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首页> 外文期刊>American Journal of Physiology >Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.
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Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.

机译：人乳腺癌抗性蛋白（BCRP / ABCG2）的跨膜螺旋1和6：鉴定对药物运输很重要的极性残基。

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摘要

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane alpha-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.
机译：人乳腺癌抗性蛋白（BCRP / ABCG2）介导药物和异种生物的外排。在这项研究中，我们调查了预测的BCRP跨膜α螺旋1和6内或附近的极性残基在药物运输中的作用。我们用Ala取代了Asn（387），Gln（398），Asn（629）和Thr（642），用Ala和Arg取代了Thr（402），并用Phe取代了Tyr（645），突变体在人类中稳定表达胚胎肾293或Flp-In-293细胞。免疫印迹和共聚焦显微镜分析表明，所有突变体均表达良好，且主要靶向质膜。尽管T402A和T402R显着降低了米托蒽醌，Hoechst 33342和BODIPY-哌唑嗪的外排量，但N629A对所有底物均显示出明显增加的外排量。 N387A和Q398A对米托蒽醌和Hoechst 33342的外排作用显着降低，但对BODIPY-吡唑嗪则没有。相反，T642A和Y645F仅显示出Hoechst 33342外流的适度降低。表达突变体的人胚胎肾293细胞的耐药性概况通常与外排数据相关。而且，N629A与BCRP ATPase活性显着增加有关，而N387A和T402A与BCRP ATPase活性显着减少有关。一些极性残基的突变可能会导致构象变化，如在存在哌唑嗪的情况下5D3抗体与BCRP的结合改变所证明的。 BCRP的向内同源模型表明跨膜1内的Thr（402）对于螺旋相互作用可能很重要，而Asn（629）可能参与BCRP-底物相互作用。总之，我们已经证明了其中某些极性残基在BCRP活性中的功能重要性。

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