Lipid Rearrangements Research Articles

Gladiolin produced by pathogenic Burkholderia synergizes with amphotericin B through membrane lipid rearrangements.

Amphotericin B (AmpB) is one of the oldest antifungal drugs in clinical use. It is an effective therapeutic, but it comes with toxicity issues due to the similarities between its fungal target (the membrane lipid ergosterol) and its mammalian counterpart (cholesterol). One strategy to improve its activity/toxicity relationship is by combinatorial therapy with potentiators, which would enable a lower therapeutic dose of AmpB. Here, we report on the discovery of the antibiotic gladiolin as a potentiator of AmpB against several priority human fungal pathogens and fungal biofilms, with no increased toxicity against mammalian cells. We show that gladiolin potentiates AmpB by increasing and accelerating membrane damage. Our findings also provide insights into the on-going debate about the mechanism of action of AmpB by indicating that both proposed mechanisms, extraction of ergosterol from membranes and pore formation, are potentiated by gladiolin.

Fission of double-membrane tubes under tension

The division of a cellular compartment culminates with the scission of a highly constricted membrane neck. Scission requires lipid rearrangements, topology changes, and transient formation of nonbilayer intermediate structures driven by curvature stress. Often, a side effect of this stress is pore-formation, which may lead to content leakage and thus breaching of the membrane barrier function. In single-membrane systems, leakage is avoided through the formation of a hemifusion (HF) intermediate, whose structure is still a subject of debate. The consequences of curvature stress have not been explored in double-membrane systems, such as the mitochondrion. Here, we combine experimental and theoretical approaches to study neck constriction and scission driven by tension in biomimetic lipid systems, namely single- and double-membrane nanotubes (sNTs and dNTs), respectively. In sNTs, constriction by high tension gives rise to a metastable HF intermediate (seen as stalk or worm-like micelle), whereas poration is universally slower in a simple neck. In dNTs, high membrane tension causes sequential rupture of each membrane. In contrast, low tension leads to the HF of both membranes, which may lead to a leaky fusion pathway, or may progress to further fusion of the two membranes along a number of transformation pathways. These findings provide a new mechanistic basis for fundamental cellular processes.

Cytotoxicity and cell membrane interactions of choline-based ionic liquids: Comparing amino acids, acetate, and geranate anions

Ionic liquids (ILs) have found diverse applications in research and industry. Biocompatible ILs, a subset considered less toxic than traditional ILs, have expanded their applications into biomedical fields. However, there is limited understanding of the toxicity profiles, safe concentrations, and underlying factors driving their toxicity. In this study, we investigated the cytotoxicity of 13 choline-based ILs using four different cell lines: Human dermal fibroblasts (HDF), epidermoid carcinoma cells (A431), cervical cancer cells (HeLa), and gastric cancer cells (AGS). Additionally, we explored the haemolytic activity of these ILs. Our findings showed that the cytotoxic and haemolytic activities of ILs can be attributed to the hydrophobicity of the anions and the pH of the IL solutions. Furthermore, utilising quartz crystal microbalance with dissipation (QCM-D), we delved into the interaction of selected ILs, including choline acetate [Cho][Ac] and choline geranate [Cho][Ge], with model cell membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The QCM-D data showed that ILs with higher toxicities exhibited more pronounced interactions with membranes. Increased variations in frequency and dissipation reflected substantial changes in membrane fluidity and mass following the addition of the more toxic ILs. Furthermore, total internal reflection fluorescence microscopy study revealed that [Cho][Ac] could cause lipid rearrangements and pore formation in the membrane, while [Cho][Ge] disrupted the bilayer packing. This study advances our understanding of the cellular toxicities associated with choline-based ILs and provides valuable insights into their mechanisms of action concerning IL-membrane interactions. These findings have significant implications for the safe and informed utilisation of biocompatible ILs in the realm of drug delivery and biotechnology.

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

Inhibition of the SREBP pathway prevents SARS-CoV-2 replication and inflammasome activation.

