微囊藻毒素及其衍生物抑制蛋白磷酸酶PP1生物活性差异机制研究
发布时间：2019-05-17 04:28
【摘要】：微囊藻毒素(Microcystins,MCs)作为一类生物毒性明显、难降解的有机污染物已引起了全社会的广泛关注。MCs进入生物体后会靶向攻击肝细胞,并特异性抑制蛋白磷酸酶(Protein Phosphatase,PPs)的生物活性,打破多种功能蛋白磷酸化-去磷酸化平衡,进而造成细胞生理功能紊乱、损伤甚至凋亡坏死。鉴于MCs存在显著的生物毒性,控制MCs浓度水平、阐明其毒性作用机制、探讨解毒机理已成为环境科学工作者的研究热点。目前关于MCs毒性作用调控的各种策略已经被广泛实施,但由于具体的分子调控机制尚未阐明,制约了相应调控策略的应用。例如:MCs存在80余种同系物,不同类别毒素对PPs的抑制作用存在一定差异,有关其致毒的种间差异机制研究尚未开展;目前饮用水消毒是调控MCs的关键技术,该过程产生的部分特征消毒副产物可能保留原毒素的结构及毒性,消毒副产物转化解毒的分子机制尚不明确;生物解毒研究证实生物体内MCs能够在谷胱甘肽S-转移酶的催化作用下与谷胱甘肽共价结合生成低毒性复合物,但其具体的解毒机制尚未阐明。本研究通过分析MCs污染特性、结构理化特征、毒性作用机制、调控策略的技术问题,以MCLR为基础研究物,通过传统氯消毒工艺和碳碳双键-巯醇的亲电加成反应分别制取MCLR-DBPs和MC-GSHs;借助分子毒性试验和分子对接技术对MCs、MCLR/MCLR-DBPs、MCs/MC-GSHs与PP1结合物的毒性作用和结构特征进行解析;将理论模拟数据与毒性试验结果相对照,探究MCs、MCLR-DBPs、MC-GSHs抑制PP1生物活性毒性作用差异机制,建立了对MCs及其衍生物毒性效应进行全面评价的新方法。主要研究工作如下:(1)微囊藻毒素同系物抑制蛋白磷酸酶PP1生物活性种间差异机制研究借助分子毒性试验获得MCs对PP1生物活性的抑制作用顺序为:MCLR(2.6μg/L)MCLF(4.4μg/L)MCLA(5.53μg/L)MCLY(7.9μg/L)MCLW(13.6μg/L)。通过MOE分子模拟对MCLR-PP1进行结构改造得到其它毒素与PP1的结合产物并进行分子对接,可以从毒物配体与PP1受体结合前后整体能量变化、结合面积、氢键、共价键、Mn~(2+)参与的离子键作用层次对MCs抑制PP1生物活性的差异机制进行阐释。结果表明:可变氨基酸位点Z4的亲疏水性与空间位阻效应能够干扰MCs对PP1的毒性抑制作用(影响配体Adda5残基与PP1的结合),使得Adda5与PP1的结合面积和毒素毒性变化存在正相关;氢键、离子键等相互作用及亲疏水性的差异与MCs-PP1结合物的能量变化具有一定的相关性(且亲水性作用对结合能量的变化影响大于疏水作用),MCs与PP1对接后整体能量下降程度与毒物毒性变化正相关;极性作用和疏水作用对MCs与PP1结合产物的氢键形成存在一定竞争且空间位阻对氢键作用存在一定影响;相对于MCLR与PP1的氢键作用而言,Z4→Glu275氢键与毒物毒性变化正相关,Leu2←Arg96、Iso Asp3←Arg96和Iso Asp3←Tyr134氢键与毒性变化负相关;对于Mn~(2+)参与的离子键作用而言,Mn~(2+)-His173、Mn~(2+)-Asn124、Mn~(2+)-Asp92及整体离子键作用与PP1活性正相关,与MCs毒性变化存在负相关性。(2)MCLR典型消毒副产物抑制蛋白磷酸酶PP1生物活性差异机制研究借鉴传统氯消毒工艺制备MCLR-DBPs,借助分子毒性试验获得MCLR及MCLR-DBPs对PP1生物活性的抑制顺序为:MCLR(2.6μg/L)P3(21.3μg/L)P1(32.7μg/L)P4(73.8μg/L)P2(113.7μg/L)P5。采用MOE分子模拟技术,对MCLR-PP1进行结构改造分别得到各MCLR-DBPs与PP1结合物,再分别对其进行对接模拟。研究发现:配体、Adda5残基与PP1的结合面积及配体与PP1的整体能量变化和毒物毒性变化正相关,表明消毒过程通过破坏毒素(特别是Adda5)与PP1的结合,从而削弱其毒性;消毒副产物参与的Leu2←Arg96氢键作用受到抑制,同时Mdha7←Glu275、Iso Asp3←Arg96、Mdha7←Gly274、Adda5←Arg221和Glu6←His125氢键作用得到强化,从而削弱了MCLR的生物毒性;配体与PP1的共价键Mdha7-Cys273和毒性变化不具有明显相关性,但PP1的分子内Cys273-Asn278和毒物毒性具有负相关性,表明消毒过程促进了该作用,从而间接削弱MCs的生物毒性;对于Mn~(2+)参与的离子键作用而言,MCLR-DBPs与PP1的离子键Mn~(2+)-His173、Mn~(2+)-His248、Mn~(2+)-Asp64及整体离子键作用明显增加,与PP1的活性正相关,表明MCLR向MCLR-DBPs的转化强化了上述指标从而实现毒素毒性的降低。(3)微囊藻毒素与谷胱甘肽结合产物抑制蛋白磷酸酶PP1生物活性差异机制研究模拟亲电加成反应制取MCLR-GSH和MCRR-GSH,通过分子毒性试验确定MCs/MC-GSHs对PP1生物活性的抑制顺序为:MCLR(2.6μg/L)MCRR(23.3μg/L)MCLR-GSH(83.4μg/L)MCRR-GSH(95.1μg/L)。利用MOE分子对接软件,首先对MCLR-PP1进行结构改造分别得到MCRR、MCLR-GSH、MCRR-GSH与PP1结合物,再分别进行对接模拟。结果表明:Adda5残基与PP1的结合面积和毒素对PP1毒性作用正相关,表明引入GSH通过破坏Mdha7与Cys273的共价结合,减弱了Adda5残基与PP1作用,削弱毒素毒性;引入GSH通过减弱MC-GSHs和PP1及H2O的共同作用、X2←Arg96、Glu6?Tyr272、Arg4→Glu275氢键或促进Mdha7-GSH→Asn278、Mdha7-GSH→Asn271、Adda5←Arg221氢键作用,降低毒素毒性;引入GSH后Cys273-Mdha7共价作用减小为零,表明GSH与MCs的Mdha7结合抑制该共价作用,从而削弱毒素毒性;对于Mn~(2+)参与的离子键作用而言,引入GSH,增强Mn~(2+)-His173和Mn~(2+)-Asp92离子键及减弱Mn~(2+)-Asn124、Mn~(2+)-His248、Mn~(2+)-Asp64和Mn~(2+)-His66离子键作用,从而降低原毒素毒性。
[Abstract]:Microcystin (MCs), as a kind of biological toxicity, is a kind of organic pollutant which is hard to degrade, which has attracted wide attention from the whole society. The MCs can target the liver cells and specifically inhibit the biological activity of the protein phosphatase (PPs) and break the phosphorylation-dephosphorylation of various functional proteins, thus causing the physiological functions of the cells to be disordered, damage and even apoptosis. In view of the significant