【摘要】：微囊藻毒素(Microcystins,MCs)作為一類(lèi)生物毒性明顯、難降解的有機(jī)污染物已引起了全社會(huì)的廣泛關(guān)注。MCs進(jìn)入生物體后會(huì)靶向攻擊肝細(xì)胞,并特異性抑制蛋白磷酸酶(Protein Phosphatase,PPs)的生物活性,打破多種功能蛋白磷酸化-去磷酸化平衡,進(jìn)而造成細(xì)胞生理功能紊亂、損傷甚至凋亡壞死。鑒于MCs存在顯著的生物毒性,控制MCs濃度水平、闡明其毒性作用機(jī)制、探討解毒機(jī)理已成為環(huán)境科學(xué)工作者的研究熱點(diǎn)。目前關(guān)于MCs毒性作用調(diào)控的各種策略已經(jīng)被廣泛實(shí)施,但由于具體的分子調(diào)控機(jī)制尚未闡明,制約了相應(yīng)調(diào)控策略的應(yīng)用。例如:MCs存在80余種同系物,不同類(lèi)別毒素對(duì)PPs的抑制作用存在一定差異,有關(guān)其致毒的種間差異機(jī)制研究尚未開(kāi)展;目前飲用水消毒是調(diào)控MCs的關(guān)鍵技術(shù),該過(guò)程產(chǎn)生的部分特征消毒副產(chǎn)物可能保留原毒素的結(jié)構(gòu)及毒性,消毒副產(chǎn)物轉(zhuǎn)化解毒的分子機(jī)制尚不明確;生物解毒研究證實(shí)生物體內(nèi)MCs能夠在谷胱甘肽S-轉(zhuǎn)移酶的催化作用下與谷胱甘肽共價(jià)結(jié)合生成低毒性復(fù)合物,但其具體的解毒機(jī)制尚未闡明。本研究通過(guò)分析MCs污染特性、結(jié)構(gòu)理化特征、毒性作用機(jī)制、調(diào)控策略的技術(shù)問(wèn)題,以MCLR為基礎(chǔ)研究物,通過(guò)傳統(tǒng)氯消毒工藝和碳碳雙鍵-巰醇的親電加成反應(yīng)分別制取MCLR-DBPs和MC-GSHs;借助分子毒性試驗(yàn)和分子對(duì)接技術(shù)對(duì)MCs、MCLR/MCLR-DBPs、MCs/MC-GSHs與PP1結(jié)合物的毒性作用和結(jié)構(gòu)特征進(jìn)行解析;將理論模擬數(shù)據(jù)與毒性試驗(yàn)結(jié)果相對(duì)照,探究MCs、MCLR-DBPs、MC-GSHs抑制PP1生物活性毒性作用差異機(jī)制,建立了對(duì)MCs及其衍生物毒性效應(yīng)進(jìn)行全面評(píng)價(jià)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生物活性種間差異機(jī)制研究借助分子毒性試驗(yàn)獲得MCs對(duì)PP1生物活性的抑制作用順序?yàn)?MCLR(2.6μg/L)MCLF(4.4μg/L)MCLA(5.53μg/L)MCLY(7.9μg/L)MCLW(13.6μg/L)。通過(guò)MOE分子模擬對(duì)MCLR-PP1進(jìn)行結(jié)構(gòu)改造得到其它毒素與PP1的結(jié)合產(chǎn)物并進(jìn)行分子對(duì)接,可以從毒物配體與PP1受體結(jié)合前后整體能量變化、結(jié)合面積、氫鍵、共價(jià)鍵、Mn~(2+)參與的離子鍵作用層次對(duì)MCs抑制PP1生物活性的差異機(jī)制進(jìn)行闡釋。結(jié)果表明:可變氨基酸位點(diǎn)Z4的親疏水性與空間位阻效應(yīng)能夠干擾MCs對(duì)PP1的毒性抑制作用(影響配體Adda5殘基與PP1的結(jié)合),使得Adda5與PP1的結(jié)合面積和毒素毒性變化存在正相關(guān);氫鍵、離子鍵等相互作用及親疏水性的差異與MCs-PP1結(jié)合物的能量變化具有一定的相關(guān)性(且親水性作用對(duì)結(jié)合能量的變化影響大于疏水作用),MCs與PP1對(duì)接后整體能量下降程度與毒物毒性變化正相關(guān);極性作用和疏水作用對(duì)MCs與PP1結(jié)合產(chǎn)物的氫鍵形成存在一定競(jìng)爭(zhēng)且空間位阻對(duì)氫鍵作用存在一定影響;相對(duì)于MCLR與PP1的氫鍵作用而言,Z4→Glu275氫鍵與毒物毒性變化正相關(guān),Leu2←Arg96、Iso Asp3←Arg96和Iso Asp3←Tyr134氫鍵與毒性變化負(fù)相關(guān);對(duì)于Mn~(2+)參與的離子鍵作用而言,Mn~(2+)-His173、Mn~(2+)-Asn124、Mn~(2+)-Asp92及整體離子鍵作用與PP1活性正相關(guān),與MCs毒性變化存在負(fù)相關(guān)性。