cells was achieved by calculating the ratio of signal intensities for the deuterated and normal forms of cysteine-containing labeled peptides in MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) spectra.

RESULTS: Several large fragments of typical cytosolic proteins, such as GAPDH, triosephosphate isomerase, and M2-type pyruvate kinase increased approximately two- to threefold in the prematurely senescent ARPE-19 cells.

CONCLUSIONS: This study is the first demonstration that large fragments of cytosolic proteins can be accumulated in prematurely senescent ARPE-19 cells, the in vitro model of AMD. These data suggest that protein degradation processes are impaired in these cells and point to a new type of "waste" material in post-mitotic cells that may contribute to the senescent phenotype.

Alternate JournalInvest. Ophthalmol. Vis. Sci.
PubMed ID19458325