Lipid droplets (LD) are newly recognized cytosolic organelles primarily involved in lipid storage. LDs are composed of a neutral lipid core, surrounded by a phospholipid monolayer and proteins. Their number, composition and size are constantly changing according to cellular needs and environmental conditions. LDs have important roles in aggressive cancer cells as they enable their survival in the hypoxic and nutrient poor tumour microenvironment. Many proteins and enzymes with different biological functions are bound to the surface of LDs, embedded within the phospholipid monolayer. Our previous studies have identified an important role of LDs in the cellular stress response to starvation. We assume that the dynamic changes in LD metabolism and function caused by cell stress are accompanied by corresponding alterations in their proteome. In this master thesis we set to explore the nutrient deficiency-induced changes in the LD proteome of aggressive MDA-MB-231 breast cancer cells. Intact LD were isolated from starved and control cells, proteins were extracted, digested to peptides and analysed the latter with mass spectrometry using the label-free quantification approach. In total, we identified 381 proteins associated with the LD fraction, 155 (41 %) of which were classified as contaminants from other cell compartments, while 49 proteins were classified as LD-associated according to previous studies. Among the latter, we detected 15 »bona fide« LD proteins, for which a clear interaction with LDs has already been directly demonstrated in previous studies. Importantly, we detected 177 (47 %) proteins without a known connection to LDs, representing a group of novel, potentially interesting targets for further investigation. We also found that the acute starvation conditions imposed on breast cancer cells had a significant effect on the composition of the LD proteome. Notably, significant change in the amounts of the following 20 LD-associated proteins were detected: G0S2, CDC42, SNX12, CBX3, UBA52, HEBP1, RAC2, AUP1, TBCB, GLRX3, CAST, DBNL, NUCKS1, PTGES3, TXN, PRDX5, TCEAL3, ACSL3, LUC7L2 and HSD17B11. Among these, there are proteins involved in fatty acid, cholesterol and LD metabolism (ACSL3, HSD17B11, HSD17B7, G0S2), ubiquitination (UBA52, AUP1), inflammation (PTGES3), cellular redox homeostasis (TXN, PRDX5), vesicular transport (TRAPPC3, LZTFL1), cytoskeletal organization (TBCB, DBNL) and nuclear function (TCEAL3, NUCKS1). These results suggest that the LD proteome is significantly altered in cancer cells exposed to acute nutrient stress and provide an impetus for future studies on the roles of novel LD-associated proteins in cancer cell metabolism and response to stress.
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