Mutations of Cys and Ser residues in the alpha 5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan

Author(s):Show less -

Leme, Janaina M. M. ^{[1, 2]} ; Ohara, Erina ^{[1, 3]} ; Santiago, Veronica F. ^{[1, 3]} ; Barros, Mario H. ^[4] ; Netto, Luis E. S. ^[2] ; Pimenta, Daniel C. ^[1] ; Mariano, Douglas O. C. ^[1] ; Oliveira, Cristiano L. P. ^[5] ; Bicev, Renata N. ^[5] ; Barreto-Chaves, Maria L. M. ^[3] ; Lino, Caroline A. ^[3] ; Demasi, Marilene ^[1]

Affiliation

^[1] Inst Butantan, Lab Biochem & Biophys, Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, IB, Dept Genet & Evolut Biol, Sao Paulo, SP - Brazil

^[3] Univ Sao Paulo, ICB, Dept Anat, Program Morfofunct Sci, Sao Paulo, SP - Brazil

^[4] Univ Sao Paulo, ICB, Dept Microbiol, Sao Paulo, SP - Brazil

^[5] Univ Sao Paulo, IF, Dept Expt Phys, Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Archives of Biochemistry and Biophysics; v. 666, p. 63-72, MAY 15 2019.
• Web of Science Citations: 1

Abstract

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the alpha 5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (alpha 5-C76S or alpha 5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that alpha 5-C76S or alpha 5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random alpha 5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., alpha-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the alpha 5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the alpha 5-subunit, and consequently impacts the lifespan of yeast. (AU)

• FAPESP's process: 14/21058-0 - Structural and functional characterization of the 20s proteasome from the yeast Saccharomyces cerevisiae after site-specific mutations of Cys residues modified by S-glutathionylation
• Grantee: Marilene Demasi
• Support Opportunities: Regular Research Grants