Regular paper on-line at: www.actabp.pl Tocopherol esters inhibit human glutathione S-transferase omega

Human glutathione S-transferase omega 1-1 (hGSTO1-1) is a newly identified member of the glutathione S-transferase (GST) family of genes, which also contains alpha, mu, pi, sigma, theta, and zeta members. hGSTO1-1 catalyzes the reduction of arsenate, monomethylarsenate (MMA(V)), and dimethylarsenate (DMA(V)) and exhibits thioltransferase and dehydroascorbate reductase activities. Recent evidence has show that cytokine release inhibitory drugs, which specifically inhibit interleukin-1b (IL-1b), directly target hGSTO1-1. We found that (+)-alpha-tocopherol phosphate and (+)-alpha-tocopherol succinate inhibit hGSTO1-1 in a concentration-dependent manner with IC50 values of 2 microM and 4 microM, respectively. A Lineweaver-Burk plot demonstrated the uncompetitive nature of this inhibition. The molecular mechanism behind the inhibition of hGSTO1-1 by alpha-tocopherol esters (vitamin E) is important for understanding neurodegenerative diseases, which are also influenced by vitamin E.


InTROduCTIOn
Glutathione S-transferases (GSTs) are a family of enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles (Hayes et al., 2005).There are seven major types of human cytosolic GSTs: alpha, mu, pi, sigma, theta, zeta, and omega.These enzymes contribute to the biotransformation of a wide range of exogenous and endogenous compounds.hGSTO1-1 is a homodimer that catalyzes the reduction of MMA(V), which is an important step in the biotransformation of inorganic arsenic (Zakharyan et al., 2001).In fact, the known MMA(V) reductase protein is identical to the protein encoded by the gene recently identified as hGSTO1-1 (Board et al., 2000;Zakharyan et al., 2001).
The omega family of human GST (GSTO1-1 and GSTO2-2) has properties unlike those of the other known GSTs (Board et al., 2000).Human GSTO1-1 exhibits both thioltransferase and dehydroascorbate reductase activities and also serves as a reductase for arsenate, MMA(V), and DMA(V) (Aposhian et al., 2004;Zakharyan et al., 1999).The hGSTO2-2 protein also possesses both MMA(V)-and DMA(V)reducing activities (Schmuck et al., 2005).In addition, hGSTO1-1 is structurally similar to the CLIC proteins, a family of intracellular chloride channels.Furthermore, hGSTO1-1 modulates the activity of a ryanodine receptor (Dulhunty et al., 2001) and plays a role in a nuclear antioxidant system (Yin et al., 2001).A wide range of human tissues including liver, macrophages, glial cells, and endocrine cells express hGSTO1-1 (Yin et al., 2001).The physiological importance of hGSTO1-1 has not been fully elucidated yet.
Recently, Laliberte et al. (2003) reported that cytokine release inhibitory drugs, which specifically inhibit IL-1b, directly target hGSTO1-1.Thus, hGS-TO1-1 may be involved in the modulation of the activity of IL-1b.This type of inflammatory response has been suggested as a contributing mechanism to the pathogenesis of both Alzheimer's and Parkinson's disease.
A. Sampayo-Reyes and R.A. Zakharyan A wide range of cell culture, animal model, and human epidemiological studies have suggested a role for vitamin E in brain function and in the prevention of neurodegeneration; however, the underlying molecular mechanisms of this activity remain largely unknown (Azzi & Stacker, 2000;Anatol et al., 2004).The antioxidant activity of vitamin E has been investigated extensively although other activities have also been identified (Traber & Packer, 1995).
Numerous compounds have been shown to inhibit GST activity.Specifically, α-tocopherol (vitamin E) has been shown to be a potent inhibitor of GSTP1-1 (van Haaften et al., 2001;2002).An extended amino terminus and cysteine residues at the active site distinguish hGSTO1-1 physically from the other GSTs, including GSTP1-1.The active site of hGSTO1-1 is more consistent with glutaredoxin activity than with GST activity (Sinning et al., 1993;Dirr et al., 1994).In addition to the structural differences, hGSTO1-1 exhibits different functional properties than other GSTs, including GSTP1-1.GSTP1-1 contributes to resistance against cytostatic drugs and apoptosis (Johansson et al., 2000).Both hGSTO1-1 and GSTO2-2 have been documented to induce inflammation and to contribute to the pathogenesis of Alzheimer's disease and apoptosis (Ogru et al., 2004;Giri et al., 2005;Wang et al., 2005).Thus, despite the marked differences between hGSTO1-1 and GSTP1-1, we examined hGSTO1-1 as a possible target for inhibition by α-tocopherol.
