Variants of peroxiredoxins expression in response to hydroperoxide stress

We examined patterns of the proteins that were expressed in human umbilical vein endothelial cells (HUVEC) in response to oxidative stress by two-dime nsional polyacrylamide gel electrophoresis (2D-PAGE). When HUVEC were expos ed to H2O2 at 100 muM for 60 min, the intensities of eight spots increased and those of eight spots decreased on 2D gels, as compared with control gel s, after staining with silver. These changes were also observed after expos ure of cells to hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide, but not after exposure to other reagents that induce oxidati ve stress such as S-alkylating compounds, nitric oxide, and salts of heavy metals. Therefore, these proteins were designated hydroperoxide responsive proteins (HPRPs). Microsequencing analysis revealed that these HPRPs corres ponded to at least six pairs of proteins. Of these, four pairs of HPRPs wer e thioredoxin peroxidase I (TPx I), TPx II, TPx III, and the product of hum an ORF06, all of which belong to the peroxiredoxin (Prx) family and all of which are involved in the elimination of hydroperoxides. The other two pair s corresponded to heat shock protein 27 (HSP27) and glyceraldehyde-3-phosph ate dehydrogenase (G3PDH), respectively. The variants that appeared in resp onse to hydroperoxides had molecular masses similar to the respective nativ e forms, but their pi values were lower by 0.2-0.3 pH units than those of t he corresponding native proteins. These variants were detected on 2D gels a fter cells had been exposed to hydroperoxides in the presence of an inhibit or of protein synthesis. All variants were generated within 30 min of expos ure to 100 muM H2O2. The variants of TPx I and TPx II appeared within 2 min of the addition of H2O2 to the culture medium. The HPRPs returned to their respective native forms after the removal of stress. Our results indicated that at least six proteins were structurally modified in response to hydro peroxides. Analysis by 2D-PAGE of P-32-labeled proteins revealed that the v ariant of HSP27 was its phosphorylated form while the other HPRPs were not modified by phosphorylation. Taken together, the results suggest that 2D-PA GE can reveal initial responses to hydroperoxide stress at the level of pro tein modification. Moreover, it is possible that the variants of four types of Prx might reflect intermediate states in the process of hydroperoxide e limination. (C) 2001 Elsevier Science.