biological toxicity of MCs, the control of the concentration of MCs, the mechanism of its toxicity, and the study of the mechanism of detoxication have become the research focus of environmental scientists. At present, the various strategies on the regulation of the toxicity of the MCs have been widely applied, but the specific molecular control mechanism has not yet been clarified, and the application of the corresponding regulation strategies has been restricted. For example, there are more than 80 homologs of MCs, and there are some differences in the inhibitory effect of different types of toxin on PPs, and the study on the mechanism of interspecific differential mechanism of its toxicity has not been carried out. At present, the disinfection of drinking water is the key technology for regulating the MCs. the part of the characteristic disinfection by-products produced by the process can retain the structure and the toxicity of the original toxin, and the molecular mechanism of the disinfection by-product transformation and detoxification is not clear; The study of biological detoxification confirmed that the MCs in the organism can be covalently bound with glutathione to form the low-toxicity complex under the catalysis of glutathione S-transferase, but the specific detoxification mechanism has not yet been clarified. In this study, MCLR-DBPs and MC-GSHs were prepared by analyzing the characteristics of MCs, the physical and chemical characteristics of the structure, the mechanism of toxicity, and the control strategy. The toxicity and structural characteristics of MCs, MCLR/ MCLR-DBPs, MCs/ MC-GSHs and PP1 conjugates were analyzed by means of molecular toxicity test and molecular docking technique. The results of the theoretical simulation data were compared with the results of the toxicity test to investigate the mechanism of the difference of the biological activity of PP1 in the MCs, MCLR-DBPs, and MC-GSHs. A new method for comprehensive evaluation of the toxic effects of MCs and its derivatives was established. The main research work is as follows: (1) The mechanism of the differential mechanism between the microcystin homolog-inhibiting protein phosphatase PP1 biological active species is: MCLR (2.6. mu.g/ L) MCLF (4.4. mu.g/ L) MCLF (4.53. mu.g/ L) MCLY (7.9. mu.g/ L) MCLW (13.mu. g/ L) MCLW (13.6. mu.g/ L). The MCLR-PP1 is structurally modified by the MOE molecular simulation to obtain the binding product of the other toxin and the PP1 and is subjected to molecular docking, and the whole energy change, the binding area, the hydrogen bond and the covalent bond can be combined before and after the combination of the poison ligand and the PP1 receptor, The effect of Mn ~ (2 +) on the mechanism of MCs inhibition of PP1 biological activity was explained. The results showed that the lipophilicity and steric hindrance effect of the variable amino acid site Z4 can interfere with the toxicity inhibition of the MCs on PP1 (the binding of the ligand Ada5 residue to the PP1), so that the binding area of the Adda5 and the PP1 and the toxic change of the toxin are positively related; and the hydrogen bond, The difference of the interaction of ionic bonds and the affinity between the affinity and the hydrophobicity