Investigating Protein Aggregation in Dictyostelium discoideum Biology and Stress Response

Abstract

Formation of toxic protein aggregates is a hallmark of neurodegenerative disorders and is detrimental to neuronal health and brain function. In some cases, this aggregation is caused by expansion of homopolymeric amino acid tracts, a notable example being the expanded polyglutamine (polyQ) tracts present in the nine polyQ diseases, including Huntington’s disease. PolyQ aggregation has been studied in a wide array of model organisms in which proteins containing expanded polyQ will form cytotoxic aggregates. However, previous work has identified the social amoeba Dictyostelium discoideum as a proteostatic outlier that is highly resistant to protein aggregation. Here, I investigated the biology of protein aggregation in this unique microorganism. The proteome of Dictyostelium discoideum contains many proteins with long polyQ tracts that would be pathogenic in other organisms. While two modulators of polyQ solubility have been identified in Dictyostelium to date, difficulties in isolating mutants of interest have limited the depth of genome coverage possible with available genetic screening techniques. To address this, I designed a method to positively select for polyQ aggregation mutant Dictyostelium using the Pyr56/5-FOA counterselection system. I then validated that this selection strategy can successfully enrich a population of mutagenized Dictyostelium for cells with polyQ protein aggregates. This technique will facilitate genetic screening approaches and increase the depth at which we can probe the Dictyostelium genome for factors that contribute to resistance to polyQ aggregation. Because genetic approaches have several limitations, I also wanted to use a biochemical approach to explore the biology of polyQ proteins in Dictyostelium. To do this, I used BioID proximity labeling to identify potential interactors of expanded polyQ in this organism. I found that proteins in proximity to expanded polyQ were often glutamine-rich and prion-like. I also found enrichment for proteins associated with transcription, suggesting a possible role for polyQ in this process. Together, these works will serve as a platform for further investigation into polyQ proteins in Dictyostelium. In addition to polyQ proteins, the Dictyostelium proteome also contains the highest percentage of prion-like proteins amongst sequenced organisms. However, little work has been done to explore the biology of these proteins in Dictyostelium. I chose to examine the behavior of the ERF3 protein, the Dictyostelium ortholog of the well-characterized yeast prion protein Sup35. From this, I found that ERF3 aggregates in Dictyostelium under multiple types of acute cellular stress. This protein can also self-assemble in vitro, and its aggregation is driven by the C-terminal GTPase domain. Interestingly, ERF3 aggregation was also induced by nutrient stress and multicellular development. Based on these observations, I wanted to further explore changes in protein solubility under stress in Dictyostelium. To do this, I used mass spectrometry to examine changes in insoluble protein following heat stress or induction of multicellular development. Each stressor resulted in distinct patterns of insoluble protein characteristics, suggesting that there may be regulation or specificity in altered protein solubility following cellular stress. I also observed differences in insoluble protein characteristics in cells deficient in the chemical chaperone polyphosphate, implicating polyphosphate as a possible modifier of protein solubility in Dictyostelium. These findings raise several questions about the role and regulation of protein aggregation in Dictyostelium, which will be an interesting avenue for future research in this unique organism.