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Proceedings of the National Academy of... May 2023The sorting of phospholipids between the inner and outer leaflets of the membrane bilayer is a fundamental problem in all organisms. Despite years of investigation, most...
The sorting of phospholipids between the inner and outer leaflets of the membrane bilayer is a fundamental problem in all organisms. Despite years of investigation, most of the enzymes that catalyze phospholipid reorientation in bacteria remain unknown. Studies from almost half a century ago in and revealed that newly synthesized phosphatidylethanolamine (PE) is rapidly translocated to the outer leaflet of the bilayer [Rothman & Kennedy, , 1821-1825 (1977)] but the identity of the putative PE flippase has eluded discovery. Recently, members of the DedA superfamily have been implicated in flipping the bacterial lipid carrier undecaprenyl phosphate and in scrambling eukaryotic phospholipids in vitro. Here, using the antimicrobial peptide duramycin that targets outward-facing PE, we show that cells lacking the DedA paralog PetA (formerly YbfM) have increased resistance to duramycin. Sensitivity to duramycin is restored by expression of PetA or homologs from other bacteria. Analysis of duramycin-mediated killing upon induction of PE synthesis indicates that PetA is required for efficient PE transport. Finally, using fluorescently labeled duramycin we demonstrate that cells lacking PetA have reduced PE in their outer leaflet compared to wildtype. We conclude that PetA is the long-sought PE transporter. These data combined with bioinformatic analysis of other DedA paralogs argue that the primary role of DedA superfamily members is transporting distinct lipids across the membrane bilayer.
Topics: Phosphatidylethanolamines; Phospholipids; Membrane Transport Proteins; Bacteria; Cell Membrane
PubMed: 37155911
DOI: 10.1073/pnas.2301979120 -
Amino Acids Nov 2011Phosphatidylethanolamine (PE) is a major component in the mammalian plasma membrane. It is present mainly in the inner leaflet of the membrane bilayer in a viable,... (Review)
Review
Phosphatidylethanolamine (PE) is a major component in the mammalian plasma membrane. It is present mainly in the inner leaflet of the membrane bilayer in a viable, typical mammalian cell. However, accumulating evidence indicates that a number of biological events involve PE externalization. For instance, PE is concentrated at the surface of cleavage furrow between mitotic daughter cells and is correlated with the dynamics of contractile ring. In apoptotic cells, PE is exposed to the cell surface, thus providing a molecular marker for detection. In addition, PE is a cofactor in the anticoagulant mechanism, and a distinct distribution profile of PE has been documented at the blood-endothelium interface. These recent discoveries were made possible using PE-specific probes derived from duramycin and cinnamycin, which are members of type B lantibiotics. This review provides an account on the features of these PE-specific lantibiotics in the context of molecular probes for the characterization of PE on a cellular and tissue level. According to the existing data, PE is likely a versatile chemical species that plays a role in the regulation of defined biological and physiological activities. The utilities of lantibiotic-based molecular probes will help accelerate the characterization of PE as an abundant, yet elusive membrane component.
Topics: Amino Acid Sequence; Animals; Bacteriocins; Humans; Molecular Imaging; Molecular Probes; Molecular Sequence Data; Phosphatidylethanolamines
PubMed: 21573677
DOI: 10.1007/s00726-009-0386-9 -
Toxicology and Applied Pharmacology Feb 2021Mathermycin, a lantipeptide isolated from marine actinomycete Marinactinospora thermotolerans, is an antibiotic that has been shown to disrupt bacterial plasma membrane....
Mathermycin, a lantipeptide isolated from marine actinomycete Marinactinospora thermotolerans, is an antibiotic that has been shown to disrupt bacterial plasma membrane. We now provide evidences that mathermycin can also disrupt cancer, but not normal, cell plasma membranes through targeting phosphatidylethanolamine (PE), which is located only in the inner leaflet of the plasma membrane in normal cells but in both the inner and outer leaflets of the membrane in tumor cells. Our data shows that mathermycin inhibits the metabolic activity and induces mainly necrotic death of all cancer cell lines with EC between 4.2 and 16.9 μM, while normal cell lines have EC between 113 and 129 μM. The cytotoxicity of mathermycin could be inhibited by exogenous PE, but not phosphoserine and phosphocholine. The formation of mathermycin-PE complexes was confirmed by in silico analysis, HPLC and MS spectrometer. Furthermore, mathermycin exhibited similar cytotoxicity toward cancer and multidrug resistant cancer cells, which could be due to its ability to inhibit mitochondrial function, as shown by our data from the Seahorse™ metabolic analyzer. This study demonstrates that mathermycin is a potentially effective class of anti-tumor chemotherapeutics that do not easily develop resistance due to a mechanism of action targeting PE.