has a certain correlation with the energy change of the MCs-PP1 conjugate (and the influence of the hydrophilic action on the combined energy is greater than that of the hydrophobic function), and the overall energy drop after the Ms and PP1 are in butt joint is positively related to the change of the toxicity of the poison; The effects of polar action and hydrophobic interaction on the formation of hydrogen bonds of the binding products of MCs and PP1 are competitive and the steric hindrance has a certain effect on the role of hydrogen bonds; for the hydrogen bonding of the MCLR and PP1, the hydrogen bond of Z4-Glu275 is positively related to the change of the toxicity of the poison, and Leu2-Arg96, There was a negative correlation between the hydrogen bond and the toxicity of Iso Asp3-Arg96 and Iso Asp3-Tyr134; for the ion-bond effect of Mn ~ (2 +), Mn ~ (2 +)-His3, Mn ~ (2 +)-Asn124, Mn ~ (2 +)-Asp92 and the total ionic bond effect were positively related to the activity of PP1, and there was a negative correlation with the change of MCs toxicity. (2) The mechanism of the differential mechanism of the MCLR typical disinfection by-product to inhibit the biological activity of the protein phosphatase PP1 is to use the traditional chlorine disinfection process to prepare the MCLR-DBPs, and the inhibition sequence of the MCLR and the MCLR-DBPs to the PP1 biological activity by means of the molecular toxicity test is: MCLR (2.6. mu.g/ L) P3 (21.3. mu.g/ L) P1 (32.7. mu.g/ L) P4 (73.8. mu.g/ L) P2 (113.7. mu.g/ L) P5. MCLR-DBPs and PP1 conjugate were obtained by using MOE molecular simulation technique, and then the MCLR-DBPs and PP1 conjugate were obtained. It was found that the binding area of the ligand, the Ada5 residue and the PP1 and the change of the total energy of the ligand to the PP1 and the change of the toxicity of the poison were positively correlated, indicating that the disinfection process reduced its toxicity by destroying the binding of the toxin (especially the Add5) to the PP1. The hydrogen bonding of Leu2-Arg96, which was involved in the disinfection by-products, was inhibited, while the hydrogen bonding of Mdha7-Glu275, Iso Asp3-Arg96, Mdha7-Gly274, Adda5-Arg221 and Glu6-His125 was enhanced, so that the biotoxicity of the MCLR was impaired; the covalent bond of the ligand to PP1, Mdha7-Cys273, and the change in toxicity did not have a significant correlation, However, there was a negative correlation between Cys273-Asn278 and the toxicity of PP1, indicating that the disinfection process promoted the effect, thus indirectly weakening the biological toxicity of the MCs; for the ionic bonding effect of Mn ~ (2 +), the ionic bond Mn ~ (2 +)-His173, Mn ~ (2 +)-His248, Mn ~ (2 +)-Asp64 and the total ionic bonding of the MCLR-DBPs and PP1 increased significantly. The activity of the MCLR to the MCLR-DBPs is positively related to the activity of PP1, indicating that the transformation of the MCLR to the MCLR-DBPs enhances the above-mentioned index to achieve a reduction in the toxicity of the toxin. (3) The inhibitory sequence of MCs/ MC-GSHs on PP1 biological activity was MCLR (2.6. mu.g/ L) MCRR (23.3. mu.g/ L) MCLR-GSH (83.4. mu.g/ L) MCRR-GSH (95.1. mu.g/ L) by molecular toxicity