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

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.

Transfection of mGSTA4 in HL-60 cells protects against 4-hydroxynonenal-induced apoptosis by inhibiting JNK-mediated signaling.

The mammalian alpha-class glutathione S-transferase (GST) isozymes mGSTA4-4, rGSTA4-4, and hGSTA4-4 are known to utilize 4-hydroxynonenal (4HNE) as a preferred substrate. During the present studies, we have examined the effect of transfecting human myeloid HL-60 cells with mGSTA4, on 4-HNE-induced apoptosis and the associated signaling mechanisms. Results of these studies show that treatment of the wild-type or vector-only-transfected HL-60 cells with 20 microM 4-HNE caused apoptosis within 2 h. The cells transfected with mGSTA4 did not undergo apoptosis under these conditions even after 4 h. In the wild-type and vector-transfected cells, apoptosis was preceded by JNK activation and c-Jun phosphorylation within 30 min, and an increase in AP-1 binding within 2 h of treatment with 20 microM 4-HNE. In mGSTA4-transfected cells, JNK activation and c-Jun phosphorylation were observed after 1 h, and increased AP-1 binding was observed after 8 h under these conditions. In the control cells, 20 microM 4-HNE caused caspase 3 activation and poly(ADP-ribose) polymerase cleavage within 2 h, while in mGSTA4-transfected cells, a lesser degree of these effects was observed even after 8 h. Transfection with mGSTA4 also provided protection to the cells from 4-HNE and doxorubicin cytotoxicity (1.6- and 2.6-fold, respectively). These results show that 4-HNE mediates apoptosis through its effects on JNK and caspase 3, and that 4-HNE metabolizing GST isozyme(s) may be important in the regulation of this pathway of oxidative-stress-induced apoptosis.

Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress.

To explore the role of lipid peroxidation (LPO) products in the initial phase of stress mediated signaling, we studied the effect of mild, transient oxidative or heat stress on parameters that regulate the cellular concentration of 4-hydroxynonenal (4-HNE). When K562 cells were exposed to mild heat shock (42 degrees C, 30 min) or oxidative stress (50 microM H2O2, 20 min) and allowed to recover for 2 h, there was a severalfold induction of hGST5.8, which catalyzes the formation of glutathione-4-HNE conjugate (GS-HNE), and RLIP76, which mediates the transport of GS-HNE from cells (Awasthi, S., Cheng, J., Singhal, S. S., Saini, M. K., Pandya, U., Pikula, S., Bandorowicz-Pikula, J., Singh, S. V., Zimniak, P., and Awasthi, Y. C. (2000) Biochemistry 39, 9327-9334). Enhanced LPO was observed in stressed cells, but the major antioxidant enzymes and HSP70 remained unaffected. The stressed cells showed higher GS-HNE-conjugating activity and increased efflux of GS-HNE. Stress-pre-conditioned cells with induced hGST5.8 and RLIP76 acquired resistance to 4-HNE and H2O2-mediated apoptosis by suppressing a sustained activation of c-Jun N-terminal kinase and caspase 3. The protective effect of stress pre-conditioning against apoptosis was abrogated by coating the cells with anti-RLIP76 IgG, which inhibited the efflux of GS-HNE from cells, indicating that the cells acquired resistance to apoptosis by metabolizing and excluding 4-HNE at a higher rate. Induction of hGST5.8 and RLIP76 by mild, transient stress and the resulting resistance of stress-pre-conditioned cells to apoptosis appears to be a general phenomenon since it was not limited to K562 cells but was also evident in lung cancer cells, H-69, H-226, human leukemia cells, HL-60, and human retinal pigmented epithelial cells. These results strongly suggest a role of LPO products, particularly 4-HNE, in the initial phase of stress mediated signaling.

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.

Role of arginine 216 in catalytic activity of murine Alpha class glutathione transferases mGSTAl-1 and mGSTA2-2 toward carcinogenic diol epoxides of polycyclic aromatic hydrocarbons.

