Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal

Abstract

4-Hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product formed during oxidative stress that can alter protein function via adduction of nucleophilic amino acid residues. 4-HNE detoxification occurs mainly via glutathione (GSH) conjugation and transporter-mediated efflux. This results in a net loss of cellular GSH, and restoration of GSH homeostasis requires de novo GSH biosynthesis. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modulatory (GCLM) subunit. The relative levels of the GCL subunits are a major determinant of cellular GSH biosynthetic capacity and 4-HNE induces the expression of both GCL subunits. In this study, we demonstrate that 4-HNE can alter GCL holoenzyme formation and activity via direct posttranslational modification of the GCL subunits in vitro. 4-HNE directly modified Cys553 of GCLC and Cys35 of GCLM in vitro, which significantly increased monomeric GCLC enzymatic activity, but reduced GCL holoenzyme activity and formation of the GCL holoenzyme complex. In silico molecular modeling studies also indicate these residues are likely to be functionally relevant. Within a cellular context, this novel posttranslational regulation of GCL activity could significantly affect cellular GSH homeostasis and GSH-dependent detoxification during periods of oxidative stress.

Figure 1. 4-HNE rapidly increases cellular GCL activity independent of de novo protein synthesis

(A) A549 cells were treated for 30 min with the indicated concentrations of 4-HNE. (B) A549 cells were treated with 50 μM 4-HNE for the times indicated. (C and D) A549 cells were pretreated for 1 h in the absence or presence of cycloheximide (CHX; 10 μg/ml) prior to treatment with 4-HNE (50 μM) for 30 min. (A-C) GCL activity and total GSH content (GSH + GSSG) of cellular extracts were assessed by the fluorescence-based NDA assay and a modified Tietze assay, respectively (*†p<0.01, **††p<0.001). (D) GCLC, GCLM, and β-actin protein expression were analyzed by immunoblotting as described in the Materials and Methods section.

Figure 2. In vitro analysis of GCL holoenzyme formation and activity using His-tagged recombinant GCL fusion proteins

Purified recombinant GCLC-His and GCLM-His fusion proteins were mixed at the indicated molar ratios. (A) GCL holoenzyme formation was analyzed by native PAGE and immunoblotting for GCLC (top panel). The relative amounts of each GCL subunit in the mixture were analyzed by SDS-PAGE and immunoblotting for GCLC or GCLM (bottom panels). (B) GCL activity was measured by the fluorescence-based NDA assay (* p<0.001, N.D. = not detectible).

Figure 3. 4-HNE-mediated adduction of the GCL subunits differentially affects GCLC and GCL holoenzyme activity

Purified recombinant (A) GCLC, (B) GCLM, or (C) GCL holoenzyme (GCL subunits pre-mixed at a 1:1 molar ratio) were incubated with the indicated molar ratio of 4-HNE for 30 min at 37°C. (A-C) 4-HNE-mediated adduction of the GCL subunits was assessed by SDS-PAGE and immunoblotting with α-4-HNE (top panels). Equivalent loading of the GCL subunits was confirmed by immunoblotting for GCLC or GCLM (bottom panels). (D) Monomeric GCLC (open bars) and GCL holoenzyme (closed bars) were treated in the absence or presence of the indicated molar ratio of 4-HNE for 30 min at 37°C. GCL activity was determined by the fluorescence-based NDA assay (* p<0.001 vs monomeric GCLC control; # p<0.05, ## p<0.001 vs GCL holoenzyme control). (E) GCL holoenzyme was treated as described above and the amount of GCLC present in the monomeric, holoenzyme, and high molecular weight fractions was assessed by native PAGE and immunoblotting for GCLC. The arrow denotes the presence of high molecular weight complexes that contain GCLC.

Figure 4. 4-HNE- and NEM-mediated adduction of monomeric GCLC partially mimics the effects of GCL holoenzyme formation on GCLC enzyme kinetics

Purified recombinant GCLC was incubated in the absence or presence of a 25-fold molar excess of 4-HNE, a 10-fold molar excess of NEM, or an equivalent molar amount of GCLM for 30 min at 37°C. GCL specific activity was determined under conditions of variable substrate concentrations and Lineweaver-Burke plots generated using GraphPad Prism 4.0 for (A) L-glutamate and (B) ATP.

Figure 5. 4-HNE-mediated adduction of the individual GCL subunits prevents heterodimerization and GCL holoenzyme formation

Purified recombinant GCLC and GCLM were incubated separately with (A and B) the indicated molar ratio of 4-HNE or (C and D) 100X molar excess of 4-HNE for 30 min at 37°C prior to mixing at a 1:1 molar ratio to form GCL holoenzyme. (A and C) GCL holoenzyme formation was assessed by native PAGE and immunoblotting for GCLC (top panel). The relative levels of the individual subunits were assessed by SDS-PAGE and immunoblotting for GCLC or GCLM (bottom panels). (B and D) GCL activity was determined by the fluorescence-based NDA assay (* p<0.001, ns = not significant).

Figure 6. Identification of GCLM(Cys35) and GCLC(Cys553) as sites for 4-HNE-mediated adduction in vitro

Monomeric GCLC and GCLM were incubated with a 25X molar excess of 4-HNE for 30 min at 37°C. Samples were reduced with NaBH₄, resolved by SDS-PAGE, and digested with trypsin, Glu-C, or chymotrypsin and peptide fragments were analyzed by MALDI-TOF/TOF mass spectrometry. (A) MS/MS spectra of the [M+H]⁺ ion at m/z = 1316.4 from the 4-HNE-modified GCLM peptide containing Cys-35, *CPSTHSEELR. (B) MS/MS spectra of the [M+H]⁺ ion at m/z = 1111.5 from the 4-HNE-modified GCLC peptide containing Cys-553, *CSILNYLK. The asterisk indicates the site of adduction. (b/y fragmentation predicted using ProteinProspector, UCSF).

Figure 7. Cys- and Lys-specific modifications differentially affect GCLC and GCL holoenzyme activity

Purified recombinant monomeric GCLC (open bars) or GCL holoenzyme (closed bars) were incubated in the absence or presence of the indicated molar excess of (A) NEM or (B) NHS for 30 min at 37°C. GCL activity was determined by the fluorescence-based NDA assay (* p<0.001 vs. respective NEM controls; *p<0.01, ** p<0.001 vs. respective NHS controls).

Figure 8. Lys-specific modification of the GCL subunits attenuates GCL holoenzyme complex formation and activity

Purified recombinant GCLC and/or GCLM were incubated separately with 100X molar excess of 4-HNE for 30 min at 37°C prior to mixing at a 1:1 molar ratio to form GCL holoenzyme as indicated. The GCL subunits were mixed prior to treatment in lanes 7 and 8. (A) GCL holoenzyme formation was assessed by native PAGE and immunoblotting for GCLC. NHS-modified GCLC migrated aberrantly upon native PAGE both as a monomer (NHS-GCLC) and a GCL holoenzyme complex (NHS-HOLO) as indicated. (B) GCL activity was determined by the fluorescence-based NDA assay (* p<0.001 vs. GCL holoenzyme activity controls).

Figure 9. Homology model of murine GCLM, GCLC, and GCL holoenzyme

The murine GCLM and GCLC protein sequences were submitted to the ModWeb protein modeling server and the resulting protein structures of (A) GCLM and (B) GCLC were rendered in Lightwave 3D 9.6. Cys residues of interest are colored in yellow and indicated with arrows. The remaining residues of GCLM are colored in green and those of GCLC in blue. (C) Prediction of protein-protein interaction between the GCLM and GCLC homology models was calculated with Escher NG and a representative solution is depicted.