Novel Function of Human RLIP76: ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin
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...
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| Veröffentlicht in: | Biochemistry (Easton) 2000-08, Vol.39 (31), p.9327-9334 |
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| creator | Awasthi, Sanjay Cheng, Jizhong Singhal, Sharad S Saini, Manjit K Pandya, Utpal Pikula, Slawomir Bandorowicz-Pikula, Joanna Singh, Shivendra V Zimniak, Piotr Awasthi, Yogesh C |
| description | 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 Ral-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. |
| doi_str_mv | 10.1021/bi992964c |
| format | Article |
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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 Ral-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.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi992964c</identifier><identifier>PMID: 10924126</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Adenosine Triphosphatases - isolation & purification ; Adenosine Triphosphatases - metabolism ; Adenosine Triphosphate - physiology ; Amino Acid Sequence ; Amino Acids - analysis ; ATP-Binding Cassette Transporters ; Biological Transport, Active - drug effects ; Carrier Proteins - chemistry ; Carrier Proteins - genetics ; Carrier Proteins - isolation & purification ; Carrier Proteins - metabolism ; Catalysis ; Cell Adhesion - drug effects ; Cell Membrane - metabolism ; Doxorubicin - metabolism ; Doxorubicin - toxicity ; Glutathione - chemistry ; Glutathione - metabolism ; GTPase-Activating Proteins ; Humans ; Intracellular Fluid - metabolism ; K562 Cells ; Molecular Sequence Data ; Peptide Fragments - isolation & purification ; Peptide Fragments - metabolism ; Phospholipids - metabolism ; Proteolipids - metabolism ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; RLIP76 protein ; Transfection ; Tumor Cells, Cultured</subject><ispartof>Biochemistry (Easton), 2000-08, Vol.39 (31), p.9327-9334</ispartof><rights>Copyright © 2000 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a380t-e7fdd91f7728b83eae11d0a4665f3e3f0696a6bafa77a57cdd492f9f9e9cb24e3</citedby><cites>FETCH-LOGICAL-a380t-e7fdd91f7728b83eae11d0a4665f3e3f0696a6bafa77a57cdd492f9f9e9cb24e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi992964c$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi992964c$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10924126$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Awasthi, Sanjay</creatorcontrib><creatorcontrib>Cheng, Jizhong</creatorcontrib><creatorcontrib>Singhal, Sharad S</creatorcontrib><creatorcontrib>Saini, Manjit K</creatorcontrib><creatorcontrib>Pandya, Utpal</creatorcontrib><creatorcontrib>Pikula, Slawomir</creatorcontrib><creatorcontrib>Bandorowicz-Pikula, Joanna</creatorcontrib><creatorcontrib>Singh, Shivendra V</creatorcontrib><creatorcontrib>Zimniak, Piotr</creatorcontrib><creatorcontrib>Awasthi, Yogesh C</creatorcontrib><title>Novel Function of Human RLIP76: ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>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 Ral-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.</description><subject>Adenosine Triphosphatases - isolation & purification</subject><subject>Adenosine Triphosphatases - metabolism</subject><subject>Adenosine Triphosphate - physiology</subject><subject>Amino Acid Sequence</subject><subject>Amino Acids - analysis</subject><subject>ATP-Binding Cassette Transporters</subject><subject>Biological Transport, Active - drug effects</subject><subject>Carrier Proteins - chemistry</subject><subject>Carrier Proteins - genetics</subject><subject>Carrier Proteins - isolation & purification</subject><subject>Carrier Proteins - metabolism</subject><subject>Catalysis</subject><subject>Cell Adhesion - drug effects</subject><subject>Cell Membrane - metabolism</subject><subject>Doxorubicin - metabolism</subject><subject>Doxorubicin - toxicity</subject><subject>Glutathione - chemistry</subject><subject>Glutathione - metabolism</subject><subject>GTPase-Activating Proteins</subject><subject>Humans</subject><subject>Intracellular Fluid - metabolism</subject><subject>K562 Cells</subject><subject>Molecular Sequence Data</subject><subject>Peptide Fragments - isolation & purification</subject><subject>Peptide Fragments - metabolism</subject><subject>Phospholipids - metabolism</subject><subject>Proteolipids - metabolism</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>RLIP76 protein</subject><subject>Transfection</subject><subject>Tumor Cells, Cultured</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpt0DtvFDEQB3ALgcgRKPgCyA2RKDbYXq99posu5CGdkhMcj87yrsewx559-BElXVq-Jp8kG20UUVCNRvObGemP0GtKDilh9H3bK8WU4N0TNKMNIxVXqnmKZoQQUY0DsodepLQZW04kf472KFGMUyZmyF6EKxjwSfFd7oPHweGzsjUef1qer6T48Pf2Dz5ar6pj2IG34DNeR-PTLsR8b0-Hkk3-OW4CXgS_KT9MhoSNt_g4XIdY2r7r_Uv0zJkhwauHuo--nHxcL86q5eXp-eJoWZl6TnIF0lmrqJOSzdt5DQYotcRwIRpXQ-2IUMKI1jgjpWlkZy1XzCmnQHUt41Dvo4Pp7i6G3wVS1ts-dTAMxkMoSVMpOWecjfDdBLsYUorg9C72WxNvNCX6PlL9GOlo3zwcLe0W7D9yynAE1QT6lOH6cW7iLy1kLRu9Xn3Wak6-8Yvvtf46-reTN13Sm1CiHzP5z-M7bx-Neg</recordid><startdate>20000808</startdate><enddate>20000808</enddate><creator>Awasthi, Sanjay</creator><creator>Cheng, Jizhong</creator><creator>Singhal, Sharad