Glutathione transferase P1 is modified by palmitate

Abstract

Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

Fig 1. Palmitoylation of GSTP1 in MCF7 cells.

MCF7 cells stably expressing GSTP1 with a V5 epitope tag at the amino- and carboxy-termini (MCF7-V5_GSTP1 or MCF7-GSTP1_V5, respectively) were metabolically labelled with ω-alkynyl-palmitate (+) or DMSO (-). Cells were lysed and GSTP1 was immunoprecipiated with agarose beads conjugated to mouse anti-V5 antibody (V5-10), subjected to click chemistry, resolved on 11% SDS-PAGE and transferred to a PVDF membrane. Palmitoylated GSTP1 was detected with neutravidin-HRP (1:20000) and total GSTP1 was detected with rabbit pAb anti-GSTP1 (GS70) (1:5000). (A) Time course of V5_GSTP1 metabolically labelled for 4, 8, 12, 24 and 48 h. (Top panel) Signal from neutravidin-HRP represents palmitoylated V5_GSTP1. (Bottom panel) Signal from anti-GSTP1 antibody represents total V5_GSTP1. (B) Densitometry on the bands detected by neutravidin-HRP and anti-GSTP1 in (A) was performed using ImageJ software. The level of palmitoylated V5_GSTP1 (neutravidin-HRP) relative to total V5_GSTP1 was expressed as a % of the 48 h time point. Symbols represent means +SD from 3 independent experiments. (C) MCF7- V5_GSTP1 and -GSTP1_V5 cells were metabolically labelled with ω-alkynyl-palmitate for 24 h (Top panel). Signal from neutravidin-HRP represents palmitoylated GSTP1. (Bottom panel) Signal from anti-GSTP1 antibody represents total GSTP1. Shown is a representative blot and similar results were obtained in three additional independent experiments.

Fig 2. Detection of cellular GSTP1 palmitoylation by a modified proximity ligation assay.

MCF7-V5_GSTP1 and MCF7-vector cells were metabolically labelled with ω-alkynyl-palmitate for 24 h, fixed, permeabilized and subjected to click chemistry. Cells were incubated with mouse mAb anti-biotin (BN-34) (1:500) and pAb rabbit anti-GSTP1(GS72) (1:250) for reaction with the biotinylated fatty acid analogue and GSTP1, respectively. Cells were then incubated with secondary antibodies PLUS and MINUS probe and ligated. The ligation product was amplified and detected with Duolink^® in situ detection reagent (orange), nuclei (blue) were detected with DAPI and GSTP1 (green) was detected with anti-rabbit Alexa488 (1:500). Cells were viewed with a Leica fluorescence microscope at a 60X magnification. (i-iii) MCF7-V5_GSTP1 cells (iv-vi) MCF7-vector control (negative control). (B) Images captured from the same experiment as A (with no Alexa Fluor® 488 secondary to detect total GSTP1) (i) in the absence of ω-alkynyl-palmitate and (ii) with no click chemistry step. Images were acquired in Volocity. White bars indicate 20 μm. Similar results were obtained for multiple cells from two independent experiments.

Fig 3. Palmitoylation status of human GSTP1 after mutation of Cys residues to Ser or Ala.

(A) Visualization of Cys residues present on GSTP1 dimer were identified in the PDB:3GSS crystal structure and visualized in Pymol; Cys15 (green), Cys48 (orange), Cys102 (blue) and Cys170 (pink). (B) pcDNA3.1-V5_GSTP1-WT, -Cys15Ser, -Cys48Ser, -Cys102Ser or-Cys170Ser were transiently transfected in HEK293T cells, metabolically labelled with ω-alkynyl-palmitate (+) or treated with DMSO (-), immunoprecipiated with mouse anti-V5 antibody (V5-10) conjugated to agarose beads and subjected to click chemistry. Samples were resolved on 11% SDS-PAGE and transferred to a PVDF membrane. (Top panel) Palmitoylated GSTP1 was detected by neutravidin-HRP (1:20000) and (bottom panel) total GSTP1 by rabbit pAb anti-GSTP1 (1:5000). (C) MCF7 cells stably expressing V5_GSTP1-WT and V5_GSTP1 with all four Cys residues mutated to Ser (Cys15/48/102/170Ser) (V5_GSTP1-4X_(C→S)) were metabolically labelled with ω-alkynyl-palmitate (+) or treated with DMSO (-) for 24–48 h, immunoprecipitated with agarose beads conjugated to mouse anti-V5 antibody (V5-10), and subjected to click chemistry. One half of each sample was treated with NaOH (0.1 M) for one h, the other half was kept at neutral pH. Samples were resolved on 11% SDS-PAGE and transferred to a PVDF membrane. (Top panel) Neutravidin-HRP (1:20000) and (bottom panel) pAb rabbit anti-GSTP1-GS72 (1:5000) were used to detect palmitoylated and total GSTP1, respectively. Similar results were obtained for an additional independent experiment.

