p38α旁路活化抑制肽关键氨基酸的确定和抑制肽的优化
发布时间：2018-08-02 10:25
【摘要】：目的：急性心肌梗塞是由于冠状动脉血栓形成后导致的组织缺血而引起。冠状动脉的再灌注对于恢复心肌功能非常重要,但刚开始恢复血液再灌注后,不但未减轻反而加重缺血所引起的细胞功能代谢障碍及结构破坏,因而将这种血液再灌注后缺血性损伤加重的现象称为缺血再灌注(ischemia-reperfusion, I/R)损伤。I/R将会带来一些不良效应,例如氧化应激,细胞内钙超载等,这些不良反应会导致心肌细胞凋亡。研究表明,在缺血再灌注过程中TAB1与p38α相互作用以及TAB1介导的p38α旁路活化在其中起关键作用。我们之前的工作通过靶向TAB1/p38α设计了一种具有细胞膜渗透性的拮抗肽PT5,并通过体内体外实验证明PT5能够选择性的抑制TAB1/p38a的相互作用,从而在大鼠心肌缺血再灌注损伤模型中减轻心肌的再灌注损伤。目前,能够有效降低心肌缺血再灌注损伤的干预策略和治疗药物还非常有限。我们之前工作所设计的PT5拮抗肽有望成为相关药物的先导化合物。然而,PT5中发挥拮抗p38α旁路活化作用的关键氨基酸还是未知的。寻找PT5拮抗肽中相应的关键位点将有助于获得更精确的结构信息并对多肽进行优化,从而为心肌再灌注损伤防治药物提供先导结构。本课题是在原有拮抗肽PT5的基础上,根据PT5和p38α的空间结构,利用距离几何学、计算机图形学技术合理判别PT5与p38α相互识别的关键位置,设计出一系列PT5的突变体,并对各突变体的p38α拮抗功能进行生物学评价。方法：1.理论推测关键位点并合成突变体多肽选择距离几何学、计算机图形学技术合理判别PT5与P38a相互识别的关键位置,将第1-15个氨基酸分段做丙氨酸替换,得到6条序列。将这6条序列委托公司进行合成,得到6条纯度在95%以上的多肽。另外,设计15个氨基酸长度的对照肽,对照肽不对p38a/TAB1相互作用产生任何影响2. GST-pull down确定关键氨基酸取一定比例的GST-TAB 1和His-p38a,溶解于Tris缓冲液中,向里面加入丙氨酸替换后的PT5突变体,进行GST-pull down实验。以对照肽为阴性对照,以PT5为阳性对照,根据不同的PT5突变体对TAB1和p38α结合能力影响的强弱来确定拮抗肽中的关键氨基酸。3.体外激酶活性分析验证肽的活性取一定比例的GST-TAB 1、His-p38a和ATP,溶于激酶缓冲液中,向其中加入上述丙氨酸替换后的PT5突变体,进行体外激酶活性分析实验。以对照肽为阴性对照,以PT5为阳性对照,根据不同的p38α拮抗肽突变体对TAB1介导的p38α磷酸化能力的影响来验证肽的活性,通过其对p38α磷酸化能力影响的强弱来验证拮抗肽中的关键氨基酸。结果与讨论：利用计算机辅助分子对接、力学优化获得了P38a与PT5相互作用的复合物结构。通过距离几何学、计算机图形学技术从理论上确定了P38a与PT5相互作用的关键区域,从理论上预测了PT5中的关键氨基酸残基为Thr11和Asp12。根据预测,设计了六个PT5的突变体,进行体外生物学功能的验证。实验结果表明,PT5-5突变体失去阻断TAB1/p38a相互作用,p38a不能自我磷酸化。此外,各突变体对p38α经典活化途径中MKK6/p38a的相互作用没有影响,证实了Thr11和Asp12是PT5的关键氨基酸。意外的是我们在此过程中同时还获得两个拮抗作用更强的多肽PT5-2和PT5-4,这两个突变体展现出对TAB1/p38a相互作用更强的阻断效应及对p38α自我磷酸化的抑制。找到该拮抗肽的关键氨基酸为将来获得结构和功能更加优化的防治心肌缺血再灌注损伤的抑制肽奠定了基础。
[Abstract]:Objective: acute myocardial infarction is caused by tissue ischemia caused by coronary artery thrombosis. The reperfusion of coronary artery is very important to restore the function of the myocardium. But after the resumption of blood reperfusion, it not only reduces the dysfunction of cell function and structural damage caused by the aggravation of ischemia, and thus the blood is caused by this kind of blood. The aggravation of ischemic injury after reperfusion is called ischemia-reperfusion (I/R) damage.I/R will bring some adverse effects, such as oxidative stress, intracellular calcium overload, and so on. These adverse reactions will lead to cardiomyocyte apoptosis. The study showed that the interaction of TAB1 with p38 alpha and TAB1 mediated during the process of ischemia reperfusion P38 alpha bypass activation plays a key role in it. Our previous work designed an antagonistic peptide PT5 with cell membrane permeability by targeting TAB1/p38 alpha. Through in vitro and in vitro experiments, it was demonstrated that PT5 could selectively inhibit the interaction of TAB1/p38a, thus reducing the myocardial reperfusion in the rat model of myocardial ischemia reperfusion injury. At present, the intervention strategies and drugs that can effectively reduce the myocardial ischemia reperfusion injury are still very limited. The PT5 antagonist designed by our previous work is expected to be the leading compound of the related drugs. However, the key amino acid, which plays an antagonistic effect on the activation of p38 a bypass, is still unknown in PT5. In search of the PT5 antagonist peptide The corresponding key points will help to obtain more accurate structural information and optimize the polypeptide so as to provide a pilot structure for the prevention and control of myocardial reperfusion injury. Based on the original antagonistic peptide PT5, this topic is based on the spatial structure of PT5 and p38 alpha, using distance geometry and computer graphics to reasonably distinguish between PT5 and p38 a. In the key position of mutual recognition, a series of PT5 mutants were designed and the p38 alpha antagonistic function of each mutant was biologically evaluated. Method: 1. theory was used to conjecture the key sites and to synthesize the mutant polypeptide selection distance geometry. The key position of the mutual recognition between PT5 and P38a was judged by computer