Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.
Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.
复制标题
DOI：
10.1371/journal.pone.0141991
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
van Veen HW
中科院分区：
文献类型：
--
作者：
Neuberger A;van Veen HW
多药转运体介导抗生素和有毒离子主动排出细胞。这种反应被认为是基于转运蛋白在两种构象状态之间的切换，在一种状态下，内部底物结合腔可用于在细胞内部结合底物，而在另一种构象状态中，空腔暴露在细胞外部，以使底物释放。与这一模型一致，半胱氨酸与乳酸乳球菌的主要促进剂超家族药物/质子逆向转运体LmrP的研究表明，转运的苯扎溴铵以内向状态与LmrP结合。荧光染料Hoechst 33342是许多多药物转运体的底物，在微生物和哺乳动物细胞中通过外排泵排出。令人惊讶的是，与其他多药转运体不同的是，LmrP被发现在乳球菌细胞中主动积聚而不是排出Hoechst 33342。与此观察一致的是，LmrP的表达与细胞敏感性有关，而不是与对Hoechst 33342的耐药性有关。因此，我们在LmrP底物中发现了一个隐藏的“Janus”，它通过与外向的LmrP结合，然后从内向的LmrP释放，以相反的方向跨膜转运。这些发现与电子顺磁共振的距离测量是一致的，在电子顺磁共振中，发现Hoechst 33342结合稳定了LmrP的外向构象。我们的数据对于使用多药物出口商选择性地将“Hoechst 33342-like”药物靶向表达这些转运蛋白的细胞和组织具有重要意义。
Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden “Janus” amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of “Hoechst 33342-like” drugs to cells and tissues in which these transporters are expressed.