Fig 4. Biochemical characterization of PAT-independent palmitoylation of human GSTP1 purified from E.coli.

GSTP1 was subjected to a series of chemical treatments pre- or post-palmitoylation in vitro and resolved on an 11% SDS-PAGE gel and transferred to a PVDF membrane. Palmitoylated GSTP1 was visualized with neutravidin-HRP (1:20000) and total GSTP1 detected with rabbit pAb anti-GSTP1 (1:1000). (A) GSTP1 was incubated in the presence or absence of NEM with ω-alkynyl-palmitoyl-CoA (lanes 1 and 2, respectively), ω-alkynyl-palmitate (lanes 3 and 4) or CoA (lanes 5 and 6). (B) Cys and Lys residues were blocked by incubating purified GSTP1 with NEM (10 mM) at pH 7 (lane 2) and pH 10 (lane 3), respectively, then in vitro palmitoylation using ω-alkynyl-palmitoyl-CoA was done. (C) In vitro palmitoylated GSTP1 was treated with NaOH (0.1 M) or MOPS at pH 7 for 1 h and then analyzed. Shown are representative blots and similar results were obtained in at least one additional independent experiment.

Fig 5. Mapping of palmitoylated amino acids on in vitro palmitoylated GSTP1 by LC-MS/MS.

Human GSTP1 purified from E.coli was palmitoylated in vitro and digested with proteases into peptides. After enriching for palmitoylated peptides, the samples were analyzed by LC-MS/MS. Shown are the annotated MS/MS spectra of GSTP1 peptides covering ⁴⁸C_palm, ¹⁰²C_palm, and ¹⁰³K_palm, with their respective backbone cleavage maps. (A) ⁴⁸C_palm-containing GSTP1 peptide obtained by Asp-N digestion, (B) tryptic GSTP1 peptides with ¹⁰²C and ¹⁰³K palmitoylation. (C) Spatial distribution of in vitro palmitoylated Cys48 (orange), Cys102 (blue), and Lys103 (red) of GSTP1 identified by LC-MS/MS was visualized in Pymol using the crystal structure (PDB 3GSS).

Fig 6. Characterization of V5_GSTP1_4XCys→Ala/Lys103Arg palmitoylation.

(A) The V5_GSTP1_4XCys→Ala/Lys103Arg mutant was generated and stably expressed in MCF7 cells. This cell line and MCF7-V5_GSTP1-WT were then metabolically labelled with ω-alkynyl-palmitate (+) or treated with DMSO (-) for >24 h. Cells were lysed and GSTP1 immunoprecipitated with agarose beads conjugated to mouse anti-V5 antibody (V5-10), subjected to click chemistry, resolved on 11% SDS-PAGE and transferred to a PVDF membrane. Palmitoylated GSTP1 was visualized with neutravidin-HRP (1:20000) and total GSTP1 with rabbit pAb anti-GSTP1 (1:5000). (B) Densitometry on neutravidin and anti-GSTP1 was performed using ImageJ software. The palmitoylated GSTP1 (neutravidin-HRP) was divided by the total GSTP1 signal and plotted normalized to WT-GSTP1. Two independent experiments were completed, bars represent means and symbols (□, ○) represent individual experiments.

Fig 7. Characterization of GSTP1 palmitoylation and activity from total, cytosolic and cellular membrane fractions of MCF7 cells.