SARS-CoV-2 induces major cellular lipid rearrangements, exploiting the host's metabolic pathways to replicate. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that control lipid metabolism. SREBP1 is associated with the regulation of fatty acids, whereas SREBP2 controls cholesterol metabolism, and both isoforms are associated with lipid droplet (LD) biogenesis. Here, we evaluated the effect of SREBP in a SARS-CoV-2-infected lung epithelial cell line (Calu-3). We showed that SARS-CoV-2 infection induced the activation of SREBP1 and SREBP2 and LD accumulation. Genetic knockdown of both SREBPs and pharmacological inhibition with the dual SREBP activation inhibitor fatostatin promote the inhibition of SARS-CoV-2 replication, cell death, and LD formation in Calu-3 cells. In addition, we demonstrated that SARS-CoV-2 induced inflammasome-dependent cell death by pyroptosis and release of IL-1β and IL-18, with activation of caspase-1, cleavage of gasdermin D1, was also reduced by SREBP inhibition. Collectively, our findings help to elucidate that SREBPs are crucial host factors required for viral replication and pathogenesis. These results indicate that SREBP is a host target for the development of antiviral strategies.

Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody

The early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies.

The Ca2+- and phospholipid-binding protein Annexin A2 is able to increase and decrease plasma membrane order

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, HII). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.

Tight docking of membranes before fusion represents a metastable state with unique properties

Membrane fusion is fundamental to biological processes as diverse as membrane trafficking or viral infection. Proteins catalyzing membrane fusion need to overcome energy barriers to induce intermediate steps in which the integrity of bilayers is lost. Here, we investigate the structural features of tightly docked intermediates preceding hemifusion. Using lipid vesicles in which progression to hemifusion is arrested, we show that the metastable intermediate does not require but is enhanced by divalent cations and is characterized by the absence of proteins and local membrane thickening. Molecular dynamics simulations reveal that thickening is due to profound lipid rearrangements induced by dehydration of the membrane surface.

Plasma membrane wound repair is characterized by extensive membrane lipid and protein rearrangements in vascular endothelial cells

Vascular endothelial cells are subject to mechanical stress resulting from blood flow and interactions with leukocytes. Stress occurs at the apical, vessel-facing cell surface and leads to membrane ruptures that have to be resealed to ensure cell survival. To mimic this process, we developed a laser ablation protocol selectively inducing wounds in the apical plasma membrane of endothelial cells. We show that Ca2+-dependent membrane resealing is initiated following this wounding protocol and that the process is accompanied by substantial membrane lipid dynamics at the wound site. Specifically, phosphatidylinositol (4,5)-bisphosphate, phosphatidylserine and phosphatidic acid rapidly accumulate at membrane wounds forming potential interaction platforms for Ca2+/phospholipid binding proteins of the annexin (Anx) family that are also recruited within seconds after wounding. Depletion of one annexin, AnxA2, and its putative binding partner S100A11 interferes with membrane resealing suggesting that Ca2+-dependent annexin-phospholipid interactions are required for efficient membrane wound repair in endothelial cells.

Bilayer-Coated Nanoparticles to Probe the Effect of Membrane Deformability on Fusion intermediates

Viral membrane fusion kinetics is dominated by the slow kinetic steps driven by viral proteins, which mask the effect of the comparatively fast lipid rearrangements on the process. To probe these lipid intermediates we have developed an experimental set up based on the use of lipid coated silica nanoparticles. This results in target membranes with decreased deformability and defined curvature without any alteration of the lipid composition. by analyzing lipid mixing events on a single particle level we were able to dissect the effects of membrane deformability and curvature on the kinetics of the fusion process and the stoichiometry of viral proteins driving this process.

SsRNA Virus and Host Lipid Rearrangements: Is There a Role for Lipid Droplets in SARS-CoV-2 Infection?

Since its appearance, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has immediately alarmed the World Health Organization for its very high contagiousness and the complexity of patient clinical profiles. The worldwide scientific community is today gathered in a massive effort in order to develop safe vaccines and effective therapies in the shortest possible time. Every day, new pieces of SARS-CoV-2 infective puzzle are disclosed. Based on knowledge gained with other related coronaviruses and, more in general, on single-strand RNA viruses, we highlight underexplored molecular routes in which lipids and lipid droplets (LDs) might serve essential functions in viral infections. In fact, both lipid homeostasis and the pathways connected to lipids seem to be fundamental in all phases of the coronavirus infection. This review aims at describing potential roles for lipid and LDs in host–virus interactions and suggesting LDs as new and central cellular organelles to be investigated as potential targets against SARS-CoV-2 infection.

