Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl‐based redox modification of the 20S proteasome

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Abstract
The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys‐SH) into cysteine sulfenic acid (Cys‐SOH), followed by S‐glutathionylation (Cys‐S‐SG). Proteasome S‐glutathionylation promotes partial loss of chymotrypsin‐like activity and post‐acidic cleavage without alteration of the trypsin‐like proteasomal activity. Here we show that the 20S proteasome purified from stationary‐phase cells was natively S‐glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively orin vitroS‐glutathionylated 20S proteasome, allowing the recovery of chymotrypsin‐like activity and post‐acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S‐glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non‐stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS–PAGE and mass spectrometry, and S‐glutathionylation was evaluated by either western blot analyses with anti‐glutathione IgG or by spectrophotometry with the thiol reactant 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole. It was also observedin vivothat glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose‐containing medium. Other cytoplasmic oxido‐reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.

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