Localization and Substrate Selectivity of Sea Urchin Multidrug (MDR) Efflux Transporters
Localization and Substrate Selectivity of Sea Urchin Multidrug (MDR) Efflux Transporters
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DOI：
10.1074/jbc.m112.424879
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发表时间：
2012-12-21
影响因子：
4.8
通讯作者：
Hamdoun, Amro
中科院分区：
文献类型：
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作者：
Goekirmak, Tufan;Campanale, Joseph P.;Hamdoun, Amro
在本研究中，我们克隆、表达并鉴定了紫毛圆线虫(Sp)ATP结合盒(ABC)转运蛋白。这项筛查确定了三种多药耐药(MDR)转运蛋白，它们与在人类中发现的主要类型的MDR转运蛋白具有功能同源性。当在胚胎中过表达时，顶端转运蛋白Sp-ABCB1a、ABCB4a和ABCG2a可以占观察到的外排活性的87%，为它们的底物选择性提供了一个可靠的检测方法。利用该方法，我们发现海胆多药耐药转运体输出典型的多药耐药物质，如钙黄素-AM、BODIPY-维拉帕米、BODIPY-长春花碱和米托蒽醌。此外，我们通过Sp-ABCB1a突变和用立体异构体环肽抑制剂(QZ59化合物)处理胚胎来表征海胆药物结合域初级序列中非保守替换对小鼠ABCB1的影响。结果表明，与小鼠ABCB1a相比，Sp-ABCB1a对QZ59对映体的立体选择性发生了逆转。这表明，转运体药物结合域初级序列的细微变化可以通过进化微调底物特异性。
In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.