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

Published

October 1, 2009

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

Pub Type

Journals

Download Paper

DOI Link

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 19, 2024)