The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine ...phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16–20:4 > DAG 18:0–20:4 > DAG 16:0–18:1 = AAG 16–18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0–18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.
Peroxiredoxin 6 (Prdx6) is a Ca2+-independent intracellular phospholipase A2 (called aiPLA2) that is localized to cytosol, lysosomes, and lysosomal-related organelles. Activity is minimal at ...cytosolic pH but is increased significantly with enzyme phosphorylation, at acidic pH, and in the presence of oxidized phospholipid substrate; maximal activity with phosphorylated aiPLA2 is ∼2 µmol/min/mg protein. Prdx6 is a “moonlighting” protein that also expresses glutathione peroxidase and lysophosphatidylcholine acyl transferase activities. The catalytic site for aiPLA2 activity is an S32-H26-D140 triad; S32-H26 is also the phospholipid binding site. Activity is inhibited by a serine “protease” inhibitor (diethyl p-nitrophenyl phosphate), an analog of the PLA2 transition state 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33), and by two naturally occurring proteins (surfactant protein A and p67phox), but not by bromoenol lactone. aiPLA2 activity has important physiological roles in the turnover (synthesis and degradation) of lung surfactant phospholipids, in the repair of peroxidized cell membranes, and in the activation of NADPH oxidase type 2 (NOX2). The enzyme has been implicated in acute lung injury, carcinogenesis, neurodegenerative diseases, diabetes, male infertility, and sundry other conditions, although its specific roles have not been well defined. Protein mutations and animal models are now available to further investigate the roles of Prdx6-aiPLA2 activity in normal and pathological physiology.
Phospholipids are major constituents of biological membranes. The fatty acyl chain composition of phospholipids determines the biophysical properties of membranes and thereby affects their impact on ...biological processes. The composition of fatty acyl chains is also actively regulated through a deacylation and reacylation pathway called Lands' cycle. Recent studies of mouse genetic models have demonstrated that lysophosphatidylcholine acyltransferases (LPCATs), which catalyze the incorporation of fatty acyl chains into the
sn
-2 site of phosphatidylcholine, play important roles in pathophysiology. Two LPCAT family members, LPCAT1 and LPCAT3, have been particularly well studied. LPCAT1 is crucial for proper lung function due to its role in pulmonary surfactant biosynthesis. LPCAT3 maintains systemic lipid homeostasis by regulating lipid absorption in intestine, lipoprotein secretion, and de novo lipogenesis in liver. Mounting evidence also suggests that changes in LPCAT activity may be potentially involved in pathological conditions, including nonalcoholic fatty liver disease, atherosclerosis, viral infections, and cancer. Pharmacological manipulation of LPCAT activity and membrane phospholipid composition may provide new therapeutic options for these conditions.
TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such ...as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.
Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane ...protein, as a Ca2+-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca2+ ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F−/− cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca2+-dependent Cl− channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca2+-dependent Cl− channels (16A and 16B) and Ca2+-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).
Background: TMEM16A and 16B work as Cl− channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown.
Results: Using TMEM16F−/− cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases.
Conclusion: Some TMEM16 members are divided into two Cl− channels and five lipid scramblases.
Significance: Learning the biochemical function of TMEM16 family members is essential to understand their physiological role.
Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining ...transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na⁺,K⁺-ATPase, and H⁺,K⁺-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.
In all animal cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. This asymmetrical phospholipid distribution is disrupted in various ...biological systems. For example, when blood platelets are activated, they expose phosphatidylserine (PtdSer) to trigger the clotting system. The PtdSer exposure is believed to be mediated by Ca2+-dependent phospholipid scramblases that transport phospholipids bidirectionally, but its molecular mechanism is still unknown. Here we show that TMEM16F (transmembrane protein 16F) is an essential component for the Ca2+-dependent exposure of PtdSer on the cell surface. When a mouse B-cell line, Ba/F3, was treated with a Ca2+ ionophore under low-Ca2+ conditions, it reversibly exposed PtdSer. Using this property, we established a Ba/F3 subline that strongly exposed PtdSer by repetitive fluorescence-activated cell sorting. A complementary DNA library was constructed from the subline, and a cDNA that caused Ba/F3 to expose PtdSer spontaneously was identified by expression cloning. The cDNA encoded a constitutively active mutant of TMEM16F, a protein with eight transmembrane segments. Wild-type TMEM16F was localized on the plasma membrane and conferred Ca2+-dependent scrambling of phospholipids. A patient with Scott syndrome, which results from a defect in phospholipid scrambling activity, was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing the premature termination of the protein.
