Novel role for glutathione S-transferase pi. Regulator of protein S-Glutathionylation following oxidative and nitrosative stress

Abstract

Glutathione S-transferase Pi (GSTpi) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTpi potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTpi is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTpi, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTpi multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTpi and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs.

FIGURE 1.

GSTπ expression enhances time-dependent S-glutathionylation of proteins. Mouse embryo fibroblast cells derived from GSTπ wild-type or knock-out animals were treated with 200 μm GSSG (A) or 25 μm PABA/NO for 0 to 240 min (B). Proteins were separated by non-reducing SDS-PAGE and S-glutathionylation evaluated by immunoblot (IB) with anti-glutathionylated protein monoclonal antibody (PSSG, Virogen, n = 4). Even loading of protein was confirmed by stripping the membrane and re-probing for actin. The kinetics of the S-glutathionylation reaction were analyzed using a standard 2 parameter exponential rise to maximum fitting procedure (Sigma Plot 10, SyStat, MA) for time dependence following GSSG and PABA/NO (C) for GSTπ^+/+ (•) and GST^-/- MEF (▴). Data represent mean ± variance, p < 0.01.

FIGURE 2.

GSTπ expression enhances dose-dependent S-glutathionylation of proteins. Mouse embryo fibroblast cells derived from GSTπ^+/+ or GSTπ^-/- animals were treated with various concentrations of GSSG (A) or PABA/NO (B) for 1 h. Proteins were resolved by non-reducing SDS-PAGE and S-glutathionylation evaluated by immunoblot (IB) with PSSG antibody (n = 4). The kinetics of the S-glutathionylation reaction were analyzed using a standard 2-parameter exponential rise to maximum fitting procedure (Sigma Plot 10, SyStat, MA) for dose dependence following GSSG or PABA/NO (C) for GSTπ^+/+ (•) and GST^-/- MEF (▴). Data represent mean ± variance; p < 0.01.

FIGURE 3.

S-Glutathionylation of actin is elevated in MEF-GST^+/+ cells. MEF-GST^+/+ and MEF-GST^-/- cells were seeded on glass coverslips and treated with: A, vehicle; B, 300 μm GSSG; C, 15 μm PABA/NO for 1 h and stained with phalloidin to visualize actin polymerization/stress fiber formation. Arrow highlights focal contacts.

FIGURE 4.

S-Glutathionylation of serum and liver proteins in GSTπ WT and KO mice. Male GSTπ wild-type (GSTP1P2^+/+) and knock-out (GSTP1P2^-/-) mice (ages 1 and 3 months) were treated with an intravenous bolus of the oxidized glutathione mimetic, NOV-002 at 25 mg/kg or PABA/NO at 5 mg/kg. Blood was collected at various time points via orbital bleed. The plasma proteins (A) were separated by non-reducing SDS-PAGE and S-glutathionylated contrapsin was evaluated by immunoblot (IB) with PSSG antibody. The ratios of S-glutathionylated contrapsin to albumin were plotted in arbitrary units (B), solid, 1-month-old; hatched, 3-month-old animals (6 animals per treatment group). Free protein sulfhydryl content was measured in the liver homogenates of 3-month-old GSTπ wild-type (+/+) and knock-out (-/-) mice (C) using the fluorescent sulfhydryl-specific probe ThioGlo-1. The ThioGlo-1 emission (at 513 nm) for each treatment group was averaged and plotted as mean ± S.D. (n = 3); solid bars represent GSTπ WT (+/+) and open bars represent KO (-/-) animals. The difference between WT and KO treated and untreated animals is statistically significant (^*, p ≤ 0.001).

FIGURE 5.

GSTπ enzymatic activity is crucial for protein S-glutathionylation. HEK293 cells were transiently transfected with vector (HEK-VA), wild-type GSTπ (HEK-WT), or an enzymatically inactive mutant form of GSTπ (HEK-Y7F). Concentration (A) and time dependence (B) effects following PABA/NO treatment illustrate that increased ectopic expression of GSTπ stimulates, whereas mutation of the catalytic tyrosine in the enzyme active site diminishes S-glutathionylation (PSSG). The corresponding relative abundance of modified proteins was plotted as the fold increase compared with untreated control for concentration (C) and time (D) dependence. Experimental data were fitted with standard sigmoid, 2-parameter exponential rise to maximum (Sigma Plot 10, SyStat). HEK-WT cells, •; HEK-VA cells, ▪; and HEK-Y7F cells, ▴; n = 3; p < 0.01. IB, immunoblot.

FIGURE 6.

Autoregulation of GSTπ occurs through the Cys⁴⁷ and Cys¹⁰¹S-glutathionylation. HEK293-WT cells were treated with 0–50 μm PABA/NO for 1 h (A). The proteins were separated by non-reducing SDS-PAGE and S-glutathionylation was evaluated by immunoblot (IB) with PSSG monoclonal primary antibody and GSTπ polyclonal primary antibody and detected simultaneously with both red (anti-mouse) and green (anti-rabbit) fluorescent secondary antibodies. The dual colored image was quantified using standard Odyssey (LI-COR, NE) software. The bars represent a relative input of red and green fluorescence in each band. MALDI-MS analysis of purified, expressed GSTπ treated with 50 μm PABA/NO and 0.5 mm GSH showed that peptides containing Cys⁴⁷ and Cys¹⁰¹ are S-glutathionylated (B). S-Glutathionylation of Cys⁴⁷ and Cys¹⁰¹ on GSTπ alters structure. Spectroscopic analysis of native (black) and PABA/NO + GSH-treated (green) GSTπ in vitro was performed using CD (C) and tryptophanyl fluorescence (D) of purified protein. According to the published crystal structure (31), the relative positions of Cys⁴⁷ and Trp³⁸ for GSTπ are depicted using RasMol 2.7.4.2 (E).

FIGURE 7.

Protein-protein interactions of GSTπ with JNK were evaluated in HEK293 cells transfected with GSTπ (WT, Y7F, C47A, and C101A mutants). JNK1/2 was immunoprecipitated from HEK293 cells and separated by SDS-PAGE followed by immunoblot for GSTπ and JNK1/2 (A). Protein S-glutathionylation (PSSG antibody) patterns were evaluated in transfected HEK293 cells treated with vehicle or 30 μm PABA/NO for 1 h (B). The membranes were stripped and reprobed for GSTπ. The relative densities of S-glutathionylated proteins bands were plotted as mean ± S.D., of at least three independent experiments (C). IB, immunoblot.

FIGURE 8.

S-Glutathionylation of GSTπ in vitro. Recombinant human purified GSTπ (WT, Y7F, C47A, and C101A) was treated with 50 μm PABA/NO and 0.5 mm GSH for 1 h (A). The proteins were separated by non-reducing SDS-PAGE and S-glutathionylation was evaluated by immunoblot (IB) with PSSG antibody. Membranes were stripped and reprobed for GSTπ. Deglutathionylation of GSTπ by hSrx1 is shown in B. GSTπ was treated with: 1, control; 2, 50 μm PABA/NO and 0.5 mm GSH for 1 h; 3, 50 μm PABA/NO and 0.5 mm GSH for 30 min followed by addition of hSrx1 and further incubation 30 min; 0–50 μm PABA/NO and 0.5 mm GSH for 30 min followed by addition of heat-inactivated hSrx1 and further incubation 30 min; 5–50 μm PABA/NO and 0.5 mm GSH for 30 min followed by the addition of C99A mutant of hSrx1 and further incubation 30 min. IP, immunoprecipitation.