Murine class Alpha glutathione (GSH) transferase A1-1 (mGSTA1-1) is unique among mammalian Alpha class GSTs due to its exceptionally high catalytic activity toward (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated metabolite of an environmentally relevant carcinogen, benzo[a] pyrene (BP). However, the molecular basis for high catalytic activity of mGSTA1-1 toward (+)-anti-BPDE is not clear. In the present study, we demonstrate that an arginine residue at position 216, which is conserved in some but not all mammalian class Alpha GSTs, plays an important role in catalytic activity of mGSTA1-1 toward (+)-anti-BPDE and carcinogenic diol epoxides of other environmentally relevant polycyclic aromatic hydrocarbons (PAHs). The catalytic efficiency (k(cat)/K(m)) of mGSTA1-1 for the GSH conjugation of (+)-anti-BPDE (108/mM/s) was reduced by about 58% upon replacement of arginine 216 with alanine (R216A). This was mainly due to a significantly lower V(max) for the R216A mutant of mGSTA1-1 compared with wild-type mGSTA1-1. The R216A mutation also resulted in a statistically significant reduction (>70%) in specific activity of mGSTA1-1 toward racemic anti-diol epoxides of chrysene and benzo[c]phenanthrene (anti-CDE and anti-B[c]PDE, respectively). The catalytic activity of mGSTA2-2, which is a close structural homologue of mGSTA1-1, was also reduced upon R216A mutation. The results of the present study clearly indicate that an arginine residue at position 216 is critical for catalytic activity of mGSTA1-1 and mGSTA2-2 toward carcinogenic diol epoxide metabolites of various PAHs that are abundant in the environment and suspected human carcinogens.

Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products.

Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties. We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation. 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity. This correlation indicates that in adult Drosophila 70 +/- 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1. The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g. GSTA4-4). The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life.

Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences.

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.

Two distinct 4-hydroxynonenal metabolizing glutathione S-transferase isozymes are differentially expressed in human tissues.

The two previously reported human glutathione S-transferase isozymes, hGST5.8 and hGSTA4-4, have been suggested to be similar because of their comparable activities toward 4-hydroxynonenal-GSH conjugation. Here, we demonstrate that hGST5.8 and hGSTA4-4 are distinct. Antibodies raised against hGSTA4-4 did not recognize hGST5.8, and antibodies raised against mouse GSTA4-4 that cross-react with hGST5.8 did not recognize hGSTA4-4. The pI value of hGSTA4-4 was found to be 8.4, as opposed to the pI value of 5.8 for hGST5.8. The two isozymes are differentially expressed in human tissues and there are significant differences in their kinetic properties. While both isozymes showed a strong expression in liver and testis, hGSTA4-4 was not detected in brain where hGST5.8 was present. In the pancreas, a strong expression of hGST5.8 was observed while hGSTA4-4 was barely detectable in this tissue.

Location of the epoxide function determines specificity of the allelic variants of human glutathione transferase Pi toward benzo[c]chrysene diol epoxide isomers.

Carcinogenic activity of many polycyclic aromatic hydrocarbons (PAHs) is mainly attributed to their respective diol epoxides, which can be classified as either bay or fjord region depending upon the location of the epoxide function. The Pi class human glutathione (GSH) transferase (hGSTP1-1), which is polymorphic in humans with respect to amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), plays an important role in the detoxification of PAH-diol epoxides. Here, we report that the location of the epoxide function determines specificity of allelic variants of hGSTP1-1 toward racemic anti-diol epoxide isomers of benzo[c]chrysene (B[c]C). The catalytic efficiency (k(cat)/K(m)) of V104,A113 (VA) and V104,V113 (VV) variants of hGSTP1-1 was approximately 2.3- and 1.7-fold higher, respectively, than that of the I104,A113 (IA) isoform toward bay region isomer (+/-)-anti-B[c]C-1,2-diol-3,4-epoxide. On the other hand, the IA variant was approximately 1.6- and 3.5-fold more efficient than VA and VV isoforms, respectively, in catalyzing the GSH conjugation of fjord region isomer (+/-)-anti-B[c]C-9,10-diol-11,12-epoxide. The results of the present study clearly indicate that the location of the epoxide function determines specificity of the allelic variants of hGSTP1-1 in the GSH conjugation of activated diol epoxide isomers of B[c]C.

Activity of allelic variants of Pi class human glutathione S-transferase toward chlorambucil.

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil, is often limited by the emergence of drug resistant tumor cells. Increased glutathione (GSH) conjugation (inactivation) of alkylating anticancer drugs or their activated metabolites due to overexpression of the Pi class GSH S-transferase (hGSTP1-1) is believed to be an important mechanism in tumor cell resistance to alkylating agents. Interestingly, the hGSTP1 locus is polymorphic in human populations and involves amino acid residues in positions 104 (isoleucine or valine) and/or 113 (alanine or valine). Here, we report that the allelic variants of hGSTP1-1 significantly differ in their efficiency in catalyzing the GSH conjugation of chlorambucil. Catalytic efficiency of the hGSTP1-1(I104,A113) isoform toward chlorambucil was approximately 2.5-, 7.5- and 15-fold higher compared with I104,V113, V104,A113 and V104,V113 variants of hGSTP1-1, respectively. The results of the present study suggest that hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to some alkylating agents.