A novel ester (RRR-bis-α-tocopheryl phosphate or RRR-di-α-tocopheryl phosphate) that consists of two α-tocopherol molecules linked by a phosphate group has recently been described.α-Tocopherol phosphate is water soluble unlike other α-tocopheryl esters.In addition, α-tocopheryl phosphate may have anti-inflammatory properties that are protective against atherosclerosis (Gianello et al., 2005).Interest in α-tocopheryl phosphate derivatives has increased after the recent discovery that this compound is present in plant and animal tissues as well as in food stuffs.These findings raised the possibilities that α-tocopheryl phosphate is a reserve form of α-tocopherol and that this compound may be capable of regulatory effects at the cellular level.Here, we describe the interactions between (+)-α-tocopherol phosphate, (+)-α-tocopherol succinate and hGSTO1-1.
recombinant hGSTO1-1 protein expression.Recombinant histidine-tagged hGSTO1-1 proteins were expressed using pQE30 constructs in Escherichia coli M15 cells containing the Rep4 plasmid (Qiagen) and purified by nickel agarose affinity chromatography.Briefly, a seed culture of the transformed cells was grown in 10 mL LB medium containing 100 μg/mL ampicillin and 25 μg/mL kanamycin.The culture was grown overnight at 37 o C with vigorous shaking.The following day, a 1 mL aliquot of the overnight culture was added to new media (500 mL) and grown to an OD 600 of 0.5-0.7.Gene expression was induced by the addition of IPTG to a final concentration of 1 mM.The cultures were then grown for an additional 4 h.The culture was then centrifuged at 4 000 × g, and the pellet was stored at -70 o C until use.The expressed 6×-His-tagged protein (hGSTO1-1) was purified with Ni-NTA resin (Qiagen, Tokyo, Japan) according to the manufacturer's instructions.The enzyme was eluted in 250 mM imidazole, 50 mM sodium phosphate, and 300 mM NaCl, pH 8.0.The purified protein was dialyzed against 20 mM Tris/HCl, 60 mM NaCl, and 5 mM dithiothreitol, pH 8.0, before storage at -70 o C (Board et al., 2000).
MMA(V) reductase assay.The reaction mixture contained 0.1 M Tris/HCl (pH 8.0), 5 mM GSH, 22 nmol and 3.4 × 10 5 c.p.m. of [ 14 C]MMA(V), and 4 μg hGSTO1-1 in a final volume of 250 μL.The reaction mixture was incubated at 37°C for 60 min.Samples were then placed on ice, and 0.5 mL of carbon tetrachloride containing 10 -2 M diethylammonium diethyldithiocarbamate (DDDC) was added.The mixture was shaken vigorously for 15 min at room temperature.After centrifugation at 3000 × g, the organic phase was separated and MMA(III) was backextracted from the organic phase with 0.5 mL of 0.1 M sodium hydroxide.After centrifugation at 3000 × g, the aqueous phase was separated and transferred into scintillation vials containing 120 μL of 0.5 M HCl.National Diagnostics Monoflow-3 scintillation Tocopherol esters inhibit human glutathione S-transferase omega cocktail (5 mL) was added, and the contents of the vials were counted in a Beckman model LS7800 liquid scintillation counter (Zakharyan & Aposhian, 1999).
MMA(V) reductase assay (spectrophotometric method).Reduction of MMA(V) with hGSTO1-1 was measured in the presence of glutathione and NADPH at 340 nm.MMA(V) reduction was assayed with 5 mM GSH, 18 mM MMA(V), 0.25 mM NADPH, 0.8 U GSH reductase, and 8 μg hGSTO1-1 in 0.1 M Tris buffer, pH 8.The reduction was monitored spectrophotometrically by recording the decrease in absorbance at 340 nm.Effects of various concentrations of (+)-α-tocopherol acid succinate (final concentration 0.5 μM to 16 μM) on hGSTO1-1 activity were determined.A stock solution of (+)-αtocopherol acid succinate was prepared in ethanol.The final concentration of ethanol in the incubation mixture (1%, v/v) had no effect on GSTO1-1 activity.The reaction mixture was incubated for 2 min at 37°C before activity measurement.
Protein assay.Protein concentrations were determined according to the Bradford method (Bradford, 1976) using bovine serum albumin as the standard.
Other methods.Kinetic parameters were calculated by linear regression of the Lineweaver-Burk plot of the reciprocal initial rate versus the reciprocal concentration.The results are given as mean ± S.E.M. and represent three independent experiments.The results represent three independent preparations of hGSTO1-1 protein.

rESuLTS
The reduction of MMA(V) by hGSTO1-1 was inhibited by α-tocopherol phosphate in a concentration-dependent manner (Fig. 1).The IC 50 for (+)-αtocopherol phosphate was in the range of 2 ± 0.072 μM.Following the addition of 8 μM tocopherol phosphate, no GST activity was detected.The GST activity was measured with increasing concentrations of MMA(V) in the presence of a fixed concentration (2 μM) of (+)-α-tocopherol phosphate.hGSTO1-1 showed characteristic Michaelis-Menten behavior with respect to MMA(V) reduction (Fig. 2).
