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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.

SUBMITTER: Paron I

PROVIDER: S-EPMC1224035 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:Roles for ubiquitin (an 8.5 kDa polypeptide) involve its conjugation to proteins as a signal to initiate degradation and as a stress protein. We investigated ubiquitin conjugation and ubiquitin-dependent proteolytic activities in cultured bovine lens epithelial cells (BLECs) upon oxidative challenge. A 44% decrease in intracellular glutathione confirmed oxidative stress upon incubation with 1 mM H2O2. After 30 min incubation, endogenous high-molecular-mass ubiquitin conjugates decreased 73%, and intracellular proteolysis decreased about 50%. In the supernatants of the oxidatively treated BLECs, the ability to form high-molecular-mass ubiquitin conjugates with exogenous 125I-labelled ubiquitin decreased 28%, and ATP-dependent degradation of oxidized alpha-crystallin decreased 36%. When the H2O2-treated BLECs were allowed to recover for 60 min, intracellular proteolysis returned to the level of control cells. There was also a subsequent transient enhancement of intracellular proteolysis and a simultaneous recovery of endogenous high-molecular-mass ubiquitin conjugates. In parallel cell-free experiments, conjugating activity with exogenous 125I-labelled ubiquitin and ATP-dependent degradation of oxidized alpha-crystallin increased 35% and 72% respectively compared with non-oxidatively treated BLECs. ATP-independent proteolysis showed little response to exposure or removal of H2O2. These results indicate that (1) the rate of intracellular proteolysis in BLECs is associated with the level of endogenous high-molecular-mass ubiquitin conjugates and (2) oxidative stress may inactivate the ubiquitin conjugation activity with coordinate depression of proteolytic capability. Enhancement in ubiquitin conjugation and proteolytic activities during recovery from oxidative stress may be important in removal of damaged proteins and restoration of normal function of BLECs. The inactivation of ubiquitin-dependent proteolysis by oxidation may be involved in the accumulation of altered proteins and other adverse sequelae in the oxidatively challenged aging lens.

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

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