Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome.
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 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.
Silva
Gustavo M
GM
Instituto Butantan, Laboratório de Bioquímica e Biofísica, São Paulo, Brazil, and Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Brazil.
Netto
Luis E S
LE
Discola
Karen F
KF
Piassa-Filho
Gilberto M
GM
Pimenta
Daniel C
DC
Bárcena
José A
JA
Demasi
Marilene
M
D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D054477:Glutaredoxins / Q000378:metabolism / Q000502:physiology*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008954:Models, Biological; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D054477:Glutaredoxins; D013879:Thioredoxins; D046988:Proteasome Endopeptidase Complex; D005978:Glutathione; D003545:Cysteine