Regulatory functions of glutathione S-transferase P1-1 unrelated to detoxification

Abstract

Glutathione S-transferase P1-1 (GSTP) is one member of the family of GSTs and is ubiquitously expressed in human tissues. The literature is replete with reports of high levels of GSTP linked either with cancer incidence or drug resistance, and yet no entirely cogent explanation for these correlations exists. The catalytic detoxification properties of the GST isozyme family have been a primary research focus for the last four decades. However, it has become apparent that they have undergone structural and functional convergence where evolutionary selective pressures have favored the emergence of noncatalytic properties of GSTP that has imbued this isozyme with expanded biological importance. For example, GSTP has now been linked with two cell-signaling functions that are critical to survival. Through protein:protein interactions, GSTP can sequester c-jun N-terminal kinase (JNK) and act as a negative regulator of this stress kinase. Pharmacologically, this activity has been linked with the activity of GSTP inhibitors in stimulating myeloproliferation. In addition, GSTP is linked with the forward S-glutathionylation reaction, a post-translational modification that impacts the function/activity of a number of proteins. Catalytic reversal of S-glutathionylation is well characterized, but the role of GSTP in catalyzing the forward reaction contributes to the "glutathionylation cycle." Moreover, GSTP is itself susceptible to S-glutathionylation, providing an autoregulatory loop for the cycle. Because oxidative stress regulates both S-glutathionylation and JNK-signaling pathways, such links may help to explain the aberrant patterns of GSTP expression in the cancer phenotype. As such, there is an ongoing preclinical and clinical platform of drug discovery and development around GSTP.

Figure 1

Scheme illustrating the forward and reverse reactions of the S-glutathionylation cycle. Modified from Tew (2007).

Figure 2

S-glutathionylation of the active-site cysteines on PDI alters protein structure. Spectroscopic analysis of native (red) and PABA/NO+GSH-treated (green) PDI in vitro using circular dichroism (A) and tryptophanyl fluorescence (B) of purified protein. The enzymatic activity of PDI was assessed using the insulin turbidity assay (C). According to the published crystal structure (Tian et al., 2006) and (D), the relative positions of the PDI C61 and 64 and W60 are depicted using Ras Mol 2.7.4.2 (http://rasmol.org last accessed February 18 2011). From Townsend et al., (2009b).

Figure 3

Autoregulation of GSTP occurs through the S-glutathionylation of Cys47 and Cys101. HEK293 cells were treated with 0–50 uM of PABA/NO for 1 hour (A). The proteins were separated by nonreducing sodium dodecyl sulfate polyacrylamide electrophoresis, and S-glutathionylation was evaluated by immunoblot with PSSG monoclonal primary antibody and GSTP polyclonal primary antibody and detected simultaneously with both red (antimouse) and green (antirabbit) fluorescent secondary antibodies. The dual-colored image was quantified, and the bars represent a relative input of red and green fluorescence in each band. Matrix-assisted laser desorption/ionization mass spectrometric analysis of purified, expressed GSTP treated with 50 μM of PABA/NO and 0.5 mM of GSH showed that peptides containing Cys47 and Cys101 are S-glutathionylated (B). S-glutathionylation of Cys47 and Cys101 on GSTP alters structure. Spectroscopic analysis of native (black) and PABA/NO+GSH-treated (green) GSTP in vitro was performed using CD (C) and tryptophanyl fluorescence (D) of purified protein. According to the published crystal structure (Ji et al., 1997), the relative positions of GSTPs C47 and W38 are depicted (E) using RasMol 2.7.4.2 (http://rasmol.org last accessed February 18 2011). From Townsend et al., (2009a).

Figure 4

S-glutathionylation of actin is elevated in mouse embryo fibroblast (MEF) from wild-type (GST^+/+) cells. MEF-GST^+/+ and ^−/− cells were seeded on glass coverslips and treated with 1) vehicle; 2) 300 μM of GSSG; or 3) 15 μM of PABA/NO for 1 hour and stained with phalloidin to visualize actin polymerization/stress fiber formation. From Townsend et al., (2009a).

Figure 5

Structure and activation of two GSTP-activated prodrugs. (A) TLK286 (Telcyta^®; canfosfamide). (B) PABA/NO.