A proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells

Abstract

Oxidative stress is one of the most relevant contributors of cataractogenesis. To identify early protein targets of oxidative stress in lens cells, we used a differential proteomics approach to CD5A human epithelial lens cells treated with 500 microM H2O2 for 30 min. This dose of H2O2 was assayed to induce efficiently a block of cellular proliferation and to activate the oxidative stress-early inducible transcription factor EGR-1 (early growth response gene product 1), previously reported as stimulated factor in a model of cataractogenesis [Nakajima, Nakajima, Fukiage, Azuma and Shearer (2002) Exp. Eye Res. 74, 231-236]. We identified nine proteins, which sensitively reacted to H2O2 treatment by using two-dimensional gel electrophoresis and matrix-assisted laserdesorption ionization-time-of-flight-MS. In addition to cytoskeletal proteins (tubulin 1alpha and vimentin) and enzymes (phosphoglycerate kinase 1, ATP synthase beta, enolase alpha, nucleophosmin and heat-shock cognate 54 kDa protein), which presented quantitative differences in expression profiles, peroxiredoxin and glyceraldehyde 3-phosphate dehydrogenase showed changes in pI as a result of overoxidation. Mass-mapping experiments demonstrated the specific modification of peroxiredoxin I active-site cysteine into cysteic acid, thus providing an explanation for the increase in negative charge measured for this protein. With respect to other global differential approaches based on gene expression analysis, our results allowed us to identify novel molecular targets of oxidative stress in lens cells. These results indicate that a combination of different approaches is required for a complete functional understanding of the biological events triggered by oxidative stress.

Nucleophosmin Is Required for DNA Integrity and p19Arf Protein Stability

“…DNA-damaging agents provoke the mobilization of nucleolar NPM into the nucleoplasm (22), and its binding to chromatin (28). Oncogene expression induces upregulation of Arf and, like the DNA-damaging agents, the partial nucleoplasmic mobilization of NPM (9).…”

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

“…UV radiation and H 2 O 2 ) [31][32][33][34]. In the present study, irradiated cells may be a source of stress factor(s) for adjacent bystander cells.…”

“…Therefore, it is interesting to note that H 2 O 2 exogenously added to human epithelial lens CD5A cells at cytotoxic concentrations (500 μM) has been reported to substantially increase NPM/B23 expression (five-six-fold) as early as 30 min after H 2 O 2 exposure [34]. Rapid stimulation of NPM/B23 expression in response to oxidative stress appears to be in concert with the fact that NPM/B23, in cooperation with nuclear factor NF-κB, induces the SOD2 gene [47].…”

Molecular Profiling of A375 Human Malignant Melanoma Cells Treated with Kojic Acid and Arbutin

“…In addition to phosphorylation, the different spots of vimentin seen on the 2-D gel may be due to association with other molecules, such as protein kinase C, integrin or other associated proteins in signal transduction pathways. Changes in vimentin expression levels are also generated by stress, such as heat shock and oxidative stress 52 . Cleavage of vimentin precedes the recognition of cytoskeletal filaments in apoptosis and programmed cell death mechanisms.…”