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Leishmania multidrug resistance (MDR) proteins of ATP-binding cassette (ABC) transporters : subcellular localization of and the role in drug resistance

ATP结合盒（ABC）转运蛋白的利什曼原虫多药耐药（MDR）蛋白：亚细胞定位及其在耐药性中的作用

基本信息

批准号：
13470055
负责人：
KATAKURA Ken
金额：
$ 6.85万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Recent progress in research for ATP-binding cassette (ABC) transporters have shown their important roles in drug resistance in various organisms, including protozoan parasites. We previously isolated two different multidrug resistance (MDR) subfamily genes of the ABC proteins, LaMDR1 and LaMDR2, in Leishmania amazonensis. We found that the LaMDR1 was a homologue to mammalian MDR1, but the LaMDR2 was a novel MDR gene. Overexpression of the LaMDR2 conferred resistance to 5-fluorouracil (5-FU), a fluoropyrimidine used as an anticancer drug, and drug uptake experiments demonstrated a decrease accumulation of 5-FU in transfectants with the LaMDR2. In the present study, we examined whether MDR2 is located in the plasma membrane and extrudes 5-FU out of the cell, or it is localized in the membrane of intracellular organelle and sequesters the drug inside the organelle. Western blot analysis using anti-LaMDR2 peptide antibodies indicated that the expression of LaMDR2 was much higher in the transfectants in the log-phase than the stationary phase. The corresponding protein was detected by RT-PCR in wild-type cells. Indirect immunofluorescence studies showed that the antibodies stained unknown cytoplasmic structures, but not the plasma membrane. Fluorescent and confocal scanning laser microscopic studies revealed that LaMDR2 tagged with green fluorescent protein (GFP) was found in the intracellular tubular, canal or round-shape structure. The GFP signal was most intense in the log-phase cells, but the signal patters varied in individual transfectants. The distribution of the GFP fluorescence was mostly overlapped with that of a lysosome marker, but not with that of a mitochondria marker. These findings suggest that LaMDR2 is located in the membrane of the lysosome and transports 5-FU into the lumen, and that the dug is extruded out of cell by subsequent exocytosis.

ATP结合盒（ABC）转运蛋白的最新研究进展表明，它们在包括寄生虫在内的多种生物的耐药性中发挥着重要作用。我们以前分离出两个不同的多药耐药（MDR）亚家族基因的ABC蛋白，LaMDR 1和LaMDR 2，亚马逊利什曼原虫。我们发现LaMDR 1与哺乳动物MDR 1同源，而LaMDR 2是一个新的MDR基因。LaMDR 2的过表达赋予了对5-氟尿嘧啶（5-FU）的抗性，5-FU是一种用作抗癌药物的氟嘧啶，药物摄取实验表明，LaMDR 2转染子中5-FU的积累减少。在本研究中，我们研究了MDR 2是否位于质膜上并将5-FU挤出细胞外，或者它是否位于细胞内细胞器的膜上并将药物隔离在细胞器内。使用抗LaMDR 2肽抗体的Western印迹分析表明，LaMDR 2的表达在对数期的转染子中比稳定期高得多。在野生型细胞中通过RT-PCR检测到相应的蛋白。间接免疫荧光研究表明，抗体染色未知的细胞质结构，但不是质膜。荧光和激光共聚焦扫描显微镜研究表明，LaMDR 2标记的绿色荧光蛋白（GFP）被发现在细胞内的管状，管道或圆形结构。GFP信号在对数期细胞中最强，但信号模式在单个转染子中变化。GFP荧光的分布主要与溶酶体标记重叠，但不与线粒体标记重叠。这些发现表明LaMDR 2位于溶酶体的膜中并将5-FU转运到管腔中，并且通过随后的胞吐作用将dug挤出细胞。