Mammalian Cell Membranes Research Articles

Cholesterol occupies the lipid translocation pathway to block phospholipid scrambling by a G protein-coupled receptor

Class A (rhodopsin-like) G protein-coupled receptors (GPCRs) are constitutive phospholipid scramblases as evinced after their reconstitution into liposomes. Yet phospholipid scrambling is not detectable in the resting plasma membrane of mammalian cells that is replete with GPCRs. We considered whether cholesterol, a prominent component of the plasma membrane, limits the ability of GPCRs to scramble lipids. Our previous Markov State Model (MSM) analysis of molecular dynamics simulations of membrane-embedded opsin indicated that phospholipid headgroups traverse a dynamically revealed hydrophilic groove between transmembrane helices (TM) 6 and 7 while their tails remain in the bilayer. Here, we present comparative MSM analyses of 150-μs simulations of opsin in cholesterol-free and cholesterol-rich membranes. Our analyses reveal that cholesterol inhibits phospholipid scrambling by occupying the TM6/7 interface and stabilizing the closed groove conformation while itself undergoing flip-flop. This mechanism may explain the inability of GPCRs to scramble lipids at the plasma membrane.

Structural, Functional and Computational Studies of Membrane Recognition by Plasmodium Perforin-Like Proteins 1 and 2

Perforin-like proteins (PLPs) play key roles in mechanisms associated with parasitic disease caused by the apicomplexan parasites Plasmodium and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1. Determination of the crystal structure of the membrane-binding APCβ domain of PvPLP1 reveals notable differences with TgPLP1, reflected in its inability to bind lipid bilayers as TgPLP1 and PvPLP2 do. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal similar tropisms for lipids enriched in the inner leaflet of the mammalian plasma membrane. In addition PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APCβ domains of apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins in parasitic life cycles and disease.

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties.

Lipid rafts are ordered lipid domains that are enriched in saturated lipids, such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have not been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here, we report that microscopic GM1-enriched domains can form, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, surprisingly, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biophysical chemistry of lipid rafts.

Curvature-based sorting of eight lipid types in asymmetric buckled plasma membrane models

Curvature is a fundamental property of biological membranes and has essential roles in cellular function. Bending of membranes can be induced by their lipid and protein compositions, as well as peripheral proteins, such as those that make up the cytoskeleton. An important aspect of membrane function is the grouping of lipid species into microdomains, or rafts, which serve as platforms for specific biochemical processes. The fluid mosaic model of membranes has evolved to recognize the importance of curvature and leaflet asymmetry, and there are efforts toward evaluating their functional roles. This work investigates the effect of curvature on the sorting of lipids in buckled asymmetric bilayers containing eight lipid types, approximating an average mammalian plasma membrane, through coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field. The simulations reveal that 1) leaflet compositional asymmetry can induce curvature asymmetry, 2) lipids are sorted by curvature to different extents, and 3) curvature-based partitioning trends show moderate to strong correlations with lipid molecular volumes and head to tail bead ratios, respectively. The findings provide unique insights into the role of curvature in membrane organization, and the curvature-based sorting trends should be useful references for later investigations and potentially interpreting the functional roles of specific lipids.

Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.

Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid β-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.

Dissociation of β2m from MHC class I triggers formation of noncovalent transient heavy chain dimers.

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (β2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for β2m.

Long-Chain Poly-d-Lysines Interact with the Plasma Membrane and Induce Protective Autophagy and Intense Cell Necrosis.

Polylysines have been frequently used in drug delivery and antimicrobial and cell adhesion studies. Because of steric hindrance, chirality plays a major role in the functional difference between poly-l-lysine (PLL) and poly-d-lysine (PDL), especially when they interact with the plasma membranes of mammalian cells. Therefore, it is speculated that the interaction between chiral polylysines and the plasma membrane may cause different cellular behaviors. Here, we carefully investigated the interaction pattern of PLL and PDL with plasma membranes. We found that PDL could be anchored onto the plasma membrane and interact with the membrane lipids, leading to the rapid morphological change and death of A549 cells (a human lung cancer cell line) and HPAEpiCs (a human pulmonary alveolar epithelial cell line). In contrast, PLL exhibited good cytocompatibility and was not anchored onto the plasma membranes of these cells. Unlike PLL, PDL could trigger protective autophagy to prevent cells in a certain degree, and the PDL-caused cell death occurred via intense necrosis (featured by increased intracellular Ca2+ content and plasma membrane disruption). In addition, it was found that the short-chain PDL with a repeat unit number of 9 (termed DL9) could locate in lysosomes and induce autophagy at high concentrations, but it could not elicit drastic cell death, which proved that the repeat unit number of polylysine could affect its cellular action. This research confirms that the interaction between chiral polylysines and the plasma membrane can induce autophagy and intense necrosis, which provides guidance for the future studies of chiral molecules/drugs.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2β and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

