1. Mutations of Cys and Ser residues in the α5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan
- Author
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Daniel Carvalho Pimenta, Veronica Feijoli Santiago, Maria Luiza Morais Barreto-Chaves, Janaina M.M. Leme, Douglas Oscar Ceolin Mariano, Renata N. Bicev, Luis Eduardo Soares Netto, Mario H. Barros, Erina Ohara, Cristiano L. P. Oliveira, Marilene Demasi, and Caroline Antunes Lino
- Subjects
0301 basic medicine ,Proteasome Endopeptidase Complex ,Saccharomyces cerevisiae Proteins ,Longevity ,Mutant ,Saccharomyces cerevisiae ,Biophysics ,Gating ,Biochemistry ,03 medical and health sciences ,Serine ,Cysteine ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Autophagy ,Wild type ,biology.organism_classification ,Glutathione ,Yeast ,Cell biology ,Oxidative Stress ,030104 developmental biology ,Proteasome ,Mutation ,Proteolysis ,Protein folding - 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 α5 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 (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-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., α-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 α5-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 α5-subunit, and consequently impacts the lifespan of yeast.
- Published
- 2019