Evaluation of TORC1 signaling cascade in mutant cells of Saccharomyces cerevisiae: implications in the chronological lifespan and proteasome activation

• Grant number: 19/07429-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2019
• End date: November 30, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marilene Demasi
• Grantee: Beatriz Marin Seixas de Carvalho
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

The present proposal is inserted in the research field developed by our group, that refers to the role of the redox modulation of the proteasome on protein metabolism and, therefore in cellular homeostasis. The proteasome is a protein complex responsible for the degradation mainly of proteins regulated by the polyubiquitination process involved in regulation and cell signaling, antigenic presentation and control of protein synthesis. It consists of a central catalytic unit named 20S proteasome (20SPT) where are located the catalytic sites and regulatory units (19S the most abundant) coupled in one or both ends of the central unit, in opposite orientations, to form the named 26S proteasome. The catalytic unit, the 20SPT, devoid of regulatory units, is also capable to degrading proteins independently of the modification with a polyubiquitin tail, as is the case of oxidized proteins among others. The 20SPT consists of a central unit formed by two heptamers called ², flanked by two others heptamers called ±. The catalytic sites are located in the ² subunits and the ± subunits regulate the opening/closing of the catalytic chamber. Our group has described a redox post-translational modification named S-glutathionylation of the 20SPT from the yeast S. cerevisiae (DEMASI; SILVA; NETTO, 2003). We have verified in later studies (SILVA et al., 2012; DEMASI et al., 2014a; DEMASI et al., 2014b) that cysteine residues of the 20SPT prone to glutathionylation are located exclusively in the ± subunits of PT20S, specifically the residues ±5-C76S and ±5-C221S. In the subsequent project, we performed functional and structural studies of 20SPT after site-specific mutation of Cys residues previously found glutathionylated, as cited above, and phenotypic assays of the of the strains carrying 20SPT mutations. The set of data obtained (LEME et al., 2019) revealed that functional and structural consequences of these mutations were the increased frequency of the closed gate conformation of the catalytic chamber in the ±5-C76S-PT20S and ±5-C221S-PT20S. Regarding the phenotype, increase in resistance to oxidative stress and extended chronological lifespan were identified in the S35P/C221S strain, where 20SPT is more active. Another important conclusion in phenotypic terms was that the chronological lifespan of the studied strains is positively associated with increased frequency of the open configuration of the 20SPT catalytic camera. Subsequently, the two mutant strains that presented opposite CLS were used for a differential proteomic analysis. The data obtained (SANTIAGO, 2018) revealed that two proteins associated with signaling TOR (Target of Rapamycin) are present in opposite concentrations in these two strains, that is, increased concentrations were observed in the mutant cell that presents CLS decreased and augmented in the mutant with the highest CLS. The TOR signaling cascade interferes with the CLS of eukaryotic cells, including yeast S. cerevisiae. Activation of this pathway is associated with decreased CLS and its inhibition to increase CLS. Based on the data obtained and continuing the studies described above, the goal of present project will be to validate the data obtained in the proteomic study. These studies involve the deletion and/or overexpression of the genes expressing the proteins in question, evaluating the activation of TOR cascade elements through immunoblotting assays and evaluating the expression of these genes by RT-PCR.