Doxorubicin Inhibits Phosphatidylserine Decarboxylase and Modifies Mitochondrial Membrane Composition in HeLa Cells.
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ABSTRACT:
SUBMITTER: Bellance N
PROVIDER: S-EPMC7072979 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.
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Project description:ObjectivePhosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo.MethodsTo determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry.ResultsPSD knockdown disrupted muscle phospholipid homeostasis leading to an ∼25% reduction in PtdEtn and an ∼45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content.ConclusionsThe PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass.
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Project description:Phosphatidylethanolamine is an abundant component of most cellular membranes whose physical and chemical properties modulate multiple aspects of organelle membrane dynamics. An evolutionarily ancient mechanism for producing phosphatidylethanolamine is to decarboxylate phosphatidylserine and the enzyme catalyzing this reaction, phosphatidylserine decarboxylase, localizes to the inner membrane of the mitochondrion. We characterize a second form of phosphatidylserine decarboxylase, termed PISD-LD, that is generated by alternative splicing of PISD pre-mRNA and localizes to lipid droplets and to mitochondria. Sub-cellular targeting is controlled by a common segment of PISD-LD that is distinct from the catalytic domain and is regulated by nutritional state. Growth conditions that promote neutral lipid storage in lipid droplets favors targeting to lipid droplets, while targeting to mitochondria is favored by conditions that promote consumption of lipid droplets. Depletion of both forms of phosphatidylserine decarboxylase impairs triacylglycerol synthesis when cells are challenged with free fatty acid, indicating a crucial role phosphatidylserine decarboxylase in neutral lipid storage. The results reveal a previously unappreciated role for phosphatidylserine decarboxylase in lipid droplet biogenesis.
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Project description:A glucose analog called 2-deoxy-D-glucose (2DG) has been successfully used to sensitize cancer cells to ROS-inducing cancer treatments such as ionizing radiation, through the inhibition of glycolysis. However, the use of 2DG can be limited by several factors such as availability, non-specific cytotoxicity, and chemoresistance under hypoxic conditions. The purpose of this study was to investigate the use of non-radioactive 2-deoxy-2-fluoro-D-glucose (19FDG), a drug that potentially addresses current limitations of 2DG. The effectiveness of using either 2DG or 19FDG in combination with doxorubicin (Dox) in HeLa cells was determined in both normoxia and hypoxia. We have also shown that under both oxygen conditions, 19FDG-treated cells produce less lactate than 2DG-treated cells, an important finding that suggests improved inhibition of glycolysis, the preferential pathway for cancerous cells. When used in combination with Dox, we have demonstrated a significant decrease in the number of viable cells, with the effect of 19FDG remaining stable across both normoxic and hypoxic conditions. Moreover, the assessment of apoptosis and necrosis revealed that 19FDG maintained its ability to sensitize HeLa cells to Dox in hypoxia, but 2DG was only effective under normoxic conditions. The retained effectiveness of 19FDG in combination with Dox under hypoxic conditions, suggests that 19FDG may be efficacious for sensitizing hypoxic regions of solid tumour masses. Importantly, the ability of 19FDG to inhibit glucose uptake in vivo was also confirmed using positron emission tomography (PET) of xenograft tumours. The results displayed here suggest 19FDG is a promising combination therapy, which may lead to decreased ROS scavenging via glycolysis, and enhanced treatment success.
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Project description:Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.
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