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

Abstract

Oxidation of protein cysteine residues into sulfenic acid (Cys-SOH) and the subsequent S-glutathionylation of these residues during enzyme catalysis and redox signaling have been increasingly accepted as commonly occurring events in redox regulation [1][2][3][4][5][6][7][8][9]. This reversible mechanism is believed to play a regulatory role in enzyme catalysis and binding of transcription factors to DNA targets, among other processes. The first step in protein-Cys-SH oxidation generates Cys-SOH, which is prone to S-glutathionylation by 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 stationaryphase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-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 observed in vivo that 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.

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

“…Proteins related to cell control were also found in our study, as two clones of a proteasome component, proteolytic complexes responsible for the degradation of many cellular proteins (Demasi et al, 2003) that play an important role in regulating the cell cycle and signalling, including apoptosis and the elimination of abnormal proteins generated by mutation and oxidative damage (Berlett & Stadtman, 1997;Bochtler et al, 1999;Coux et al, 1996;Demasi et al, 2003;Giulivi et al, 1994;Ullrich et al, 1999). Moreover, proteasomes are controlled by glutathione S-transferase and the related control of oxidation-reduction reactions and production of transcription factors (Demasi et al, 2003;Silva et al, 2008). Finally, it has been observed that the proteolytic activity controlled by proteasomes is increased in the proliferative (logarithmic) phase and gradually decreases on passing into the stationary phase (Bajorek et al, 2003;Laporte et al, 2008).…”

“…In Schizosaccharomyces pombe, alterations in expression of the rds1 gene were observed when the fungus was subjected to various conditions such as glucose, ammonia and phosphate deprivation and changes in CO 2 concentration and temperature (Ludin et al, 1995). Kraus et al (2004), using a microarray technique, found increased expression of RDS1 in Cryptococcus neofomans maintained at 37 u C and, similarly, Rosa e Silva et al (2008), comparing the yeast at 25 and 37 u C, verified an increase in expression of this gene using RDA.…”

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

“…Both preparations were incubated with glutaredoxin 2 (Grx2). As previously described [21], this oxidoreductase provoked deglutathionylation of the 20S, as revealed using 2-D PAGE followed by anti-GSH immunolabeling ( Fig. 1E and F).…”

“…Anti-Grx2, anti-Trr1 and anti-Trx3 antibodies were generated in the Instituto Butantan via rabbit immunization with yeast recombinant proteins, as previously described [21].…”

“…3). Based on our previous data, cytosolic Trxs and Grx2 were hypothesized to play important roles in keeping the 20S deglutathionylated [21]. Trx3 was also evaluated because it is representative of mitochondrial metabolism.…”