Topics: 3T3 Cells; A549 Cells; Animals; Antineoplastic Agents; Cell Membrane; Drug Resistance, Neoplasm; Energy Metabolism; Hep G2 Cells; Humans; MCF-7 Cells; Mice; Necrosis; Neoplasms; Phosphatidylethanolamines
PubMed: 33476679
DOI: 10.1016/j.taap.2021.115410 -
Biophysical Journal Jan 2018We measured the effect of intrinsic lipid curvature, J, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J<0) and...
We measured the effect of intrinsic lipid curvature, J, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J<0) and palmitoyl-oleoyl-phosphatidylcholine (POPC; J∼0). Electron microscopy and dynamic light scattering were used to determine vesicle size and morphology, and x-ray and neutron scattering, combined with calorimetric experiments and solution NMR, yielded insights into leaflet-specific lipid packing and melting processes. Below the lipid melting temperature we observed strong interleaflet coupling in asymmetric vesicles with POPE inner bilayer leaflets and outer leaflets enriched in POPC. This lipid arrangement manifested itself by lipids melting cooperatively in both leaflets, and a rearrangement of lipid packing in both monolayers. On the other hand, no coupling was observed in vesicles with POPC inner bilayer leaflets and outer leaflets enriched in POPE. In this case, the leaflets melted independently and did not affect each other's acyl chain packing. Furthermore, we found no evidence for transbilayer structural coupling above the melting temperature of either sample preparation. Our results are consistent with the energetically preferred location of POPE residing in the inner leaflet, where it also resides in natural membranes, most likely causing the coupling of both leaflets. The loss of this coupling in the fluid bilayers is most likely the result of entropic contributions.
Topics: Lipid Bilayers; Mechanical Phenomena; Phosphatidylcholines; Phosphatidylethanolamines
PubMed: 29320681
DOI: 10.1016/j.bpj.2017.11.009 -
SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis.Molecular Oncology Nov 2021SMAC/Diablo, a pro-apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid...
SMAC/Diablo, a pro-apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid synthesis during cancer cell proliferation and development. Here, we explore the molecular mechanism through which SMAC/Diablo regulates phospholipid synthesis. We showed that SMAC/Diablo directly interacts with mitochondrial phosphatidylserine decarboxylase (PSD) and inhibits its catalytic activity during synthesis of phosphatidylethanolamine (PE) from phosphatidylserine (PS). Unlike other phospholipids (PLs), PE is synthesized not only in the endoplasmic reticulum but also in mitochondria. As a result, PSD activity and mitochondrial PE levels were increased in the mitochondria of SMAC/Diablo-deficient cancer cells, with the total amount of cellular PLs and phosphatidylcholine (PC) being lower as compared to SMAC-expressing cancer cells. Moreover, in the absence of SMAC/Diablo, PSD inhibited cancer cell proliferation by catalysing the overproduction of mitochondrial PE and depleting the cellular levels of PC, PE and PS. Additionally, we demonstrated that both SMAC/Diablo and PSD colocalization in the nucleus resulted in increased levels of nuclear PE, that acts as a signalling molecule in regulating several nuclear activities. By using a peptide array composed of 768-peptides derived from 11 SMAC-interacting proteins, we identified six nuclear proteins ARNT, BIRC2, MAML2, NR4A1, BIRC5 and HTRA2 Five of them also interacted with PSD through motifs that are not involved in SMAC binding. Synthetic peptides carrying the PSD-interacting motifs of these proteins could bind purified PSD and inhibit the PSD catalytic activity. When targeted specifically to the mitochondria or the nucleus, these synthetic peptides inhibited cancer cell proliferation. To our knowledge, these are the first reported inhibitors of PSD acting also as inhibitors of cancer cell proliferation. Altogether, we demonstrated that phospholipid metabolism and PE synthesis regulated by the SMAC-PSD interaction are essential for cancer cell proliferation and may be potentially targeted for treating cancer.
Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Proliferation; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Neoplasms; Phosphatidylethanolamines
PubMed: 33794068
DOI: 10.1002/1878-0261.12959 -
Biochimica Et Biophysica Acta.... Feb 2018Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the...
Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.
Topics: Cell Membrane; Cytoplasmic Vesicles; Lipid Bilayers; Membrane Fusion; Models, Chemical; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipase D; Phospholipids; Spectrometry, Fluorescence
PubMed: 29032301
DOI: 10.1016/j.bbamem.2017.10.011 -
Journal of Virology Jan 2020To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent...
To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism. For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.
Topics: Aedes; Animals; Bacteriocins; Cell Line; Membrane Microdomains; Phosphatidylethanolamines; Virus Diseases; Viruses
PubMed: 31666384
DOI: 10.1128/JVI.01471-19 -
Nature Communications Jan 2018Macroautophagy (autophagy) is a crucial cellular stress response for degrading defective macromolecules and organelles, as well as providing bioenergetic intermediates...