Effects of cholesterol on the mechanism of fengycin, a biofungicide

Fengycins are a class of antifungal lipopeptides synthesized by the bacteria Bacillus subtilis, commercially available as the primary component of the agricultural fungicide Serenade. They are toxic to fungi but far less to mammalian cells. One key difference between mammalian and fungal cell membranes is the presence of cholesterol only in the former; recent experimental work showed that the presence of cholesterol reduces fengycin-induced membrane leakage. Since our previous all-atom and coarse-grained simulations suggested that aggregation of membrane-bound fengycin is central to its ability to disrupt membranes, we hypothesized that cholesterol might reduce fengycin aggregation. Here, we test this hypothesis using coarse-grained molecular dynamics simulations, with sampling enhanced via the weighted ensemble method. The results indicate that cholesterol subtly alters the size distribution for fengycin aggregates, limits the lateral range of their membrane disordering, and reduces the ability of aggregates to bend the membrane. Taken together, these phenomena may account for cholesterol’s effects on fengycin activity.

Inhibiting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants: Targeting the Spike and Envelope Proteins Using Nanomaterial Like Peptides.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused significant death, economic crisis, and the world to almost completely shut down. This present study focused on targeting the novel SARS-CoV-2 envelope protein, which has not been frequently mutating, and the S protein with a much larger peptide capable of inhibiting virus-mammalian cell attraction. In doing so, molecular dynamics software was used here to model six peptides including: NapFFTLUFLTUTE, NapFFSLAFLTATE, NapFFSLUFLSUTE, NapFFTLAFLTATE, NapFFSLUFLSUSE, and NapFFMLUFLMUME. Results showed that two of these completely hydrophobic peptides (NapFFTLUFLTUTE and NapFFMLUFLMUME) had a strong ability to bind to the virus, preventing its binding to a mammalian cell membrane, entering the cell, and replicating by covering many cell attachment sites on SARS-CoV-2. Further cell modeling results demonstrated the low toxicity and suitable pharmacokinetic properties of both peptides making them ideal for additional in vitro and in vivo investigation. In this manner, these two peptides should be further explored for a wide range of present and future COVID-19 therapeutic and prophylactic applications.

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes.

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane–saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air–aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.

N-(3-Oxododecanoyl) Homoserine Lactone Is a Generalizable Plasma Membrane Lipid-Ordered Domain Modifier.

A mammalian plasma membrane is a structure on which several layers of complexity are built. The first order of complexity comes from the heterogeneity of lipid-ordered domains. Gangliosides in concert with cholesterol are preferentially packed on the outer leaflet and form lipid-ordered domains, commonly known as lipid rafts. The formation and dynamics of these domains impact nearly all membrane protein functions and are an intensely studied topic. However, tools suited for lipid domain alteration are extremely limited. Currently, methyl-β-cyclodextrin (MβCD) appears to be the most common way to disrupt lipid domains, which is believed to operate via cholesterol extraction. This significantly limits our ability in membrane biophysics research. Previously, we found that N-(3-oxo-dodecanoyl) homoserine lactone (3oc), a small signaling chemical produced by Pseudomonas aeruginosa, is highly efficient in altering lipid-ordered domains. In this study, 3oc was compared with MβCD in a series of biochemical, biophysical, and cell biological analyses. Per molarity, 3oc is more efficient than MβCD in domain alteration and appears to better retain membrane lipids after treatment. This finding will provide an essential reagent in membrane biophysics research.

The Applications of Metabolic Glycoengineering.

Mammalian cell membranes are decorated by the glycocalyx, which offer versatile means of generating biochemical signals. By manipulating the set of glycans displayed on cell surface, it is vital for gaining insight into the cellular behavior modulation and medical and biotechnological adhibition. Although genetic engineering is proven to be an effective approach for cell surface modification, the technique is only suitable for natural and genetically encoded molecules. To circumvent these limitations, non-genetic approaches are developed for modifying cell surfaces with unnatural but functional groups. Here, we review latest development of metabolic glycoengineering (MGE), which enriches the chemical functions of the cell surface and is becoming an intriguing new tool for regenerative medicine and tissue engineering. Particular emphasis of this review is placed on discussing current applications and perspectives of MGE.