test. MCRR, MCLR-GSH, MCRR-GSH and PP1 conjugate were obtained from MCLR-PP1 respectively by using MOE molecular docking software. The results showed that the binding area and toxin of Ada5 residue and PP1 were positively related to the toxicity of PP1, indicating that the introduction of GSH was related to the covalent binding of Mdha7 and Cys273, and the effect of Ada5 residue and PP1 was reduced, and the toxicity of toxin was reduced; and GSH was introduced to reduce the co-action of MC-GSHs and PP1 and H2O, X2-Arg96, Glu6-Tyr272, The hydrogen bonding of Arg4-Glu275 or the promotion of the hydrogen bonding of Mdha7-GSH-Asn278, Mdha7-GSH-Asn271 and Ada5-Arg221 reduced the toxicity of the toxin, and the covalent interaction of the Cys273-Mdha7 after the introduction of GSH was reduced to zero, indicating that the combination of GSH and Mdha7 of the MCs inhibited the covalent interaction, thereby impairing the toxin toxicity; and for the ion-bonding effect of the participation of Mn-(2 +), The effects of GSH, Mn ~ (2 +)-His173 and Mn ~ (2 +)-Asp92 ionic bonding and the reduction of Mn ~ (2 +)-Asn124, Mn ~ (2 +)-His248, Mn ~ (2 +)-Asp64 and Mn ~ (2 +)-His66 were introduced to reduce the toxicity of the original toxin.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TU991.25
本文编号：2478804
[Abstract]:Microcystin (MCs), as a kind of biological toxicity, is a kind of organic pollutant which is hard to degrade, which has attracted wide attention from the whole society. The MCs can target the liver cells and specifically inhibit the biological activity of the protein phosphatase (PPs) and break the phosphorylation-dephosphorylation of various functional proteins, thus causing the physiological functions of the cells to be disordered, damage and even apoptosis. In view of the significant biological toxicity of MCs, the control of the concentration of MCs, the mechanism of its toxicity, and the study of the mechanism of detoxication have become the research focus of environmental scientists. At present, the various strategies on the regulation of the toxicity of the MCs have been widely applied, but the specific molecular control mechanism has not yet been clarified, and the application of the corresponding regulation strategies has been restricted. For example, there are more than 80 homologs of MCs, and there are some differences in the inhibitory effect of different types of toxin on PPs, and the study on the mechanism of interspecific differential mechanism of its toxicity has not been carried out. At present, the disinfection of drinking water is the key technology for regulating the MCs. the part of the characteristic disinfection by-products produced by the process can retain the structure and the toxicity of the original toxin, and the molecular mechanism of the disinfection by-product transformation and detoxification is not clear; The study of biological detoxification confirmed that the MCs in the organism can be covalently bound with glutathione to form the low-toxicity complex under the catalysis of glutathione S-transferase, but the specific detoxification mechanism has not yet been clarified. In this study, MCLR-DBPs and MC-GSHs were prepared by analyzing the characteristics of MCs, the physical and chemical characteristics