Coupling of proteasomal regulatory and catalytic units and mitochondrial functionality in yeast strains after site-specific mutations in the proteasomal catalytic unit

• Grant number: 22/08453-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2022
• End date: November 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marilene Demasi
• Grantee: Natália Mori Avellaneda Penatti
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

The proteasome is a protein complex of utter importance for cellular homeostasis, as it is one of the main mechanisms of intracellular proteolysis. The proteasome is made up of a catalytic unit called 20S, which can sometimes be coupled to the 19S regulatory unit at one end (forming the 26S proteasome) or at both ends (forming the 30S proteasome). When associated with the 19S regulatory unit, either in the 26S or 30S conformation, the proteasome is able to recognize and degrade proteins previously labeled for catalysis by a long polyubiquitin tail. In its free form, the 20S catalytic unit is mainly responsible for the proteolysis of substrates damaged by oxidative stress or that present naturally destructured sequences. It is currently known that the coupling between the two units of the proteasome depends, among other factors, on good mitochondrial functionality, which is guaranteed in part by the proteasome, responsible for the quality control of mitochondrial protein imports.In yeasts of the species S. cerevisiae, the research group of Dr. Marilene Demasi described a post-translational redox modification called S-glutathionylation at Cys residues of the ±5 subunit of the 20S catalytic unit of the proteasome, especially ±5-C76 and ±5-C221. The ±5-C76S and ±5-C221S strains presented as a phenotypic alteration a higher frequency of the closed conformation of the catalytic chamber of the 20S unit and a shorter chronological life span (CLS: chronological life span). A double random mutation in the ±5 subunit (±5-S35P/C221S) induced the opening of the 20S catalytic chamber and also increased the cell's CLS. It was observed that in the strains with reduced CLS there was a lower degree of coupling between the 20S catalytic and 19S regulatory units, in addition to a decreased mitochondrial activity. The present project aims to deepen the investigation about the coupling relationship between both proteasome units and mitochondrial functionality, verifying the quality control of mitochondrial protein import and the activity of mitochondrial enzymes essential in energy production in each of the studied strains.