Redox control of 20S proteasome gating

Abstract

The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities.

Aims: The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation.

Results: Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal α5 subunit of yeast cells grown to the stationary phase in glucose-containing medium.

Innovation: We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity.

Conclusion: S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.

FIG. 1.

Protein degradation by redox-modified 20S catalytic unit of the proteasome (20SPT) preparations. Representative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of (A) oxidized bovine serum albumin (20 μg; BSA_ox), (B) casein (20 μg), and (C) glutaredoxin 2 (15 μg; Grx2) after incubation for 120, 15, and 60 min, respectively, with natively S-glutathiolated 20S proteasome (5 μg; nPT-SG) and dithiothreitol (DTT)-treated proteasome (5 μg; PT-SH). After this incubation, the samples were filtered through YM-100 microfilters (Millipore) to remove the 20SPT, and the filtrates were used to load the gels. To test the integrity of the preparations, 0.0125 % SDS-containing buffer (+SDS) was utilized as a positive control. BSA was oxidized in the presence of 5 mM H₂O₂ and 100 μM diethylene triamine pentaacetic acid (DTPA) for 30 minutes at room temperature, and the remaining H₂O₂ was removed by cycles of filtration and redilution through YM-10 microfilters (Millipore). All incubations were performed at 37°C. St, standard proteins not incubated with 20SPT; MW, molecular weight standard.

FIG. 2.

Quantitative protein degradation by redox-modified 20SPT preparations. (A) BSA_ox that had reacted with dinitrophenylhydrazine (DNPH), a carbonyl protein reactant (31), was incubated with the 20SPT preparations for 60 min followed by the addition of 20% trichloroacetic acid. The supernatant was retained for spectrometric measurement at 370 nm. (B) Fluorescein isothiocyanate (FITC)-modified casein (casein-FITC) was incubated with the proteasomal preparations for 15 min followed by the addition of 20% trichloroacetic acid. The supernatant was sampled for fluorometric determination (excitation, 492 nm; emission, 515 nm). Both the casein-FITC and DNPH-treated BSA_ox samples were processed using the same conditions in the absence of the proteasome as controls. The results shown represent the mean±SD and are expressed as arbitrary units of absorbance (hydrazone adducts) or fluorescence (FITC). *p<0.000021; **p<0.000003.

FIG. 3.

20S proteasomal gating control is dependent on the cysteine (Cys) redox state. (A) Representative images obtained by transmission electron microscopy of nPT-SG in the open conformation. (B) nPT-SG samples analyzed immediately after treatment with 20 mM DTT for 30 min followed by a washing procedure to eliminate DTT, as described in the Materials and Methods section. The squares were amplified as shown on the right. The combined conformations (open and closed) were observed in both 20SPT preparations (nPT-SG and PT-SH), as shown in Supplementary Fig. S1A and B (Supplementary Data).

FIG. 4.

Representative spectra of the thiol-modified proteasomal Cys residues obtained by LC-ESI-MS/MS. LC-ESI-Q-TOF (Waters Synapt HDMS) analysis of the tryptic peptide from the 20SPT α5 subunit containing Cys221. (A) MS/MS spectrum of a triply charged ion [M+3H]³⁺ with an m/z ratio of 585.28 containing a glutathione moiety (+305.1) attached to the Cys residue. The monoisotopic mass of the deprotonated peptide (LDENNAQLSCITK) is equal to 1752.82 Da. (B) MS/MS spectrum corresponding to the same peptide shown in A from DTT-treated samples. The doubly charged ion [M+2H]²⁺ possesses an m/z ratio of 724.84 and a monoisotopic mass of 1447.67 Da, indicating the reduced form of the Cys residue. The respective fragmentation series is shown above each spectrum.

FIG. 5.

Location of Cys221 in the α5 subunit in the 3D proteasomal structure. (A) Surface structure of the α-ring highlighting the solvent-accessible sulfur atom (yellow) of Cys221 from the α5 subunit and the surface oxygen (red) from the Cys66 residue of the α6 subunit. (B) Modeling of glutathione docking onto Cys221 of the α5 subunit was performed by Gold 4.1–Protein-Ligand Docking (Cambridge Crystallographic Data Centre). The proteasome is shown by a surface representation, and the glutathione is represented by sticks. The proteasomal residues interacting with the GSH-charged groups are highlighted in white in the surface representation and are depicted as blue sticks underneath. The sulfur atoms are depicted in yellow. The distances (Å) between the GSH-charged groups and the lateral chains of the proteasomal amino acids are shown. The graphical images were generated using Pymol software (DeLano Scientific).