graphics, and the 1-15 amino acid was used. 6 sequences were obtained by subsection of alanine substitution. The 6 sequences were synthesized to obtain 6 polypeptides with a purity above 95%. In addition, a control peptide of 15 amino acid lengths was designed. The control peptide did not produce any effect on the p38a/TAB1 interaction between 2. GST-pull down and GST-TAB 1 and His-p38a with a certain proportion of the key amino acids. The PT5 mutant after alanine replacement was dissolved in the Tris buffer solution, and the GST-pull down experiment was carried out. The control peptide was negative control, PT5 was used as the positive control, and the effect of the PT5 mutants on the binding capacity of TAB1 and p38 alpha was determined to determine the peptide of the key amino acid.3. extracorporeal kinase activity in the antagonist peptide. The activity took a certain proportion of GST-TAB 1, His-p38a and ATP, dissolved in the kinase buffer solution, and added the PT5 mutant after the alanine replacement. The activity of the kinase was analyzed in vitro. The control peptide was negative control and PT5 was the positive control. The effect of the p38 alpha antagonist mutant on the TAB1 mediated p38 alpha phosphorylation ability To verify the activity of the peptide and to verify the key amino acids in the antagonistic peptide by its influence on the phosphorylation of p38 a. Results and discussion: the interaction between P38a and PT5 is obtained by using computer assisted molecular docking. The structure of the interaction of PT5 is obtained by mechanical optimization. P38a and PT are theoretically determined by distance geometry, computer graphics technology. 5 the key region of interaction, theoretically predicted that the key amino acid residues in PT5 were Thr11 and Asp12., according to the prediction, six PT5 mutants were designed to verify the biological function in vitro. The experimental results showed that the PT5-5 mutant lost the interaction of blocking TAB1/p38a and the p38a could not be phosphorylated. In addition, the mutants were p38 a to p38 a. The interaction of MKK6/p38a in the classical activation pathway has not been affected, which confirms that Thr11 and Asp12 are the key amino acids of PT5. In this process we also have two stronger antagonistic peptides PT5-2 and PT5-4, and these two mutants show a stronger blocking effect on TAB1/p38a interaction and the self phosphorylation of p38 alpha. Finding the key amino acid of the antagonist peptide has laid the foundation for the more optimized structure and function of the inhibitory peptide for the prevention and treatment of myocardial ischemia reperfusion injury in the future.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.22
本文编号：2159129
[Abstract]:Objective: acute myocardial infarction is caused by tissue ischemia caused by coronary artery thrombosis. The reperfusion of coronary artery is very important to restore the function of the myocardium. But after the resumption of blood reperfusion, it not only reduces the dysfunction of cell function and structural damage caused by the aggravation of ischemia, and thus the blood is caused by this kind of blood. The aggravation of ischemic injury after reperfusion is called ischemia-reperfusion (I/R) damage.I/R will bring some adverse effects, such as oxidative stress, intracellular calcium overload, and so on. These adverse reactions will lead to cardiomyocyte apoptosis. The study showed that the interaction of TAB1 with p38 alpha and TAB1 mediated during the process of ischemia reperfusion P38 alpha bypass activation plays a key role in it. Our previous work designed an antagonistic peptide PT5 with cell membrane permeability by targeting TAB1/p38 alpha. Through in vitro and in vitro experiments, it was demonstrated that PT5 could selectively inhibit the interaction of TAB1/p38a, thus reducing the myocardial reperfusion in the rat model of myocardial ischemia reperfusion injury. At present, the intervention