Membrane Engineering: Phase Separation in Polymeric Giant Vesicles.

Cell membranes exhibit elaborate lipidic patterning to carry out a myriad of functions such as signaling and trafficking. Domain formation in giant unilamellar vesicles (GUVs) is thus of interest for understanding fundamental biological processes and to provide new prospects for biocompatible soft materials. Lipid rearrangements in lipidic GUVs and lipid/polymer GUVs are extensively studied whereas polymer/polymer hybrid GUVs remain evasive. Here, the focus is on the thermodynamically driven phase separation of amphiphilic polymers in GUVs. It is demonstrated that polymer phase separation is entropically dictated by hydrophobic block incompatibilities and that films topology can help to determine the outcome of polymeric phase separation in GUVs. Lastly, Janus-GUVs are obtained and GUVs exhibit a single large domain by using a compatibilizing hydrophobic block copolymer.

Membranes with Decreased Deformability to Study the Kinetics of Fusion Intermediates

While the initial protein activation steps involved in influenza membrane fusion have been relatively well resolved, the subsequent lipid rearrangements are much more challenging to study. This is because the formation of lipid intermediates is faster than protein activation and thus masked in kinetic measurements of intact virus. To unmask these intermediates, we have developed methods that utilize target membranes with decreased deformability. By using supported lipid bilayers on silica nanoparticles of a defined diameter we are able to alter the deformability of the target membrane without changing its lipid composition. In conjunction with single-virus fusion kinetics, this allows us to examine the effect of membrane deformability on the kinetics of influenza membrane fusion. Our results show that decreasing target membrane deformability does indeed slow lipid mixing between virus and target. Further extensions to this platform then allow stepwise dissection of how membrane mechanical properties affect influenza membrane fusion.

Cold acclimation triggers lipidomic and metabolic adjustments in the spotted wing drosophila Drosophila suzukii (Matsumara).

Chronic cold exposure is detrimental to chill susceptible insects that may accumulate chill injuries. To cope with deleterious effects of cold temperature, insects employ a variety of physiological strategies and metabolic adjustments, such as production of cryoprotectants, or remodeling of cellular membranes. Cold tolerance is a key element determining the fundamental niche of species. Because Drosophila suzukii is an invasive fruit pest, originating from East Asia, knowledge about its thermal biology is urgently needed. Physiological mechanisms underlying cold tolerance plasticity remain poorly understood in this species. Here, we explored metabolic and lipidomic modifications associated with the acquisition of cold tolerance in D. suzukii using Omics technologies (LC- and GC-MS/MS). In both cold-acclimated males and females, we observed physiological changes consistent with homeoviscous/homeophasic adaptation of membranes: reshuffling of phospholipid head groups and increasing unsaturation rate of fatty acids. Modification of fatty acids unsaturation were also observed in triacylglycerides, which would likely increase accessibility of lipid reserves. At the metabolic level, we observed clear-cut differentiation of metabolic profiles with cold-acclimated metabotypes showing accumulation of several potential cryoprotectants (sugars and amino acids). Metabolic pathway analyses indicated a remodeling of various processes, including purine metabolism and aminoacyl tRNA biosynthesis. These data provide a large-scale characterization of lipid rearrangements and metabolic pathway modifications in D. suzukii in response to cold acclimation and contribute to characterizing the strategies used by this species to modulate cold tolerance.

The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment

Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2TM22, or a peptide mutated at the central residues G100/C103 (VAMP2TM22VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2TM22, in terms of α-helices and β-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G100/C103 within the transmembrane domain during lipid rearrangements in membrane fusion.