Ferroptosis, a cell death process driven by iron-dependent phospholipid peroxidation, has been implicated in various diseases. There are two major surveillance mechanisms to suppress ferroptosis: one ...mediated by glutathione peroxidase 4 (GPX4) that catalyzes the reduction of phospholipid peroxides and the other mediated by enzymes, such as FSP1, that produce metabolites with free radical-trapping antioxidant activity. In this study, through a whole-genome CRISPR activation screen, followed by mechanistic investigation, we identified phospholipid-modifying enzymes MBOAT1 and MBOAT2 as ferroptosis suppressors. MBOAT1/2 inhibit ferroptosis by remodeling the cellular phospholipid profile, and strikingly, their ferroptosis surveillance function is independent of GPX4 or FSP1. MBOAT1 and MBOAT2 are transcriptionally upregulated by sex hormone receptors, i.e., estrogen receptor (ER) and androgen receptor (AR), respectively. A combination of ER or AR antagonist with ferroptosis induction significantly inhibited the growth of ER+ breast cancer and AR+ prostate cancer, even when tumors were resistant to single-agent hormonal therapies.
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•MBOAT1/2 suppress ferroptosis through phospholipid remodeling independently of GPX4•MBOAT1 and MBOAT2 are regulated by ER and AR signaling, respectively•ER antagonists sensitize ER+ breast cancer to ferroptosis by downregulating MBOAT1•AR antagonists sensitize AR+ prostate cancer to ferroptosis by downregulating MBOAT2
MBOAT1 and MBOAT2 are sex hormone-dependent ferroptosis regulators with therapeutic implications for ER+ breast cancer and AR+ prostate cancer, respectively.
Cytosolic lipid droplets (LDs) are the main storage organelles for metabolic energy in most cells. They are unusual organelles that are bounded by a phospholipid monolayer and specific surface ...proteins, including key enzymes of lipid and energy metabolism. Proteins targeting LDs from the cytoplasm often contain amphipathic helices, but how they bind to LDs is not well understood. Combining computer simulations with experimental studies in vitro and in cells, we uncover a general mechanism for targeting of cytosolic proteins to LDs: large hydrophobic residues of amphipathic helices detect and bind to large, persistent membrane packing defects that are unique to the LD surface. Surprisingly, amphipathic helices with large hydrophobic residues from many different proteins are capable of binding to LDs. This suggests that LD protein composition is additionally determined by mechanisms that selectively prevent proteins from binding LDs, such as macromolecular crowding at the LD surface.
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•Lipid droplet surfaces are characterized by phospholipid packing defects•Packing defects recruit amphipathic helices to the lipid droplet surface•Large, hydrophobic residues of amphipathic helices initially bind packing defects•In isolation, many amphipathic helices accumulate on lipid droplets
Prévost, Sharp, et al. explore how cytosolic proteins target the surface of lipid droplets (LDs). A distinctive surface structure makes LDs suited to recruiting large hydrophobic amino acid-enriched protein motifs. Rather than specific LD protein-targeting mechanisms, regulation is likely at the level of preventing promiscuous association by non-LD proteins.
Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, ...cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P4-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P4-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P4-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
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► Up to date review on phospholipid transport across cell membranes ► Review of the role of P4-ATPases in the flipping of aminophospholipids across cell membranes ► Review of the role of ABC transporters in the efflux and flipping of phospholipids across cell membranes ► Overview of the role of P4-ATases and ABC transporters in various cellular processes and diseases
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