Macroautophagy (autophagy) is a crucial cellular stress response for degrading defective macromolecules and organelles, as well as providing bioenergetic intermediates during hypoxia and nutrient deprivation. Here we report a thiol-dependent process that may account for impaired autophagy during aging. This is through direct oxidation of key autophagy-related (Atg) proteins Atg3 and Atg7. When inactive Atg3 and Atg7 are protected from oxidation due to stable covalent interaction with their substrate LC3. This interaction becomes transient upon activation of Atg3 and Atg7 due to transfer of LC3 to phosphatidylethanolamine (lipidation), a process crucial for functional autophagy. However, loss in covalent-bound LC3 also sensitizes the catalytic thiols of Atg3 and Atg7 to inhibitory oxidation that prevents LC3 lipidation, observed in vitro and in mouse aorta. Here findings provide a thiol-dependent process for negatively regulating autophagy that may contribute to the process of aging, as well as therapeutic targets to regulate autophagosome maturation.
Topics: Aging; Animals; Aorta; Autophagosomes; Autophagy; Autophagy-Related Protein 7; Autophagy-Related Proteins; HEK293 Cells; Humans; Hydrogen Peroxide; Male; Mice, Inbred C57BL; Microtubule-Associated Proteins; Myocytes, Smooth Muscle; Oxidation-Reduction; Phosphatidylethanolamines; Primary Cell Culture; Rats; Sulfhydryl Compounds; Ubiquitin-Conjugating Enzymes
PubMed: 29311554
DOI: 10.1038/s41467-017-02352-z -
Structure (London, England : 1993) Jul 2020In both prokaryotes and eukaryotes, phosphatidylethanolamine (PE), one of the most abundant membrane phospholipids, plays important roles in various membrane functions...
In both prokaryotes and eukaryotes, phosphatidylethanolamine (PE), one of the most abundant membrane phospholipids, plays important roles in various membrane functions and is synthesized through the decarboxylation of phosphatidylserine (PS) by PS decarboxylases (PSDs). However, the catalysis and substrate recognition mechanisms of PSDs remain unclear. In this study, we focused on the PSD from Escherichia coli (EcPsd) and determined the crystal structures of EcPsd in the apo form and PE-bound form at resolutions of 2.6 and 3.6 Å, respectively. EcPsd forms a homodimer, and each protomer has a positively charged substrate binding pocket at the active site. Structure-based mutational analyses revealed that conserved residues in the pocket are involved in PS decarboxylation. EcPsd has an N-terminal hydrophobic helical region that is important for membrane binding, thereby achieving efficient PS recognition. These results provide a structural basis for understanding the mechanism of PE biosynthesis by PSDs.
Topics: Binding Sites; Carboxy-Lyases; Cell Membrane; Conserved Sequence; Escherichia coli; Escherichia coli Proteins; Mutation; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding
PubMed: 32402247
DOI: 10.1016/j.str.2020.04.006 -
Biochimica Et Biophysica Acta.... Aug 2021Recently, we reported that a ternary lipid bilayer comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), which were both derived from chicken egg, and...
Recently, we reported that a ternary lipid bilayer comprising phosphatidylethanolamine (PE), phosphatidylcholine (PC), which were both derived from chicken egg, and cholesterol (Chol) generates microdomains that function as specific fusion sites for proteoliposomes. Chol-induced microdomain formation in a completely miscible lipid bilayer is an exceptional phenomenon. Numerous studies have elucidated the formation of domains in liquid ordered (L) and liquid disordered (L) phases of ternary bilayers, which comprise two partially miscible lipids and Chol. Herein, we investigated the composition and mechanism of formation of these unique microdomains in supported lipid bilayers (SLBs) using a fluorescence microscope and an atomic force microscope (AFM). We prepared ternary SLBs using egg-derived PC (eggPC), Chol and three different types of PE: egg-derived PE, 1-palmitoyl-2-oleoyl-PE, and 1,2-didocosahexaenoyl-PE (diDHPE). Fluorescence microscopy observations revealed that fluid and continuous SLBs were formed at PE concentrations (C) of ≥6 mol%. Fluorescence recovery after photobleaching measurement revealed that the microdomain was more fluid than the surrounding region that showed typical diffusion coefficient of the L phase. The microdomains were observed as depressions in the AFM topographies. Their area fraction (θ) increased with C, and diDHPE produced a significantly large θ among the three PEs. The microdomains in the PE+eggPC+Chol-SLBs were rich in polyunsaturated PE and were in the L-like phase. Associating eggPC and Chol caused polyunsaturated PE to segregate, resulting in a microdomain formation by conferring the umbrella effect on Chol, entropic effect of disordered acyl chains, and π-π interactions in the hydrophobic core.
Topics: Cholesterol; Lipid Bilayers; Membrane Microdomains; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids
PubMed: 33901442
DOI: 10.1016/j.bbamem.2021.183626