摘要
糖尿病是一种常见的易发性代谢疾病。越来越多的证据表明,活性氧和活性氮介导糖尿病及其并发症的病理过程。蛋白质酪氨酸硝化是一种重要的蛋白质翻译后修饰,它与炎症、心血管疾病和神经退行性疾病等多种病症相关。过氧亚硝酸根(ONOO-)路径是促使蛋白质硝化最主要的一种途径,其反应为自由基机理。与糖尿病相关的蛋白质硝化已有较多文献报道,这不仅表现在硝基酪氨酸(NT)水平的增高,也有特定的硝化蛋白被发现。胰岛素是由脊椎动物胰腺β细胞分泌的一种多功能蛋白质激素,是治疗糖尿病最有效的药物。ONOO-可能会在β细胞内形成,对细胞造成损伤,而胰岛素也可能成为氧化应激条件下ONOO-的一个靶点。目前有关ONOO-对胰岛素及其信号转导系统影响的报道还较少。
本文研究了体外ONOO-对胰岛素的硝化反应,探讨了硝化对胰岛素结构、功能的影响,并对抗氧化剂等因素对胰岛素硝化作用的影响进行了研究,取得了以下主要结果:
⑴采用紫外可见光谱、凝胶电泳、免疫印迹以及质谱等对胰岛素的硝化产物进行了表征,并考察了pH值、CO2、铁配合物等因素以及白蛋白对该反应的影响。结果显示ONOO-可以硝化胰岛素酪氨酸(Tyr)残基,且随浓度的不断增大,胰岛素4个酪氨酸残基都有可能被硝化。ONOO-硝化胰岛素的反应与pH值密切相关,在生理pH值条件下,ONOO-的硝化作用最强。CO2对胰岛素的硝化具有催化作用。乙二胺四乙酸铁配合物可催化胰岛素硝化,而二乙三胺五乙酸铁配合物对胰岛素硝化无影响。白蛋白对胰岛素的硝化具有竞争抑制作用,但在高浓度的白蛋白存在时,胰岛素依然可以被硝化。
⑵采用高效液相色谱分离制备得到了胰岛素的单硝化和二硝化组分。运用凝胶电泳、高效液相色谱和质谱等技术,结合A、B链拆分和V8蛋白酶酶解方法,对硝化胰岛素的硝化位点进行了分析。还原条件下硝化组分的常规聚丙烯酰胺凝胶电泳以及色谱分离出的单硝化胰岛素A链的质谱结果表明胰岛素的A链优先B链被硝化。V8蛋白酶酶切单硝化胰岛素后的质谱结果表明单硝化胰岛素的硝化位点是Tyr-A14。结合电泳的结果和晶体结构分析,Tyr-A19较B链上的Tyr残基更容易硝化,而Tyr-B26可能较Tyr-B16更容易硝化。
⑶运用光谱学方法研究了硝化对胰岛素二级结构的影响。综合荧光光谱、圆二色光谱和傅立叶变换红外光谱的测试结果,发现单硝化和二硝化胰岛素与胰岛素相比α-螺旋的含量下降,A链的硝化可能使A(12-17)的310螺旋肽段逐渐向不规则螺旋和无规结构方向变化,但对胰岛素与受体结合面的影响不大。同时硝基的引入使得单硝化和二硝化硝化胰岛素趋向于聚集,而二硝化胰岛素中Tyr-A19的硝化可能会改变胰岛素与受体的结合面疏水区的疏水性质。
采用循环伏安法研究了Tyr、NT以及Tyr、NT与白蛋白相互作用后的伏安行为,结果表明硝基的引入将会改变酚羟基氢键的作用方式,影响Tyr与白蛋白的相互作用,这从一个侧面反映了硝化可能会影响胰岛素与受体的结合。
⑷测定了单硝化胰岛素的生物活力,并对硝化胰岛素刺激胰岛素受体磷酸化进行了初步的讨论。单硝化胰岛素的受体结合能力约为胰岛素的70%,动物实验与此结果相一致。
⑸采用紫外可见光谱测定胰岛素与ONOO-反应后NT产量的变化,研究了谷胱甘肽(GSH)和Ebselen等抗氧化剂对ONOO-硝化胰岛素的影响,并采用高效液相色谱和质谱等手段对GSH和Ebselen之间相互作用及其对ONOO-硝化胰岛素的影响机理进行了分析。结果表明,油酸和花生四烯酸对胰岛素的硝化具有很强的抑制作用,不饱和脂肪酸的抑制作用随其不饱和度的增大而增强。在实验浓度范围内,亚硒酸钠对胰岛素的硝化无明显影响;单独的GSH、抗坏血酸、Ebselen和维生素E等抗氧化剂对胰岛素的硝化均有不同程度的抑制作用;但在一定浓度范围内,GSH和Ebselen之间存在相互拮抗作用,原因是GSH和Ebselen可以生成加合物Ebselen-Se-SG。
Diabetes is a common kind of metabolic diseases. There is emerging evidence that RNS/ROS make a significant contribution to the progression of diabetes and its complications. Protein tyrosine nitration is an important posttranslational modification, and involved in a variety of diseases. Nitration by peroxynitrite is a principal pathway, and nitrotyrosine is formed by free radical reaction. Diabetes-associated protein nitration has been reported by several recent publications. Insulin is one of the most important versatile hormones, and it may be a potential target of peroxynitrite during conditions of oxidative stress in pancreatic isletβ-cells.
