Accumulation of large protein fragments in prematurely senescent ARPE-19 cells.
OBJECTIVE
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.
Liao
Wei-Li
WL
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA.
Turko
Illarion V
IV
D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology*; D002853:Chromatography, Liquid; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D018384:Oxidative Stress / Q000187:drug effects; D010446:Peptide Fragments / Q000378:metabolism*; D010641:Phenotype; D011770:Pyruvate Kinase / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D005136:Eye Proteins; D010446:Peptide Fragments; D020122:tert-Butylhydroperoxide; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D011770:Pyruvate Kinase; D014305:Triose-Phosphate Isomerase