Study of lipid-protein interactions in Oncogene Induced Senescence (OIS)

Abstract

Cellular senescence is a state of irreversible cell cycle arrest aimed at cellular maintenance. This state is triggered by numerous stressors such as cellular replication or oncogene activation, among others. Inducing cellular senescence upon oncogenic activation, called oncogene-induced senescence (OIS), is a tumour suppressor mechanism that impairs cancer transformation. For this project, we use IMR-90 primary human cells, a well-characterized and established Rasinduced senescence model. One of the hallmarks of senescence is an altered cellular metabolism, including lipid metabolism. In a screen to identify metabolic regulators, the host group has identified the largely overlooked ceramide synthase 4 (CERS4) as a critical regulator of OIS. Furthermore, the group has seen an increase in CERS4-mediated ceramide synthesis upon Ras activation. This master's thesis aims to identify signals linking CERS4-derived lipids and key proteins regulating OIS. The use of functionalized lipids allows the visualization of lipid cellular localization and protein interactions. Our results with functionalized sphingosine show changes in the subcellular localization of CERS-derived sphingolipids using fluorescence microscopy. Interestingly, we have observed an accumulation of these lipids in mitochondria during OIS. The use of photo-click chemistry enables the binding of the functionalized lipid to nearby proteins through UV-crosslinking and pulldown using biotin-labelled molecules. The discovery of proteins bound to the functionalized lipid would provide an initial insight about ceramides interactome. Using KD-CERS4 cells, available in the lab, we could investigate the main binding proteins of CERS4-derived sphingolipids, which could be a big step in the study of metabolism regulation in senescence and therefore in cancer.