Orchestration of transcriptome, proteome and metabolome in the diatom Phaeodactylum tricornutum during nitrogen limitation

Nitrogen deprivation increases the triacylglycerol (TAG) content in microalgae but also severely decreases the growth rate. Most approaches that attempted to increase TAG productivity by overexpression or knockdown of specific genes related to the regulation of the lipid synthesis have reported only little success. More insight into the molecular mechanisms related to lipid accumulation and impaired growth rate is needed to find targets for improving TAG productivity. By using the emerging “omics” approach, we comprehensively profiled the physiology, transcriptome, proteome and metabolome of the diatom Phaeodactylum tricornutum during steady state growth at both nitrogen limited and replete levels during light:dark cycles. Under nitrogen limited conditions, 22% (2699) of the total identified transcripts, 17% (543) of the proteins and 44% (345) of the metabolites were significantly differentially regulated compared to nitrogen replete growth conditions. Although nitrogen limitation was responsible for the majority of significant differential transcript, protein and metabolite accumulation, we also observed differential expression over a diurnal cycle. Nitrogen limitation mainly induced an upregulation of nitrogen fixation, central carbon metabolism and TCA cycle, while photosynthetic and ribosomal protein synthesis are mainly downregulated. Regulation of the lipid metabolism and the expression of predicted proteins involved in lipid processes suggest that lipid rearrangements may substantially contribute to TAG distribution. However, TAG synthesis is also limited by the reduced carbon flux through central metabolism. Future strain improvements should therefore focus on understanding and improving the carbon flux through central carbon metabolism, selectivity and activity of DGAT isoforms and lipase enzymes.

The gut microbiota drives the impact of bile acids and fat source in diet on mouse metabolism

BackgroundAs the gut microbiota contributes to metabolic health, it is important to determine specific diet-microbiota interactions that influence host metabolism. Bile acids and dietary fat source can alter phenotypes of diet-induced obesity, but the interplay with intestinal microorganisms is unclear. Here, we investigated metabolic consequences of diets enriched in primary bile acids with or without addition of lard or palm oil, and studied gut microbiota structure and functions in mice.ResultsIn combination with bile acids, dietary lard fed to male C57BL/6N mice for a period of 8 weeks enhanced fat mass accumulation in colonized, but not in germ-free mice when compared to palm oil. This was associated with impaired glucose tolerance, lower fasting insulin levels, lower counts of enteroendocrine cells, fatty liver, and elevated amounts of hepatic triglycerides, cholesteryl esters, and monounsaturated fatty acids. Lard- and bile acid-fed mice were characterized by shifts in dominant gut bacterial communities, including decreased relative abundances of Lachnospiraceae and increased occurrence of Desulfovibrionaceae and the species Clostridium lactatifermentans and Flintibacter butyricus. Metatranscriptomic analysis revealed shifts in microbial functions, including lipid and amino acid metabolism.ConclusionsCaution is required when interpreting data from diet-induced obesity models due to varying effects of dietary fat source. Detrimental metabolic consequences of a diet enriched with lard and primary bile acids were dependent on microbial colonization of the host and were linked to hepatic lipid rearrangements and to alterations of dominant bacterial communities in the cecum.

High Hydrostatic Pressure Induces a Lipid Phase Transition and Molecular Rearrangements in Low-Density Lipoprotein Nanoparticles.

Low-density lipoproteins (LDL) are natural lipid transporter in human plasma whose chemically modified forms contribute to the progression of atherosclerosis and cardiovascular diseases accounting for a vast majority of deaths in westernized civilizations. For the development of new treatment strategies, it is important to have a detailed picture of LDL nanoparticles on a molecular basis. Through the combination of X-ray and neutron small-angle scattering (SAS) techniques with high hydrostatic pressure (HHP) this study describes structural features of normolipidemic, triglyceride-rich and oxidized forms of LDL. Due to the different scattering contrasts for X-rays and neutrons, information on the effects of HHP on the internal structure determined by lipid rearrangements and changes in particle shape becomes accessible. Independent pressure and temperature variations provoke a phase transition in the lipid core domain. With increasing pressure an inter-related anisotropic deformation and flattening of the particle are induced. All LDL nanoparticles maintain their structural integrity even at 3000 bar and show a reversible response toward pressure variations. The present work depicts the complementarity of pressure and temperature as independent thermodynamic parameters and introduces HHP as a tool to study molecular assembling and interaction processes in distinct lipoprotein particles in a nondestructive manner.