Development of Duramycin-Based Molecular Probes for Cell Death Imaging.

Cell death is involved in numerous pathological conditions such as cardiovascular disorders, ischemic stroke and organ transplant rejection, and plays a critical role in the treatment of cancer. Cell death imaging can serve as a noninvasive means to detect the severity of tissue damage, monitor the progression of diseases, and evaluate the effectiveness of treatments, which help to provide prognostic information and guide the formulation of individualized treatment plans. The high abundance of phosphatidylethanolamine (PE), which is predominantly confined to the inner leaflet of the lipid bilayer membrane in healthy mammalian cells, becomes exposed on the cell surface in the early stages of apoptosis or accessible to the extracellular milieu when the cell suffers from necrosis, thus representing an attractive target for cell death imaging. Duramycin is a tetracyclic polypeptide that contains 19 amino acids and can bind to PE with excellent affinity and specificity. Additionally, this peptide has several favorable structural traits including relatively low molecular weight, stability to enzymatic hydrolysis, and ease of conjugation and labeling. All these highlight the potential of duramycin as a candidate ligand for developing PE-specific molecular probes. By far, a couple of duramycin-based molecular probes such as Tc-99m-, F-18-, or Ga-68-labeled duramycin have been developed to target exposed PE for in vivo noninvasive imaging of cell death in different animal models. In this review article, we describe the state of the art with respect to in vivo imaging of cell death using duramycin-based molecular probes, as validated by immunohistopathology.

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.

Cholesterol stiffening of model mammalian and microbial membranes

Cholesterol plays an essential role in mammalian cell membranes by controlling membrane fluidity, organization, and other physicochemical parameters. Such material properties emerge from atomistic level interactions of the lipids with cholesterol [1,2]. Membrane stiffness plays a central role in various biological processes, including vesiculation, protein-membrane interactions, viral budding, and the mechanical stability of cells against pathogen attacks. Our studies show that the degree of membrane stiffening by cholesterol in model di-monounsaturated phosphatidylcholine and phosphatidylethanolamine (DOPC versus DOPE)lipid bilayers depends on both headgroup size and acyl chain unsaturation.

Challenging old dogma with new tech: asymmetry of lipid abundances within the plasma membrane

In the 1970s, it was established that the plasma membrane (PM) is asymmetric in the phospholipid compositions of its two leaflets—the exoplasmic one is enriched in phospholipids with uncharged headgroups, compared to the endoplasmic leaflet whose phospholipids are negatively charged. Repurposing classical strategies and coupling them with high-resolution shotgun lipidomics, we quantified the detailed lipid compositions of the two leaflets of the mammalian PM. In addition to largely recapitulating classical headgroup asymmetries, we found that the endoplasmic leaflet contained 1.7-2-fold more phospholipids compared to the exoplasmic leaflet.

Rivalry of cholesterol and antimicrobial peptides as seen by molecular simulations and NMR spectroscopy

Antimicrobial peptides (AMPs) exhibit cell selectivity and activity against microorganisms and are promising candidates as pharmaceutical agents. In mammalian cell membranes cholesterol plays a regulatory function in antibiotic drug resistance and the immune response. Our hypothesis is that differences in the peptide-membrane interactions versus cholesterol affect the bilayer properties giving a possible framework for selectivity of AMPs for bacterial membranes. Here we employed solid-state 2H NMR to compare the degree of softening or stiffening by AMPs compared to cholesterol in model di-monounsaturated phosphatidylcholine and phosphatidylethanolamine lipids (DOPC vs.

Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A

Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes to form nanopores spanning the lipid bilayer, which causes cell lysis and promotes further infections. Cytolysin A (ClyA), an alpha (α)-PFT, is known to undergo one of the largest conformational changes during its transition from a water-soluble monomeric form to a membrane-embedded dodecameric pore complex. Despite extensive work on ClyA structure and assembly, what features of the protein drive this transformation is poorly understood.

Structure, Function and Regulation of the Plasma Membrane Calcium Pump in Health and Disease.

In this review, I summarize the present knowledge of the structural and functional properties of the mammalian plasma membrane calcium pump (PMCA). It is outlined how the cellular expression of the different spliced isoforms of the four genes are regulated under normal and pathological conditions.