The transporter associated with antigen processing (TAP) plays a key role in adaptive immunity by translocating proteasomal degradation products from the cytosol into the endoplasmic reticulum lumen ...for subsequent loading onto major histocompatibility (MHC) class I molecules. For functional and structural analysis of this ATP-binding cassette complex, we established the overexpression of TAP in the methylotrophic yeast Pichia pastoris. Screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex, yielding 30 mg of TAP/liter of culture. Detailed analysis of TAP function in the membrane, solubilized, purified, and reconstituted states revealed a direct influence of the native lipid environment on activity. TAP-associated phospholipids, essential for function, were profiled by liquid chromatography Fourier transform mass spectrometry. The antigen translocation activity is stimulated by phosphatidylinositol and -ethanolamine, whereas cholesterol has a negative effect on TAP activity.
•Antibiotics are among the most frequently prescribed medications in modern medicine.•Biophysical interaction studies constitute a platform for antibiotics development.•Drug-membrane interaction ...studies allow to predict the ADME-Tox of new molecules.•Biophysical studies permit to estalish drug’structure/membrane’activity correlation.
Lipidomics and proteomics have undergone a tremendous revolution, and the knowledge about drugs’ mechanism of action in biological membranes has been deepened. Methods to study the interactions of drugs with biological membranes have opened new perspectives to rational drug design, based not only in the pharmacological target of the drugs but also on the interaction with biological membranes. These methods expand our ability to acquire the ADME-Tox profile of drugs, simplifying the complexity of biological membranes. Particularly, antibiotic resistance is considered one of the greatest threats to human health, being the prospects for replacing current antimicrobial drugs extremely scarce. With the decline of the discovery and the emergence of multidrug resistant pathogens to the existing arsenal, the objective in the development of new drugs to combat the resistance to antibiotics has been replaced by the modification of existing antibiotics. Therefore, drug-membrane interaction studies using membrane models of the eukaryotic and prokaryotic cell membranes, associated with a broad of complementary methods, may contribute to a deep picture concerning the effect of antibiotics upon their intake until their pharmacological target. This critical review will discuss the relevance of a range of different methods to study the interaction of antibiotic drugs using liposomes as biological membranes models. The advantages and the limitations of these methods will be discussed and future perspectives in this field will be proposed.
Cholesterol is a crucial component of mammalian cell membranes that takes part in many vital processes. It is generally accepted that cholesterol stabilizes the membrane and induces transitions into ...ordered states. In contrast to expectations, we demonstrate that cholesterol can destabilize the membrane by creating a nanodomain around a perpendicularly embedded ultrashort carbon nanotube (CNT), and we show that cholesterol triggers the translocation of an ultrashort CNT through the cell membrane. Using atomistic simulations, we report the existence of a nanoscale domain around an ultrashort carbon nanotube within a crossover distance of 0.9 nm from the surface of the nanotube, where the properties of the bilayer are different from the bulk: the domain is characterized by increased fluctuations, increased thickness, and increased order of the lipids with respect to the bulk. Cholesterol decreases the thickness and order of lipids and increases the fluctuations with respect to a pure lipid bilayer. Experimentally, we confirm that cholesterol nanodomains provoke spontaneous translocation of nanotubes through a lipid bilayer even for low membrane tensions. A specially designed microfluidic device allows us to trace the kinetic pathway of the translocation process and establish the threshold cholesterol concentration of 20% for translocation. The reported nanoscale cholesterol-induced membrane restructuring near the ultrashort CNT in lipid membranes enables precise control and specific targeting of a membrane using cholesterol. As an example, it may allow for specific targeting between cholesterol-rich mammalian cells and cholesterol-poor bacterial cells.
Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating ...proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR’s intracellular module with the membrane are critical in maintaining this coupling.
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► Full-length EGFRs are modeled in a realistic membrane environment ► The models show how EGF binding controls the extracellular domains in EGFR dimers ► The trans- and juxtamembrane segments alternate between two dimer forms as a result ► Anionic lipids in the membrane are critical to the regulation of the kinase domains
Atomistic models of full-length EGFR based on computer simulations illustrate the conformational coupling between the extra- and intracellular components and show that the receptor’s interactions with the anionic lipids in the inner leaflet of the cell membrane are critical to its regulation.
Membrane lipids and cell signaling Sunshine, Hannah; Iruela-Arispe, Maria Luisa
Current opinion in lipidology
28, Issue:
5
Journal Article
Peer reviewed
Open access
Reception and transmission of signals across the plasma membrane has been a function generally attributed to transmembrane proteins. In the last 3 years, however, a growing number of reports have ...further acknowledged important contributions played by membrane lipids in the process of signal transduction.
In particular, the constituency of membrane lipids can regulate how proteins with SH2 domains and molecules like K-Ras expose their catalytic domains to the cytosol and interact with effectors and second messengers. Recent reports have also shown that the degree of saturation of phospholipids can reduce the activation of certain G-protein-coupled receptors, and signaling downstream to Toll-like receptor 4 with consequences to nuclear factor kappa B activation and inflammation. Levels of specific gangliosides in the membrane were reported to activate integrins in a cell-autonomous manner affecting tumor cell migration. Furthermore, high resolution of the association of cholesterol with the smoothened receptor has clarified its participation in sonic hedgehog signaling. These are some of the key advancements that have further propelled our understanding of the broad versatile contributions of membrane lipids in signal transduction.
As we gain definitive detail regarding the impact of lipid-protein interactions and their consequences to cell function, the options for therapeutic targeting expand with the possibility of greater specificity.
Glycerolipid metabolism of plants responds dynamically to changes in light intensity and temperature, leading to the modification of membrane lipid composition to ensure optimal biochemical and ...physical properties in the new environment Although multiple posttranscriptional regulatory mechanisms have been reported to be involved in the process, the contribution of transcriptional regulation remains largely unknown. Here, we present an integrative analysis of transcriptomic and lipidomic data, revealing largescale coordination between gene expression and changes in glycerolipid levels during iheArabidopsis thaliana response to light and temperature stimuli. Using a murtivariate regression technique called O2PLS, we show that the gene expression response is strictly coordinated at the biochemical pathway level and occurs in parallel with changes of specific glycerolipid pools. Five interesting candidate genes were chosen for further analysis from a larger set of candidates identified based on their close association with various groups of glycerolipids. Lipidomic analysis of knockout mutant lines of these five genes showed a significant relationship between the coordination of transcripts and glycerolipid levels in a changing environment and the effects of single gene perturbations.
In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. ...Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.
The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. ...The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15-32%. When the same quenchers have access to both leaflets, they quench 70-80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60-70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes approximately 40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.
The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We ...recently developed a chemical–genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.
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