Glutathione Conjugate Transporter RLIP76
Synonyms
Definition
RLIP76 is a multifunctional protein important for utilizing the energy currency adenosine triphosphate ( ATP) to transport various chemical materials outside the cell. Through this transport process this transporter protein converges many cell signaling processes through the modulation of substrate levels or the contribution of protein–protein interactions to stimulate cell signaling. Overexpression of RLIP76 has been correlated to contribute to the phenomenon known as multidrug resistance. This protein is also known to have a clinical impact in addition to multidrug resistance toward inflammation, heat and oxidative stress, radiation, and a culmination of these stressors allowing it to contribute to general stress resistance.
Characteristics
(a) At normal levels or absence of increased stress, a baseline level of lipid hydroperoxide generation from membrane lipids gives rise to radicals. 4-hydroxynonenal (4-HNE) and other lipid aldehydes as well as peroxides are processed by GSTs to glutathione conjugate (GS-E) which are transported in an ATP-dependent manner by RLIP76 and other transporter proteins. The concentration of GS-E is low. Because of low concentration of GS-E, RLIP76 is balancing between transport and GAP (toward cdc42), thus inhibiting this proapoptotic pathway. Under conditions of low HNE, formation of DNA adducts is kept low and cell proliferation (marked bold) is favored. (b) Under the conditions of stress, lipid peroxidation is increased resulting in a greater amount of toxicity and greater amount of HNE formation, which is catalyzed to GS-E by the various GSTs. Because of the rise in GS-E product, some product-inhibition of GSTs occurs until RLIP76 activity is increased. If xenobiotic toxins (chemotherapy drugs, carcinogens) are also present under these condition, a competition arises between lipid metabolites and activated P450 metabolites (XO*) for GST-mediated conjugation with GSH, the concentration of XO* rises more rapidly, and genotoxicity is enhanced, particularly because XO* can propagate themselves via lipid peroxidation. Because of increased transport substrate, GS-E, the transport activity of RLIP76 is favored over its GAP activity toward cdc42, partially releasing that inhibition. The net effect, depending on the severity of stress, would result in decreased proliferative signals and increased tendency to apoptosis (marked bold), both as a result of loss of cdc42 inhibition, and as a result of increased electrophilic lipid or xenobiotic oxidation products. (c) If RLIP76 is inhibited under conditions of stress, not only is there rapid accumulation of GS-E, but inhibition of GSTs by GS-E results in accumulation of XO*, which are also formed more rapidly because of increased substrate xenobiotic due to lack of the xenobiotic efflux activity represented by RLIP76. Under these conditions, genotoxicity and apoptosis signaling are greatly increased and proliferation is diminished
Expression of RLIP76 is everywhere in mammalian tissues including kidney, liver, brain, muscle, eye, erythrocytes, and leukocytes. Its presence has also been demonstrated in cancer cell lines of various tissue origins. In cells, RLIP76 is primarily associated with the membrane but its presence in cytosol has been demonstrated, which is consistent with its known functions.
The primary structure of RLIP76 using N-terminal sequencing revealed that RLIP76 possess a GAP activity for cdc42 and the Ral-binding domain. In addition, it also contains a GTP-binding domain. RLIP76 was demonstrated to function as a Ral GEF (guanidine exchange factor), Rho- GAP (GTP-activating protein) and is responsible for signal propagation downstream to proteins such as POB1 (partner of RalBP1).
Discovery of RLIP76
A protein initially discovered as an ATP-dependent GS-E transporter was designated as DNP-SG ATPase. This protein stimulated ATPase activity in the presence of DNP-SG (dinitrophenyl S-glutathione a known conjugate of the cofactor glutathione and 1-chloro-2, 4-dinitrobenzene). ATP-dependent transport of DNP-SG and other GS-E was demonstrated in liposomes reconstituted with purified DNP-SG ATPase protein. Immunoscreening of a human bone marrow cDNA library using antibodies specific to DNP-SG ATPase resulted in cloning of a previously described protein RLIP76. It was established that the structural, immunological, and kinetic properties of tissue-purified DNP-SG ATPase were identical and that in addition to DNP-SG, other GS-E including LTC4, GS-HNE, and various chemotherapeutic drugs such as doxorubicin also stimulated the ATPase activity of RLIP76. Proteoliposomes reconstituted with recombinant RLIP76 catalyzed the ATP-dependent transport of these chemicals with kinetic parameters similar to that of DNP-SG ATPase. Additional immunological studies with tissue-purified DNP-SG ATPase and recombinant RLIP76 established the identities of these two proteins and that this transporter was distinct from the known transporters including the classical family of transporters that are members of the ATP-binding cassette transporter superfamily ( ABC transporter proteins).
