Identification of the major oxidatively damaged proteins in Escherichia coli cells exposed to oxidative stress.
In the present study we have analyzed protein oxidation on Escherichia coli when these cells were submitted to different stress conditions such as hydrogen peroxide, superoxide-generating compounds, and iron overloading. Carbonyl groups on oxidized cell proteins were examined by Western blot immunoassay. When anaerobically grown E. coli cells were exposed to hydrogen peroxide stress, alcohol dehydrogenase E, elongation factor G, the heat shock protein DNA K, oligopeptide-binding protein A, enolase, and the outer membrane protein A were identified as the major protein targets. A similar immunostained band pattern was found when cells were shifted from anaerobic to aerobic conditions in the presence of different concentrations of iron; it is relevant to note that oxidation of outer membrane protein C, not observed in peroxide stress conditions, was clearly detected as the concentration of iron was increased in the culture media. The hydrogen peroxide stress performed under aerobic conditions affected the beta-subunit of F0F1-ATPase; the rest of the oxidized protein pattern was very similar to that found for anaerobic conditions, with the exception of alcohol dehydrogenase E, a protein not synthesized aerobically. Cells submitted to superoxide stress using menadione showed a more specific pattern in which elongation factor G and the beta-subunit of F0F1-ATPase were affected significantly. When paraquat was used, although the degree of oxidative damage was lower, the same two modified proteins were detected, and DNA K was also clearly damaged. Cell viability was affected to different extents depending on the type of stress exerted. The results described in this paper provide data about the in vivo effects of oxidative stress on protein oxidation and give insights into understanding how such modifications can affect cellular functions.
Tamarit
J
J
Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Spain.
Cabiscol
E
E
Ros
J
J
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000378:metabolism*; D002470:Cell Survival; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005297:Ferrozine / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D005297:Ferrozine; D006861:Hydrogen Peroxide; D007501:Iron; C063842:dnaK protein, E coli; D006180:Proton-Translocating ATPases