| Exposure of proteins to reactive oxygen species (ROS) and reactive nitrogen species (RNS) results in oxidative and nitrative modifications of amino acid residues, altering the protein structure and function. Protein tyrosine nitration is becoming increasingly recognized as a prevalent post-translational modification that could serve as a biomarker of pathological process and oxidative stress. Meanwhile, oxidative damage of protein is always accompanied, which makes it difficult to interpret the single effect of nitrative modification on protein function. Moreover, iron is known to play an important role in catalyzing the formation of free radicals and protein nitrative/oxidative modifications. However, the relationship between iron and protein nitration/oxidation, and the respective function of protein nitrative and oxidative modifications remain poorly understood. In this paper, the respective effects of iron-induced nitrative and oxidative modifications on enolase function were firstly investigated in vitro. Subsequently, the iron chelating agent desferrioxamine (DFO) was used to affect the development of nitrative and oxidative stress. Finally, experimental diabetic rat model was established to investigate the effect of nitrative and oxidative modifications on enolase function in vivo. The main results are as follows:(1) Effects of iron on enolase nitrative and oxidative modificationsEffects of different forms of iron on NO2--H2O2 or ONOO--induced enolase nitrative and oxidative modifications were investigated in vitro. The results showed that ferric citrate and ferritin exhibited ineffective activity in catalyzing protein 3-nitrotyrosine and carbonyl formation in both nitrating models. EDTA-Fe showed a promotive effect on ONOO--induced enolase tyrosine nitration, whereas exhibited ineffectively effect on NO2--H2O2-induced enolase nitrative and oxidative modifications. Moreover, both hemin and hemoglobin exhibited significant effect on catalyzing NO2--H2O2-triggered protein nitration and oxidation, while they promoted ONOO--induced protein nitration, but inhibited ONOO--induced protein oxidation. Meanwhile, free iron (Fe2+, Fe3+) could clearly promote NO2--H2O2-triggered protein carbonyls formation, and effectively inhibited ONOO--induced enolase nitration. The more protein carbonyls generated, the more significant enolase inactivation was. In hemin-NO2--H2O2-induced enolase nitrative and oxidative modifications, protein oxidation formation (not thiol oxidation), rather than protein tyrosine nitration, might make a major contribution to the inactivation of enolase. These findings indicate that the different forms of iron exhibit different activities in catalyzing enolase nitration and oxidation, suggesting that iron-induced protein nitrative/oxidative modifications would play an important role in protein dysfunction.(2) Effects of DFO on protein nitration and oxidationIn the hemin-NO2--H2O2-induced protein nitration and oxidation model, protein was analyzed for 3-nitrotyosine and carbonyl groups measured by spectrophotometry and Western blotting upon exposure to the iron chelating agent DFO. The results showed a significant inhibitory effect of DFO on hemin-NO2--H2O2-induced protein (bovine serum albumin, L-glutamic dehydrogenase, human plasma proteins) nitration, while an enhancement on oxidation was surprisingly observed at lower concentration (0.01-O.lmmol/L). However, DFO exhibited protective effect on protein 3-nitrotyrosine and carbonyl formation when the higher concentration was used. In addition, the abnormal effect of DFO on promoting protein oxidation was probably originated from heme-DFO complex which needs further study. These results indicate the completely effects of DFO on hemin-NO2--H2O2-induced protein nitration and oxidation first time, suggesting that the toxicity should be taken into account when DFO is used in clinical and medical application.(3) Preliminary study on the promoting mechanism of hemin-DFO complexThe promoting mechanism of hemin-DFO complex on protein oxidation was studied based on free radials formation. The results showed that the promoting effect of DFO on hemin-H2O2-induced oxidative stress was dependent on the coexistence of hemin and H2O2. Moreover, the additive O2 formation would be the important contributing factor to the promotion effect of DFO, while the integrity mechanism needs further study.(4) Nitrative and oxidative modifications of a-enolase in diabetic rat proteinsBy means of immunoprecipitation and Western blotting analysis, the levels of 3-nitrotyrosine residues and protein carbonyls in a-enolase from streptozotocin-induced diabetic rats were determined and compared with age-matched control. Moreover, by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, the nitrated site of tyrosine residues in a-enolase from diabetic rat heart was identified to link nitrative and oxidative modifications to protein dysfunction in diabetic damage. The cardiac proteins from diabetic rats showed that the total level of protein tyrosine nitration was clearly elevated, while the total protein carbonylation was slightly increased, but showed no statistical significance. However, both protein tyrosine nitration and protein carbonylation were clearly elevated in diabetic rat liver and serum. By means of immunoprecipitation analysis, a-enolase was identified as the important target for nitrative and oxidative modifications in diabetic rats. The levels of a-enolase expression and nitration were clearly increased in diabetic group, whereas the enolase activity and oxidation status were not significantly changed. By means of HPLC-MS/MS analysis, it was found that Tyr257 and Tyr131 of a-enolase were the most susceptible to nitration in diabetic rat heart. The addition of catechin selectively inhibited ONOO--induced tyrosine nitration (Tyr259 and Tyr191) and partially recovered the enzyme activity. These results suggested that tyrosine nitration was a more susceptive parameter for oxidative damage in diabetic state. Although protein oxidation may play a major role in enzyme inactivation, there is also a significant contribution of protein tyrosine nitration to the inactivation of enolase.
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