Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin
Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin
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DOI：
10.1021/bi992964c
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发表时间：
2000-08-08
期刊：
影响因子：
2.9
通讯作者：
Awasthi, YC
中科院分区：
文献类型：
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作者：
Awasthi, S;Cheng, JZ;Awasthi, YC
主动运输的共轭和非共轭的亲电试剂的细胞是必不可少的细胞稳态。我们之前在人体组织中发现了一种转运体DNP-SG [S-（2,4-二硝基苯）谷胱甘肽]atp酶，能够执行这种功能[Awasthi等人，（1998）生物化学37,5231-5238,5239-5248]。我们现在报道DNP-SG atp酶的克隆。cDNA克隆的序列与人类已知的pal结合蛋白RLIP76相同。用DNP-SG亲和层析纯化大肠杆菌中表达的RLIP76。纯化的重组RLIP76:(1)具有DNP-SG或阿霉素（DOX）刺激的ATP酶活性，并且RLIP76对ATP、DOX和DNP-SG的K-m值与报道的DNP-SG ATP酶相似；(2)在与凝集素和定义的脂质重构后，催化DNP-SG和DOX的atp依赖性转运，其动力学参数与DNP-SG atp酶相似；(3)转染到K562细胞后，导致对DOX的抗性增强，并增加了DNP-SG和DOX通过转染细胞的内外膜囊泡的atp依赖性转运；(4)哺乳动物细胞从供体蛋白脂质体中直接摄取纯化的RLIP76蛋白可产生DOX抗性。这些结果表明，RLIP76除了在信号转导中发挥作用外，还可以催化转运。谷胱甘肽缀合物和外源性药物，并可能有助于多药耐药现象。
Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2,4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Pal-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K-m values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport. of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.