Project Details
Projekt
Investigations on the process and relevance of aggregation of misfolded proteins during aging in Saccharomyces Cerevisiae
Applicant
Dr. Arthur Fischbach
Subject Area
Cell Biology
Term
from 2018 to 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 396975076
Aging is a ubiquitous process in life. It affects most living organisms, in particular animals but also many unicellular organisms. Aging is characterized by a time-dependent functional decline leading ultimately to death of the organism. The molecular causes of aging are still weakly understood. One of the organisms that contributed the most to aging research is the budding yeast Saccharomyces cerevisiae. Despite its unicellular nature and simplicity, it exhibits an aging phenotype, which shares many properties with more complex organisms. During aging of yeast, protein aggregates accumulate with time, which is considered a hallmark of aging, giving rise to a collapse of essential cellular functions. This can be attributed to a combined effect of a decreased capacity of the protein quality control system (PQC) to remove aggregated proteins together with elevated levels of damaging agents during aging. As an additional protection mechanism to the PQC, the coalescence and sequestration of aggregates into large inclusions at certain protective cellular positions has evolved. However, the ability of yeast cells for aggregate coalescence and to form inclusions declines with aging, as well. Why this is the case is still unknown and could be the results of a simultaneous collapse of multiple systems.The first objective of this proposal is to investigate the process of protein aggregation during aging in more detail, especially regarding the question of how the coalescence of protein aggregates declines in its function during aging. The second objective is to investigate how relevant aggregated proteins are as a potential cause of the aging process in S. cerevisiae, which will be investigated by engineering new strains and approaches allowing the removal of protein aggregates from mother cells. To address the first objective, a misfolding, temperature sensitive allele of Pyrroline-5-carboxylate reductase (PRO3), i.e. pro3-1 will be employed. It was observed that pro3-1 is able to be efficiently incorporated into 1-2 inclusion bodies during heat stress. However, this mutant protein does not efficiently coalesce in aging cells and instead forms a large number of small aggregates, indicating a failure of specific protein quality control pathways. The goal will be to identify these defective pathways using a high content microscopy screen of the yeast knockout library modified with pro3-1-mRuby and Hsp104-GFP. Hsp104 is a disaggregase and can be used to monitor inclusion formation and disassembly. To investigate the relevance of aggregated proteins during aging and if they constitute a cause of aging, aggregates will be removed from the mother cell in an engineered, artificial way into the daughter cell. It will be investigated how this influences the lifespan of the mother cell. The artificial targeting will be achieved by using a motor protein. To specifically recognize protein aggregates, the disaggregase Hsp104p will be fused to such a motor protein.
DFG Programme
Research Fellowships
International Connection
Sweden
