Pdr5p跨膜螺旋6和7边界位点突变引起Pdr5p功能受损的机制研究

发布时间：2018-04-14 02:12

本文选题：酿酒酵母 + 多药耐药转运蛋白　；参考：《浙江大学》2012年硕士论文

【摘要】：微生物耐药是抗菌治疗中面临的严峻挑战之一。近年来,致病真菌的多药物耐药现象日益增多,导致免疫缺陷、器官移植等病人抗感染治疗难度增大,治疗费用昂贵,临床致死率较高。导致多药物耐药现象的一个重要原因是细胞中ABC转运蛋白过量表达。Pdr5p是酿酒酵母中重要的ABC转运蛋白,它与许多致病酵母菌株中发现的ABC转运蛋白在结构和功能上高度相似。以Pdr5p为模型,研究ABC转运蛋白介导的多药物耐药性分子机制已成为近年的研究热点。本论文采用PCR介导的随机突变方法,对高表达质粒YEplac195：BPDR5进行突变,在氟康唑药物板上筛选到两个能导致Pdr5p外排泵功能严重受损的单一位点突变C793F和S1230F。其中C793位于预测的Pdr5p跨膜螺旋6(TMH6)的胞内边界上,而S1230位于跨膜螺旋7(TMH7)的胞外边界处。通过研究两个突变株的耐药性,发现其在含有放线菌酮、罗丹明6G、红四氮唑的固体药物板上具有和PDR5缺失菌株相同的耐药表型。在含有上述药物不同浓度的液体培养基中,通过测定相对生长率,发现C793F和S1230F突变株对药物的外排能力也显著下降。Pdr5p特异性ATP酶活性实验和Western blot实验表明793和1230的突变没有导致Pdr5p特异性ATP酶活性改变,突变的Pdr5p依然能够正确定位在细胞膜上且蛋白表达量基本没有受到影响,说明这两个位点突变造成Pdr5p功能受损的原因是氨基酸的差异性。为了进一步研究Pdr5p功能受损的机制,我们通过定点突变分别构建了C793S, C793M,C793Y, S1230A,S1230N, S1230Y等单一位点系列突变株。通过研究这些突变菌株的药物敏感性、Pdr5p特异性ATP酶活以及蛋白定位、表达等,发现所有突变菌株均具有和WT菌株相似的的ATP酶活性和膜定位,但其药物敏感性则与替代的氨基酸的性质相关。实验的结果表明在793位点,氨基酸空间体积改变是导致Pdr5p功能受损的主要因素；在1230位点,氨基酸的空间体积和极性的变化共同影响Pdr5p的外排泵功能。本论文在随机突变的基础上筛选出能导致Pdr5p功能严重受损的两个新突变位点,通过药物筛选和生化证据证实位于跨膜区两端的位点对于Pdr5p的功能至关重要。这些结果为进一步明晰ABC转运蛋白的分子机制提供了新的证据。
[Abstract]:Microbial resistance is one of the severe challenges in antimicrobial therapy.In recent years, the phenomenon of multidrug resistance of pathogenic fungi is increasing day by day, which leads to more difficult anti-infection treatment of patients such as immune deficiency, organ transplantation, high cost of treatment and high clinical mortality.One of the important reasons for multidrug resistance is that the overexpression of ABC transporter. Pdr5p is an important ABC transporter in Saccharomyces cerevisiae. It is highly similar to the ABC transporter found in many pathogenic yeast strains in structure and function.Using Pdr5p as a model to study the molecular mechanism of multidrug resistance mediated by ABC transporter has become a hot topic in recent years.In this paper, PCR mediated random mutation was used to mutate the high expression plasmid YEplac195:BPDR5. Two single site mutations, C793F and S1230F, were screened on fluconazole drug plate, which could seriously damage the function of Pdr5p efflux pump.C793 is located at the intracellular boundary of the predicted Pdr5p transmembrane helix 6 (TMH6), while S1230 is located at the extracellular boundary of the transmembrane helix 7 (TMH7).By studying the drug resistance of two mutants, it was found that they had the same drug resistance phenotype as PDR5 deficient strains on the solid drug plate containing actinomycin, Rhodamine 6G and erythrotetrazole.In liquid media containing different concentrations of the above drugs, the relative growth rate was measured.It was found that the efflux ability of C793F and S1230F mutants also decreased significantly. Pdr5p-specific ATP enzyme activity test and Western blot assay showed that the mutation of 793 and 1230 did not result in the change of Pdr5p specific ATP enzyme activity.The mutated Pdr5p can still be located correctly on the cell membrane and the protein expression is not affected, which indicates that the difference of amino acid is the reason of the damage of Pdr5p function caused by the mutation of these two loci.In order to further study the mechanism of Pdr5p function impairment, we constructed C793S, C793MN C793Y, S1230Agna S1230N, S1230Y single locus series by site-directed mutagenesis (S793S), C793Y, S1230Y and S1230Y, respectively.By studying the drug-sensitive Pdr5p specific ATP enzyme activity and protein localization and expression of these mutant strains, it was found that all mutant strains had similar ATP enzyme activity and membrane localization to WT strain.However, its drug sensitivity is related to the properties of the substituted amino acids.The results showed that at site 793, the change of amino acid spatial volume was the main factor leading to the impairment of Pdr5p function, and at site 1230, the spatial volume and polarity of amino acid affected the function of Pdr5p efflux pump.On the basis of random mutation, two new mutation sites were screened out which can seriously damage the function of Pdr5p. The results of drug screening and biochemical evidence confirm that the sites located at the two ends of the transmembrane region are very important for the function of Pdr5p.These results provide new evidence for further understanding the molecular mechanism of ABC transporter.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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