大鼠心肌蛋白巯基亚硝基化修饰的组学研究

发布时间：2018-05-12 03:15

本文选题：蛋白质巯基亚硝化修饰 + Biotin　；参考：《浙江大学》2008年硕士论文

【摘要】： 蛋白质巯基(S-)亚硝基化修饰是一种新近发现的蛋白质翻译后修饰方式,即一氧化氮(nitric oxide,NO)作用于蛋白质半胱氨酸巯基(-SH)生成巯亚硝基(-SNO)的过程。研究表明NO通过蛋白质S-亚硝基化修饰影响蛋白质的活性与功能,属于一种氧化还原信号的转导机制。特别是在缺血性心脏病中,提高心肌组织中的蛋白S-亚硝基化修饰水平,能发挥一定的心肌保护作用。目前,对心肌组织中潜在的S-亚硝基化修饰靶点还缺乏系统全面的研究。一些在临床上广泛应用,且能提高组织中NO水平的抗心肌缺血药物,其药理效应是否与心肌蛋白S-亚硝基化修饰水平的改变有关也尚不清楚。为此,本文开展了以下研究工作: 采用NO供体—巯基-亚硝基-谷胱甘肽(GSNO)对大鼠心肌蛋白进行体外孵育,采用Biotin switch法纯化S-亚硝基化蛋白,经双向电泳(2-DE)分离,基质辅助电离激光解吸-飞行时间-串联二级质谱(MALDI—TOF-MS/MS)以及候选Western blot技术进行蛋白鉴定。结果共确定了10种蛋白,其中GAPDH用Westernblot法进行了验证。其中,腺苷酸激酶1(AK1)是一个新发现的S-亚硝酰化修饰靶点,其活性可以被GSNO,而不是GSSG所抑制;且这种抑制作用可以被DTT逆转,说明S-亚硝基化修饰可能是一种新的AK活性的调节机制。采用10mg·kg~(-1)剂量的硝酸甘油腹腔注射,诱导大鼠心肌蛋白S-亚硝基化修饰。采用Biotin switch法纯化S-亚硝基化修饰蛋白,2-DE分离,MALDI—TOF-MS/MS鉴定,共确定17种蛋白。其中磷酸甘油酸激酶1、醛缩酶A、抗氧化蛋白1、3和6、NDPK、transthyretin等为首次发现的S-亚硝基化修饰蛋白。这些所发现的蛋白在调节心肌收缩,清除氧自由基,改善能量代谢等方面发挥着重要作用,而且S-亚硝基化修饰往往是调控这些酶活性的关键。因此推测硝酸甘油在体内发挥的药理作用与调节蛋白S-亚硝基化修饰有关。采用大鼠心肌缺血/再灌注损伤模型,以血清肌酸激酶(CK)、乳酸脱氢酶(LDH)为指标,考察参麦方抗心肌缺血再灌注损伤的作用,Griess法测定血清NO含量,Western blot法检测心肌组织内皮型一氧化氮合酶(eNOS)的表达变化;并分别采用Biotin switch法和DAN荧光法进行半定量和定量检测心肌蛋白S-亚硝基化水平。结果表明,与模型组相比,参麦方给药组血清损伤指标CK、LDH均明显下降;与假手术组和模型组相比,参麦方给药组血清NO含量显著升高,心肌组织eNOS表达上调,心肌蛋白S-亚硝基化水平由4.42+0.60 nmol·mg~(-1)上升至8.78±1.37nmol·mg~(-1),以及在90-117 kD分子量范围发生S-亚硝基化的心肌蛋白明显增多,说明参麦方可能通过促进蛋白S-亚硝基化而发挥心肌保护作用。本研究首次运用蛋白质组学方法对大鼠心肌可能发生S-亚硝基修饰的蛋白质进行了较为全面的分析,共发现22个S-亚硝基修饰靶点,且S-亚硝基化修饰可能是一种新的AK活性的调节机制,并发现参麦方抗心肌缺血/再灌注损伤的作用可能与提高心肌蛋白S-亚硝基化修饰水平有关。
[Abstract]:Nitroso modification of protein sulfhydryl (S-) is a newly discovered post-translational modification of protein, that is, nitric oxide (NO) acts on protein cysteine sulfhydryl (-SH) to produce mercapto nitroso (-SNO). The study shows that NO can affect the activity and function of protein through the modification of protein S-, which belongs to a kind of oxygen. The transduction mechanism of reduced signal, especially in ischemic heart disease, improves the level of S- nitroso modification in myocardial tissue and can play a certain role in myocardial protection. At present, a systematic and comprehensive study of potential S- nitroso modification targets in myocardial tissue is still lacking. Some are widely used in clinical and can improve tissue. It is not clear whether the NO level of anti myocardial ischemia drugs is related to changes in the level of S- nitroso modification. The following work has been carried out in this paper.
The NO donor - thiol - nitroso - glutathione (GSNO) was used to incubate the rat cardiac muscle protein in vitro. The Biotin switch method was used to purify the S- nitroso protein and be separated by two-dimensional electrophoresis (2-DE). The matrix assisted ionization laser desorption - flight time series two - mass spectrometry (MALDI - TOF-MS/MS) and the candidate Western blot technology were used to identify the protein. Results a total of 10 proteins were identified, of which GAPDH was verified by Westernblot. Adenylate kinase 1 (AK1) was a newly discovered S- subnitrosation target, and its activity could be inhibited by GSNO rather than GSSG; and this inhibition could be reversed by DTT, suggesting that S- nitroso modification may be a new AK activity. Section mechanism.
