p38α旁路活化抑制肽關(guān)鍵氨基酸的確定和抑制肽的優(yōu)化
發(fā)布時(shí)間：2018-08-02 10:25
【摘要】：目的：急性心肌梗塞是由于冠狀動(dòng)脈血栓形成后導(dǎo)致的組織缺血而引起。冠狀動(dòng)脈的再灌注對(duì)于恢復(fù)心肌功能非常重要,但剛開始恢復(fù)血液再灌注后,不但未減輕反而加重缺血所引起的細(xì)胞功能代謝障礙及結(jié)構(gòu)破壞,因而將這種血液再灌注后缺血性損傷加重的現(xiàn)象稱為缺血再灌注(ischemia-reperfusion, I/R)損傷。I/R將會(huì)帶來一些不良效應(yīng),例如氧化應(yīng)激,細(xì)胞內(nèi)鈣超載等,這些不良反應(yīng)會(huì)導(dǎo)致心肌細(xì)胞凋亡。研究表明,在缺血再灌注過程中TAB1與p38α相互作用以及TAB1介導(dǎo)的p38α旁路活化在其中起關(guān)鍵作用。我們之前的工作通過靶向TAB1/p38α設(shè)計(jì)了一種具有細(xì)胞膜滲透性的拮抗肽PT5,并通過體內(nèi)體外實(shí)驗(yàn)證明PT5能夠選擇性的抑制TAB1/p38a的相互作用,從而在大鼠心肌缺血再灌注損傷模型中減輕心肌的再灌注損傷。目前,能夠有效降低心肌缺血再灌注損傷的干預(yù)策略和治療藥物還非常有限。我們之前工作所設(shè)計(jì)的PT5拮抗肽有望成為相關(guān)藥物的先導(dǎo)化合物。然而,PT5中發(fā)揮拮抗p38α旁路活化作用的關(guān)鍵氨基酸還是未知的。尋找PT5拮抗肽中相應(yīng)的關(guān)鍵位點(diǎn)將有助于獲得更精確的結(jié)構(gòu)信息并對(duì)多肽進(jìn)行優(yōu)化,從而為心肌再灌注損傷防治藥物提供先導(dǎo)結(jié)構(gòu)。本課題是在原有拮抗肽PT5的基礎(chǔ)上,根據(jù)PT5和p38α的空間結(jié)構(gòu),利用距離幾何學(xué)、計(jì)算機(jī)圖形學(xué)技術(shù)合理判別PT5與p38α相互識(shí)別的關(guān)鍵位置,設(shè)計(jì)出一系列PT5的突變體,并對(duì)各突變體的p38α拮抗功能進(jìn)行生物學(xué)評(píng)價(jià)。方法：1.理論推測(cè)關(guān)鍵位點(diǎn)并合成突變體多肽選擇距離幾何學(xué)、計(jì)算機(jī)圖形學(xué)技術(shù)合理判別PT5與P38a相互識(shí)別的關(guān)鍵位置,將第1-15個(gè)氨基酸分段做丙氨酸替換,得到6條序列。將這6條序列委托公司進(jìn)行合成,得到6條純度在95%以上的多肽。另外,設(shè)計(jì)15個(gè)氨基酸長(zhǎng)度的對(duì)照肽,對(duì)照肽不對(duì)p38a/TAB1相互作用產(chǎn)生任何影響2. GST-pull down確定關(guān)鍵氨基酸取一定比例的GST-TAB 1和His-p38a,溶解于Tris緩沖液中,向里面加入丙氨酸替換后的PT5突變體,進(jìn)行GST-pull down實(shí)驗(yàn)。以對(duì)照肽為陰性對(duì)照,以PT5為陽性對(duì)照,根據(jù)不同的PT5突變體對(duì)TAB1和p38α結(jié)合能力影響的強(qiáng)弱來確定拮抗肽中的關(guān)鍵氨基酸。3.體外激酶活性分析驗(yàn)證肽的活性取一定比例的GST-TAB 1、His-p38a和ATP,溶于激酶緩沖液中,向其中加入上述丙氨酸替換后的PT5突變體,進(jìn)行體外激酶活性分析實(shí)驗(yàn)。以對(duì)照肽為陰性對(duì)照,以PT5為陽性對(duì)照,根據(jù)不同的p38α拮抗肽突變體對(duì)TAB1介導(dǎo)的p38α磷酸化能力的影響來驗(yàn)證肽的活性,通過其對(duì)p38α磷酸化能力影響的強(qiáng)弱來驗(yàn)證拮抗肽中的關(guān)鍵氨基酸。結(jié)果與討論：利用計(jì)算機(jī)輔助分子對(duì)接、力學(xué)優(yōu)化獲得了P38a與PT5相互作用的復(fù)合物結(jié)構(gòu)。通過距離幾何學(xué)、計(jì)算機(jī)圖形學(xué)技術(shù)從理論上確定了P38a與PT5相互作用的關(guān)鍵區(qū)域,從理論上預(yù)測(cè)了PT5中的關(guān)鍵氨基酸殘基為Thr11和Asp12。根據(jù)預(yù)測(cè),設(shè)計(jì)了六個(gè)PT5的突變體,進(jìn)行體外生物學(xué)功能的驗(yàn)證。實(shí)驗(yàn)結(jié)果表明,PT5-5突變體失去阻斷TAB1/p38a相互作用,p38a不能自我磷酸化。此外,各突變體對(duì)p38α經(jīng)典活化途徑中MKK6/p38a的相互作用沒有影響,證實(shí)了Thr11和Asp12是PT5的關(guān)鍵氨基酸。意外的是我們?cè)诖诉^程中同時(shí)還獲得兩個(gè)拮抗作用更強(qiáng)的多肽PT5-2和PT5-4,這兩個(gè)突變體展現(xiàn)出對(duì)TAB1/p38a相互作用更強(qiáng)的阻斷效應(yīng)及對(duì)p38α自我磷酸化的抑制。找到該拮抗肽的關(guān)鍵氨基酸為將來獲得結(jié)構(gòu)和功能更加優(yōu)化的防治心肌缺血再灌注損傷的抑制肽奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.22
本文編號(hào)：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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.22
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