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.
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