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Structural and functional characterization of the 20s proteasome from the yeast Saccharomyces cerevisiae after site-specific mutations of Cys residues modified by S-glutathionylation

Grant number: 14/21058-0
Support Opportunities:Regular Research Grants
Duration: March 01, 2015 - February 28, 2017
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Marilene Demasi
Grantee:Marilene Demasi
Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil


The proteasome is a multicatalytic and multimeric protease component of the ubiquiitin-proteasome system (UPS). The proteasome is formed by a catalytic unit named 20S (20SPT) flanked or not by regulatory units, being the 19S the most abundant. When interacting to the 19S complex it is termed 26S proteasome (26SPT), responsible for the degradation of poly-ubiquitinylated proteins involved in the regulation of cellular cycle and signalling, antigen presentation and in the control of protein synthesis. However, oxidized proteins are degraded by the 20SPT independently of the 19S regulatory unit so, of poly-ubiquitinylation and ATP consumption. Increased hydrophobicity of oxidized proteins seems to underlie their interaction with the 20SPT. The removal of oxidized proteins by the 20SPT is considered an important cellular defense and prevention of protein aggregation. The 20SPT is formed by four heptameric rings, as follows: ±²²±. The catalytic sites are located in three ²-subunits and the ±-rings are responsible for the regulation of proteasomal gating. Among the post-translational modifications of the 20SPT, S-glutathionylation modifies the ±-annulus gating. As demonstrated by Silva et al (2012), two Cys residues located in the ±5-subunit, among the 32 Cys residues present in the 20S proteasomal structure of the yeast Saccharomyces cerevisiae, were found S-glutathiolated in preparations of the 20SPT purified from cells grown to stationary phase into glucose medium. The S-glutathionylated 20SPT degrades more efficiently oxidized proteins when compared to the reduced counterpart, most likely because of the opening of the catalytic chamber as demonstrated earlier. The goals in the present project are to explore phenotype and the ability to remove oxidized proteins of yeast strains harboring the 20SPT mutated-Cys residues, those previously found S-glutathionylated C76 and C221 in the ±5 subunit), and to structurally and functionally characterize the 20SPT mutated forms. (AU)

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(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
LEME, JANAINA M. M.; OHARA, ERINA; SANTIAGO, VERONICA F.; BARROS, MARIO H.; NETTO, LUIS E. S.; PIMENTA, DANIEL C.; MARIANO, DOUGLAS O. C.; OLIVEIRA, CRISTIANO L. P.; BICEV, RENATA N.; BARRETO-CHAVES, MARIA L. M.; et al. Mutations of Cys and Ser residues in the alpha 5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan. Archives of Biochemistry and Biophysics, v. 666, p. 63-72, . (14/21058-0)

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