Roles of P-glycoprotein genes in drug resistance and intracellular parasitism in Leishmania

我们在寄生虫中发现了两种不同的P-糖蛋白基因，亚马松线虫。LaMDR 1基因编码1341氨基酸是由两个相似的半部分组成的蛋白质组成的，每个都含有一个ATP绑定和六个跨膜域。L.L.野生型promastigotes中LaMDR 1基因的转染和过度表达。亚马逊菌会议对文布拉司汀、多氯联苯素和actinomycin D的抵抗力，表明LaMDR 1基因是人类多程抵抗力MDR 1基因的同源性。LaMDR 2基因编码为1267氨基酸，仅揭示LaMDR 1蛋白的47%氨基酸身份。LaMDR 2基因的转染剂显示出对5-fluorouracil（5-FU）的抗性，一种用于癌症化疗的氟吡啶，但不是用于其他抗癌症药物的描述。A slightly higher intracellular accumulation of [イイD13エD1H] vinblastine was observed in the LaMDR1-transfected cells与control transfectant cells比较。在与LaMDR 2基因的转染中，[D13 YD 1H] 5-FU增加了在药物暴露后的头30分钟内测量了，但药物暴露后的头120分钟内，药物累积被研究减少并达到了20- 30%的控制水平。这5-FU累积动力学与LaMDR 2基因的转染者有关的建议，即LaMDR 2蛋白可能位于细胞塑料容器的膜和运输5-FU在初始阶段期间在容器内部，然后药物在后期阶段由exocytosis保密。这些结果揭示了LaMDR 2基因是ATP-绑定盒（ABC）的新成员，其中含有P-糖蛋白质结构的蛋白家族，并在宿主细胞中扮演一种新颖的寄生虫存活作用。使用LaMDR 2蛋白的亚细胞本地化-LaMDR 2抗体是研究中的一种。基因干扰技术对LaMDR 2基因的功能性分析也在过程中。

We isolated and characterized two different P-glycoprotein genes in a parasitic protozoan, Leishmania amazonensis. The LaMDR1 gene encodes 1341 amino acids for a protein consisting of two similar halves, each containing one ATP-binding and six transmembrane domains. Transfection and over-expression of the LaMDR1 gene in wild-type promastigotes of L. amazonensis conferred resistance to vinblastine, doxorubicin and actinomycin D, indicating that the LaMDR1 gene is a homologue of human multidrung resistance MDR1 gene. The LaMDR2 gene encodes 1267 amino acids, revealing only 47% amino acid identity to the LaMDR1 protein. Transfectants with the LaMDR2 gene showed resistance to 5-fluorouracil (5-FU), a fluoropyrimidine used for cancer chemotherapy, but not to other anti-cancer drugs described above. A slightly higher intracellular accumulation of [ィイD13ィエD1H] vinblastine was observed in the LaMDR1-transfected cells compared with control transfectant cells. In transfectants with the LaMDR2 gene, an increased accumulation of [ィイD13ィエD1H]5-FU was measured in the first 30 min after drug exposure, but the drug accumulation was gradually decreased and reached to 20-30% of the control level at 120 min after the exposure. This 5-FU accumulation kinetics in transfectants with the LaMDR2 gene suggests that the LaMDR2 protein may locate on the membrane of cytoplasmic vesicles and transport 5-FU inside the vesicles during the initial phase, and then drugs were secreted by exocytosis during the later phase. These results revealed that the LaMDR2 gene is a new member of the ATP-binding cassette (ABC) family of protein with the P-glycoprotein structure and play a novel role in survival of the parasites in the host cells. Subcellular localization of the LaMDR2 protein using anti-LaMDR2 antibodies is under study. Functional analysis of the LaMDR2 gene by gene-disruption technology is also in process.