The Lineweaver-Burk plot shows that the inhibition of hGSTO1-1 with MMA(V) as a substrate by 2 μM of (+)-α-tocopherol phosphate is uncompetitive (Fig. 3).The regression line represents a K m of about 42.18 ± 3.4 × 10 -3 M with a V max = 0.78 ± 0.039 μmol/mg min for MMA(V) alone while the K m is 32.17 ± 1.91 × 10 -3 M with a V max = 0.53 ± 0.023 μmol/mg min in the presence of 2 μM tocopherol phosphate.

dISCuSSIOn
The previously reported inhibitors of GST activity exert their action by binding to the hydrophobic pocket of the active site of GST (H-site) (Burg et al., 2002).Others have reported that RRRα-tocopherol, the naturally occurring form of vitamin E, probably binds to a lipophilic pit-like structure in GST.Binding of RRR-α-tocopherol to this site induces a conformational change of the GST molecule and thus diminishes the activity of the enzyme (van Haaften et al., 2002).Conformational changes could be caused by an increase in protein volume and/or structural modifications of the lipophilic regions between the two monomers of GST.Binding of a compound to this region could, in fact, change the activity of the enzyme (van Haaften et al., 2002).In our experiments, the addition of 2 μM (+)-α-tocopherol to a solution of purified hGSTO1-1 resulted in a dramatic inhibition of the enzyme (the IC 50 value for (+)-α-tocopherol phosphate was 2 μM).Thus, (+)-α-tocopherol phosphate is a potent inhibitor of human GSTO1-1.Similar effects were observed for (+)-α-tocopherol succinate.While the effects of tocopherol phosphate and tocopherol succinate on hGSTO1-1 were specific, other compounds such as CDNB, deoxycholate, which is a nonsubstrate inhibitor of GSTs, and ethacrynic acid were inhibitory with IC 50 values of 900 ± 75, 1020 ± 82 and 25 ± 1 μM, respectively.
A novel natural form of vitamin E, tocopheryl phosphate, has been detected in biological tissues as well as in a variety of foods (Gianello et al., 2005).This molecule is a water-soluble tocopherol ester and thus may be valuable in cellular functions (Gianello et al., 2005).Particularly, α-tocopherol phosphate was shown to exert a protective effect against ultraviolet-induced skin damage.This protection was greater than that seen with -tocopherol acetate (Nakayama et al., 2003).
Studies encompassing cell culture, animal model, and human epidemiological approaches have indicated a role for vitamin E both in normal brain function and in the prevention of neurodegeneration; however, the underlying molecular mechanisms of this association are not understood (Rota et al., 2005).In a placebo-controlled trial, administration of vitamin E (2000 IU/day for 2 years) slowed (by 53%) the functional deterioration in patients with moderate Alzheimer's disease.These findings suggest that the use of α-tocopherol may actually delay clinically important functional deterioration in patients with Alzheimer's disease (Sano et al., 1997).The prevention of Alzheimer's disease progression has been linked to vitamin E from food sources but not from supplements (Clare et al., 2005).One potential explanation for this difference stems from the fact that vitamin E supplements consist of α-tocopherol alone while naturally occurring vitamin E consists of four different tocopherol forms (α, β, γ, and δ) and four corresponding tocotrienols.Thus, the authors of the above mentioned study propose that the effects of vitamin E on Alzheimer's disease are not due to α-tocopherol alone but to either another tocopherol form or a combination of tocopherol forms (Clare et al., 2005).
The nervous, immune, and endocrine systems are closely linked due, in part, to the presence of molecules such as the interleukins.IL-1 and IL-6, for example, are involved in the inflammatory responses as well as the immune responses that occur in Alzheimer's disease (Serrano-Sanchez & Diaz-Arnesto, 2001).Inhibitors of inflammation may be important for preventing progression of Alzheimer's disease and other neurodegenerative diseases and thus require more in-depth investigations (Laliberte et al., 2003).Studies have suggested that IL-1b is overexpressed in the brains of both Alzheimer's and Parkinson's patients (Laliberte et al., 2003).Furthermore, hGSTO1-1 and possibly hGSTO2-2 have been correlated with an inflammatory response and the age-at onset for Alzheimer's disease (Laliberte et al., 2003).Cytokine release in- A K m of 42.18 ± 3.4 × 10 -3 M with a V max of 0.78 ± 0.039 μmol/mg min was obtained by linear regression for MMA(V) alone (n).A K m of 32.17 ± 1.91 × 10 -3 M with V max of 0.53 ± 0.023 μmol/mg min was obtained by linear regression following the addtion of 2 μM tocopherol phosphate (•).Each point denotes the mean of three independent experiments (± S.E.M.).
Tocopherol esters inhibit human glutathione S-transferase omega hibitory drugs, which inhibit IL-1b directly, target hGSTO1-1.A possible mechanism for the effect of vitamin E on the delay of clinically important deterioration in Alzheimer's patients may be the direct inhibition of hGSTO1-1 and the subsequent inhibition of the activity of IL-1b, which is one of the key signalling that modulate inflammation.Therefore, vitamin E, which we have shown here to be a potent inhibitor of hGSTO1-1, should be evaluated for its role in inflammatory inhibition in neurodegenerative diseases.