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Accumulation of Large Protein Fragments in Prematurely Senescent ARPE-19 Cells

From the Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, University of Maryland Biotechnology Institute, Rockville, Maryland.

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 D₃-acrylamide, and control cells were treated with normal D₀-acrylamide. The D₃ and D₀ 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.