10mg / kg~ (-1) dose of nitroglycerin was intraperitoneally injected to induce S- nitroglycerin modification in rat cardiac muscle protein. The Biotin switch method was used to purify the S- nitroso modified protein, 2-DE separation, MALDI TOF-MS/MS identification, and 17 proteins were identified, including glyceric acid kinase 1, aldehyde contraction enzyme A, antioxidant protein 1,3 and 6 S- nitroso modified proteins are found. These proteins play an important role in regulating myocardial contraction, removing oxygen free radicals, and improving energy metabolism, and S- nitroso modification is often the key to regulate the activity of these enzymes. Therefore, the pharmacological effects of nitroglycerin in the body and the regulation of protein S- nitroso are suggested. It is related to chemical modification.
The rat myocardial ischemia / reperfusion injury model was used to investigate the effect of Shenmai recipe (CK) and lactate dehydrogenase (LDH) on the anti myocardial ischemia and reperfusion injury. The content of serum NO was measured by Griess method. The expression of eNOS in myocardial tissue was detected by Western blot, and Biotin switch was used respectively. The method of semi quantitative and quantitative determination of the level of S- nitroso of myocardial protein was carried out by method and DAN fluorescence method. The results showed that compared with the model group, the serum damage indexes of the Shenmai prescription group, CK, LDH were obviously decreased, and the serum NO content of the Shenmai prescription group was significantly higher than that of the sham operation group and the model group, the expression of eNOS in the myocardial tissue was up and the myocardial protein S- subnitrate was increased. The level of 4.42+0.60 nmol. Mg~ (-1) increased to 8.78 + 1.37nmol. Mg~ (-1), as well as the increase of myocardial protein of S- nitroso in the range of 90-117 kD molecular weight, indicating that Shenmai Fang may play a myocardial protective effect by promoting protein S- nitroylation.
In this study, the proteomics method was used for the first time to analyze the possible S- nitroso modified protein in rat myocardium. 22 S- nitroso modification targets were found, and S- nitroso modification may be a new regulation mechanism for the activity of AK, and the effect of Shenmai Fang on myocardial ischemia / reperfusion injury may be found. It is related to improving the level of myocardial protein S- nitroso modification.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341

【引证文献】