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1

Transport of glutathione-conjugates in human erythrocytes

100%

Sharma R., Awasthi S., Zimniak P., Awasthi Y. C.

Acta Biochimica Polonica

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2000

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tom 47

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nr 3

The last step of detoxification of both endogenous and environmental toxicants is typically a conjugation that produces a bulky hydrophilic molecule. The excretion of such conjugates out of cells is of sufficient biological importance to have led to the evolution of ATP-driven export pumps for this purpose. The substrate specificity of such transporters is broad, and in some cases it has been shown to include not only anionic conjugates but also neutral or weakly cationic drugs. In the present article, we review the molecular identity, functional and structural characteristics of these pumps, mainly on the example of human erythrocytes, and discuss their physiological role in detoxification and in the multidrug resistance phenotype of cancer cells.

2

Lipid peroxidation and cell cycle signalling: 4-hydroxynonenal, a key molecule in stress mediated signalling

88%

Yang Y., Sharma R., Sharma A., Awasthi S., Awasthi Y. C.

Acta Biochimica Polonica

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2003

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tom 50

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nr 2

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the a-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H2O2 or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H2O2 with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.

3

Functional reconstitution of Ral-binding GTPase activating protein, RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal

76%

Sharma R., Sharma A., Yang Y., Awasthi S., Singhal S. S., Zimniak P., Awasthi Y. C.

Acta Biochimica Polonica

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2002

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tom 49

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nr 3

Earlier studies from our laboratories have shown that RLIP76, a previously described Ral-binding GTPase activating protein (Jullien-Flores et al., 1995, J. Biol. Chem. 270: 22473), is identical with the xenobiotic transporter DNP-SG ATPase, and can catalyze ATP-dependent transport of glutathione-conjugates as well as doxorubin (Awasthi et al., 2000, Biochemistry, 39: 9327). We have now reconstituted purified bacterially expressed RLIP76 in proteoliposomes, and have studied ATP-dependent uptake of the glutathione conjugate of 4-hydroxynonenal (GS-HNE) by these vesicles. Results of these studies show that RLIP76 reconstituted in proteoliposomes catalyzes ATP-dependent transport of GS-HNE against a concentration gradient. The transport of GS-HNE is saturable with respect to ATP as well as GS-HNE with Km values of 1.4 mM and 2.5 ,«M, respectively. These studies demonstrate that RLIP76 mediates active transport of GS-HNE, and are consistent with our previous work showing that RLIP76-mediated efflux of GS-HNE regulates the intracellular concentration of 4-HNE and thereby affects 4-HNE mediated signaling.

4

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein [RLIP76]

64%

Awasthi S., Sharma R., Yang Y., Singhal S. S., Pikula S., Bandorowicz-Pikula J., Singh S. V., Zimniak P., Awasthi Y. C.

Acta Biochimica Polonica

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2002

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tom 49

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nr 4

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi etal., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.

5

Purification and functional reconstitution of intact ral-binding GTPase activating protein, RLIP76, in artificial liposomes

64%

Singhal S. S., Singhal J., Cheng J., Pikula S., Sharma R., Zimniak P., Awasthi Y. C., Awasthi S.

Acta Biochimica Polonica

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2001

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tom 48

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nr 2

We have recently shown that RLIP76, a ral-bind ing GTPase ac ti vat ing pro tein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi eta/., Biochemistry39l9327l2000).TransportfunctionofRLIP76 was found to be in tact de spite con sid er able proteolytic frag men ta tion in prep a ra tions used for those stud ies, sug gest ing ei ther that the re sid ual in tact RLIP76 was re spon si ble for trans port ac tiv ity, or that the trans port ac tiv ity could be re con sti tuted by fragments of RLIP76. If the for mer were true, in tact RLIP76 would have a much higher spe cific ac tiv ity for ATP-hydrolysis than the frag mented pro tein. We have ad dressed this ques tion by com par ing trans port prop er ties of re com bi nant RLIP76 and hu man eryth ro cyte mem brane RLIP76 pu ri fied in buff ers treated with either 100 or 500uM serine pro te ase in hib i tor, PMSF. The pu rity and iden tity of re com bi nant and hu man eryth ro cyte RLIP76 was es tab lished by SDS/PAGE and West ern-blot anal y sis. These studies con firmed the or i gin of the 38 kDa pro tein, pre vi ously re ferred to as DNP-SG ATPase, from RLIP76. Higher PMSF con cen tra tion re sulted in lower yield of the 38 kDa band and higher yield of in tact RLIP76 from both hu man and re com bi nant source. In con trast, the sub strate-stimulated ATPase ac tiv ity in pres ence of DNP-SG, doxorubicin, daunorubicin, or colchicine were un af fected by in creased PMSF; similarly, ATP-dependent trans port of doxorubicin in proteo liposomes re con sti tuted with RLIP76 was un af fected by higher PMSF. These re sults in di cated that lim ited proteolysis by serine pro teas es does not ab ro gate the trans port func tion of RLIP76. Com parison of trans port ki net ics for daunorubicin be tween re com bi nant vs hu man erythrocyte RLIP76 re vealed higher spe cific ac tiv ity of trans port for tis sue pu ri fied RLIP76, in di cat ing that ad di tional fac tors pres ent in tis sue pu ri fied RLIP76 can mod u late its transport activity.

Ograniczanie wyników

5 Acta Biochimica Polonica

5 Awasthi S.

5 Awasthi Y. C.

5 Sharma R.

4 Zimniak P.

3 Singhal S. S.

3 Yang Y.

2 Pikula S.

2 Sharma A.

1 Bandorowicz-Pikula J.

1 Cheng J.

1 Singh S. V.

1 Singhal J.

1 2003

2 2002

1 2001

1 2000

Transport of glutathione-conjugates in human erythrocytes

Lipid peroxidation and cell cycle signalling: 4-hydroxynonenal, a key molecule in stress mediated signalling

Functional reconstitution of Ral-binding GTPase activating protein, RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein [RLIP76]

Purification and functional reconstitution of intact ral-binding GTPase activating protein, RLIP76, in artificial liposomes