MCF7-V5_GSTP1, MCF7-GSTP1, or MCF7-vector cells were disrupted by N₂ cavitation, nuclei and unbroken cells were removed by centrifuging at 2000 g. Supernatant (total) was collected and cytosolic fraction was separated from cellular membranes by centrifugation at 100000 g for 1 h. (A) 10 μg of protein sample from total input, cytosol, and membrane fractions from MCF7-GSTP1 cells were resolved on 11% SDS-PAGE and tranferred to a PVDF membrane. Total GSTP1 was detected with rabbit pAb anti-GSTP1 (1:5000) and equal protein loading was confirmed with amido black staining. (B) Densitometry of anti-GSTP1 in panel A was performed using ImageJ software. The level of GSTP1 from each fraction relative to cytosolic was plotted with bars representing means (±SD) from 5 independent experiments. Significant difference is indicated with **** p < 0.0001 (one-way ANOVA with Dunnett multiple comparisons test). (C) GSTP1 was immunoprecipitated from cytosolic and membrane fractions of MCF7-V5_GSTP1 cells metabolically labelled with ω-alkynyl palmitate (+) or DMSO containing medium (—) using agarose beads conjugated to mouse anti-V5 antibody (V5-10), and subjected to click chemistry, resolved on a 11% SDS-PAGE and tranferred onto a PVDF membrane. (top panel) Palmitoylated GSTP1 was detected with neutravidin-HRP (1:20000) and (bottom panel) total GSTP1 with rabbit pAb anti-GSTP1 (1:5000). Palmitoylated GSTP1 from cytosolic and membrane fractions (neutravidin-HRP) and total GSTP1(anti-GSTP1) from MCF7-V5_GSTP1 are shown. Shown is a representative blot and similar results were found in one additional experiment. (D) GSTP1 activity of total cell lysate, cytosol and membrane fraction from MCF7-vector and MCF7-GSTP1. Three μg of total cell lysate, cytosol, and membranes were incubated with 1 mM CDNB and 2.5 mM GSH. Formation of the DNP-GS conjugate was measured at 340 nm. Abssorbance values were normalized for blanks and spontaneous formation of DNP-GS in the absence of cellular fraction. Rates were normalized for relative GSTP1 levels estimated by densitometry, with bars representing means (±SD) of three independent experiments. Significant differences compared to MCF7-GSTP1 membranes are indicated with **** p ≤ 0.0001, (one-way ANOVA with Dunnett multiple comparisons test).

Fig 8. Association of purifed GSTP1 with the MCF7 plasma membrane-enriched fraction.

Human GSTP1 grown in and purified from (A, B) E. coli or (C-F) MCF7-GSTP1 cells grown in the presence of (C, D) regular FBS or (E, F) charcoal stripped fatty acid free FBS were tested for their ability to interact with the MCF7 plasma membrane-enriched fraction. GSTP1 from the different sources were left untreated or in vitro labelled with ω-alkynyl-palmitoyl-CoA. Untreated and in vitro palmitoylated GSTP1 (2 μg) were incubated with the MCF7 plasma membrane-enriched fraction (20 μg) for 30 min at 37°C. The MCF7 plasma membrane-enriched fraction without GSTP1 were incubated under identical conditions. Cellular membranes were pelleted by ultracentrifugation at 100000 g for 1 h, washed with PBS 1X + Triton-X-100 0.1%, resolved by 11% SDS-PAGE, and transferred onto a PVDF membrane. Membranes were immunoblotted for GSTP1 using the pAb rabbit anti-GSTP1(GS72) (1:5000). Equality of plasma membrane fraction loading was determined by probing for the integral plasma membrane protein Na⁺/K⁺-ATPase using the rabbit pAb anti- Na⁺/K⁺-ATPase (1:10000). (A, C, E) A representative blot is shown for each source of GSTP1 and similar results were observed in at least three additional independent experiments. (B, D, F) Densitometry analysis of GSTP1 and Na⁺/K⁺-ATPase bands was performed with ImageJ software. GSTP1 associated with the membrane was normalized for plamsa membrane fraction protein loading using the Na⁺/K⁺-ATPase signal. Bars represent the mean ± SD of 4 independent experiments with data points from individual determinations shown. Signficant differences are indicated with * p<0.05, ** p<0.01, *** p<0.001 and non-significant differences are indicated with a p-value, (one-way ANOVA followed by a Tukey multiple comparisons test).