Investigation of molecular mechanisms of lipid droplets catabolic pathways and their interactions in ichthyosis disease

Official URL: https://risc01.sabanciuniv.edu/record=b2708503_ (Table of contents)

In cells, excessive lipids are stored as neutral lipids in an organelle called lipid droplets (LDs) to prevent lipotoxicity. In recent years, the importance of these droplets in lipid metabolism, cell signaling, and membrane trafficking was proven with the identification of over 150 proteins on LD surfaces. Additionally, LDs can be catabolized and used as an energy source in the need of energy by three mechanisms, namely lipolysis, chaperonemediated autophagy (CMA), and lipophagy. Autosomal recessive congenital ichthyosis (ARCI) is a non-syndromic form of ichthyosis, resulting in scaly skin, dehydration, and electrolytic imbalance. Many genes, causing ARCI disease, have been proven to be involved in abnormal lipid metabolism and associated with LD accumulation. Patatinlike phospholipase domain containing-1 (PNPLA1) is a protein that involves in phospholipid metabolism and a mutation in this gene causes the development of ARCI pathology. For a better understanding of the effect of PNPLA1 gene mutations on LD metabolism, LD catabolic pathways and their interactions were investigated in this master thesis. Therefore, lipolysis, CMA, and lipophagy mechanisms of fibroblast cells derived from ARCI patients that have p.D172N mutation were analyzed. Localization of LDs with lipolysis-, lipophagy- and CMA-related proteins in fibroblast cells compared to healthy fibroblasts were checked under confocal microscopy by labeling protein of interest with different fluorophores. In addition, the amount of protein expression was analyzed by immunoblotting, and the change in the gene regulation was determined by RT-qPCR. As a result, it was found that PNPLA1 gene mutation in ARCI patient cells shows aberrant LD accumulation and sizes due to the defective lipolysis and lipophagy mechanism, demonstrating the crucial role of PNPLA1 protein in catabolic regulation of LDs.