Clinical Significance
Drug Resistance
The role of RLIP76 in drug resistance of cancer cells is suggested by studies showing that its overexpression confers resistance and drug accumulation by increasing the rate of transport from cancer cells. In contrast, inhibition of RLIP76 by antibodies reverses the protein’s overexpression effects. More importantly, depletion of the normal levels of RLIP76 in cancer cells by small interfering RNA (siRNA) sensitizes these cells to chemotherapy treatment and promotes apoptosis. The ability of RLIP76 to transport foreign material as well as their metabolites suggests that it is one of the major components of the phase III detoxification systems. This contention is supported by studies showing that in mice as well as in cancer cells in culture, RLIP76 is responsible for more than 70% of the total doxorubicin transport as opposed to MRP1 and other transporters which are responsible for transporting only less than 30% of the drug.
Heat-Shock and Oxidative Stress
Increases in oxidants can cause genomic insult leading to carcinogenesis. Oxidative stress usually leads to enhanced generation of endogenous electrophiles, which are metabolized to GSK-3 by GSTs. Besides being toxic, GS-E metabolites are known to be important contributors to cell signaling. Competitive inhibition of GS-E transport by xenobiotics implies that cell signaling pathways may be modulated by such compounds, particularly during oxidative stress-induced signaling. Alternatively, pharmacological effects of such xenobiotics could be modulated by oxidative stress. A major role of RLIP76 in defense against oxidative stress is suggested by studies showing that short exposure of mild heat-shock, oxidative stress, or UV radiation to human cell lines leads to induction of RLIP76 as an early stress responder even prior to the proteins known for stress response (e.g., the heat-shock proteins). Under stress conditions, formation of electrophilic products (e.g., 4-HNE) from the breakdown of lipids is observed and consequently GS-E levels rise. With induced expression of RLIP76, the stress-preconditioned cells acquire at least threefold higher capacity to transport endogenous GS-E as compared with untreated controls. This increased efflux of GS-E can be blocked by coating the cells with antibodies against RLIP76, indicating that GS-E are specifically transported by RLIP76. Cells, preconditioned to stress, display an enhanced ability to exclude GS-E due to induced RLIP76. Preconditioned cells are resistant to 4-hydroxynonenal (4-HNE), superoxide anion (O2-), hydrogen peroxide (H2O2), or UV radiation-mediated toxicity. These cells also acquire resistance to apoptosis by suppressing Jun N-terminal kinase and caspase-3. Inhibiting GS-E transport can abrogate the protective effect of this stress preconditioning. These studies suggest that the intracellular concentrations of endogenous GS-E, such as the conjugate between glutathione and 4-hydroxynonenal (GS-HNE), can affect stress-mediated signaling and that RLIP76 and GSTs are the major determinants of the intracellular levels of 4-HNE and GS-HNE.
Radiation Toxicity
Generation of reactive-oxygen species is one of the common effects of using high-energy radiation on biological tissues. RLIP76 is capable of providing protection against oxidative stress, primarily due to its ability to remove various electrophilic toxicants generated during lipid peroxidation caused by the reactive-oxygen species. This implies that RLIP76 should provide protection against radiation toxicity because lipid peroxidation is an unavoidable consequence of exposure to radiation. It has been demonstrated that purified RLIP76 incorporated into liposomes is eagerly taken up by cultured cells resulting in increased cellular RLIP76 levels. When lung cancer cell lines in culture were treated with RLIP76 liposomes prior to the exposure to radiation, these cells with increased levels of RLIP76 were found to be more resistant to radiation toxicity as compared with the control cells. Efflux of GS-E formed from the reactive products of lipid peroxidation contributes significantly to radiation toxicity and the increased efflux of GS-E from RLIP76-enriched cells rendered these cells resistance to radiation. Significance of GS-E efflux and role of RLIP76 against X-radiation has also been confirmed in the in vivo model.
The ability of RLIP76 to modulate stress from chemical, heat, radiation sources in addition to amphiphilic xenobiotic toxins such as vinca alkaloids used in chemotherapy indicates that RLIP76 plays a positive role in preventing oncogenesis but a negative role as the major contributor to resistance in cancerous cells.
References
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