Membrane Phosphatidylethanolamine Research Articles

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy.

Elaborate viral replication organelles (VROs) are formed to support positive-strand RNA virus replication in infected cells. VRO formation requires subversion of intracellular membranes by viral replication proteins. Here, we showed that the key ATG8f autophagy protein and NBR1 selective autophagy receptor were co-opted by Tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus. Knockdown of ATG8f or NBR1 in plants led to reduced tombusvirus replication, suggesting pro-viral function for selective autophagy. BiFC and proximity-labeling experiments showed that the TBSV p33 replication protein interacted with ATG8f and NBR1 to recruit them to VROs. In addition, we observed that several core autophagy proteins, such as ATG1a, ATG4, ATG5, ATG101 and the plant-specific SH3P2 autophagy adaptor proteins were also re-localized to TBSV VROs, suggesting that TBSV hijacks the autophagy machinery in plant cells. We demonstrated that subversion of autophagy components facilitated the recruitment of VPS34 PI3 kinase and enrichment of phospholipids, such as phosphatidylethanolamine and PI3P phosphoinositide in the VRO membranes. Hijacking of autophagy components into TBSV VROs led to inhibition of autophagic flux. We also found that a fraction of the subverted ATG8f and NBR1 was sequestered in biomolecular condensates associated with VROs. We propose that the VRO-associated condensates trap those autophagy proteins, taking them away from the autophagy pathway. Overall, tombusviruses hijack selective autophagy to provide phospholipid-rich membranes for replication and to regulate the antiviral autophagic flux.

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.

Composition of phospholipids of erythrocyte membranes in newborns from mothers with virus pneumonia caused by SARS-CoV-2 in the third trimester of pregnancy

Aim. To study the composition of phospholipids in erythrocyte membranes of newborns from mothers who had viral pneumonia caused by SARS-CoV-2 in the third trimester of pregnancy.Materials and methods. The study included newborns who were born to mothers diagnosed with COVID-19 and community-acquired pneumonia of viral etiology in the third trimester (main group). Of the total number of newborns (n=67), groups of children were formed from mothers with moderate pneumonia (group 1, n=34), and with severe pneumonia (group 2, n=33). The control group consisted of 35 newborns from practically healthy mothers. The quantitative composition of phospholipids in the membranes of erythrocytes of venous blood of the umbilical cord of newborns was determined by the method of two-dimensional thin-layer chromatography according to Kirchner.Results. The study found a statistically significant decrease in the concentration of phosphatidylethanolamine and phosphatidylcholine in erythrocyte membranes in group 1 by 19 and 20%, respectively (p<0.001), in group 2 – by 31 and 29%, respectively (p<0.001); a significant increase in the concentration of lysophosphatidylcholine, sphingomyelin, phosphatidylserine and phosphatidylinositol in group 1 by 43, 8, 15 and 26%, respectively (p<0.001), in group 2 – by 67, 14, 23 and 35%, respectively (p<0.001), compared with their concentration in newborns of the control group.Conclusion. Infection of the mother with SARS-CoV-2 during pregnancy is accompanied by structural disintegration of the erythrocyte membranes of their newborns, manifested by a change in the phospholipid profile in favor of an increase in the fractions of sphingo-, lyso-, inositoland serine phospholipids, which, ultimately, can disrupt the work of the oxygen transport system of the blood, contributing to the development of hypoxia.

The DedA superfamily member PetA is required for the transbilayer distribution of phosphatidylethanolamine in bacterial membranes

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 Bacillus subtilis and Bacillus megaterium revealed that newly synthesized phosphatidylethanolamine (PE) is rapidly translocated to the outer leaflet of the bilayer [Rothman & Kennedy, Proc. Natl. Acad. Sci. U.S.A. 74, 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 invitro. Here, using the antimicrobial peptide duramycin that targets outward-facing PE, we show that Bacillus subtilis cells lacking the DedA paralog PetA (formerly YbfM) have increased resistance to duramycin. Sensitivity to duramycin is restored by expression of B. subtilis 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.

Mechanisms Limiting Renal Tissue Protection and Repair in Glomerulonephritis.

