Secreted phospholipase A2 (sPLA2) enzymes bind to the surface of various membrane structures and cleave the sn-2 ester bond of glycerophospholipids, releasing free fatty acids (FFAs) and lysophospholipids. The action of the human group X (hGX) sPLA2 on MDA-MB-231 breast cancer cells induces lipid droplet (LD) biogenesis and enables cell survival during starvation. FFAs, in particular oleic acid (OA), liberated from membrane phospholipids by the enzymatic activity of hGX sPLA2 are responsible for LD formation and the pro-survival effect of the enzyme. The released FFAs are incorporated into growing LDs, but also induce other changes in lipid metabolism and signalling that endow cancerous cells with a growth advantage in the hypoxic and nutrient-poor tumor microenvironment. LDs accumulate in stressed cancer cells, acting as sources of energy and providing protection against lipotoxic and oxidative stress. The functions of LDs are inherently determined by the proteins present on their surface, bound to the LD phospholipid monolayer. Dynamic alterations in the LD protein coat are crucial for the adaptation of LDs to fluctuating metabolic states. Currently, the high confidence lipid droplet proteome consists of 100–150 proteins in a prototypical mammalian cell, whereby the presence on LDs has been experimentally validated for more than 50 proteins. Interestingly, numerous proteins belonging to different functional groups, mostly not related to lipid metabolism, associate with LDs. Here we investigated the changes in the LD proteome of MDA-MB-231 breast cancer cells induced by treatments with hGX and one of its products, OA. In order to improve the reproducibility and reliability of our proteomic analyses of isolated LDs, we performed several technical and biological replicates. Changes in LD proteome were quantified with two different proteome profiling strategies (SILAC, "stable isotope labeling by amino acids in cell culture" and LFQ, "label free quantification"). In total, we identified 432 proteins in LDs isolated from MDA-MB-231 breast cancer cells. At least 12% (50/432) were classified as high confidence LD-associated proteins, including 17 already well recognized, "bona fide" LD proteins in mammalian cells. Based on the current literature, we could not assign a clear association with LDs for almost half of the detected proteins (49%, 213/432), suggesting that these could potentially represent new LD proteins. The rest of the detected proteins, however, were likely contaminants from other cell compartments (39%, 169/432). Interestingly, proteins involved in lipid and redox metabolism were the most abundant protein classes observed on LDs. Importantly, cells treated with hGX contained more high confidence LD proteins than untreated cells and those treated with OA. However, several well-known LD proteins were also detected in untreated cells that were not exposed to excess amounts of lipids. With the expection of perilipin 3, the addition of OA or hGX to breast cancer cells caused a significant increase in the amounts of high confidence LD proteins. A similar trend in the determined quantitative changes in the LD proteome were observed for cells treated with hGX and those with OA. The observed protein diversity and dynamics of the LD proteome suggest an involvement of LDs in homeostatic processes and the cancer cell stress response.
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