Substitution of threonine-1351 in the multidrug transporter Cdr1p of Candida albicans results in hypersusceptibility to antifungal agents and threonine-1351 is essential for synergic effects of calcineurin inhibitor FK520.
Substitution of threonine-1351 in the multidrug transporter Cdr1p of Candida albicans results in hypersusceptibility to antifungal agents and threonine-1351 is essential for synergic effects of calcineurin inhibitor FK520.
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白色念珠菌多药转运蛋白 Cdr1p 中苏氨酸-1351 的取代会导致对抗真菌药物过敏，而苏氨酸-1351 对于钙调神经磷酸酶抑制剂 FK520 的协同作用至关重要。
DOI：
10.1093/jac/dkh308
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Prasad,Rajendra
中科院分区：
文献类型：
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作者：
Shukla,Suneet;Ambudkar,SureshV;Prasad,Rajendra
目的：方法：我们在S. ParaeAD 1 - 8 u −宿主中过表达绿色荧光蛋白标记的Cdr 1 p，并引入一个点突变，将Cdr 1 p中的T1351替换为F。使用最小抑菌浓度、斑点试验和氟康唑外排分析表达T1351 F突变体Cdr 1 p的细胞的功能活性。结果：T1351 F突变型Cdr 1 p表达细胞对茴香霉素、放线菌酮、氟康唑、咪康唑和制霉菌素敏感。突变体蛋白表达水平与天然Cdr 1 p在S. gae细胞中的表达水平相同，并适当地定位于细胞表面。突变体变体和天然蛋白质结合ATP的光亲和类似物、8-叠氮基ATP或放射性标记的光亲和剂碘芳基叠氮吡唑嗪和叠氮平的能力之间也没有差异。然而，T1351的取代导致其输出荧光底物罗丹明6 G的能力显著降低。结论：Cdr 1 p跨膜结构域（TMD）11中的T1351不仅在药物-底物转运中起重要作用，而且在FK 520协同作用中起重要作用。
Objectives: Functional characterization of a mutantCandida albicansdrug resistance protein (Cdr1p) by overexpression inSaccharomyces cerevisiae.Methods: We overexpressed green fluorescent protein-tagged Cdr1p inS. cerevisiaeAD1-8u−host and introduced a point mutation to substitute T1351 with F in Cdr1p. The cells expressing T1351F mutant Cdr1p were analysed for their functional activity using minimum inhibitory concentration, spot assay, and fluconazole efflux. The binding activity of photoaffinity analogues 8-azidoATP, iodoarylazidoprazosin and azidopine to the mutant T1351F Cdr1p was also characterized.Results: The T1351F mutant Cdr1p-expressing cells were susceptible to anisomycin, cycloheximide, fluconazole, miconazole and nystatin. The mutant protein was expressed to the same level as that of native Cdr1p inS. cerevisiaecells and was properly localized to the cell surface. There was also no difference between the mutant variant and the native protein's ability to bind a photoaffinity analogue of ATP, 8-azidoATP, or the radiolabelled photoaffinity agents iodoarylazidoprazosin and azidopine. However, the substitution of T1351 resulted in considerable reduction in its ability to export fluorescent substrate rhodamine 6G. The synergy between calcineurin inhibitors FK520 and azoles was abrogated in cells expressing the T1351F mutant variant of Cdr1p.Conclusions: The results from this study suggest that the T1351 in the predicted transmembrane domain (TMD) 11 of Cdr1p is not only important for drug-substrate transport but also has a role in governing synergy of FK520.