Assembly of intermediates for rapid membrane fusion

Membrane fusion is essential for intracellular protein sorting, cell growth, hormone secretion, and neurotransmission. Rapid membrane fusion requires tethering and Sec1-Munc18 (SM) function to catalyze R-, Qa-, Qb-, and Qc-SNARE complex assembly in trans, as well as SNARE engagement by the SNARE-binding chaperone Sec17/αSNAP. The hexameric vacuolar HOPS (homotypic fusion and vacuole protein sorting) complex in the yeast Saccharomyces cerevisiae tethers membranes through its affinities for the membrane Rab GTPase Ypt7. HOPS also has specific affinities for the vacuolar SNAREs and catalyzes SNARE complex assembly, but the order of their assembly into a 4-SNARE complex is unclear. We now report defined assembly intermediates on the path to membrane fusion. We found that a prefusion intermediate will assemble with HOPS and the R, Qa, and Qc SNAREs, and that this assembly undergoes rapid fusion upon addition of Qb and Sec17. HOPS-tethered membranes and all four vacuolar SNAREs formed a complex that underwent an even more dramatic burst of fusion upon Sec17p addition. These findings provide initial insights into an ordered fusion pathway consisting of the following intermediates and events: 1) Rab- and HOPS-tethered membranes, 2) a HOPS:R:Qa:Qc trans-complex, 3) a HOPS:4-SNARE trans-complex, 4) an engagement with Sec17, and 5) the rapid lipid rearrangements during fusion. In conclusion, our results indicate that the R:Qa:Qc complex forms in the context of membrane, Ypt7, HOPS, and trans-SNARE assembly and serves as a functional intermediate for rapid fusion after addition of the Qb-SNARE and Sec17 proteins.

Antivirals acting on viral envelopes via biophysical mechanisms of action

Most antivirals target viral proteins and are specific for only one virus, or viral type. Whereas viral proteins are encoded in the plastic viral genome, virion lipids are not and their rearrangements during fusion are conserved among otherwise unrelated enveloped viruses. Antivirals that inhibit these lipid rearrangements could thus pose a high barrier to resistance and have broad-spectrum activity.Fusion occurs through a hemifusion stalk in which only the outer leaflets are fused and thus curved with a smaller radius for the polar heads than for the hydrophobic tails (negative curvature). Outer leaflets enriched in phospholipids with head groups of larger cross sections than their lipid tails (“inverted cone”) disfavor negative curvature, inhibiting fusion. The rigid amphipathic fusion inhibitors (RAFIs) are synthetic compounds of inverted cone molecular geometry. They inhibit infectivity of otherwise unrelated enveloped viruses. The leading RAFI, aUY11, has an ethynyl-perylene hydrophobic and an uracil-arabinose polar moiety. aUY11 intercalates in viral envelopes and inhibits virion-to-cell fusion of a broad spectrum of otherwise unrelated enveloped viruses. Previous studies showed that amphipathicity, rigidity, and inverted cone molecular geometry were required. We propose that the inverted cone molecular geometry of the RAFIs increases the energy barrier for the hemifusion stalk, inhibiting fusion. Then, chemically distinct compounds with similar amphipathicity, rigidity, and inverted cone shape would have similar antiviral potencies, regardless of specific chemical groups. Alternatively, the perylene group exposed to visible light may induce viral lipid peroxidation. Then, the perylene group and absorbance at visible spectrum would be required. We now evaluated twenty-five chemically distinct RAFIs. The perylene moiety and absorption at visible spectrum were not required, but a minimum length of the hydrophobic moiety was, 10.3 Å. The arabino moiety could be modified or replaced by other groups. Cytidine was not tolerated. Bilayer intercalation was required but not sufficient. The vast majority of RAFIs had no overt cytotoxicity (CC50 > 20 μM; TI > 250–1200). Carbonyl or butylamide substitutions for arabino, or cytidine replacement for uracil, increased cytotoxicity. Cytotoxicity was mainly determined by the polar moiety and there was no correlation between antiviral and cytostatic activities.The definition of the effects of shape and chemical groups of the RAFIs opens the possibility to the rational design of lipid-acting antivirals active against a broad spectrum of enveloped viruses.