Glomerulonephritis are renal disorders resulting from different pathogenic mechanisms (i.e., autoimmunity, complement, inflammatory activation, etc.). Clarifying details of the pathogenic cascade is basic to limit the progression from starting inflammation to degenerative stages. The balance between tissue injury, activation of protective systems and renal tissue repair determines the final outcome. Induction of an oxidative stress is part of glomerular inflammation and activation of protective antioxidant systems has a crucial role in reducing tissue effects. The generation of highly reactive oxygen species can be evaluated in vivo by tracing the inner-layer content of phosphatidyl ethanolamine and phosphatidyl serine in cell membranes. Albumin is the major antioxidant in serum and the level of oxidized albumin is another indirect sign of oxidative stress. Studies performed in Gn, specifically in FSGS, showed a high degree of oxidation in most contexts. High levels of circulating anti-SOD2 antibodies, limiting the detoxyfing activity of SOD2, have been detected in autoimmune Gn(lupus nephritis and membranous nephropathy) in association with persistence of proteinuria and worsening of renal function. In renal transplant, high levels of circulating anti-Glutathione S-transferase antibodies have been correlated with chronic antibody rejection and progressive loss of renal function. Annexins, mainly ANXA1 and ANXA2, play a general anti-inflammatory effect by inhibiting neutrophil functions. Cytosolic ANXA1 is decreased in apoptotic neutrophils of patients with glomerular polyangitis in association with delayed apoptosis that is considered the mechanism for polyangitis. High circulating levels of anti-ANXA1 and anti-ANXA2 antibodies characterize lupus nephritis implying a reduced anti-inflammatory effect. High circulating levels of antibodies targeting Macrophages (anti-FMNL1) have been detected in Gn in association with proteinuria. They potentially modify the intra-glomerular presence of protective macrophages (M2a, M2c) thus acting on the composition of renal infiltrate and on tissue repair.

Peculiarities of phospholipiid changes in erythrocyte membranes in parturient women with COVID-19-associated community-acquired pneumonia

Aim. To evaluate the phospholipid composition of erythrocyte membranes in parturient women who had COVID-19-associated community-acquired pneumonia (CAP) in the third trimester.Materials and methods. The material for the study was erythrocytes of peripheral blood of 65 parturient women diagnosed with COVID-19, moderate/severe course, CAP of viral etiology (main group). Patients of the main group, depending on the severity of CAP, were divided into two subgroups: subgroup 1 – moderate course of pneumonia (n=33), subgroup 2 – severe course of pneumonia (n=32). The control group consisted of 35 healthy parturient women. The quantitative composition of phospholipids was studied by two-dimensional thin-layer chromatography according to Kirchner.Results. In subgroup 1, the concentration of phosphatidylethanolamine and phosphatidylcholine in erythrocyte membranes was below the standard values by 38% and 29%, respectively (p<0.001), in subgroup 2, these indicators decreased by 32% and 48%, respectively (p<0.001). At the same time, a significant increase in the concentration of lysophosphatidylcholine was found in patients of subgroup 1 by 92% (p<0.001) and in patients of subgroup 2 by 110% (p<0.001), compared with the group of healthy individuals. In addition, structural changes in the lipid bilayer of erythrocyte membranes under conditions of COVID-19associated CAP were characterized by a pronounced increase in the concentration of minor fractions of phospholipids: phosphatidylserine and phosphatidylinositol in subgroup 1 by 63% and 53%, respectively (p<0.001), in subgroup 2 by 79% and 68%, respectively (p<0.001), compared with similar indicators in the control group.Conclusion. With COVID19-associated CAP in maternity women, structural disorganization of the phospholipid components of erythrocyte membranes is determined, manifested by a decrease in the concentration of phosphatidylethanolamine and phosphatidylcholine with a simultaneous increase in the level of lysophosphatidylcholine, phosphatidylserine and phosphatidylinositol. These disorders increase with increasing severity of pulmonary inflammation. The revealed changes in the lipid spectrum of peripheral blood and the composition of erythrocyte membrane phospholipids in COVID-19-associated CAP indicate the need to develop methods for their correction.

Bilayer Forming Phospholipids as Targets for Cancer Therapy.

Phospholipids represent a crucial component for the structure of cell membranes. Phosphatidylcholine and phosphatidylethanolamine are two phospholipids that comprise the majority of cell membranes. De novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine occurs via the Kennedy pathway, and perturbations in the regulation of this pathway are linked to a variety of human diseases, including cancer. Altered phosphatidylcholine and phosphatidylethanolamine membrane content, phospholipid metabolite levels, and fatty acid profiles are frequently identified as hallmarks of cancer development and progression. This review summarizes the research on how phospholipid metabolism changes over oncogenic transformation, and how phospholipid profiling can differentiate between human cancer and healthy tissues, with a focus on colorectal cancer, breast cancer, and non-small cell lung cancer. The potential for phospholipids to serve as biomarkers for diagnostics, or as anticancer therapy targets, is also discussed.

