Phenotypic screening of genes and compounds modifying lipotoxicity in Saccharomyces cerevisiae

Lipotoxicity is where lipids accumulate in non-adipose tissues and cause apoptosis or necrosis that can lead to diseases such as obesity, diabetes, and non-alcohol fatty liver disease. One in 3 adults in Aotearoa are considered obese and rates of these diseases are predicted to increase, which will put extra pressure on an already struggling healthcare system. It is thus paramount to identify a compound that prevents lipotoxicity.

In chapter 2, I screened 3,300 compounds for their efficacy to suppress lipotoxicity in a lipotoxic yeast model (dga1∆lro1∆) that lacks the DGAT enzymes to tolerate the addition of the fatty acid palmitoleate. I identified 18 compounds that increased the amount of palmitoleate (PO) required for lipotoxicity. Of these 18 compounds, six increased lipid droplet production in dga1∆lro1∆ cells that otherwise produce only one lipid droplet per cell. To further understand the impacts of four compounds (pregnenolone, hexestrol, estradiol and NBI-27914) on lipid metabolism, levels of neutral lipids and phospholipids were monitored using thin-layer chromatography, albeit no significant changes were detected using this methodology. Abundance of proteins involved in various aspects of lipid metabolism was measured using fluorescent microscopy analysis of GFP-tagged proteins, which pointed to the potential importance of the sphingolipid pathway in reducing lipotoxic stress.

In chapter 3, I aimed to find a compound that mimicked the deletion of the SLC1 acyltransferase that previously rescued dga1∆lro1∆ from lipotoxicity, and to characterize the lipid changes seen with slc1Δ. Since slc1∆ale1∆ is synthetic lethal, I predict that a compound toxic to ale1∆ may be an inhibitor of SLC1. Out of the 3,300 compounds screened in chapter 3, nine compounds caused inviability in ale1∆. Four of these nine compounds were further investigated via protein abundance and thin-layer chromatography, which suggested upregulation of sphingolipid synthesis and downregulation of neutral lipid and phospholipid synthesis pathways.

In summary, both chapters implicated the sphingolipid pathway in rescuing dga1Δlro1Δ from lipotoxicity. Compounds that rescued dga1Δlro1Δ seemingly activated the sphingolipid pathway, while slc1Δ seemingly restored sphingolipid synthesis.