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 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.

Catalytic efficiencies of allelic variants of human glutathione S-transferase Pi in the glutathione conjugation of alpha, beta-unsaturated aldehydes.

The catalytic efficiencies of the allelic variants of human glutathione (GSH) S-transferase Pi (hGSTP1-1), which differ in their primary structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), in the GSH conjugation (detoxification) of acrolein and crotonaldehyde have been determined. The k(cat)/K(m) values for hGSTP1-1 isoforms I104,A113 (IA), I104, V113 (IV), V104,A113 (VA) and V104,V113 (VV) toward acrolein were 129+/-3, 116+/-3, 128+/-4 and 92+/-3 mM(-1) s(-1), respectively. The catalytic efficiencies of the hGSTP1-1 variants IA, IV, and VA in the GSH conjugation of acrolein were statistically significantly higher (at P=0.05) compared with the VV isoform. On the other hand, the catalytic efficiencies of the hGSTP1-1 isoforms IA, IV, VA and VV toward crotonaldehyde (16+/-2, 12+/-1, 17+/-2, and 12+/-2 mM(-1)s(-1), respectively) were not statistically significantly different from each other. Our results suggest that hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to the toxic effects of acrolein, which is a widely spread environmental pollutant and generated endogenously during metabolic activation of anticancer drug cyclophosphamide.

Transport of glutathione-conjugates in human erythrocytes.

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.

Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells.

Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.

Crystal structure of a murine glutathione S-transferase in complex with a glutathione conjugate of 4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence of signaling across the dimer interface.

mGSTA4-4, a murine glutathione S-transferase (GST) exhibiting high activity in conjugating the lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) with glutathione (GSH), was crystallized in complex with the GSH conjugate of 4-HNE (GS-Hna). The structure has been solved at 2.6 A resolution, which reveals that the active site of one subunit of the dimeric enzyme binds GS-Hna, whereas the other binds GSH. A marked asymmetry between the two subunits is evident. Most noticeable are the differences in the conformation of arginine residues 69 and 15. In all GST structures published previously, the guanidino groups of R69 residues from both subunits stack at the dimer interface and are related by a (pseudo-) 2-fold axis. In the present structure of mGSTA4-4, however, the two R69 side chains point in opposite directions, although their guanidino groups remain in contact. In the subunit with bound GSH, R69 also interacts with R15, and the guanidino group of R15 points away from the active site, whereas in the subunit that binds GS-Hna, R15 pivots into the active site, which breaks its interaction with R69. According to our previous results [Nanduri et al. (1997) Arch. Biochem. Biophys. 335, 305-310], the availability of R15 in the active site assists the conjugation of 4-HNE with GSH. We propose a model for the catalytic mechanism of mGSTA4-4 in conjugating 4-HNE with GSH-i.e., the guanidino group of R15 is available in the active site of only one subunit at any given time and the stacked pair of R69 residues act as a switch that couples the concerted movement of the two R15 side chains. The alternate occupancy of 4-HNE in the two subunits has been confirmed by our kinetic analysis that shows the negative cooperativity of mGSTA4-4 for 4-HNE. Disruption of the signaling between the subunits by mutating the R69 residues released the negative cooperativity with 4-HNE.

Structure and function of residue 104 and water molecules in the xenobiotic substrate-binding site in human glutathione S-transferase P1-1.

Two variants of human class pi glutathione (GSH) S-transferase 1-1 with either isoleucine or valine in position 104 (hGSTP1-1[I104] and hGSTP1-1[V104]) have distinct activity toward (+)-anti-7, 8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. To elucidate their structure-function relationship, we determined the crystal structures of the two variants in complex with GSBpd, the GSH conjugate of (+)-anti-BPDE, at 2.1 and 2.0 A resolution, respectively. The crystal structures reveal that residue 104 in the xenobiotic substrate-binding site (H-site) dictates the binding modes of the product molecule GSBpd with the following three consequences. First, the distance between the hydroxyl group of Y7 and the sulfur atom of GSBpd is 5.9 A in the hGSTP1-1[I104].GSBpd complex versus 3.2 A in the V104 variant. Second, one of the hydroxyl groups of GSBpd forms a direct hydrogen bond with R13 in hGSTP1-1[V104].GSBpd; in contrast, this hydrogen bond is not observed in the I104 complex. Third, in the hydrophilic portion of the H-site of the I104 complex, five H-site water molecules [Ji, X., et al. (1997) Biochemistry 36, 9690-9702] are observed, whereas in the V104 complex, two of the five have been displaced by the Bpd moiety of GSBpd. Although there is no direct hydrogen bond between Y108 (OH) and the hydroxyl groups of GSBpd, indirect hydrogen bonds mediated by water molecules are observed in both complexes, supporting the previously suggested role of the hydroxyl group of Y108 as an electrophilic participant in the addition of GSH to epoxides.

