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

Published

Author(s)

Illarion V. Turko, Wei-Li Liao

Abstract

Senescence of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of age-related macular degeneration (AMD). This study was design to improve the understanding of changes that underlie the RPE senescence. The levels of various protein fragments of prematurely senescent ARPE-19 cells were quantitatively compared with that of control cells. Premature senescence of human ARPE-19 cells was generated by repeated treatments with 6 mM tert-butylhydroperoxide. The whole prematurely senescent cells were then treated with deuterated D3-acrylamide while the control cells were treated with normal D0-acrylamide. The D3- and D0-samples were mixed at 1:1 ratio and the proteins were separated by FPLC and 2D-PAGE. After in-gel trypsinolysis, the relative quantification of selected proteins in the prematurely senescent cells versus control ARPE-19 cells was achieved by calculating the ratio of signal intensities for the deuterated and normal forms of a cysteine-containing labeled peptide in MALDI-MS spectra. Several large fragments of typical cytosolic proteins, GAPDH, triosephosphate isomerase, and M2-type pyruvate kinase were found ≈2-3 fold increased in the prematurely senescent ARPE-19 cells. This study is the first demonstration that rather big fragments of cytosolic proteins can be accumulated in the prematurely senescent ARPE-19 cells, the in vitro model of the AMD. These data point to a new type of a “waste” material in post-mitotic cells that may contribute to the senescent phenotype.
Citation
Investigative Ophthalmology & Visual Science
Volume
50
Issue
10

Keywords

acrylamide, fragments, MALDI, relative quantification, retina

Citation

Turko, I. and Liao, W. (2009), Accumulation of Large Protein Fragments in Prematurely Senescent ARPE-19 Cells, Investigative Ophthalmology & Visual Science, [online], https://doi.org/10.1167/iovs.09-3671 (Accessed April 12, 2024)
Created October 1, 2009, Updated November 10, 2018