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）转运蛋白研究的最新进展表明，在包括原生动物寄生虫在内的各种生物体中，它们在耐药性中的重要作用。我们以前在亚马逊利什曼尼亚（Leishmania）隔离了ABC蛋白LAMDR1和LAMDR2的两个不同的多药耐药性（MDR）亚家族基因。我们发现LAMDR1是哺乳动物MDR1的同源物，但LAMDR2是一种新型的MDR基因。 LAMDR2对5-氟尿嘧啶（5-FU）的耐药性（一种用作抗癌药物）的过表达，药物摄取实验表明，在使用LAMDR2的转染剂中，5-FU的积累降低。在本研究中，我们检查了MDR2是否位于质膜中，并从细胞中挤出5-FU，还是将其定位在细胞内细胞器的膜中，并在细胞器内部隔离药物。使用抗LAMDR2肽抗体的蛋白质印迹分析表明，在对数相的转染剂中LAMDR2的表达高于固定相。通过RT-PCR在野生型细胞中检测到相应的蛋白质。间接免疫荧光研究表明，抗体染色未知的细胞质结构，而不是质膜。荧光和共聚焦扫描激光显微镜研究表明，在细胞内管状，运河或圆形结构中发现了用绿色荧光蛋白（GFP）标记的LAMDR2。 GFP信号在对数相细胞中最强烈，但是单个转染物中的信号模式变化。 GFP荧光的分布主要与溶酶体标记物的分布重叠，但与线粒体标记物的分布不同。这些发现表明LAMDR2位于溶酶体的膜中，并将5-FU运输到管腔中，并且通过随后的胞吐作用将DUG挤出了细胞。