Differential catalytic efficiency of allelic variants of human glutathione S-transferase Pi in catalyzing the glutathione conjugation of thiotepa.

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.

Differential protection against benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced DNA damage in HepG2 cells stably transfected with allelic variants of pi class human glutathione S-transferase.

The pi class glutathione S-transferase (GSTP1-1), which is polymorphic in human populations, is believed to play an important role in detoxification of the ultimate carcinogen of widespread environmental pollutant benzo[a]pyrene [(+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-BPDE]]. The allelic variants of human GSTP1-1 (hGSTP1-1) differ in their structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (valine or alanine). Here, we have determined the protective effect of overexpression of allelic variants of hGSTP1-1, through stable transfection in HepG2 cells, against (+)-anti-BPDE-induced DNA modification. Clonal transfectants of HepG2 cells corresponding to the three allelic variants of hGSTP1-1 [(I104,A113), (V104,A113), and (V104,V113), denoted hGSTP1(IA), hGSTP1(VA), and hGSTP1(VV), respectively] with similar levels of hGSTP1 protein were identified and characterized for their GST activity and (+)-anti-BPDE-induced DNA modification. The glutathione S-transferase activity toward (+)-anti-BPDE was significantly higher (approximately 3.0-3.6-fold) in cells transfected with hGSTP1(VA) [HepG2(VA)] and hGSTP1(VV) [HepG2(VV)] compared with hGSTP1(IA) transfectant [HepG2(IA)]. The formation of (+)-anti-BPDE-DNA adducts was significantly reduced in HepG2(VA) and HepG2(VV) cells compared with cells transfected with insert-free vector (HepG2-vect). Maximum protection against (+)-anti-BPDE-induced DNA damage was afforded by the hGSTP1(VV) isoform. The results of this study indicate that the allelic variants of hGSTP1-1 significantly differ in their ability to provide protection against (+)-anti-BPDE-induced DNA damage. Thus, hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to tumorigenesis induced by benzo[a]pyrene.

ATP-Dependent colchicine transport by human erythrocyte glutathione conjugate transporter.

We have recently demonstrated mutually inhibitory ATP-dependent transport of dinitrophenyl-S-glutathione (DNP-SG) and doxorubicin by DNP-SG ATPase purified from human erythrocyte membranes (S. Awasthi et al., 1998a,b). Our previous studies indicate a broad substrate specificity for this transport mechanism, including some P-glycoprotein substrates. Present studies were carried out to determine whether colchicine (COL), a classical P-glycoprotein substrate, could be transported by purified human erythrocyte DNP-SG ATPase reconstituted in artificial liposomes. We also investigated whether leukotriene C4 (LTC4), an endogenous proinflammatory glutathione-conjugate derived from arachidonic acid, would inhibit colchicine transport. Uptake of COL was compared in proteoliposomes reconstituted with the purified DNP-SG ATPase as well as control liposomes in the presence or absence of ATP. Increased colchicine uptake was observed upon addition of ATP to proteoliposomes, but not control liposomes. Uptake was linear with respect to the amount of vesicle protein used. Sensitivity to osmolarity was consistent with intravesicular COL accumulation. The ATP-dependent colchicine uptake was sensitive to temperature in a manner consistent with a protein-mediated transport process with activation energy of 7.3 kcal/mol. Time-dependent COL uptake by proteoliposomes in the presence of ATP was consistent with a single compartment model with an apparent rate constant of 0.21 +/- 0.02 min-1. Kinetic studies indicated a saturable behavior with respect to ATP (Km 2.3 +/- 0.7 mM) and colchicine (Km 4.3 +/- 0.2 microM). LTC4 was found to be a competitive inhibitor of COL transport (Kis 16.4 microM). Since DNP-SG ATPase is present in many tissues, it may play an important role in determining colchicine accumulation in cells. Increased LTC4 would tend to increase cellular COL accumulation.