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use 19F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF3-Phe47 labeled M2 peptide and measuring F-CF3 cross peaks in 2D 19F19F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. 1H19F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static 31P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.

Plasma membrane rigidity effects of 4-hydroxy-2-nonenal in Leishmania, erythrocyte and macrophage

4-hydroxy-2-nonenal (HNE) is a reactive aldehyde produced by cells under conditions of oxidative stress, which has been shown to react with proteins and phosphatidylethanolamine in biological membranes. Using electron paramagnetic resonance (EPR) spectroscopy of a spin label it was demonstrated that 2 h of treatment with HNE causes membrane rigidity in promastigotes of Leishmania (L.) amazonensis, J774.A1 macrophages and erythrocytes. Remarkable fluidity-reducing effects on the parasite membrane were observed at HNE concentrations approximately 4-fold lower than in the case of erythrocyte and macrophage membranes. Autofluorescence of the parasites in PBS suspension (1 × 107 cell/mL) with excitation at 354 nm showed a linear increase of intensity in the range of 400 to 600 nm over 3 h after treatment with 30 μM HNE. Parasite ghosts prepared after this period of HNE treatment showed a high degree of membrane rigidity. Bovine serum albumin (BSA) in PBS treated with HNE for 2 h showed an increase in molecular dynamics and suffered a decrease in its ability to bind a lipid probe. In addition, the antiproliferative activity of L. amazonensis promastigotes, macrophage cytotoxicity and hemolytic potential were assessed for HNE. An IC50 of 24 μM was found, which was a concentration > 10 times lower than the cytotoxic and hemolytic concentrations of HNE. These results indicate that the action of HNE has high selectivity indices for the parasite as opposed to the macrophage and erythrocyte.

Axonal degeneration induces distinct patterns of phosphatidylserine and phosphatidylethanolamine externalization

Axonal degeneration is a common feature of multiple neurodegenerative diseases, yet the mechanisms underlying its various manifestations are incompletely understood. We previously demonstrated that axonal degeneration is associated with externalization of phosphatidylserine (PS), which precedes morphological evidence of degeneration, is redox-sensitive, and is delayed in Wallerian degeneration slow (WldS) mutant animals. Phosphatidylethanolamine (PE) is the other major membrane phospholipid in the inner leaflet of the cell membrane, and given that PS signals apoptosis, phagocytosis, and degeneration, we hypothesized that PS and PE membrane dynamics play distinct roles in axonal degeneration. To test this hypothesis, axonal degeneration was induced with calcium ionophores in postnatal rat retinal ganglion cells, and PS- and PE-specific fluorescent probes used to measure their externalization over time. In untreated cells, cell-surface PS was prominent in the cell body alone. Elevation of intracellular calcium with calcium ionophores resulted in significantly increased levels of PS externalization in the cell body, axon, and axon growth cone. Unlike PS, cell-surface PE was diffusely distributed in untreated cells, with comparable levels across the soma, axons, and axon terminals. After exposure to calcium ionophores, PE externalization significantly increased in the cell body and axon. Elevated intracellular calcium also resulted in the formation of axonal blebs which exclusively contained externalized PS, but not PE. Together, these results indicated distinct patterns of externalized PS and PE in normal and degenerating neurons, suggesting a differential role for these phospholipids in transducing neuronal injury.

Metallomic and lipidomic analysis of S. cerevisiae response to cellulosic copper nanoparticles uncovers drivers of toxicity.

Nanotechnology is a promising new technology, of which antimicrobial metal nanocomposites are predicted to become valuable in medical and food packaging applications. Copper is a redox-active antimicrobial metal that can become increasingly toxic depending on the target biomolecule's donor atom selectivity and the chemical species of copper present. Mass is the traditional measurement of the intrinsic elemental chemistry, but this practice fails to reflect the morphology and surface area reactivity of nanotechnology. The carboxymethyl cellulose copper nanoparticles (CMC-Cu) investigated in this study have unique and undefined toxicity to Saccharomyces cerevisiae that is different from CuSO4. Cellular surface damage was found in scanning electron micrographs upon CMC-Cu exposure. Further investigation into the lipids revealed altered phosphatidylcholine and phosphatidylethanolamine membrane composition, as well as depleted triacylglycerols, suggesting an impact on the Kennedy lipid pathway. High levels of reactive oxygen species were measured which likely played a role in the lipid peroxidation detected with CMC-Cu treatment. Metal homeostasis was affected by CMC-Cu treatment. The copper sensitive yeast strain, YJM789, significantly decreased cellular zinc concentrations while the copper concentrations increased, suggesting a possible ionic mimicry relationship. In contrast to other compounds that generate ROS, no evidence of genotoxicity was found. As commonplace objects become more integrated with nanotechnology, humanity must look forward past traditional measurements of toxicity.

