分子对接模拟酵母Pdr5p蛋白E574K突变致外排功能障碍的机制

发布时间：2018-11-02 16:14

【摘要】：近年来,由于器官移植病例的大量增加、癌症的化学治疗和广谱抗生素的大量使用等原因,真菌感染风险逐年增加,已成为许多疾病致死的主要原因。同时,抗真菌药物的大量使用也使得真菌的耐药性日益增强,为临床治疗带来了极大的挑战。而ABC转运蛋白能够将多种药物外排出细胞,是真菌多药耐药性的主要原因,也是导致药物治疗效果下降的主要原因。目前,真菌转运蛋白的转运机制还不是很清楚,也为药物设计增加了难度。在本论文中,我们把酵母跨膜蛋白Pdr5p作为研究对象。酵母Pdr5p蛋白在结构和功能上与白色念珠菌以及哺乳动物药物耐受相关的ABC转运蛋白都有较高的相似性,是比较理想的研究对象。而且有研究证明跨膜螺旋末端特定位点的突变会使Pdr5p失去转运活性。根据合作实验室的结果,新发现了两个突变——E574K和E580K,能够在不破坏Pdr5p表达、膜定位和ATP酶活性的情况下,使酵母对多个药物敏感性显著增加。为了进一步解释现象背后的原理,我们借助生物信息学工具(分子对接)模拟了蛋白质与药物分子的结合。发现E574K突变会破坏Pdr5p与底物间的氢键和静电相互作用力,干扰底物与受体活性位点的结合；而E580K突变可能是通过干扰底物被Pdr5p识别的过程,使底物进出转运通道受阻。E574和E580都在TMH2上,证明TMH2在药物外排过程中发挥着重要作用。生物学的实验方法和生物信息学的计算方法基于不同的研究条件和策略,他们可以很好的互补。而且近年来随着各自技术的发展,两种方法的交叉越来越多。计算可以为实验节约成本和时间,实验可以修正和优化计算方法和策略。两种方法的结合使用不仅能够帮助我们更好地理解一些生物学问题,更可以为合理药物设计提供指导,有着广阔的应用前景。
[Abstract]:In recent years, the risk of fungal infection has increased year by year because of the increase of organ transplantation cases, chemotherapy of cancer and extensive use of broad-spectrum antibiotics, which has become the main cause of death of many diseases. At the same time, the extensive use of antifungal drugs also makes the drug resistance of fungi increasing day by day, which brings great challenges to the clinical treatment. ABC transporter is the main reason of drug resistance and the main reason of drug treatment. At present, the mechanism of fungal transporter transport is not very clear, which makes drug design more difficult. In this thesis, we studied yeast transmembrane protein Pdr5p. Yeast Pdr5p protein has high similarity in structure and function with Candida albicans and mammalian drug resistance-related ABC transporter, which is an ideal research object. Moreover, some studies have shown that transmembrane helical terminal specific site mutation may cause Pdr5p to lose its transport activity. Two new mutations, E574K and E580K, were found according to the results of co-laboratory, which could significantly increase the sensitivity of yeast to many drugs without destroying Pdr5p expression, membrane localization and ATP enzyme activity. To further explain the underlying principles of the phenomenon, we use bioinformatics tools (molecular docking) to simulate the binding of proteins to drug molecules. It was found that the E574K mutation would destroy the hydrogen bond and electrostatic interaction between Pdr5p and substrate and interfere with the binding of substrate to receptor active site. The E580K mutation may be caused by interfering the substrate to be recognized by Pdr5p, which obstructs the transport channel of the substrate. E574 and E580 are both on TMH2, which indicates that TMH2 plays an important role in drug efflux. Experimental methods of biology and computational methods of bioinformatics can complement each other well based on different research conditions and strategies. In recent years, with the development of their respective technologies, the two methods are more and more intersected. The calculation can save the cost and time of the experiment, and the experiment can modify and optimize the calculation method and strategy. The combination of the two methods can not only help us understand some biological problems better, but also provide guidance for rational drug design.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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