Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression.
Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression.
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DOI：
10.1074/jbc.m111.324319
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发表时间：
2012-03-23
期刊：
影响因子：
--
通讯作者：
Biagini GA
中科院分区：
文献类型：
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作者：
Fisher N;Abd Majid R;Antoine T;Al-Helal M;Warman AJ;Johnson DJ;Lawrenson AS;Ranson H;O'Neill PM;Ward SA;Biagini GA
背景：细胞色素 b 突变导致阿托伐醌耐药，导致抗疟药物失败。结果：突变 Y268S 降低了 bc1 催化周转率和稳定性。结论：寄生虫 Y268S bc1 中催化转化和铁硫蛋白含量的减少会带来适应成本。这些结果不是使用酵母模型预测的。意义：数据将有助于新型 bc1 抑制剂的设计，并为阿托伐醌耐药性的流行病学研究提供信息。阿托伐醌是一种抗疟疾药物，与氯胍（例如 MalaroneTM）联合使用，用于治疗和预防疟疾。 Atovaquone 是一种 2-羟基萘醌，是线粒体细胞色素 bc1 复合物对苯二酚氧化 (Qo) 位点的竞争性抑制剂。抑制这种酶会导致线粒体膜电位崩溃、嘧啶生物合成中断以及随后的寄生虫死亡。现场对阿托伐醌的耐药性与细胞色素 b Qo 口袋中的点突变有关，最显着的是靠近 ef 环中保守的 Pro260-Glu261-Trp262-Tyr263 (PEWY) 区域。这种突变的影响已在模式生物中得到广泛研究，但迄今为止尚未在寄生虫本身中进行研究。在这里，我们对阿托伐醌抗性野外分离株 TM902CB 进行了分子和生化表征。该菌株的分子分析揭示了细胞色素 b 中存在 Y268S 突变。 Y268S 突变导致阿托伐醌的抑制常数 (Ki) 发生 270 倍的变化，同时 bc1 复合物的 Vmax 降低约 40%，观察到的癸联苯醌 Km 增加 3 倍。蛋白质印迹分析显示 Y268S bc1 中铁硫蛋白含量降低，表明该亚基与细胞色素 b 之间的相互作用减弱。与 3D7（阿托伐醌敏感）对照菌株相比，TM902CB 菌株的基因表达分析显示 bc1 和细胞色素 c 氧化酶基因的表达水平更高。据推测，观察到的这些和其他关键基因的差异表达抵消了 bc1 活性降低导致的适应度成本。
Background: Cytochrome b mutations confer atovaquone resistance, resulting in antimalarial drug failures. Results: Mutation Y268S reduces bc1 catalytic turnover and stability. Conclusion: Reduction of catalytic turnover and iron-sulfur protein content in parasite Y268S bc1 confers a fitness cost. These results were not predicted using yeast models. Significance: Data will aid novel bc1 inhibitor design and inform epidemiological studies of atovaquone resistance. Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. MalaroneTM) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc1 complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Qo pocket of cytochrome b, most notably near the conserved Pro260-Glu261-Trp262-Tyr263 (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (Ki) for atovaquone with a concomitant reduction in the Vmax of the bc1 complex of ∼40% and a 3-fold increase in the observed Km for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc1 suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc1 and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc1 activity.