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 a5 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 posttranslational 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.

20S proteasome activity is modified via S-glutathionylation based on intracellular redox status of the yeast Saccharomyces cerevisiae: Implications for the degradation of oxidized proteins

“…Small number of Cys residues of the 20S is redox modulated inside cells [15]. Indeed, S-glutathionylation of two Cys residues on the a5 subunit correlates with the opening of the catalytic chamber, thereby facilitating protein entrance, as evidenced by the increased degradation of oxidized proteins.…”

“…Later preparations displayed discrete labeling probably of a1 or of b7 subunits, which were not detected in the 20S that had been extracted from cells grown either in synthetic medium containing glucose (Fig. 1C) or YPD [15]. Both preparations were incubated with glutaredoxin 2 (Grx2).…”

“…The second dimension was performed on a 12.5% SDS-PAGE, and the protein spots were visualized using Coomassie Brilliant Blue R-250 staining. The proteasomal subunits were identified using Maldi-TOF-MS fingerprinting, as described by Silva et al [15]. When specified, the subunits that had been separated using 2-D PAGE electrophoresis were transferred to nitrocellulose membranes for anti-GSH labeling.…”

“…S-glutathionylation of the a5 subunit of the 20S is related to gate opening [15]. Therefore, gate status in 20S preparations was analyzed using TEM.…”

“…All of the free 20S core particles within the cells were previously assumed to be in the closed gating conformation, although evidence has indicated that oxidized and disordered proteins can be degraded by the 20S proteasome in vitro and in vivo [7,14]. We recently demonstrated that S-glutathionylation of the 20S proteasomal a-subunits promotes gate opening in a 19S-independent manner, increasing the degradation of oxidized proteins [15].…”