DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

描述：（改编自P.I.的摘要）。在这次修改后的竞争中， 更新申请，研究提出了一种新的特点， 外源性/谷胱甘肽结合转运蛋白，DNP-SG ATP酶，存在于人类 红细胞膜在上一个融资期间，P.I.具有 证明：1）纯化的DNP-SG ATP酶结合8-叠氮基ATP，催化ATP 在两亲性阳离子药物多柔比星（DOX）的存在下水解， 以及阴离子GSH-缀合物。2)它不同于药物外排 泵、P-糖蛋白（Pgp）和多药耐药相关蛋白 （MRP）。3)纯化的DNP-SG ATP酶在蛋白脂质体中重组， GSH结合物以及DOX的ATP依赖性主动转运。4)使用 DNP-SG ATP酶抗体、P.I.从人类cDNA文库中克隆了一个cDNA 其产生具有类似于以下的性质的重组蛋白（RLip 76）： DNP-SG ATP酶。私家侦探因此，假设DNP-SG ATP酶， 积极地从细胞中输出有毒的外源性物质及其代谢物， 代表了结构多样的异生物质的主要解毒系统， 正常细胞私家侦探计划进一步表征DNP-SG ATP酶/Rlip 76， 并提出了三个具体目标。在第一个具体的目标，P.I.将 从cDNA中异源表达获得重组RLip 76蛋白， e.大肠杆菌中用于结构和动力学研究。私家侦探将取消H-69 和K-562细胞，以检查转染的细胞是否 耐受由外源性物质/内生性物质介导的细胞毒性， 此（RLip76）DNP-SG ATP酶的底物。在具体目标#2中，P.I.将 通过重建组织纯化的DNP-SG ATP酶， 和重组（RLip76）DNP-SG ATP酶在蛋白脂质体中的动力学研究 生理性阴离子结合物（如白三烯、4-HNE GSH）的转运 胆红素的缀合物）和药物（例如DOX、道诺霉素等）。在特定 目标三，私家侦探。建议将RLip76共转染H-69和K-562细胞， 谷胱甘肽S-转移酶（GST）同工酶，mGSTA4 - 4，以检验假设 DNP-SG ATP酶（RLip76）与GST结合，在 细胞中内源性和外源性亲电体的解毒。这些研究 将提供DNP-SG ATP酶在以下方面作用的临床相关信息： 细胞解毒过程和多药耐药机制 不表达Pgp和/或MRP的癌细胞。

DESCRIPTION: (Adapted from the P.I.'s Abstract). In this revised competing renewal application, studies are proposed to characterize a novel xenobiotic/GSH-conjugate transporter, DNP-SG ATPase, present in human erythrocyte membranes. During the prior funding period the P.I. has demonstrated that: 1) Purified DNP-SG ATPase binds 8-azido ATP, catalyze ATP hydrolysis in the presence of an amphiphilic cationic drug, doxorubicin (DOX), as well as anionic GSH-conjugates. 2) It is distinct from the drug efflux pumps, P-glycoprotein (Pgp), and multi-drug resistance associated protein (MRP). 3) Purified DNP-SG ATPase reconstituted in proteoliposomes mediates ATP-dependent, active transport of GSH-conjugates as well as DOX. 4) Using DNP-SG ATPase antibodies, the P.I. has cloned a cDNA from a human cDNA library which yields a recombinant protein (RLip 76) with properties similar to that of DNP-SG ATPase. The P.I. therefore hypothesizes that DNP-SG ATPase, which actively exports from the cell toxic xenobiotics and their metabolites, represents a major detoxication system for structurally diverse xenobiotics in normal cells. The P.I. plans to further characterize the DNP-SG ATPase/Rlip 76, and proposes three Specific Aims. In the first Specific Aim, the P.I. will obtain the recombinant RLip 76 protein from cDNA by heterologous expression in E. coli for use in structural and kinetic studies. The P.I. will transfect H-69 and K-562 cells with RLip 76 cDNA to examine whether the transfected cells are resistant to cytotoxicity mediated by xenobiotics/endobiotics which are substrates for this (RLip76) DNP-SG ATPase. In Specific Aim #2, the P.I. will functionally characterize the DNP-SG ATPase by reconstituting tissue-purified and recombinant (RLip76) DNP-SG ATPase in proteoliposomes to study the kinetics of transport of physiological anionic conjugates (e.g. leukotrienes, 4-HNE GSH conjugates of bilirubin), and drugs (e.g. DOX, daunomycin, etc.). In Specific Aim #3, the P.I. proposes to co-transfect RLip76 into H-69 and K-562 cells with the glutathione S-transferase (GST) isozyme, mGSTA4-4, to test the hypothesis that DNP-SG ATPase (RLip76) in conjunction with GSTs, plays a major role in the detoxication of endogenous and exogenous electrophiles in cells. These studies will provide clinically relevant information on the role of DNP-SG ATPase in cellular detoxication processes and on the mechanisms of multidrug resistance of cancer cells which do not express Pgp and/or MRP.