In eukaryotic cells, the proteasome is the protein complex responsible for the degradation of damaged and nonfunctional proteins, regulating various cellular processes, such as metabolism, signaling, division, and cell death. The accumulation of damaged proteins, among which the oxidized ones, is related to aging and neurodegenerative diseases. Studies have shown that proteasome integrity is required for cells to recover from stress or extend their lifespan.

In a study carried out with yeast Saccharomyces cerevisiae mutants, the team of Marilene Demasi, a researcher at the Instituto Butantan and a member of the RIDC Redoxoma, showed that the activation of the 20S proteasome via the opening of the catalytic chamber is related to the yeast increase of chronological lifespan and resistance to oxidative stress. Researchers studied strains of S. cerevisiae with mutations in the cysteine and serine residues in the alpha-5 subunit of the 20S proteasome.

According to Demasi, this work reveals a sophisticated regulatory mechanism that controls the opening of the 20S proteasome and affects yeast lifespan, based on the interactions between several amino acids of the alpha-5 subunit. “We have already identified the cysteines in the alpha-5 subunit that are modified by a redox process called glutathionylation and thus promote the opening of the 20S proteasome gate. We have now made mutant yeasts to make unequivocal the importance of the redox modification of these cysteines for cell survival”.

The study, published in the Archives of Biochemistry and Biophysics, was conducted in collaboration with the teams of researchers Mario H. Barros, ICB-USP, and Luis E.S. Netto, from IB-USP, both members of Redoxoma, Daniel C. Pimenta, from the Butantan Institute, and Cristiano L.P. Oliveira from IF-USP. RIDC Redoxoma is a Research, Innovation and Dissemination Center supported by FAPESP.

Chronological lifespan

Protein degradation is a process that involves recognition of the protein by attaching it to ubiquitin molecules. Oxidized proteins, however, are degraded in an ubiquitin-independent manner by the free 20S proteasome - the catalytic core of the proteasome dissociated from regulatory units. The 20S proteasome has a cylindrical-shaped structure with four heptameric rings: two inner beta rings, which form the catalytic chamber, and two alpha rings at the ends, responsible for opening and closing the protease. Regulatory units are coupled to one or both sides of the 20S core particle, with the 19S unit being the most abundant. When coupled to the 19S regulatory unit, the proteasome is called 26S. The free 20S proteasome is estimated to account for more than one-third of the total proteasomes pool of mammalian and yeast cells. In addition, it has been shown that only 20% of the pool contains doubly capped 19S-20SPT-19S complexes, indicating that, in most of these complexes, the 20S unit is free.

In 2012, Demasi’s team revealed for the first time a redox mechanism of proteasome regulation, showing that S-glutathionylation of two cysteine residues, Cys76 and Cys221, in the alpha-5 subunit promotes 20S free proteasome gate opening and, consequently, increases degradation of oxidized proteins.

For this new study, the researchers constructed yeast strains with site-specific mutations in these previously identified cysteines, which were replaced by serines - α5-C76S and α5-C221S. “We made single mutation, in which only one of the cysteines is substituted, and double mutation, replacing both cysteines. The first result we obtained was that cells without these two cysteines are nonviable, which demonstrates the importance of these residues in the proper functioning of the proteasome,” explained Demasi. In addition, yeasts with simple mutations had a shorter chronological lifespan and the 20S proteasome was presented predominantly with the gate in a closed conformation.

The most interesting result, however, came from a random mutation. During the experiments performed to obtain the strain carrying the α5-C221S mutation, the researchers observed that a strain presented increased chronological lifespan. After sequencing the mutated gene, they discovered an unexpected secondary mutation: the serine 35 had been replaced by a proline (S35P) along with the expected C221S mutation. The strain carrying the double mutation α5-S35P/C221S, with the Cys76 preserved, presented increased lifespan and resistance to oxidative stress.

To investigate this result, the researchers performed phenotypic and proteasome structure studies. Using the transmission electron microscopy technique, they observed that in the double mutant yeast the frequency with which the proteasome is found with the gate in an open conformation is 80%, while in wild-type yeast the frequency is 65%. In chronological lifespan (CLS) assays, the α5-S35P/C221S strain had a lifespan increase of 25% compared to the wild-type strain, whereas α5-C76S and α5-C221S were decreased by 50% and 25%, respectively. According to the researcher, these results indicate that the increase in yeast lifespan is due to the proteasome gate opening. Likewise, when yeasts are treated with hydrogen peroxide, the mutant double strain is more resistant to oxidative stress because it has a higher frequency of the proteasome with the gate in an open conformation, and, therefore, has greater efficiency in the removal of oxidized proteins.

WT 65%	α5-C76S 30%	α5-S35P/C221S 80%
Transmission electron microscopy shows frequency of 20S proteasome with the gate in the open conformation in a strain of wild type yeast, a strain carrying the Cys76 mutation, and a strain carrying the S35P/C221S double mutation Adapted from Leme at al., 2019 (doi: 10.1016/j.abb.2019.03.012)

Demasi explains that in the double-mutant 20S proteasome, cysteine 76 is glutathionylated, as shown by mass spectrometric analyses. When that proteasome is treated with a reductant, that is, when the glutathione is withdrawn, the frequency with which the proteasome gate is opened drops to 20%. In the case of single mutations, when one of the cysteines is mutated, the other does not suffer glutathionylation, which indicates that both are necessary for this modification to occur. Cys221 is located on the outer surface of the proteasome, being accessible to the solvent and therefore more susceptible to a redox modification; Cys76 is facing the central part of the subunit and is not exposed to the solvent.

The researchers hypothesized that when Cys221 is S-glutathionylated, a structural rearrangement of the proteasome exposes and promotes the modification of Cys76. In the case of the α5-S35P/C221S double mutant, proline likely induces a conformational change, exposing Cys76, and thereby facilitates its glutathionylation and consequent opening of the proteasome gate. Mass spectrometric analyses of the mutants showed that Cys76 is the main determinant for the opening of the catalytic chamber of the 20S proteasome and for cell survival. This cysteine, the researcher recalls, is highly conserved throughout evolution, occurring from yeast to the man.

According to Demasi, the next steps of the research involve studies of molecular dynamics and the detailing of the proteasome structure by cryo-electron microscopy.

The article Mutations of Cys and Ser Residues in the 5-Subunit of The 20s Proteasome from Saccharomyces Cerevisiae Affects Gating and Chronological Lifespan, by Janaína M.M. Leme, Erina Ohara, Verônica F. Santiago, Mario H. Barros, Luis E.S. Netto, Daniel C. Pimenta, Douglas O. C. Mariano, Cristiano L.P. Oliveira, Renata N. Bicev, Maria L. M. de Chaves, Caroline A. Lino and Marilene Demasi, can be accessed here.