of the structure, the mechanism of toxicity, and the control strategy. The toxicity and structural characteristics of MCs, MCLR/ MCLR-DBPs, MCs/ MC-GSHs and PP1 conjugates were analyzed by means of molecular toxicity test and molecular docking technique. The results of the theoretical simulation data were compared with the results of the toxicity test to investigate the mechanism of the difference of the biological activity of PP1 in the MCs, MCLR-DBPs, and MC-GSHs. A new method for comprehensive evaluation of the toxic effects of MCs and its derivatives was established. The main research work is as follows: (1) The mechanism of the differential mechanism between the microcystin homolog-inhibiting protein phosphatase PP1 biological active species is: MCLR (2.6. mu.g/ L) MCLF (4.4. mu.g/ L) MCLF (4.53. mu.g/ L) MCLY (7.9. mu.g/ L) MCLW (13.mu. g/ L) MCLW (13.6. mu.g/ L). The MCLR-PP1 is structurally modified by the MOE molecular simulation to obtain the binding product of the other toxin and the PP1 and is subjected to molecular docking, and the whole energy change, the binding area, the hydrogen bond and the covalent bond can be combined before and after the combination of the poison ligand and the PP1 receptor, The effect of Mn ~ (2 +) on the mechanism of MCs inhibition of PP1 biological activity was explained. The results showed that the lipophilicity and steric hindrance effect of the variable amino acid site Z4 can interfere with the toxicity inhibition of the MCs on PP1 (the binding of the ligand Ada5 residue to the PP1), so that the binding area of the Adda5 and the PP1 and the toxic change of the toxin are positively related; and the hydrogen bond, The difference of the interaction of ionic bonds and the affinity between the affinity and the hydrophobicity has a certain correlation with the energy change of the MCs-PP1 conjugate (and the influence of the hydrophilic action on the combined energy is greater than that of the hydrophobic function), and the overall energy drop after the Ms and PP1 are in butt joint is positively related to the change of the toxicity of the poison; The effects of polar action and hydrophobic interaction on the formation of hydrogen bonds of the binding products of MCs and PP1 are competitive and the steric hindrance has a certain effect on the role of hydrogen bonds; for the hydrogen bonding of the MCLR and PP1, the hydrogen bond of Z4-Glu275 is positively related to the change of the toxicity of the poison, and Leu2-Arg96, There was a negative correlation between the hydrogen bond and the toxicity of Iso Asp3-Arg96 and Iso Asp3-Tyr134; for the ion-bond effect of Mn ~ (2 +), Mn ~ (2 +)-His3, Mn ~ (2 +)-Asn124, Mn ~ (2 +)-Asp92 and the total ionic bond effect were positively related to the activity of PP1, and there was a negative correlation with the change of MCs toxicity. (2) The mechanism of the differential mechanism of the MCLR typical disinfection by-product to inhibit the biological activity of the protein phosphatase PP1 is to use the traditional chlorine disinfection process to prepare the MCLR-DBPs, and the inhibition sequence of the MCLR and the MCLR-DBPs to the PP1 biological activity by means of the molecular toxicity test is: MCLR (2.6. mu.g/ L) P3 (21.3. mu.g/ L) P1 (32.7. mu.g/ L) P4 (73.8. mu.g/ L) P2 (113.7. mu.g/ L) P5. MCLR-DBPs and PP1 conjugate were obtained by using MOE molecular simulation technique, and then the MCLR-DBPs and PP1 conjugate were obtained. It was found that the binding area of the ligand, the Ada5 residue and the PP1 and the change of the total energy of the ligand to the PP1 and the change of the toxicity of the poison were positively correlated, indicating that the disinfection process reduced its toxicity by destroying the binding of the toxin (especially the Add5) to the PP1. The hydrogen bonding of Leu2-Arg96, which was involved in the disinfection by-products, was inhibited, while the hydrogen bonding of Mdha7-Glu275, Iso Asp3-Arg96, Mdha7-Gly274, Adda5-Arg221 and Glu6-His125 was enhanced, so that the biotoxicity of the MCLR was impaired; the covalent bond of the ligand to PP1, Mdha7-Cys273, and the change in toxicity did not have a significant correlation, However, there was a negative correlation between Cys273-Asn278 and the toxicity of PP1, indicating that the disinfection process promoted the effect, thus indirectly weakening the biological toxicity of the MCs; for the ionic bonding effect of Mn ~ (2 +), the ionic bond Mn ~ (2 +)-His173, Mn ~ (2 +)-His248, Mn ~ (2 +)-Asp64 and the total ionic bonding of the MCLR-DBPs and PP1 increased significantly. The activity of the MCLR to the MCLR-DBPs is positively related to the activity of PP1, indicating that the transformation of the MCLR to the MCLR-DBPs enhances the above-mentioned index to achieve a reduction in the toxicity of the toxin. (3) The inhibitory sequence of MCs/ MC-GSHs on PP1 biological activity was MCLR (2.6. mu.g/ L) MCRR (23.3. mu.g/ L) MCLR-GSH (83.4. mu.g/ L) MCRR-GSH (95.1. mu.g/ L) by molecular toxicity test. MCRR, MCLR-GSH, MCRR-GSH and PP1 conjugate were obtained from MCLR-PP1 respectively by using MOE molecular docking software. The results showed that the binding area and toxin of Ada5 residue and PP1 were positively related to the toxicity of PP1, indicating that the introduction of GSH was related to the covalent binding of Mdha7 and Cys273, and the effect of Ada5 residue and PP1 was reduced, and the toxicity of toxin was reduced; and GSH was introduced to reduce the co-action of MC-GSHs and PP1 and H2O, X2-Arg96, Glu6-Tyr272, The hydrogen bonding of Arg4-Glu275 or the promotion of the hydrogen bonding of Mdha7-GSH-Asn278, Mdha7-GSH-Asn271 and Ada5-Arg221 reduced the toxicity of the toxin, and the covalent interaction of the Cys273-Mdha7 after the introduction of GSH was reduced to zero, indicating that the combination of GSH and Mdha7 of the MCs inhibited the covalent interaction, thereby impairing the toxin toxicity; and for the ion-bonding effect of the participation of Mn-(2 +), The effects of GSH, Mn ~ (2 +)-His173 and Mn ~ (2 +)-Asp92 ionic bonding and the reduction of Mn ~ (2 +)-Asn124, Mn ~ (2 +)-His248, Mn ~ (2 +)-Asp64 and Mn ~ (2 +)-His66 were introduced to reduce the toxicity of the original toxin.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TU991.25
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2 赵慧;微囊藻毒素的树脂吸附与原位监测方法研究[D];中国海洋大学;2012年
3 张琼;微囊藻毒素MC-LR致小鼠糖代谢紊乱的作用过程和机理[D];南京大学;2012年
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