(2)MCLR典型消毒副產(chǎn)物抑制蛋白磷酸酶PP1生物活性差異機(jī)制研究借鑒傳統(tǒng)氯消毒工藝制備MCLR-DBPs,借助分子毒性試驗(yàn)獲得MCLR及MCLR-DBPs對(duì)PP1生物活性的抑制順序?yàn)?MCLR(2.6μg/L)P3(21.3μg/L)P1(32.7μg/L)P4(73.8μg/L)P2(113.7μg/L)P5。采用MOE分子模擬技術(shù),對(duì)MCLR-PP1進(jìn)行結(jié)構(gòu)改造分別得到各MCLR-DBPs與PP1結(jié)合物,再分別對(duì)其進(jìn)行對(duì)接模擬。研究發(fā)現(xiàn):配體、Adda5殘基與PP1的結(jié)合面積及配體與PP1的整體能量變化和毒物毒性變化正相關(guān),表明消毒過(guò)程通過(guò)破壞毒素(特別是Adda5)與PP1的結(jié)合,從而削弱其毒性;消毒副產(chǎn)物參與的Leu2←Arg96氫鍵作用受到抑制,同時(shí)Mdha7←Glu275、Iso Asp3←Arg96、Mdha7←Gly274、Adda5←Arg221和Glu6←His125氫鍵作用得到強(qiáng)化,從而削弱了MCLR的生物毒性;配體與PP1的共價(jià)鍵Mdha7-Cys273和毒性變化不具有明顯相關(guān)性,但PP1的分子內(nèi)Cys273-Asn278和毒物毒性具有負(fù)相關(guān)性,表明消毒過(guò)程促進(jìn)了該作用,從而間接削弱MCs的生物毒性;對(duì)于Mn~(2+)參與的離子鍵作用而言,MCLR-DBPs與PP1的離子鍵Mn~(2+)-His173、Mn~(2+)-His248、Mn~(2+)-Asp64及整體離子鍵作用明顯增加,與PP1的活性正相關(guān),表明MCLR向MCLR-DBPs的轉(zhuǎn)化強(qiáng)化了上述指標(biāo)從而實(shí)現(xiàn)毒素毒性的降低。(3)微囊藻毒素與谷胱甘肽結(jié)合產(chǎn)物抑制蛋白磷酸酶PP1生物活性差異機(jī)制研究模擬親電加成反應(yīng)制取MCLR-GSH和MCRR-GSH,通過(guò)分子毒性試驗(yàn)確定MCs/MC-GSHs對(duì)PP1生物活性的抑制順序?yàn)?MCLR(2.6μg/L)MCRR(23.3μg/L)MCLR-GSH(83.4μg/L)MCRR-GSH(95.1μg/L)。利用MOE分子對(duì)接軟件,首先對(duì)MCLR-PP1進(jìn)行結(jié)構(gòu)改造分別得到MCRR、MCLR-GSH、MCRR-GSH與PP1結(jié)合物,再分別進(jìn)行對(duì)接模擬。結(jié)果表明:Adda5殘基與PP1的結(jié)合面積和毒素對(duì)PP1毒性作用正相關(guān),表明引入GSH通過(guò)破壞Mdha7與Cys273的共價(jià)結(jié)合,減弱了Adda5殘基與PP1作用,削弱毒素毒性;引入GSH通過(guò)減弱MC-GSHs和PP1及H2O的共同作用、X2←Arg96、Glu6?Tyr272、Arg4→Glu275氫鍵或促進(jìn)Mdha7-GSH→Asn278、Mdha7-GSH→Asn271、Adda5←Arg221氫鍵作用,降低毒素毒性;引入GSH后Cys273-Mdha7共價(jià)作用減小為零,表明GSH與MCs的Mdha7結(jié)合抑制該共價(jià)作用,從而削弱毒素毒性;對(duì)于Mn~(2+)參與的離子鍵作用而言,引入GSH,增強(qiáng)Mn~(2+)-His173和Mn~(2+)-Asp92離子鍵及減弱Mn~(2+)-Asn124、Mn~(2+)-His248、Mn~(2+)-Asp64和Mn~(2+)-His66離子鍵作用,從而降低原毒素毒性。
[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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TU991.25

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