ATP-Dependent Human Erythrocyte Glutathione-Conjugate Transporter. II. Functional Reconstitution of Transport Activity

Awasthi, Sanjay; Singhal, Sharad S.; Pikula, Sławomir; Piper, John T.; Srivastava, Sanjay; Torman, Robert T.; Bandorowicz‐Pikuła, Joanna; Lin, Julian; Singh, Shivendra V.; Zimniak, Piotr; Awasthi, Yogesh C.

doi:10.1021/bi972131r

Cited by 46 publications

(84 citation statements)

References 22 publications

(35 reference statements)

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“…Surprisingly, DNP-SG ATPase-mediated ATP hydrolysis was stimulated not only by the organic anions (e.g., DNP-SG), but also by weak cations such as doxorubicin (DOX) and its metabolites (Awasthi et al, 1994(Awasthi et al, , 1998b. Furthermore, our studies with membrane vesicles (Awasthi et al, 1994) as well as with reconstituted proteoliposomes (Awasthi et al, 1998a) demonstrated that DNP-SG ATPase catalyzed transport of anionic GSH conjugates as well as of weakly cationic drugs such as DOX and colchicine (Awasthi et al, 1994;1998a,b;. ATPdependent transport of both DNP-SG and DOX against a concentration gradient was demonstrated in proteoliposomes reconstituted with highly purified DNP-SG ATPase (Awasthi et al, 1998a).…”

Section: Dnp-sg Atpase a Transporter For Anionic As Well As Cationicmentioning

confidence: 78%

“…Furthermore, our studies with membrane vesicles (Awasthi et al, 1994) as well as with reconstituted proteoliposomes (Awasthi et al, 1998a) demonstrated that DNP-SG ATPase catalyzed transport of anionic GSH conjugates as well as of weakly cationic drugs such as DOX and colchicine (Awasthi et al, 1994;1998a,b;. ATPdependent transport of both DNP-SG and DOX against a concentration gradient was demonstrated in proteoliposomes reconstituted with highly purified DNP-SG ATPase (Awasthi et al, 1998a). The transport was temperature-dependent and sensitive to the osmolarity of the assay medium.…”

Section: Dnp-sg Atpase a Transporter For Anionic As Well As Cationicmentioning

confidence: 79%

“…In these preparations, a 38 kDa peptide fragment was, however, consistently observed. This initially led us to an erroneous conclusion that this peptide was the intact DNP-SG ATPase because purified preparations highly enriched in this peptide mediated ATP-dependent, uphill transport of DNP-SG as well as DOX in reconstituted proteoliposomes (Awasthi et al, 1998a).…”

Section: Cloning Of Dnp-sgatpase and Its Identity With Rlip76mentioning

confidence: 99%

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RLIP76, a Novel Transporter Catalyzing ATP-Dependent Efflux of Xenobiotics

Awasthi

Sharma²,

Singhal³

et al. 2002

Drug Metab Dispos

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ABSTRACT:Transport of xenobiotics and their metabolites by ATP-binding cassette (ABC) transporters particularly P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP1) has been extensively studied during last decade. Our recent studies demonstrate that RLIP76, a previously known GTPase-activating protein catalyzes ATP-dependent, uphill transport of anionic glutathione conjugates as well as of weakly cationic anthracyclines including doxorubicin (Adriamycin), a widely used drug in cancer chemotherapy. RLIP76 has inherent ATPase activity, which is stimulated by doxorubicin and glutathione conjugates. RLIP76 does not meet the criteria for classical ABC proteins such as MRP1 or Pgp, but similar to ABC proteins, it has two ATP-binding sequences, 69 with RLIP76 also acquire resistance to radiation toxicity. RLIP76 also catalyzes the transport of physiologic ligands such as leukotrienes (LTC4) and the conjugate of 4-hydroxynonenal and glutathione. In some cells (e.g., erythrocytes and lung cancer cells), the majority of transport activity for Adriamycin and glutathione conjugates including LTC4 is accounted for by RLIP76. These studies strongly suggest that RLIP76-mediated transport of organic ions has physiological and toxicological relevance and that it may play an important role in the mechanism of drug resistance.

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Section: Dnp-sg Atpase a Transporter For Anionic As Well As Cationicmentioning

confidence: 78%

Section: Dnp-sg Atpase a Transporter For Anionic As Well As Cationicmentioning

confidence: 79%

Section: Cloning Of Dnp-sgatpase and Its Identity With Rlip76mentioning

confidence: 99%

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RLIP76, a Novel Transporter Catalyzing ATP-Dependent Efflux of Xenobiotics

Awasthi

Sharma²,

Singhal³

et al. 2002

Drug Metab Dispos

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“…Liposome formation was initiated by the addition of SM2 Biobeads (200 mg/ml). We have shown that vesiculation is complete within 4 h and yields primarily unilammelar vesicles with a median diameter of 0.25 m and intravesicular/extravesicular volume ratio of 18 l/ml (8,22). The liposomes reconstituted with purified recombinant Hsf-1, POB1, and Ralbp1 proteoliposomes were used for delivery of these proteins to cells in culture.…”

