【作者】 卢乃浩；

【导师】 高中洪；

【作者基本信息】 华中科技大学 ， 无机化学， 2010， 博士

【摘要】 蛋白质酪氨酸硝化是一种重要的蛋白质翻译后修饰,蛋白质上的氨基酸残基一旦发生翻译后修饰,就会对蛋白质的结构和功能产生重要的影响,进而与多种疾病的发生发展紧密联系。在病理条件下,蛋白质容易受到各种活性氧和活性氮自由基的损伤,发生蛋白质酪氨酸硝化。蛋白质酪氨酸硝化常常伴随有蛋白质氧化,使得对蛋白质酪氨酸硝化功能的研究变得复杂。此外,在蛋白质硝化和氧化的过程中,生物体内广泛存在的微量元素铁起着非常重要的催化作用。到目前为止,蛋白质硝化和氧化修饰与铁的关系,及其对蛋白质功能的不同影响尚未完全明了。本文从体外化学体系和动物模型两个方面入手,首先以烯醇酶为研究对象,在体外化学体系中研究了由铁诱导的蛋白质硝化和氧化修饰对烯醇酶功能的影响；并在此基础上考察了抗氧化剂去铁胺(DFO)对蛋白质硝化和氧化损伤的干预作用；最后,在动物模型上研究了糖尿病大鼠组织中烯醇酶硝化和氧化的情况,探讨了生物体内的硝化和氧化修饰对烯醇酶功能的影响。主要研究内容包括以下几个方面：(1)铁在烯醇酶硝化和氧化修饰中的作用选取酵母烯醇酶为研究对象,研究了不同形态铁催化NO2--H2O2和ONOO-对烯醇酶硝化、氧化及酶活的影响。实验结果显示,在催化NO2--H2O2和ONOO"导致的烯醇酶硝化／氧化损伤中,实验浓度(10μmol/L)下的柠檬酸铁(Citrate-Fe)和铁蛋白(ferritin)对两种损伤体系没有显著影响,而乙二胺四乙酸铁(EDTA-Fe)能促进ONOO-导致的烯醇酶硝化,但对NO2--H2O2体系导致的蛋白质硝化和氧化无显著作用。氯高铁血红素(hemin)和血红蛋白(hemoglobin)不仅能有效催化NO2--H2O2硝化和氧化蛋白质,同时它们也能有效促进ONOO-导致的蛋白质酪氨酸硝化,但对ONOO-导致的蛋白质氧化有显著的抑制作用。游离的二价铁(Fe2+)和三价铁(Fe3+)能有效促进NO2--H2O2导致的烯醇酶氧化,但同时显著地抑制ONOO-导致的烯醇酶硝化。此外,与蛋白质硝化相比,烯醇酶活性与蛋白质羰基化关系更密切,蛋白质氧化程度越强,酶活力下降越明显。在hemin-NO2--H2O2导致的烯醇酶硝化／氧化损伤中,蛋白质氧化(非巯基氧化)对酶活的影响要显著强于蛋白质酪氨酸硝化,但酪氨酸修饰对酶活也有一定的影响。这些结果表明,不同硝化途径导致的烯醇酶硝化／氧化修饰受铁的影响不同,除储存态铁如ferritin外,其他形态的铁都可能通过促进氧化或硝化影响烯醇酶的功能。(2)去铁胺对蛋白质硝化和氧化修饰的干预作用研究了铁螯合剂DFO对hemin-NO2--H2O2体系中蛋白质硝化和氧化的影响,并对其作用机理进行初步探讨。在hemin-NO2--H2O2体系中,这种抗氧化剂在低浓度(0.01-0.1mmol/L)时对蛋白质(牛血清白蛋白、谷氨酸脱氢酶、人血浆蛋白)的硝化和氧化有完全不同的作用,它能有效抑制蛋白质硝化,而对蛋白质的羰基化却有促进作用。在高浓度时,DFO则会发挥其抗氧化作用,抑制蛋白质的硝化和氧化。进一步研究证实,这种促氧化作用与heme-DFO复合物的形成密切相关。以上结果首次阐明了低浓度的DFO对heme-NO2--H2O2体系导致的蛋白质硝化和氧化有完全不同的干预作用,研究结果对于深入认识DFO在临床和医药上的毒副作用有重要的生理意义。(3)Hemin-DFO复合物促氧化机理的初步研究从自由基产生的角度初步研究了hemin-DFO复合物的促氧化机制。实验结果表明,在DFO促进hemin-H2O2体系导致的氧化损伤中,DFO促氧化作用的发挥需要与hemin和H2O2共存,其促氧化机理可能是促进了O2·-的生成。DFO完善的促氧化机制,有待进一步研究。(4)糖尿病大鼠组织中α-烯醇酶的硝化和氧化修饰通过腹腔注射链脲霉素建立糖尿病大鼠模型,同时选取α-烯醇酶作为靶蛋白,利用免疫沉淀和Western blotting技术检测糖尿病组织中α-烯醇酶的硝化和氧化,并采用高效液相色谱-质谱联用技术鉴定糖尿病大鼠心肌中α-烯醇酶的硝化位点,初步探讨生物体内的蛋白质酪氨酸硝化和氧化对α-烯醇酶功能的影响。实验结果显示,糖尿病大鼠心肌组织中蛋白质硝化水平明显提高,而其羰基化水平无显著变化,同时,糖尿病肝脏和血清中蛋白质硝化和羰基化水平均显著性提高。通过免疫沉淀的方法,确证了糖尿病组织和血清中α-烯醇酶是一种易于发生酪氨酸硝化的蛋白质。与正常大鼠相比,糖尿病心肌、肝脏和血清中α-烯醇酶的表达和硝化水平显著上升,同时其氧化水平和酶活略微提高,但无统计学上的差异。此外,还通过高效液相色谱-质谱联用技术,鉴定了糖尿病大鼠心肌中α-烯醇酶的硝化位点在Tyr257和Tyr131。而在体外ONOO-体系中,儿茶素选择性地抑制烯醇酶的硝化(Tyr259和Tyr191),并部分恢复酶活。这些结果表明,相比于蛋白质氧化,糖尿病组织中α-烯醇酶的硝化是一种更敏感的氧化损伤。尽管蛋白质的羰基化对酶活的影响要强于蛋白质硝化,但酪氨酸残基的硝化修饰对烯醇酶失活也有一定的贡献。更多还原

【Abstract】 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.更多还原