In this paper the nitration of insulin by peroxynitrite in vitro and the effects of nitration on the structure and function of insulin were investigated. The influence of antioxidants and some facts on insulin nitration were also studied. The main results obtained were as follows:
(1) The products of insulin nitration by peroxynitrite were characterized by UV-Vis, Native-PAGE, Western blotting and MALDI-TOF-MS, and the influence of the pH value, carbon dioxide, iron complexes and albumin was also investigated. The results showed that insulin could be nitrated by peroxynitrite in vitro, and with increasing the concentration of peroxynitrite all of the four tyrosine residues in insulin could be nitrated. The nitration reaction was correlated with the pH value of the solution, and at physiological pH peroxynitrite has the maximum nitration capability. Carbon dioxide and Fe(III)-EDTA could catalyze the nitration reaction, but Fe(III)-DTPA had no effect. Albumin could competitively inhibit the nitration of insulin by peroxynitrite, but in the presence of high concentrations of albumin insulin could still be nitrated.
(2) Mononitrated and dinitrated insulin were purified by RP-HPLC. The preferential nitration site of the four tyrosine residues in insulin was confirmed by Native-PAGE, RP-HPLC and MALDI-TOF-MS. Following reduction of insulin disulfide bridges, Native-PAGE indicated that A-chain was preferentially nitrated. Combination of enzymatic digestion of mononitrated insulin with endoproteinase Glu-C, mass spectrometry confirmed that Tyr-A14 was the preferential nitration site when insulin was treated with peroxynitrite. Tyr-A19 maybe was the next preferential nitration site. According to the crystal structure, Tyr-B26 between the two tyrosine residues in insulin B-chain was likely easier to be nitrated by peroxynitrite.
(3) The effects of nitration on the secondary structures of insulin were investigated by analysis of the fluorescence spectra, circular dichroism spectra and fourier transformation infrared spectra of the nitrated insulin. The contents ofα-helix of the mono and dinitrated insulin were decreased, and the nitration of A-chain could make the 310 helix of A(12-17) segment convert to irregular helix or unordered structures, but it didn’t influence the main part of the insulin binding surface with insulin receptor, and the nitration of Tyr-A19 may alter the hydrophobicity of the insulin binding surface. In the mean time the introduction of nitro group could incline the mono and dinitrated insulin to aggregation.
The voltammetric response of Tyr and NT was investigated by cyclic voltammetry, and the results showed that introduction of nitro group could alter the hydrogen bonding manner of the phenol hydroxyl and influence the interaction between Tyr and albumin. These reflected that nitration could affect the binding between insulin and insulin receptor to some extent.
(4) The biological activity of mononitrated insulin was assayed, and the phosphorylation of insulin receptor by stimulation of nitrated insulin was also investigated. The receptor binding capability of mononitrated insulin was about 70% of that of insulin, and the biological activity in vivo accorded with it.
(5) The effects of antioxidants on the nitration of insulin by peroxynitrite were evaluated by determination of the production of nitrotyrosine by UV-Vis, and the interaction of GSH and Ebselen and the effects on the nitration of insulin were investigated by RP-HPLC and ESI-MS. Oleic acid and arachidonic acid have strong inhibition effects on nitration of insulin, and the inhibition capability of unsaturated fatty acids were increasing with the unsaturation degree. At the experimental concentration range sodium selenite had no effect on nitration of insulin. Individual GSH, VC, Ebselen and VE had different inhibition capabilities on nitration of insulin, but at a definite concentration range antagonism was occurred between GSH and Ebselen because an adduct, Ebselen-Se-SG, could be formed.
引文
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