Characterization of Oxidized Lens Proteins Cells Which Are Degraded by the Proteasome

Abstract

Abstract: : Purpose: We previously demonstrated that oxidized proteins in lens epithelial cells(LEC) are degraded by the ubiquitin-proteasome pathway (UPP). The objective of this work is to characterize and identify the oxidatively damaged proteins which are degraded by theUPP. Methods: LEC in culture were exposed to mild oxidation (30-50 µM H₂O₂) for 4 h in the presence or absence of proteasome inhibitor. Proteins isolated from the cells were reacted with dinitrophenylhydrazine (DNPH) and separated by 2D-gels. The oxidized proteins were probed with antibodies to DNPH-derivatives. The carbonyl-containing proteins which were stabilized by proteasome inhibitor were further analyzed by LC-MS/MS. Results:In the absence of proteasome inhibitor, this mild oxidation only caused a marginal increase in levels of carbonyl-containing proteins in the cells. As shown previously, inhibition of the proteasome resulted in a dramatic increase in levels of carbonyl-containing proteins. 2D-gel Western blot analysis showed that some carbonyl-containing proteins were accumulated more than others in the presence of proteasome inhibitor. The apparent masses and pIs of the five most abundant carbonyl-containing proteins which were stabilized by proteasome inhibitor were 62 kDa/pI 4.5, 62 kDa/pI 5.3, 75 kDa/pI 5.0, 75 kDa/pI 5.5 and 92 kDa/pI 4.7, respectively. Preliminary MS analysis indicates that the 75 kDa/pI 5.0 protein is enolase, an enzyme involved in glycolysis. Conclusions: Most of the oxidized proteins in LEC are susceptible to UPP-mediated degradation, although some oxidized proteins are more susceptible than others. Identificating these proteins will allow us to further elucidate the protein quality control mechanism in the lens.

Keywords: oxidation/oxidative or free radical damage • proteolysis • enzymes/enzyme inhibitors