S</creator><creator>Saini, Manjit K</creator><creator>Pandya, Utpal</creator><creator>Pikula, Slawomir</creator><creator>Bandorowicz-Pikula, Joanna</creator><creator>Singh, Shivendra V</creator><creator>Zimniak, Piotr</creator><creator>Awasthi, Yogesh C</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope></search><sort><creationdate>20000808</creationdate><title>Novel Function of Human RLIP76: ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin</title><author>Awasthi, Sanjay ; Cheng, Jizhong ; Singhal, Sharad S ; Saini, Manjit K ; Pandya, Utpal ; Pikula, Slawomir ; Bandorowicz-Pikula, Joanna ; Singh, Shivendra V ; Zimniak, Piotr ; Awasthi, Yogesh C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a380t-e7fdd91f7728b83eae11d0a4665f3e3f0696a6bafa77a57cdd492f9f9e9cb24e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Adenosine Triphosphatases - isolation & purification</topic><topic>Adenosine Triphosphatases - metabolism</topic><topic>Adenosine Triphosphate - physiology</topic><topic>Amino Acid Sequence</topic><topic>Amino Acids - analysis</topic><topic>ATP-Binding Cassette Transporters</topic><topic>Biological Transport, Active - drug effects</topic><topic>Carrier Proteins - chemistry</topic><topic>Carrier Proteins - genetics</topic><topic>Carrier Proteins - isolation & purification</topic><topic>Carrier Proteins - metabolism</topic><topic>Catalysis</topic><topic>Cell Adhesion - drug effects</topic><topic>Cell Membrane - metabolism</topic><topic>Doxorubicin - metabolism</topic><topic>Doxorubicin - toxicity</topic><topic>Glutathione - chemistry</topic><topic>Glutathione - metabolism</topic><topic>GTPase-Activating Proteins</topic><topic>Humans</topic><topic>Intracellular Fluid - metabolism</topic><topic>K562 Cells</topic><topic>Molecular Sequence Data</topic><topic>Peptide Fragments - isolation & purification</topic><topic>Peptide Fragments - metabolism</topic><topic>Phospholipids - metabolism</topic><topic>Proteolipids - metabolism</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>RLIP76 protein</topic><topic>Transfection</topic><topic>Tumor Cells, Cultured</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Awasthi, Sanjay</creatorcontrib><creatorcontrib>Cheng, Jizhong</creatorcontrib><creatorcontrib>Singhal, Sharad S</creatorcontrib><creatorcontrib>Saini, Manjit K</creatorcontrib><creatorcontrib>Pandya, Utpal</creatorcontrib><creatorcontrib>Pikula, Slawomir</creatorcontrib><creatorcontrib>Bandorowicz-Pikula, Joanna</creatorcontrib><creatorcontrib>Singh, Shivendra V</creatorcontrib><creatorcontrib>Zimniak, Piotr</creatorcontrib><creatorcontrib>Awasthi, Yogesh C</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Awasthi, Sanjay</au><au>Cheng, Jizhong</au><au>Singhal, Sharad S</au><au>Saini, Manjit K</au><au>Pandya, Utpal</au><au>Pikula, Slawomir</au><au>Bandorowicz-Pikula, Joanna</au><au>Singh, Shivendra V</au><au>Zimniak, Piotr</au><au>Awasthi, Yogesh C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Novel Function of Human RLIP76: ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2000-08-08</date><risdate>2000</risdate><volume>39</volume><issue>31</issue><spage>9327</spage><epage>9334</epage><pages>9327-9334</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>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 Ral-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.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>10924126</pmid><doi>10.1021/bi992964c</doi><tpages>8</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 2000-08, Vol.39 (31), p.9327-9334 |
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| source | MEDLINE; American Chemical Society Journals |
| subjects | Adenosine Triphosphatases - isolation & purification Adenosine Triphosphatases - metabolism Adenosine Triphosphate - physiology Amino Acid Sequence Amino Acids - analysis ATP-Binding Cassette Transporters Biological Transport, Active - drug effects Carrier Proteins - chemistry Carrier Proteins - genetics Carrier Proteins - isolation & purification Carrier Proteins - metabolism Catalysis Cell Adhesion - drug effects Cell Membrane - metabolism Doxorubicin - metabolism Doxorubicin - toxicity Glutathione - chemistry Glutathione - metabolism GTPase-Activating Proteins Humans Intracellular Fluid - metabolism K562 Cells Molecular Sequence Data Peptide Fragments - isolation & purification Peptide Fragments - metabolism Phospholipids - metabolism Proteolipids - metabolism Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism RLIP76 protein Transfection Tumor Cells, Cultured |
| title | Novel Function of Human RLIP76: ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin |
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