Author:

Abstract:

Copper is an essential trace metal for many organisms, but toxic amounts are detrimental to individual cells and organisms. Such toxic accumulation of copper is a key feature of the human disease “Wilson's disease”. In these patients, copper accumulates in liver and brain cells due to a defect in a copper exporter. This causes liver damage and neurodegeneration, and clearly has severe consequences for patients. Current therapies are insufficient, and therefore development of novel therapies is of high importance. In this context, at the “Plant-Fungi Interactions” group of the “Centre of Microbial and Plant Genetics” (CMPG-PFI), the cytoprotective or survival-promoting peptide OSIP108 was identified. This peptide increases survival of yeast and human cells during copper stress. This survival-promoting activity was further demonstrated in several cellular models for Wilson's disease as well as a zebrafish larvae model. In this PhD, we aimed to further unravel OSIP108’s activity using yeast as a model. In a first instance we aimed to get insight in copper toxicity and determined how the cells alter gene expression in response to toxic copper. A gene expression analysis suggested that glutathione production is increased in yeast treated with copper. Moreover, treatment with copper induced expression of genes that are important in protein folding. In contrast, these cells have decreased expression of genes involved in lipid biosynthesis. Microscopy analysis revealed that these changes in the lipid balance are associated with an accumulation of lipid droplets. All of these observations indicate that copper damages the cell’s endoplasmic reticulum (ER), the organelle important for lipid and protein biosynthesis, leading to ER stress and lipid droplet accumulation. A structure-activity relationship analysis was performed to study how the amino acid sequence of OSIP108 translates into its survival-promoting activity. Based on these results, we selected an improved OSIP108 variant which has increased activity on copper-treated yeast and HepG2 cells. This improved variant and the native OSIP108 internalized into HeLa cells suggesting that survival-promoting activity is regulated via an intracellular interaction. Therefore, we selected possible OSIP108 interacting yeast proteins using a peptide-protein interaction screen. This, combined with experimental validations in yeast, suggested that OSIP108 interacts with a yeast protein kinase and that its survival-promoting activity is conferred via a downstream signaling cascade. We also showed that treatment with the peptide reduces the accumulation of lipid droplets which links OSIP108 activity, the lipid droplet content and cell survival. Moreover, based on its interaction with the protein kinase, we also selected OSIP108 variants with improved survival-promoting activity. Similarly to the original peptide, such improved variants have potential in treatment development for Wilson’s disease.