Identification of ubiquitin ligases (E3) involved in cotranslational degradation in Saccharomyces cerevisiae

• Grant number: 17/24244-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2018
• End date: July 31, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Mário Henrique Bengtson
• Grantee: Alexandre Seiji Maekawa
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Cells are continuously synthesizing new proteins. About 6% to 30% of all new proteins have some kind of mistake that prone them to be marked with ubiquitin chains even before they are released from ribosomes, leading to their cotranslational degradation. Problems on the elimination of these proteins result in an excessive accumulation of defective proteins, what can have serious consequences to cells and organisms. Ltn1 mutations, the main ubiquitin ligase identified as a participant of this process, promote neurodegenerative disease development in mice. However, only around 20% of the cotranslational degradation events are catalyzed by LNT1 pathway (Ribosome Quality Control - RQC pathway). It is still unknown what ubiquitin ligases and what other pathways contribute with the remaining 80%. The present project objective is to discover others E3s that participate on this process in Saccharomyces Cerevisiae. We propose to optimize a previously described protocol that measures cotranslational ubiquitination, to make it more quantitative and suitable for high throughput screening. Once the new protocol is established, we will used it to perform a screening on a deletion library which comprise deletions of all yeast genome coded ubiquitin ligases (80 in total), to identify which ones are involved in cotranslational ubiquitination. These enzymes are extremely conserved through evolution, allowing a) research in yeast to later search for homologous enzymes in humans and b) later utilization of the same protocol to analyze if there are defects on cotranslational degradation pathways in patients with neurodegenerative diseases. (AU)