利用分子模拟方法对DHCR24抑制剂的抑制作用及基因致病点突变的分子机制研究

发布时间：2018-05-10 16:59

本文选题：DHCR24 + U18666a　；参考：《辽宁大学》2016年硕士论文

【摘要】：3p-脱氢胆固醇-△24还原酶(DHCR24),是人体内胆固醇合成最终阶段的酶,它催化胆甾醇还原为胆固醇,同时也具有抗细胞凋亡的作用。U18666a是DHCR24催化胆甾醇生成胆固醇反应的有效非竞争性抑制剂,但抑制机制不明。在人体内DHCR24基因变异(Y471S、N294T、K306N、E191K)引起的疾病称作胆甾醇血症,呈现多发性先天畸形与发育异常,但变异位点如何影响DHCR24活性目前尚无报道。本研究用分子模拟的方法系统考察U18666a与DHCR24相互作用的分子机制；同时,用点突变及分子模拟的方法研究单点突变Y471S、N294T、K306N、E191K和两点突变N294T/K306N对DHCR24结构和活性的影响。在U18666a与DHCR24相互作用研究中,使用同源建模、分子对接和分子动力学模拟方法的综合结果显示,与不加入U18666a的复合物相比较,加U18666a后,底物胆甾醇(desmosterol,des)与DHCR24结合能(亲和性)增加。二级结构变化分析表明,加U18666a后,DHCR24二级结构发生较显著的变化,进而影响FAD、des与DHCR24相互作用。这些结果暗示U18666a的添加,通过引起DHCR24的二级结构变化,使des与DHCR24亲和性发生改变从而抑制酶催化活性,即从分子水平证明了U18666a对DHCR24酶的抑制作用为别构抑制,这与先前发表的酶活性测定的湿实验结论一致。在突变位点对DHCR24结构影响的研究中,自由能计算结果显示,与野生型DHCR24-fad-des体系相比,五个突变体系中的底物des与蛋白复合物的结合能力均有增加的趋势。二级结构变化结果分析,N294T/K306N突变体系的二级结构变化最大,也最大程度影响了酶与底物的结合活性,这与文献报道的此突变体抑制DHCR24酶活性降低以及临床引起的胆甾醇血症的症状最严重这一发现相一致。
[Abstract]:3p- dehydrogencholesterol-24 reductase DHCR24, which catalyzes the reduction of cholesterol from cholesterol to cholesterol, is the last stage of cholesterol synthesis in human body. U18666a is an effective non-competitive inhibitor of cholesterol-producing reaction catalyzed by DHCR24, but its mechanism is unclear. The disease caused by the mutation of DHCR24 gene (Y471SU N294TK306NP191K) in human body is called cholesterolemia, which presents multiple congenital malformation and abnormal development. However, there is no report on how the mutation site affects the activity of DHCR24. In this study, the molecular mechanism of the interaction between U18666a and DHCR24 was systematically investigated by molecular simulation, and the effects of point mutation and molecular simulation on the structure and activity of DHCR24 were studied by single point mutation Y471SN 294T K306NU E191K and two point mutated N294T/K306N. In the study of the interaction between U18666a and DHCR24, the synthesis results of homologous modeling, molecular docking and molecular dynamics simulation showed that compared with the complexes without U18666a, the binding energy (affinity) between the substrate and DHCR24 increased after the addition of U18666a. The analysis of secondary structure changes showed that the secondary structure of DHCR24 changed significantly after U18666a, which affected the interaction between Faddes and DHCR24. These results suggest that the addition of U18666a inhibits the catalytic activity of des by causing changes in the secondary structure of DHCR24 and the affinity of des to DHCR24, that is, the inhibition of U18666a on DHCR24 enzyme is shown to be allosteric inhibition at the molecular level. This is consistent with the previously published results of wet experiments for the determination of enzyme activity. In the study of the effect of mutation sites on the structure of DHCR24, the results of free energy calculation showed that the binding ability of substrates des and protein complexes in the five mutant systems was increased compared with the wild type DHCR24-fad-des system. Analysis of the results of secondary structure changes in the N294T / K306N mutation system, the secondary structure changes were the largest, and the binding activity of the enzyme to the substrate was affected to the greatest extent. This is consistent with the reported findings that the mutant inhibits the decrease of DHCR24 enzyme activity and the most severe symptoms of clinically induced choleridemia.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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