Study and characterization of the S-glutathiolation and deglutathiolation of the 20S proteasome core from the yeast Saccharomyces cerevisiae: Implications on the intracellular redox metabolism and peptide generation.

Author(s)

Gustavo Monteiro Silva

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2010-10-15

Examining board members

Marilene Demasi; Mário Henrique de Barros; Marcelo Damario Gomes; Fábio Cesar Gozzo; Francisco Rafael Martins Laurindo

• Advisor: Luis Eduardo Soares Netto; Marilene Demasi

Abstract

The proteasome is the protease of the Ubiquitin-Proteasome System (UPS) responsible for the breakdown of intracellular ubiquitin-tagged proteins. However, the catalytic particle of the proteasome (20SPT) is capable of hydrolyzing some substrates in an ubiquitin-independent fashion. The S-glutathiolation of the 20SPT was described among several post-translational modifications and according to Demasi et. al. (2003), the chymotrypsin-like activity of proteasome from yeast Saccharomyces cerevisiae is regulated by glutathione. The mechanism of S-glutathiolation is dependent on the formation of the sulfenic acid intermediate in the cisteine residues of the 20SPT. In this present work, we identified in vitro and in vivo, a total of seven different S-glutathiolated proteasomal cysteine residues by mass spectrometry studies (MS/MS) and, by analyzing the 3D structure of the 20SPT, the modified cysteine residues are not located either on the entrance of the catalytic core or near to the active sites, indicating an allosteric mechanism of proteasomal modulation. During protein degradation, the natively S-glutathiolated 20SPT produces different patterns of peptide products when compared to the DTT-reduced particle through distinct site-specific cleavage of the protein substrates, as herein demonstrated by HPLC and MS/MS analyses. Furthermore, by electron microscopy, we showed that the entrance of the natively glutathiolated 20SPT is in the open conformation that immediately shifts to the closed conformation in the presence of DTT. We have also characterized the deglutathiolase role of the oxidoreductases Glutaredoxin 2 and Citosolic Thioredoxins 1 and 2 which recover the partially inhibited 20SPT activities. The deglutathiolation mechanism also includes the oxidoreductase degradation dependent on the 20SPT activation. The proteasome Sglutathiolation emerges as a new physiological post-translational modification correlated to the cellular redox state. Moreover, the S-glutathiolation of the 20SPT increases its proteolytic activity suggesting an antioxidant role by removing oxidized proteins generated during oxidative challenges. (AU)

• FAPESP's process: 05/50079-6 - Study and characterization of the S-glutathiolation and deglutathiolation of the 20S proteasome core from the yeast Saccharomyces cerevisiae: implications on the intracellular redox metabolism and peptide generation
• Grantee: Gustavo Monteiro Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)