Significance of bilayer-forming phospholipids for skeletal muscle insulin sensitivity and mitochondrial function.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which make up the bulk of mammalian cell membrane phospholipids, are recognized for their importance in metabolic health. Perturbations in the ratio of PC:PE can affect membrane integrity and function, which thus have serious health consequences. Imbalance in the hepatic PC and PE membrane content can be linked to metabolic disturbances such as ER stress, fatty liver and insulin resistance. Given that impaired insulin sensitivity underlies the pathology of many metabolic disorders and skeletal muscle is a significant regulator of energy metabolism, it is likely that aberrant phospholipid metabolism in skeletal muscle affects whole-body insulin sensitivity. Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity and mitochondrial function respond to alterations in PC:PE ratio and are associated with glucose homeostasis. Moreover, PC and PE content within the mitochondrial membrane influence mitochondrial respiration and biogenesis and thus, metabolic function. As skeletal muscle phospholipids respond to stimuli such as diet and exercise, understanding the implications of imbalances in PC:PE ratio is of great importance in the face of the rising epidemic of obesity related diseases. This review will summarize the current state of knowledge signifying the links between skeletal muscle PC:PE ratio and insulin sensitivity with respects to PC and PE metabolism, SERCA activity, mitochondrial function and exercise.

Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a β-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a β-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module

The TRPV1 cation nonselective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular pH, high temperature, or naturally occurring pungent molecules such as allicin, capsaicin, or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here, we used a combination of approaches to try to understand the role of the ankyrin repeat domain (ARD) in channel behavior. First, a computational modeling approach by coarse-grained molecular dynamics simulation of the whole TRPV1 embedded in a phosphatidylcholine and phosphatidylethanolamine membrane provides insight into the dynamics of this channel domain. Global analysis of the structural ensemble shows that the ARD is a region that sustains high fluctuations during dynamics at different temperatures. We then performed biochemical and thermal stability studies of the purified ARD by the means of circular dichroism and tryptophan fluorescence and demonstrate that this region undergoes structural changes at similar temperatures that lead to TRPV1 activation. Our data suggest that the ARD is a dynamic module and that it may participate in controlling the temperature sensitivity of TRPV1.

Membrane Remodeling by the Lytic Fragment of SticholysinII: Implications for the Toroidal Pore Model

Sticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and, together with the full-length toxins, have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this article, we combine molecular dynamics simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1–30, a peptide derived from the N-terminus of sticholysin II (StII). With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1–30. Pore formation by this peptide was enhanced by the presence of the negatively curved lipid phosphatidylethanolamine in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1–30 pore that is recruited to the ring upon its assembly. Collectively, these, to our knowledge, new findings support a toroidal model for the architecture of the pore formed by StII1–30 and provide new molecular insight into the role of phosphatidylethanolamine as a membrane component that can easily integrate into the ring of toroidal pores, thus probably aiding in their stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1–30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules.

Covalent modification of phosphatidylethanolamine by 4-hydroxy-2-nonenal increases sodium permeability across phospholipid bilayer membranes

Reactive aldehydes (RAs), such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), produced by cells under conditions of oxidative stress, were shown to react with phosphatidylethanolamine (PE) in biological and artificial membranes. They form RA-PE adducts, which affect the function of membrane proteins by modifying various biophysical properties of the membrane. The ratio of protein to lipid in biological membranes is different, but can reach 0.25 in the membranes of oligodendrocytes. However, the impact of RA-PE adducts on permeability (P) of the neat lipid phase and molecular mechanism of their action are poorly understood. In this study, we showed that HNE increased the membrane P for ions, and in particular for sodium. This effect depended on the presence of DOPE, and was not recorded for the more toxic compound, ONE. Molecular dynamics simulations suggested that HNE-PE and ONE-PE adducts anchored different positions in the lipid bilayer, and thus changed the membrane lipid area and bilayer thickness in different ways. Sodium permeability, calculated in the presence of double HNE-PE adducts, was increased by three to four orders of magnitude when compared to PNa in adduct – free membranes. A novel mechanism by which HNE alters permeability of the lipid membrane may explain the multiple toxic or regulative effects of HNE on the function of excitable cells, such as neurons, cardiomyocytes and neurosensory cells under conditions of oxidative stress.

Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes

Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.

Structures of monomeric and oligomeric forms of the Toxoplasma gondii perforin-like protein 1.

Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.