Inhibition of caspase-3 activity and activation by protein glutathionylation

Abstract

Protein glutathionylation is a post-translational modification that may account for a broad mechanism of redox signaling. The caspase family of cysteine proteases represents a potential target for regulation by glutathionylation. To examine this, caspase proteins, derived from HL-60 cells after activation with actinomycin D, were incubated with GSSG. Total protein glutathionylation was enhanced and caspase-3 activity was inhibited in a dose- and time-dependent manner by GSSG. Caspase inhibition was reversible by thiol-specific reducing reagents. Proteolytic activation of caspases was also affected, as the activation of procaspase-3 and procaspase-9 in HL-60 cell extracts induced by cytochrome c and dATP was inhibited by pre-incubation with GSSG. When biotin-labeled GSSG was incubated with recombinant caspase-3, biotin label was found associated with both p12 and p17 subunits of active caspase-3 by non-reducing SDS-PAGE. Caspase-3 glutathionylation was confirmed by matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis of GSSG-treated recombinant caspase-3. Specific sites of glutathionylation were identified as Cys(135) of the p17 protein (the active site) and Cys(45) of the p12 protein. These results indicate that glutathionylation of caspase can occur at physiologically relevant concentrations of GSSG and results in the inhibition of caspase activation and activity.

Figure 1

Time Course, Dose Response and Reversibility of Inhibition of Caspase-3 Activity by GSSG. (A) Time course of caspase-3 Inhibition by GSSG. HL-60 cell lysates (100 µg protein) were incubated with (solid circle) or without (open circle) 1 mM GSSG at 37°C for various times (0–120 min). Caspase-3 activity was measured according to Experimental Procedures. 1 unit=1 ΔO.D./min. (B) Dose Response of Caspase-3 Inhibition by GSSG. HL-60 cell lysates (100 µg protein) were incubated with GSSG (0–1 mM) for 90 min at 37°C. Caspase-3 activity was measured according to Experimental Procedures. (C) Reversibility of Caspase Inhibition by GSSG is Thiol Specific. HL-60 apoptotic cell lysates (100 µg protein) were preincubated with 1 mM GSSG for 90 min at 37°C, then incubated with various concentrations of DTT, cysteine, ascorbic acid and GSH for 30 min. Caspase-3 activity was measured as described in Experimental Procedures. Results are presented as means ± SEM (n=3). * Significantly different from untreated control, (P<0.01). ** Significantly different from GSSG-treated control, (P<0.01).

Figure 2

Inhibition of Caspase-9 and Caspase-3 by GSSG in a Cell-Free System. (A) Activation of Caspase-9 and Caspase-3 in HL-60 Cytosolic Cell Lysates by Cytochrome c and dATP. HL-60 cytosolic cell lysates (100 µg protein) were incubated with +/− 10 µM cytochrome c, +/− 1 mM dATP and +/− 1 mM DTT for 60 min at 37°C. Reactions were stopped by addition of caspase activity buffer. Activities of caspase-9 and caspase-3 were measured using synthetic substrates Ac-LEHD-pNA and Ac-DEVD-pNA, respectively. (B) Inhibition of caspase-3 activation by GSSG. HL-60 cytosolic cell lysates (100 µg protein) were preincubated with different concentrations of GSSG for 90 min at room temperature. Cytochrome c and dATP were added to activate caspases. After 60 min at 37°C, reactions were stopped by addition of caspase activity buffer. Caspase-3 activity was measured in the presence (solid square) or absence of 5 mM DTT (open square) using the synthetic tetrapeptide substrate. (C) Inhibition of caspase cleavage by GSSG. Resulting reaction solutions (100 µl) after caspase activation (above) were directly subjected to western blot analysis for caspase-3 and caspase-9 using rabbit anti-caspase-3 and anti-caspase-9, respectively. Lane 1 (labeled “-“) contained no cytochrome C and dATP.

Figure 3

Glutathionylation of Native and Recombinant Caspase-3 by Biotinylated GSSG. (A) Structure of biotinylated GSSG. (B) Inhibition of Caspase-3 activity in HL-60 Cell Lysate by GSSG (open square) and Biotinylated GSSG (solid square). (C) Glutathionylation of Native Caspase-3 in HL-60 Cell Lysates. HL-60 apoptotic cell lysates containing 10 mg protein were incubated with or without 50 µM biotinylated GSSG for 90 min. Proteins were dialyzed to remove unbound biotinylated GSSG and then incubated with streptavidin-agarose beads. The bound proteins were eluted by addition of 50 mM DTT, then subjected to western blot analysis and probed with rabbit anti-caspase-3 antibody. (D) Glutathionylation of Recombinant Caspase-3 by Biotinlyated GSSG. Human recombinant caspase-3 (150 ng) was incubated with increasing concentrations of biotinylated GSSG for 90 min. Western blot analysis of caspase-3 was performed under reducing and non-reducing conditions using SDS-PAGE and probed with avidin peroxidase.

Figure 4

Mass Spectrometric Analysis of GSSG-treated Recombinant Caspase-3. Human recombinant caspase-3 (2 µg) was treated with (A) 0 µM GSSG, (B) 50 µM GSSG, (C) 1 mM GSSG, or (D) 1 mM GSSG and subsequently with 1 mM DTT at 37°C. The exposure times for GSSG and DTT treatments were 90 and 30 min, respectively. All samples were subjected to mass spectrometric analyses as described in Experimental Procedures.

Figure 5

Glutathionylation of Procaspase-3 in Liver. Protein extracts from rat liver were prepared as described in Methods. Portions were reduced with 20 mM DTT for 30 min at 37°C and then dialyzed over night. Portions of the DTT-treated samples were incubated with either 1 mM GSSG or 1 mM cystine at 37°C for 90 min. All samples were subjected to IEF and immunoblot. Lane 1, the original liver extract; lane 2, reduced liver extract; lane 3, GSSG-treated liver extract; lane 4, cystine-treated liver extract.