strategies and drugs that can effectively reduce the myocardial ischemia reperfusion injury are still very limited. The PT5 antagonist designed by our previous work is expected to be the leading compound of the related drugs. However, the key amino acid, which plays an antagonistic effect on the activation of p38 a bypass, is still unknown in PT5. In search of the PT5 antagonist peptide The corresponding key points will help to obtain more accurate structural information and optimize the polypeptide so as to provide a pilot structure for the prevention and control of myocardial reperfusion injury. Based on the original antagonistic peptide PT5, this topic is based on the spatial structure of PT5 and p38 alpha, using distance geometry and computer graphics to reasonably distinguish between PT5 and p38 a. In the key position of mutual recognition, a series of PT5 mutants were designed and the p38 alpha antagonistic function of each mutant was biologically evaluated. Method: 1. theory was used to conjecture the key sites and to synthesize the mutant polypeptide selection distance geometry. The key position of the mutual recognition between PT5 and P38a was judged by computer graphics, and the 1-15 amino acid was used. 6 sequences were obtained by subsection of alanine substitution. The 6 sequences were synthesized to obtain 6 polypeptides with a purity above 95%. In addition, a control peptide of 15 amino acid lengths was designed. The control peptide did not produce any effect on the p38a/TAB1 interaction between 2. GST-pull down and GST-TAB 1 and His-p38a with a certain proportion of the key amino acids. The PT5 mutant after alanine replacement was dissolved in the Tris buffer solution, and the GST-pull down experiment was carried out. The control peptide was negative control, PT5 was used as the positive control, and the effect of the PT5 mutants on the binding capacity of TAB1 and p38 alpha was determined to determine the peptide of the key amino acid.3. extracorporeal kinase activity in the antagonist peptide. The activity took a certain proportion of GST-TAB 1, His-p38a and ATP, dissolved in the kinase buffer solution, and added the PT5 mutant after the alanine replacement. The activity of the kinase was analyzed in vitro. The control peptide was negative control and PT5 was the positive control. The effect of the p38 alpha antagonist mutant on the TAB1 mediated p38 alpha phosphorylation ability To verify the activity of the peptide and to verify the key amino acids in the antagonistic peptide by its influence on the phosphorylation of p38 a. Results and discussion: the interaction between P38a and PT5 is obtained by using computer assisted molecular docking. The structure of the interaction of PT5 is obtained by mechanical optimization. P38a and PT are theoretically determined by distance geometry, computer graphics technology. 5 the key region of interaction, theoretically predicted that the key amino acid residues in PT5 were Thr11 and Asp12., according to the prediction, six PT5 mutants were designed to verify the biological function in vitro. The experimental results showed that the PT5-5 mutant lost the interaction of blocking TAB1/p38a and the p38a could not be phosphorylated. In addition, the mutants were p38 a to p38 a. The interaction of MKK6/p38a in the classical activation pathway has not been affected, which confirms that Thr11 and Asp12 are the key amino acids of PT5. In this process we also have two stronger antagonistic peptides PT5-2 and PT5-4, and these two mutants show a stronger blocking effect on TAB1/p38a interaction and the self phosphorylation of p38 alpha. Finding the key amino acid of the antagonist peptide has laid the foundation for the more optimized structure and function of the inhibitory peptide for the prevention and treatment of myocardial ischemia reperfusion injury in the future.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.22
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