Section: Prokaryotic Expression and Purification Of Pob1 And Pob1-(1-mentioning

confidence: 99%

Hsf-1 and POB1 Induce Drug Sensitivity and Apoptosis by Inhibiting Ralbp1

Singhal

Yadav

Drake

et al. 2008

Journal of Biological Chemistry

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Hsf-1 (heat shock factor-1) is a transcription factor that is known to regulate cellular heat shock response through its binding with the multispecific transporter protein, Ralbp1. Results of present studies demonstrate that Hsf-1 causes specific and saturable inhibition of the transport activity of Ralbp1 and that the combination of Hsf-1 and POB1 causes nearly complete inhibition through specific bindings with Ralbp1. Augmentation of cellular levels of Hsf-1 and POB1 caused dramatic apoptosis in non-small cell lung cancer cell line H358 through Ralbp1 inhibition. These findings indicate a novel model for mutual regulation of Hsf-1 and Ralbp1 through Ralbp1-mediated sequestration of Hsf-1 in the cellular cytoskeleton and Hsf-1-mediated inhibition of the transport activity of membranebound Ralbp1.In response to heat stress, human cells respond by activation of Hsf-1 (heat shock factor-1), a transcription factor that binds to NGAAN repeats of the promoter of heat shock genes, augmenting transcription (1-5). Considered the master regulator of the heat shock response (1-3), Hsf-1 binds DNA constitutively, and its binding affinity is based upon its phosphorylation in response to heat shock (1-5). In the unstressed state, Hsf-1 is sequestered in a complex with tubulin, HSP90, and Ralbp1 (6). Stress or constitutively active Ral-GTP binding to Ralbp1 triggers the release of Hsf-1 and its migration to the nucleus, where its transcription factor activity is important for the expression of heat shock proteins (6, 7). Although these studies focused on Ralbp1 present in the cytoplasm bound to the cytoskeleton and nuclear membrane, several previous and subsequent reports have clearly demonstrated the presence of Ralbp1 in nuclear as well as plasma membranes (8 -12). In several recent studies, we have conclusively demonstrated that Ralbp1 is a transmembrane protein with a defined cell surface domain (8 -11) and that it catalyzes in ATP hydrolysis-dependent trans-membrane anti-gradient efflux of toxic xenobiotics as well as endogenous metabolites. The preferred physiological substrates for transport by Ralbp1 are glutathione-electrophile conjugates of electrophilic lipid metabolites that arise from stress or heat shockinduced lipid peroxidation (13). The cell surface domain of Ralbp1 can be targeted by highly specific antibodies that inhibit the transport activity of Ralbp1 and result in dramatic regression of tumor in syngeneic and xenograft models of melanoma, lung cancer, and colon cancer (14, 15). The membrane functionality of Ralbp1 is also evident from its crucial role in endocytosis as a rate-regulatory element (12, 16 -18).An endocytosis-linked protein POB1 that binds Ralbp1 in a similar region as Hsf-1 has been shown to be a specific and saturable inhibitor of the glutathione-electrophile conjugates and doxorubicin (DOX) 2 transport activity of membrane-reconstituted purified Ralbp1 (19). We proposed that just as POB1 could function as an inhibitor of the transport activity of Ralbp1, Hsf-1 could also function as a tr...

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“…We cloned RLIP76 independently, in the search for glutathione-electrophile conjugates (GS-Es) and chemotherapy drug transporters, from a human bone-marrow cDNA library using polyclonal antibodies raised against purified human erythrocyte putative membrane transporter, DNPSG-ATPase (28)(29)(30). The identity of RLIP76 with DNPSG-ATPase was demonstrated by showing that both the human and recombinant protein could be purified by the same GS-E affinity chromatography method (9,(28)(29)(30). Tissue-purified and recombinant RLIP76 have demonstrated constitutive ATPase activity that was stimulated by anionic (e.g., DNP-SG, GS-HNE,) as well as cationic (e.g., DOX) ligands (9,31).…”

Section: Rlip76: a Non-abc Transportermentioning

confidence: 99%

Role of RLIP76 in doxorubicin resistance in lung cancer (Review)

Vatsyayan

Chaudhary²,

Lelsani³

et al. 2009

Int J Oncol

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Abstract. Lung cancer is still a major cause of cancer deaths in spite of considerable efforts in its systemic therapy. Chemotherapy, along with local irradiation is frequently employed but as a palliative therapy. Inherent and acquired resistance in NSCLC and SCLC towards chemotherapeutic agents further makes chemotherapy an incommodious problem. The resistance mechanisms responsible for inherent DOX-resistance of NSCLC and acquired DOX-resistance in SCLC have been the subject of numerous investigations. This review will focus on the recent studies done for understanding the mechanism(s) of inherent and acquired resistance in NSCLC and SCLC and how these can be exploited for the future development of more effective novel biologic agents for the treatment of lung cancer.

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ATP-Dependent Human Erythrocyte Glutathione-Conjugate Transporter. II. Functional Reconstitution of Transport Activity

Cited by 46 publications

References 22 publications

RLIP76, a Novel Transporter Catalyzing ATP-Dependent Efflux of Xenobiotics

RLIP76, a Novel Transporter Catalyzing ATP-Dependent Efflux of Xenobiotics

Hsf-1 and POB1 Induce Drug Sensitivity and Apoptosis by Inhibiting Ralbp1

Role of RLIP76 in doxorubicin resistance in lung cancer (Review)

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